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tyo:4503
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Astellas Pharma
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Apr 26th, 2022 12:00AM
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Jul 15th, 2021 12:00AM
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https://www.uspto.gov?id=US11312966-20220426
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Nucleic acid for treating mite allergy
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[Problem] To provide a nucleic acid expected to be useful for treating mite allergy.
[Means to be solved] Provided is a nucleic acid comprising a nucleotide sequence encoding a chimeric protein, wherein the nucleic acid comprises a nucleotide sequence encoding a signal peptide, a nucleotide sequence encoding an intra-organelle stabilizing domain of LAMP, a nucleotide sequence encoding an allergen domain comprising Der p 1, Der p 2, Der p 23, and Der p 7, a nucleotide sequence encoding a transmembrane domain and a nucleotide sequence encoding an endosomal/lysosomal targeting domain of LAMP in this order.
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11312966
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1. A nucleic acid encoding a chimeric protein, wherein the nucleic acid comprises the following nucleotide sequences in this order:
a nucleotide sequence encoding a signal peptide;
a nucleotide sequence encoding an intra-organelle stabilizing domain of lysosome-associated membrane proteins (LAMP) consisting of amino acids 28 to 380 of SEQ ID NO: 2;
a nucleotide sequence encoding an allergen domain consisting of amino acids 383 to 1002 of SEQ ID NO: 2;
a nucleotide sequence encoding a transmembrane domain of LAMP-1 consisting of amino acids 1006 to 1028 of SEQ ID NO: 2; and
a nucleotide sequence encoding an endosomal/lysosomal targeting domain of LAMP-1 consisting of amino acids 1037 to 1040 of SEQ ID NO: 2.
2. The nucleic acid according to claim 1, wherein the signal peptide is a signal peptide of LAMP.
3. The nucleic acid according to claim 1, wherein the signal peptide consists of amino acid numbers 1 to 27 of SEQ ID NO: 2.
4. An expression vector comprising:
the nucleic acid according to claim 1.
5. A pharmaceutical composition comprising:
the expression vector according to claim 4 and a pharmaceutically acceptable excipient.
6. A host cell transformed with the nucleic acid according to claim 1.
7. A method for producing a chimeric protein, comprising:
culturing a host cell transformed with the nucleic acid according to claim 1.
8. A nucleic acid encoding a chimeric protein consisting of an amino acid sequence having at least 90% identity to SEQ ID NO: 2.
9. A nucleic acid comprising:
a) a nucleotide sequence encoding a chimeric protein consisting of the nucleic acid sequence of SEQ ID NO: 2; or
b) a nucleotide sequence encoding a chimeric protein consisting of an amino acid sequence of SEQ ID NO: 2 in which 1 to 10 amino acids are deleted, substituted, inserted and/or added.
10. A nucleic acid comprising:
a nucleotide sequence encoding a chimeric protein consisting of the amino acid sequence of SEQ ID NO: 2.
11. An expression vector comprising:
the nucleic acid according to claim 10.
12. A pharmaceutical composition comprising:
the expression vector according to claim 11 and a pharmaceutically acceptable excipient.
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CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a Continuation of U.S. application Ser. No. 17/054,340, filed Nov. 10, 2020, which is the U.S. National Stage application of PCT/JP2019/018657, filed May 10, 2019, which claims priority to JP 2018-091963, filed May 11, 2018. The entire contents of each of the aforementioned applications are incorporated herein by reference.
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-WEB and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Nov. 9, 2020, is named sequence.txt and is 32,768 bytes.
TECHNICAL FIELD
The present invention relates to a nucleic acid which is expected to be useful as an active ingredient of a pharmaceutical composition, for example, a nucleic acid which is expected to be useful for treating mite allergy.
BACKGROUND ART
Mite allergy is an allergic disease that occurs in response to mite-derived allergens. The allergic disease is caused by the following steps: 1) allergens taken into a body are phagocytosed by antigen-presenting cells and presented to naive T cells, 2) the naive T cells are differentiated into Th2 cells, 3) cytokine such as IL-4 is produced from an immune cell such as the Th2 cell, 4) B cells produce IgE by IL-4, and 5) IgE binding to the allergens binds to mast cells. It has been known that in allergic disease patients, antagonism between Th1-type immunity involving Th1 cells producing IFN-γ or the like and Th2-type immunity involving Th2 cells producing IL-4 or the like shifts to Th2-type dominant which results in Th2-type inflammatory immune response (Middleton's Allergy Seventh edition Principles & Practice, 2009). Thus, IFN-γ can be used as an indicator of Th1-type immunity, and IL-4 can be used as an indicator of Th2-type immunity. Further, in mice, IFN-γ causes a preferential class switch to IgG2a isotype in activated B cells, while suppresses responses to all the other isotypes. That is, IgG2a can also be used as an indicator of Th1-type immunity. For example, it has been known that production of IgG2a is promoted in IL-4-deficient mice and that IgG2a production is suppressed in IFN-γ-deficient mice (Arthritis Res., 2002, Vol. 4, p. 54-58). There is also a report that antibodies produced from B cells are involved in the mechanism of action of allergen immunotherapy. For example, it has been known that in humans, IgG antagonizes IgE binding to an allergen to inhibit formation of allergen-IgE complex and thereby inhibit histamine release from mast cells (J Allergy Clin Immunol., 2017, Vol. 140, p. 1485-1498).
Until now, development of multiple immunotherapies for mite allergy has been advanced (J Allergy Clin Immunol., 2013, Vol. 132, p. 1322-1336; WO 2014/195803; Expert Rev Vaccines., 2014, Vol. 13, p. 1427-1438). Further, Der p 1, Der p 2, Der p 7, Der p 23, and the like have been known as allergens related to the mite allergy (Patent Documents 1 and 2, and Non-Patent Document 1). However, for example, subcutaneous immunotherapy (SCIT) and sublingual immunotherapy (SLIT) have problems such as possibility of anaphylaxis and long treatment period over several years.
As one of the techniques for nucleic acid vaccines, nucleic acid vaccines for treating allergy using lysosome-associated membrane proteins (LAMP) have been studied.
Further, a plasmid comprising a nucleic acid encoding a chimeric protein comprising LAMP-1, which is a member of LAMP family, and Cry J1 and/or Cry J2, which are allergens of Cryptomeria japonica, was constructed (Patent Document 3 and Non-Patent Document 2). It has been reported that such a plasmid does not cause systemic release of free allergen which causes anaphylaxis, but induces a Th1-type immune response. Furthermore, it has been reported that a plasmid comprising a nucleic acid encoding a chimeric protein comprising LAMP-1 and peanut allergens Ara H1, Ara H2 and Ara H3 reduced production of IgE in a mouse model (Patent Document 4). In a field of mite allergy, a vaccine comprising a nucleic acid encoding a chimeric protein comprising Der p 1 and a transmembrane domain of LAMP-1 and an endosomal/lysosomal targeting domain has been constructed (Patent Document 5 and Non-Patent Document 3). However, a nucleic acid vaccine for treating mite allergy comprising multiple mite allergen antigens, and an intra-organelle stabilizing domain of LAMP-1 and an endosomal/lysosomal targeting domain has not been reported.
RELATED ART
Patent Document
[Patent Document 1] WO 1988/010297
[Patent Document 2] WO 2007/124524
[Patent Document 3] WO 2013/187906
[Patent Document 4] WO 2015/200357
[Patent Document 5] WO 2004/019978
Non-Patent Document
[Non-Patent Document 1] “Clinical & Experimental Allergy”, (UK), 1995; 25: 416-422
[Non-Patent Document 2] “Journal of Immunology Research”, (Egypt), 2016; Article ID 4857869
[Non-Patent Document 3] “Vaccine”, (Netherlands), 2006; 24 (29-30): 5762-5771
SUMMARY OF INVENTION
Problems to Be Solved by the Invention
An object of the present invention is to provide a nucleic acid which is expected to be useful for treating mite allergy.
Means for Solving the Problems
As a result of repeated investigation with considerable creativity in the preparation of nucleic acids for treating mite allergy, the present inventors have prepared LAMP-Der p 1-Der p 2-Der p 23-Der p 7 plasmid (Example 1), confirmed that a chimeric protein is expressed from the plasmid (Example 2), and found that a Th1-type immune response is induced in mice to which the plasmid is administered (Examples 3 and 4). As a result, a nucleic acid which is expected to be useful for treating mite allergy is provided, and thereby the present invention has been completed.
That is, the present invention relates to the following [1] to [17].
[1]
A nucleic acid comprising:
a nucleotide sequence encoding a chimeric protein,
wherein the nucleotide sequence is a nucleotide sequence comprising the following nucleotide sequences in this order:
a nucleotide sequence encoding a signal peptide;
a nucleotide sequence encoding an intra-organelle stabilizing domain of LAMP;
a nucleotide sequence encoding an allergen domain comprising Der p 1, Der p 2, Der p 23, and Der p 7;
a nucleotide sequence encoding a transmembrane domain; and
a nucleotide sequence encoding an endosomal/lysosomal targeting domain of LAMP.
[2]
A nucleic acid comprising:
a nucleotide sequence encoding a chimeric protein,
wherein the nucleotide sequence is a nucleotide sequence comprising the following nucleotide sequences in this order:
a nucleotide sequence encoding a signal peptide;
a nucleotide sequence encoding an intra-organelle stabilizing domain of LAMP;
a nucleotide sequence encoding an allergen domain comprising Der p 1, Der p 2, Der p 23, and Der p 7 in this order;
a nucleotide sequence encoding a transmembrane domain; and
a nucleotide sequence encoding an endosomal/lysosomal targeting domain of LAMP.
[3]
The nucleic acid described in [1] or [2], wherein the signal peptide is a signal peptide of LAMP.
[4]
The nucleic acid described in any one of [1] to [3], wherein the transmembrane domain is a transmembrane domain of LAMP.
[5]
The nucleic acid described in any one of [1] to [4], wherein the signal peptide consists of the amino acid sequence of amino acid numbers 1 to 27 of SEQ ID NO: 2, the intra-organelle stabilizing domain consists of an amino acid sequence of amino acid numbers 28 to 380 of SEQ ID NO: 2, the allergen domain is an allergen domain comprising Der p 1 consisting of the amino acid sequence of amino acid numbers 383 to 594 of SEQ ID NO: 2, Der p 2 consisting of the amino acid sequence of amino acid numbers 599 to 727 of SEQ ID NO: 2, Der p 23 consisting of the amino acid sequence of amino acid numbers 732 to 800 of SEQ ID NO: 2, and Der p 7 consisting of the amino acid sequence of amino acid numbers 805 to 1002 of SEQ ID NO: 2, the transmembrane domain consists of the amino acid sequence of amino acid numbers 1006 to 1028 of SEQ ID NO: 2, and the endosomal/lysosomal targeting domain consists of the an amino acid sequence of amino acid numbers 1037 to 1040 of SEQ ID NO: 2.
[6]
A nucleic acid comprising:
a nucleotide sequence encoding a chimeric protein consisting of an amino acid sequence having at least 90% identity to the amino acid sequence shown by SEQ ID NO: 2, wherein the nucleic acid has an action of inducing Th1-type immunity to an allergen selected from the group consisting of Der p 1, Der p 2, Der p 23, and Der p 7.
[7]
A nucleic acid comprising:
a) a nucleotide sequence encoding a chimeric protein consisting of the amino acid sequence shown by SEQ ID NO: 2; or
b) a nucleotide sequence encoding a chimeric protein consisting of an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted and/or added in the amino acid sequence shown by SEQ ID NO: 2, wherein the nucleic acid has an action of inducing Th1-type immunity to an allergen selected from the group consisting of Der p 1, Der p 2, Der p 23, and Der p 7.
[8]
A nucleic acid comprising:
a nucleotide sequence encoding a chimeric protein consisting of an amino acid sequence shown by SEQ ID NO: 2.
[9]
An expression vector comprising:
the nucleic described in any one of [1] to [8].
[10]
An expression vector comprising:
the nucleic acid described in [8].
A host cell transformed with the nucleic acid described in any one of [1] to [8].
[12]
A method for producing a nucleic acid, comprising: culturing a host cell transformed with the nucleic acid described in any one of [1] to [8].
[13]
A pharmaceutical composition comprising:
the expression vector described in [10] and a pharmaceutically acceptable excipient.
[14]
The pharmaceutical composition described in [13], which is a pharmaceutical composition for preventing or treating mite allergy.
[15]
A method for preventing or treating mite allergy, comprising:
administering a prophylactically effective or therapeutically effective amount of the expression vector described in [10].
[16]
The expression vector described in [10], for use in preventing or treating mite allergy.
Use of the expression vector described in [10] for the manufacture of a pharmaceutical composition for preventing or treating mite allergy.
Effects of the Invention
The nucleic acid of the present invention can be used for preventing or treating mite allergy.
BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 is a diagram illustrating production of IgG2a specific to Der p 1, Der p 2, Der p 23, and Der p 7, which is induced when the nucleic acid of the present invention is administered to a mouse. The vertical axis indicates absorbance at 450 nm, and the horizontal axis indicates each administration group. The horizontal lines indicate arithmetic mean values.
FIG. 2 illustrates IFN-γ production when spleen cells of mice to which the nucleic acid of the present invention has been administered were stimulated with Der p 1 protein, Der p 2 protein, Der p 7 protein, or Der p 23 protein. The vertical axis indicates the concentration of IFN-γ in the culture supernatant (pg/mL), and the horizontal axis indicates each administration group. The horizontal lines indicate arithmetic mean values. The dotted line indicates the value of lower limit of detection (LLOD).
FIG. 3 illustrates IL-4 production when spleen cells of mice to which the nucleic acid of the present invention has been administered were stimulated with Der p 1 protein, Der p 2 protein, Der p 7 protein, or Der p 23 protein. The vertical axis indicates the concentration of IL-4 in the culture supernatant (pg/mL), and the horizontal axis indicates each administration group. The horizontal lines indicate arithmetic mean values. The dotted line indicates the value of lower limit of detection (LLOD).
EMBODIMENTS FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in detail.
<Nucleic Acid of the Present Invention>
Examples of the nucleic acid of the present invention include a nucleic acid having the following features:
a nucleic acid comprising a nucleotide sequence encoding a chimeric protein, wherein the nucleotide sequence is a nucleotide sequence comprising the following nucleotide sequences in this order:
a nucleotide sequence encoding a signal peptide,
a nucleotide sequence encoding an intra-organelle stabilizing domain of LAMP,
a nucleotide sequence encoding an allergen domain comprising Der p 1, Der p 2, Der p 23, and Der p 7,
a nucleotide sequence encoding a transmembrane domain, and
a nucleotide sequence encoding an endosomal/lysosomal targeting domain of LAMP.
In the present invention, the nucleic acid is a polymer which is formed by polymerization of nucleotides and consists of a nucleotide sequence with an arbitrary length. The nucleotides can include deoxyribonucleotides, ribonucleotides, and/or their analogs. The nucleic acid of the present invention is DNA, RNA or modified a nucleic acid thereof. In one embodiment, the nucleic acid of the present invention is DNA.
In one embodiment, the nucleic acid of the present invention is a nucleic acid introduced into an expression vector. In one embodiment, the nucleic acid of the present invention is a nucleic acid introduced into a plasmid vector.
In the specification, “chimeric protein” means a protein encoded by a nucleotide sequence in which two or more genes are fused by using genetic recombination technology. The nucleic acid of the present invention includes a nucleotide sequence encoding chimeric protein comprising a signal peptide, an intra-organelle stabilizing domain of LAMP, an allergen domain comprising Der p 1, Der p 2, Der p 23, and Der p 7, a transmembrane domain, and an endosomal/lysosomal targeting domain of LAMP in this order (hereinafter, referred to as “chimeric protein relating to the present invention”).
LAMP is well-known protein to those skilled in the art (J Biol Chem., 1991, Vol.266, p.21327-21330). In the present specification, LAMP is not particularly limited, but examples thereof include LAMP-1, LAMP-2, CD63/LAMP-3, DC-LAMP, and LIMP II, and homologs, orthologs, paralogs, variants, and modified proteins thereof. In one embodiment of the present invention, LAMP is LAMP-1. In the present invention, an animal from which LAMP is derived is not particularly limited, but in one embodiment, LAMP is human LAMP. In one embodiment, human LAMP is human LAMP-1. Examples of an amino acid sequence of human LAMP-1 include an amino acid sequence in which the amino acid sequence shown by amino acid numbers 1005 to 1040 of SEQ ID
NO: 2 is bound to a C-terminal of the amino acid sequence shown by amino acid numbers 1 to 380 of SEQ ID NO: 2.
The general structure of the signal peptide is well known to those skilled in the art (Annu Rev Biochem., 2003, Vol. 72, p. 395 to 447). The signal peptide has a function of directing transport and localization of a protein. As the signal peptide used in the present invention, any suitable signal peptide can be selected as long as it has a function of directing transport and localization of the protein. In one embodiment, the signal peptide used in the present invention is a signal peptide of LAMP. In one embodiment, the signal peptide of LAMP used in the present invention is a signal peptide of LAMP-1.
In one embodiment, the signal peptide used in the present invention consists of the following amino acid sequence of (a) or (b):
(a) an amino acid sequence having at least 90% identity to the amino acid sequence of amino acid numbers 1 to 27 of SEQ ID NO: 2; or
(b) the amino acid sequence of amino acid numbers 1 to 27 of SEQ ID NO: 2, or an amino acid sequence in which 1 to 3 amino acids are deleted, substituted, inserted and/or added in the amino acid sequence of amino acid numbers 1 to 27 of SEQ ID NO: 2.
The term of “identity” in the present specification means a value of Identity obtained by using an EMBOSS Needle (Nucleic Acids Res., 2015, Vol. 43, p.W580-W584; ebi.ac.uk/Tools/psa/emboss needle/) with a parameter prepared by default.
The above parameters are as follows.
Gap Open Penalty=10
Gap Extend Penalty=0.5
Matrix=EBLOSUM62
End Gap Penalty=false
In one embodiment, the signal peptide used in the present invention consists of the amino acid sequence of amino acid numbers 1 to 27 of SEQ ID NO: 2.
The sequence of the intra-organelle stabilizing domain of LAMP is well known to those skilled in the art (WO 2013/187906). The intra-organelle stabilizing domain of LAMP has a function of protecting the allergen domain from proteases, low pH, and other substances and conditions that destabilize a protein. As the intra-organelle stabilizing domain of LAMP used in the present invention, any suitable intra-organelle stabilizing domain of LAMP can be selected as long as it has a function of protecting the allergen domain from proteases, low pH, and other substances and conditions that destabilize a protein. In one embodiment, the intra-organelle stabilizing domain of LAMP used in the present invention is an intra-organelle stabilizing domain of LAMP-1.
In one embodiment, the intra-organelle stabilizing domain of LAMP used in the present invention consists of the following amino acid sequence of (a) or (b):
(a) an amino acid sequence having at least 90% identity to the amino acid sequence of amino acid numbers 28 to 380 of SEQ ID NO: 2; or
(b) the amino acid sequence of amino acid numbers 28 to 380 of SEQ ID NO: 2, or an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted and/or added in the amino acid sequence of amino acid numbers 28 to 380 of SEQ ID NO: 2.
In one embodiment, the intra-organelle stabilizing domain of LAMP used in the present invention consists of the amino acid sequence of amino acid numbers 28 to 380 of SEQ ID NO: 2.
The allergen domain used in the present invention includes Der p 1, Der p 2, Der p 23, and Der p 7 as allergens. Der p 1, Der p 2, Der p 23, and Der p 7 are allergens that can be observed in mites (WO 1988/010297; WO 2007/124524; and Clin Exp Allergy., 1995, Vol. 25, p. 416-422). Der p 1, Der p 2, Der p 23, and Der p 7 used in the present invention may be variants thereof as long as they have antigenicity. The antigenicity of any protein can be confirmed, for example, by observing that administration to an animal elicits antibody production or T cell response to that protein (Bioanalysis., 2012, Vol. 4, p. 397-406). In one embodiment, Der p 1, Der p 2, Der p 23, and Der p 7 used in the present invention lack the signal peptide.
In one embodiment, Der p 1 consists of the following amino acid sequence of (a) or (b):
(a) an amino acid sequence having at least 90% identity to the amino acid sequence of amino acid numbers 383 to 594 of SEQ ID NO: 2; or
(b) the amino acid sequence of amino acid numbers 383 to 594 of SEQ ID NO: 2, or an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted and/or added in the amino acid sequence of amino acid numbers 383 to 594 of SEQ ID NO: 2.
In one embodiment, Der p 1 consists of the amino acid sequence of amino acid numbers 383 to 594 of SEQ ID NO: 2.
In one embodiment, Der p 2 consists of the following amino acid sequence of (a) or (b):
(a) an amino acid sequence having at least 90% identity to the amino acid sequence of amino acid numbers 599 to 727 of SEQ ID NO: 2; or (b) the amino acid sequence of amino acid numbers 599 to 727 of SEQ ID NO: 2, or an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted and/or added in the amino acid sequence of amino acid numbers 599 to 727 of SEQ ID NO: 2.
In one embodiment, Der p 2 consists of the amino acid sequence of amino acid numbers 599 to 727 of SEQ ID NO: 2.
In one embodiment, Der p 23 consists of the following amino acid sequence of (a) or (b):
(a) an amino acid sequence having at least 90% identity to the amino acid sequence of amino acid numbers 732 to 800 of SEQ ID NO: 2; or
(b) the amino acid sequence of amino acid numbers 732 to 800 of SEQ ID NO: 2, or an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted and/or added in the amino acid sequence of amino acid numbers 732 to 800 of SEQ ID NO: 2.
In one embodiment, Der p 23 consists of the amino acid sequence of amino acid numbers 732 to 800 of SEQ ID NO: 2.
In one embodiment, Der p 7 consists of the following amino acid sequence of (a) or (b):
(a) an amino acid sequence having at least 90% identity to the amino acid sequence of amino acid numbers 805 to 1002 of SEQ ID NO: 2; or
(b) the amino acid sequence of amino acid numbers 805 to 1002 of SEQ ID NO: 2, or an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted and/or added in the amino acid sequence of amino acid numbers 805 to 1002 of SEQ ID NO: 2.
In one embodiment, Der p 7 consists of the amino acid sequence of amino acid numbers 805 to 1002 of SEQ ID NO: 2.
In one embodiment, the allergen domain used in the present invention comprises Der p 1, Der p 2, Der p 23, and Der p 7 in any order. In addition, in one embodiment, the allergen domain used in the present invention comprises Der p 1, Der p 2, Der p 23, and Der p 7 in this order.
In one embodiment, the allergen domain used in the present invention consists of the amino acid sequence of amino acid numbers 383 to 1002 of SEQ ID NO: 2.
The general structure of the transmembrane domain is well known to those skilled in the art (Annu Rev Biochem., 2007, Vol. 76, p. 125 to 140). The transmembrane domain has a function of anchoring proteins to biological membranes. As the transmembrane domain used in the present invention, any suitable transmembrane domain protein can be selected as long as it has a function of anchoring proteins to biological membranes. In one embodiment, the transmembrane domain used in the present invention is a transmembrane domain of LAMP. In one embodiment, the transmembrane domain of LAMP used in the present invention is a transmembrane domain of LAMP-1.
In one embodiment, the transmembrane domain used in the present invention consists of the following amino acid sequence of (a) or (b):
(a) an amino acid sequence having at least 90% identity to the amino acid sequence of amino acid numbers 1006 to 1028 of SEQ ID NO: 2; or
(b) the amino acid sequence of amino acid numbers 1006 to 1028 of SEQ ID NO: 2, or an amino acid sequence in which 1 to 2 amino acids are deleted, substituted, inserted and/or added in the amino acid sequence of amino acid numbers 1006 to 1028 of SEQ ID NO: 2.
In one embodiment, the transmembrane domain used in the present invention consists of the amino acid sequence of amino acid numbers 1006 to 1028 of SEQ ID NO: 2.
The structure of the endosomal/lysosomal targeting domain of LAMP is well known to those skilled in the art (WO 1994/017192). The endosomal/lysosomal targeting domain of LAMP has a function of transporting a protein to lysosome. As the endosomal/lysosomal targeting domain of LAMP used in the present invention, any suitable endosomal/lysosomal targeting domain of LAMP can be selected as long as it has a function of transporting the protein to lysosome. In one embodiment, endosomal/lysosomal targeting domain of LAMP used in the present invention is an endosomal/lysosomal targeting domain of LAMP-1.
In one embodiment, the endosomal/lysosomal targeting domain of LAMP used in the present invention consists of the amino acid sequence of amino acid numbers 1037 to 1040 of SEQ ID NO: 2, or an amino acid sequence in which 1 amino acid is deleted, substituted, inserted and/or added in the amino acid sequence of amino acid numbers 1037 to 1040 of SEQ ID NO: 2.
In one embodiment, the endosomal/lysosomal targeting domain of LAMP used in the present invention consists of an amino acid sequence in a range of amino acid numbers 1037 to 1040 of SEQ ID NO: 2.
In the chimeric protein relating to the present invention, the signal peptide, the intra-organelle stabilizing domain of LAMP, each allergen comprised in the allergen domain, the transmembrane domain, and the endosomal/lysosomal targeting domain of LAMP may be directly linked or may be indirectly linked via a linker peptide. The linker peptide to be used can be appropriately selected by those skilled in the art. In one embodiment, the linker peptide consists of 10 or less amino acids. In one embodiment, a linker peptide used between the intra-organelle stabilizing domain of LAMP and the allergen domain, between allergens, and between the allergen domain and the transmembrane domain is a linker peptide selected from the group consisting of LeuGlu, GlyGlyGlyGly, and GluPheThr. In one embodiment, the linker peptide used between the transmembrane domain and the endosomal/lysosomal targeting domain of LAMP is a linker peptide consisting of the amino acid sequence of amino acid numbers 1029 to 1036 of SEQ ID NO: 2.
In one embodiment, the nucleic acid of the present invention is the following nucleic acid:
a nucleic acid comprising a nucleotide sequence encoding a chimeric protein, wherein the nucleotide sequence is a nucleotide sequence comprising the following nucleotide sequences in this order:
a nucleotide sequence encoding a signal peptide of LAMP, a nucleotide sequence encoding an intra-organelle stabilizing domain of LAMP, a nucleotide sequence encoding an allergen domain comprising Der p 1, Der p 2, Der p 23, and Der p 7,
a nucleotide sequence encoding a transmembrane domain of LAMP, and
a nucleotide sequence encoding an endosomal/lysosomal targeting domain of LAMP.
In one embodiment, the nucleic acid of the present invention is the following nucleic acid:
a nucleic acid comprising a nucleotide sequence encoding a chimeric protein, wherein the nucleotide sequence is a nucleotide sequence comprising the following nucleotide sequences in this order:
a nucleotide sequence encoding a signal peptide of LAMP-1,
a nucleotide sequence encoding the intra-organelle stabilizing domain of LAMP-1,
a nucleotide sequence encoding an allergen domain comprising Der p 1, Der p 2, Der p 23, and Der p 7,
a nucleotide sequence encoding a transmembrane domain of LAMP-1, and
a nucleotide sequence encoding an endosomal/lysosomal targeting domain the of LAMP-1.
In one embodiment, the nucleic acid of the present invention is the following nucleic acid:
a nucleic acid comprising a nucleotide sequence encoding a chimeric protein, wherein the nucleotide sequence is a nucleotide sequence comprising the following nucleotide sequences in this order:
a nucleotide sequence encoding a signal peptide consisting of the amino acid sequence of amino acid numbers 1 to 27 of SEQ ID NO: 2,
a nucleotide sequence encoding an intra-organelle stabilizing domain of LAMP consisting of the amino acid sequence of amino acid numbers 28 to 380 of SEQ ID NO: 2,
a nucleotide sequence encoding an allergen domain comprising Der p 1 consisting of the amino acid sequence of amino acid numbers 383 to 594 of SEQ ID NO: 2, Der p 2 consisting of the amino acid sequence of amino acid numbers 599 to 727 of SEQ ID NO: 2, Der p 23 consisting of the amino acid sequence of amino acid numbers 732 to 800 of SEQ ID NO: 2, and Der p 7 consisting of the amino acid sequence of amino acid numbers 805 to 1002 of SEQ ID NO: 2,
a nucleotide sequence encoding a transmembrane domain consisting of the amino acid sequence of amino acid numbers 1006 to 1028 of SEQ ID NO: 2, and
a nucleotide sequence encoding an endosomal/lysosomal targeting domain of LAMP consisting of the amino acid sequence of amino acid numbers 1037 to 1040 of SEQ ID NO: 2.
In one embodiment, the nucleic acid of the present invention is the following nucleic acid:
a nucleic acid comprising a nucleotide sequence encoding a chimeric protein, wherein the nucleotide sequence is a nucleotide sequence comprising the following nucleotide sequences in this order:
a nucleotide sequence encoding a signal peptide consisting of the amino acid sequence of amino acid numbers 1 to 27 of SEQ ID NO: 2,
a nucleotide sequence encoding an intra-organelle stabilizing domain of LAMP consisting of the amino acid sequence of amino acid numbers 28 to 380 of SEQ ID NO: 2,
a nucleotide sequence encoding an allergen domain comprising Der p 1 consisting of the amino acid sequence of amino acid numbers 383 to 594 of SEQ ID NO: 2, Der p 2 consisting of the amino acid sequence of amino acid numbers 599 to 727 of SEQ ID NO: 2, Der p 23 consisting of the amino acid sequence of amino acid numbers 732 to 800 of SEQ ID NO: 2, and Der p 7 consisting of the amino acid sequence of amino acid numbers 805 to 1002 of SEQ ID NO: 2,
a nucleotide sequence encoding a transmembrane domain consisting of the amino acid sequence of amino acid numbers 1006 to 1028 of SEQ ID NO: 2,
a nucleotide sequence encoding a peptide linker consisting of the amino acid sequence of amino acid numbers 1029 to 1036 of SEQ ID NO: 2, and
a nucleotide sequence encoding an endosomal/lysosomal targeting domain of LAMP consisting of the amino acid sequence of amino acid numbers 1037 to 1040 of SEQ ID NO: 2.
In one embodiment, the nucleic acid of the present invention is the following nucleic acid:
a nucleic acid comprising a nucleotide sequence encoding a chimeric protein, wherein the nucleotide sequence is a nucleotide sequence comprising the following nucleotide sequences in this order:
a nucleotide sequence encoding a signal peptide,
a nucleotide sequence encoding an intra-organelle stabilizing domain of LAMP,
a nucleotide sequence encoding an allergen domain comprising Der p 1, Der p 2, Der p 23, and Der p 7 in this order,
a nucleotide sequence encoding a transmembrane domain, and
a nucleotide sequence encoding an endosomal/lysosomal targeting domain of LAMP.
In one embodiment, the nucleic acid of the present invention is the following nucleic acid:
a nucleic acid comprising a nucleotide sequence encoding a chimeric protein, wherein the nucleotide sequence is a nucleotide sequence comprising the following nucleotide sequences in this order:
a nucleotide sequence encoding a signal peptide of LAMP,
a nucleotide sequence encoding an intra-organelle stabilizing domain of LAMP,
a nucleotide sequence encoding an allergen domain comprising Der p 1, Der p 2, Der p 23, and Der p 7 in this order,
a nucleotide sequence encoding a transmembrane domain of LAMP, and a nucleotide sequence encoding an endosomal/lysosomal targeting domain of LAMP.
In one embodiment, the nucleic acid of the present invention is the following nucleic acid:
a nucleic acid comprising a nucleotide sequence encoding a chimeric protein, wherein the nucleotide sequence is a nucleotide sequence comprising the following nucleotide sequences in this order:
a nucleotide sequence encoding a signal peptide of LAMP-1,
a nucleotide sequence encoding the intra-organelle stabilizing domain of LAMP-1,
a nucleotide sequence encoding an allergen domain comprising Der p 1, Der p 2, Der p 23, and Der p 7 in this order,
a nucleotide sequence encoding a transmembrane domain of LAMP-1, and
a nucleotide sequence encoding an endosomal/lysosomal targeting domain of LAMP-1.
In one embodiment, the nucleic acid of the present invention is the following nucleic acid:
a nucleic acid comprising a nucleotide sequence encoding a chimeric protein, wherein the nucleotide sequence is a nucleotide sequence comprising the following nucleotide sequences in this order:
a nucleotide sequence encoding a signal peptide consisting of the amino acid sequence of amino acid numbers 1 to 27 of SEQ ID NO: 2,
a nucleotide sequence encoding an intra-organelle stabilizing domain of LAMP consisting of the amino acid sequence of amino acid numbers 28 to 380 of SEQ ID NO: 2,
a nucleotide sequence encoding an allergen domain comprising Der p 1 consisting of the amino acid sequence of amino acid numbers 383 to 594 of SEQ ID NO: 2, Der p 2 consisting of the amino acid sequence of amino acid numbers 599 to 727 of SEQ ID NO: 2, Der p 23 consisting of the amino acid sequence of amino acid numbers 732 to 800 of SEQ ID NO: 2, and Der p 7 consisting of the amino acid sequence of amino acid numbers 805 to 1002 of SEQ ID NO: 2 in this order,
a nucleotide sequence encoding a transmembrane domain consisting of the amino acid sequence of amino acid numbers 1006 to 1028 of SEQ ID NO: 2, and
a nucleotide sequence encoding an endosomal/lysosomal targeting domain of LAMP consisting of the amino acid sequence of amino acid numbers 1037 to 1040 of SEQ ID NO: 2.
In one embodiment, the nucleic acid of the present invention is the following nucleic acid:
a nucleic acid comprising a nucleotide sequence encoding a chimeric protein, wherein the nucleotide sequence is a nucleotide sequence comprising the following nucleotide sequences in this order:
a nucleotide sequence encoding a signal peptide consisting of the amino acid sequence of amino acid numbers 1 to 27 of SEQ ID NO: 2,
a nucleotide sequence encoding an intra-organelle stabilizing domain of LAMP consisting of the amino acid sequence of amino acid numbers 28 to 380 of SEQ ID NO: 2,
a nucleotide sequence encoding an allergen domain comprising Der p 1 consisting of the amino acid sequence of amino acid numbers 383 to 594 of SEQ ID NO: 2, Der p 2 consisting of the amino acid sequence of amino acid numbers 599 to 727 of SEQ ID NO: 2, Der p 23 consisting of the amino acid sequence of amino acid numbers 732 to 800 of SEQ ID NO: 2, and Der p 7 consisting of the amino acid sequence of amino acid numbers 805 to 1002 of SEQ ID NO: 2 in this order,
a nucleotide sequence encoding a transmembrane domain consisting of the amino acid sequence of amino acid numbers 1006 to 1028 of SEQ ID NO: 2,
a nucleotide sequence encoding a peptide linker consisting of the amino acid sequence of amino acid numbers 1029 to 1036 of SEQ ID NO: 2, and
a nucleotide sequence encoding an endosomal/lysosomal targeting domain of LAMP consisting of the amino acid sequence of amino acid numbers 1037 to 1040 of SEQ ID NO: 2.
The nucleic acid of the present invention is not particularly limited as long as it encodes the chimeric protein relating to the present invention, and has an action of inducing Th1-type immunity with respect to the allergen selected from the group consisting of Der p 1, Der p 2, Der p 23, and Der p 7, when the nucleic acid is administered to a human or an animal. One can confirm whether or not a certain nucleic acid has an action of inducing Th1-type immunity, when the nucleic acid is administered to a human or an animal, by the method described in, for example, Example 3 and/or Example 4. In addition, the nucleic acid of the present invention may be a nucleic acid having an action of inducing Th1 cell dominant immune response, when the nucleic acid is administered to a human or an animal. One can confirm whether or not a certain nucleic acid has an action of inducing Th1 cell dominant immune response, when the nucleic acid is administered to human or animal, by the method described in, for example, Example 4.
In one embodiment, the nucleic acid of the present invention is the following nucleic acid:
a nucleic acid comprising a nucleotide sequence encoding a chimeric protein consisting of an amino acid sequence having at least 90%, 92%, 94%, 96%, 98%, or 99% identity to the amino acid sequence shown by SEQ ID NO: 2.
In one embodiment, the nucleic acid of the present invention is the following nucleic acid:
a nucleic acid comprising a nucleotide sequence encoding a chimeric protein consisting of an amino acid sequence having at least 90% identity to the amino acid sequence shown by SEQ ID NO: 2.
In one embodiment, the nucleic acid of the present invention is the following nucleic acid:
a nucleic acid comprising a nucleotide sequence encoding a chimeric protein consisting of the amino acid sequence shown by SEQ ID NO: 2, or a chimeric protein consisting of an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted and/or added in the amino acid sequence shown by SEQ ID NO: 2.
In one embodiment, the nucleic acid of the present invention is the following nucleic acid:
a nucleic acid comprising a nucleotide sequence encoding a chimeric protein consisting of an amino acid sequence having at least 90% identity to the amino acid sequence shown by SEQ ID NO: 2,
wherein the nucleic acid has an action of inducing Th1-type immunity to the allergen selected from the group consisting of Der p 1, Der p 2, Der p 23, and Der p 7.
In one embodiment, the nucleic acid of the present invention is the following nucleic acid:
a) a nucleic acid comprising a nucleotide sequence encoding a chimeric protein consisting of the amino acid sequence shown by SEQ ID NO: 2 or
b) a nucleic acid comprising a nucleotide sequence encoding a chimeric protein consisting of an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted and/or added in the amino acid sequence shown by SEQ ID NO: 2, wherein the nucleic acid has an action of inducing Th1-type immunity to an allergen selected from the group consisting of Der p 1, Der p 2, Der p 23, and Der p 7.
In one embodiment, the nucleic acid of the present invention is the following nucleic acid:
a nucleic acid comprising a nucleotide sequence encoding a chimeric protein consisting of an amino acid sequence having at least 90% identity to the amino acid sequence shown by SEQ ID NO: 2, wherein the nucleic acid has an action of inducing Th1-type immunity to Der p 1, Der p 2, Der p 23, and Der p 7.
In one embodiment, the nucleic acid of the present invention is the following nucleic acid:
a) a nucleic acid comprising a nucleotide sequence encoding a chimeric protein consisting of the amino acid sequence shown by SEQ ID NO: 2 or
b) a nucleic acid comprising a nucleotide sequence encoding a chimeric protein consisting of an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted and/or added in the amino acid sequence shown by SEQ ID NO: 2, wherein the nucleic acid has an action of inducing Th1-type immunity to Der p 1, Der p 2, Der p 23, and Der p 7.
In one embodiment, the nucleic acid of the present invention is the following nucleic acids
a nucleic acid comprising a nucleotide sequence encoding a chimeric protein consisting of the amino acid sequence shown by SEQ ID NO: 2.
In one embodiment, the nucleotide sequence encoding the chimeric protein consisting of the amino acid sequence shown by SEQ ID NO: 2 means the nucleotide sequence shown by SEQ ID NO: 1.
Based on the nucleotide sequence, the nucleic acid of the present invention can be easily prepared by those skilled in the art by using methods known in the art. For example, the nucleic acid of the present invention can be synthesized by using gene synthesis methods known in the art. As such a gene synthesis method, various methods known to those skilled in the art such as a method for synthesizing an antibody gene described in WO 90/07861 can be used.
Once being synthesized, the nucleic acid of the present invention can be easily replicated by those skilled in the art using methods known in the art. For example, the nucleic acid of the present invention can be replicated by the method described later in
<Method for Producing the Nucleic Acid of the Present Invention and Nucleic Acid Which can be Produced by the Method>.
<Expression Vector of the Present Invention>
The expression vector of the present invention includes an expression vector comprising the nucleic acid of the present invention.
The expression vector used to express a chimeric protein from the nucleic acid of the present invention is not particularly limited as long as it can express the chimeric protein from the nucleic acid of the present invention in the animal cells. In one embodiment, the expression vector used to express a chimeric protein from the nucleic acid of the present invention is an expression vector which can be used for expressing the chimeric protein in a human body. Examples of the expression vector used in the present invention include a plasmid vector, a viral vector (for example, adenovirus, retrovirus, adeno-associated virus) and the like. In one embodiment, the expression vector of the present invention is a plasmid vector. In the present specification, “plasmid” means the plasmid vector.
The expression vector of the present invention may comprise a promoter operably linked to the nucleic acid of the present invention. Examples of the promoter for expressing the chimeric protein from the nucleic acid of the present invention in animal cells include a virus-derived promoter such as CMV (cytomegalovirus), RSV (respiratory syncytial virus), and SV40 (simian virus 40), an actin promoter, EF (elongation factor) 1α promoter, a heat shock promoter and the like. In one embodiment, the promoter comprised in the expression vector of the present invention is a CMV promoter. The expression vector of the present invention may comprise a start codon and a stop codon. In this case, an enhancer sequence, an untranslated region, a splicing junction, a polyadenylation site, or a replicable unit may be comprised.
In one embodiment, the expression vector of the present invention is an expression vector comprising the following nucleic acid:
a nucleic acid comprising a nucleotide sequence encoding a chimeric protein, wherein the nucleotide sequence is a nucleotide sequence comprising the following nucleotide sequences in this order:
a nucleotide sequence encoding a signal peptide consisting of the amino acid sequence of amino acid numbers 1 to 27 of SEQ ID NO: 2,
a nucleotide sequence encoding an intra-organelle stabilizing domain of LAMP consisting of the amino acid sequence of amino acid numbers 28 to 380 of SEQ ID NO: 2,
a nucleotide sequence encoding an allergen domain comprising Der p 1 consisting of the amino acid sequence of amino acid numbers 383 to 594 of SEQ ID NO: 2, Der p 2 consisting of the amino acid sequence of amino acid numbers 599 to 727 of SEQ ID NO: 2, Der p 23 consisting of the amino acid sequence of amino acid numbers 732 to 800 of SEQ ID NO: 2, and Der p 7 consisting of the amino acid sequence of amino acid numbers 805 to 1002 of SEQ ID NO: 2,
a nucleotide sequence encoding a transmembrane domain consisting of the amino acid sequence of amino acid numbers 1006 to 1028 of SEQ ID NO: 2, and
a nucleotide sequence encoding an endosomal/lysosomal targeting domain of LAMP consisting of the amino acid sequence of amino acid numbers 1037 to 1040 of SEQ ID NO: 2.
In one embodiment, the expression vector of the present invention is an expression vector comprising the following nucleic acid:
a nucleic acid comprising a nucleotide sequence encoding a chimeric protein, wherein the nucleotide sequence is a nucleotide sequence comprising the following nucleotide sequences in this order:
a nucleotide sequence encoding a signal peptide consisting of the amino acid sequence of amino acid numbers 1 to 27 of SEQ ID NO: 2,
a nucleotide sequence encoding an intra-organelle stabilizing domain of LAMP consisting of the amino acid sequence of amino acid numbers 28 to 380 of SEQ ID NO: 2,
a nucleotide sequence encoding an allergen domain comprising Der p 1 consisting of the amino acid sequence of amino acid numbers 383 to 594 of SEQ ID NO: 2, Der p 2 consisting of the amino acid sequence of amino acid numbers 599 to 727 of SEQ ID NO: 2, Der p 23 consisting of the amino acid sequence of amino acid numbers 732 to 800 of SEQ ID NO: 2, and Der p 7 consisting of the amino acid sequence of amino acid numbers 805 to 1002 of SEQ ID NO: 2,
a nucleotide sequence encoding a transmembrane domain consisting of the amino acid sequence of amino acid numbers 1006 to 1028 of SEQ ID NO: 2,
a nucleotide sequence encoding a peptide linker consisting of the amino acid sequence of amino acid numbers 1029 to 1036 of SEQ ID NO: 2, and
a nucleotide sequence encoding an endosomal/lysosomal targeting domain of LAMP consisting of the amino acid sequence of amino acid numbers 1037 to 1040 of SEQ ID NO: 2.
In one embodiment, the expression vector of the present invention is an expression vector comprising the following nucleic acid:
a nucleic acid comprising a nucleotide sequence encoding a chimeric protein, wherein the nucleotide sequence is a nucleotide sequence comprising the following nucleotide sequences in this order:
a nucleotide sequence encoding a signal peptide consisting of the amino acid sequence of amino acid numbers 1 to 27 of SEQ ID NO: 2,
a nucleotide sequence encoding an intra-organelle stabilizing domain of LAMP consisting of the amino acid sequence of amino acid numbers 28 to 380 of SEQ ID NO: 2,
a nucleotide sequence encoding an allergen domain comprising Der p 1 consisting of the amino acid sequence of amino acid numbers 383 to 594 of SEQ ID NO: 2, Der p 2 consisting of the amino acid sequence of amino acid numbers 599 to 727 of SEQ ID NO: 2, Der p 23 consisting of the amino acid sequence of amino acid numbers 732 to 800 of SEQ ID NO: 2, and Der p 7 consisting of the amino acid sequence of amino acid numbers 805 to 1002 of SEQ ID NO: 2 in this order,
a nucleotide sequence encoding a transmembrane domain consisting of the amino acid sequence of amino acid numbers 1006 to 1028 of SEQ ID NO: 2, and a nucleotide sequence encoding an endosomal/lysosomal targeting domain of LAMP consisting of the amino acid sequence of amino acid numbers 1037 to 1040 of SEQ ID NO: 2.
In one embodiment, the expression vector of the present invention is an expression vector comprising the following nucleic acid:
a nucleic acid comprising a nucleotide sequence encoding a chimeric protein, wherein the nucleotide sequence is a nucleotide sequence comprising the following nucleotide sequences in this order:
a nucleotide sequence encoding a signal peptide consisting of the amino acid sequence of amino acid numbers 1 to 27 of SEQ ID NO: 2,
a nucleotide sequence encoding an intra-organelle stabilizing domain of LAMP consisting of the amino acid sequence of amino acid numbers 28 to 380 of SEQ ID NO: 2,
a nucleotide sequence encoding an allergen domain comprising Der p 1 consisting of the amino acid sequence of amino acid numbers 383 to 594 of SEQ ID NO: 2, Der p 2 consisting of the amino acid sequence of amino acid numbers 599 to 727 of SEQ ID NO: 2, Der p 23 consisting of the amino acid sequence of amino acid numbers 732 to 800 of SEQ ID NO: 2, and Der p 7 consisting of the amino acid sequence of amino acid numbers 805 to 1002 of SEQ ID NO: 2 in this order,
a nucleotide sequence encoding a transmembrane domain consisting of the amino acid sequence of amino acid numbers 1006 to 1028 of SEQ ID NO: 2,
a nucleotide sequence encoding a peptide linker consisting of the amino acid sequence of amino acid numbers 1029 to 1036 of SEQ ID NO: 2, and
a nucleotide sequence encoding an endosomal/lysosomal targeting domain of LAMP consisting of the amino acid sequence of amino acid numbers 1037 to 1040 of SEQ ID NO: 2.
In one embodiment, the expression vector of the present invention is an expression vector comprising a nucleic acid comprising a nucleotide sequence encoding a chimeric protein consisting of the amino acid sequence shown by SEQ ID NO: 2.
In one embodiment, the expression vector of the present invention is an expression vector comprising a nucleic acid comprising the nucleotide sequence shown by SEQ ID NO: 1.
In one embodiment, the expression vector of the present invention is an expression vector comprising a nucleic acid consisting of the nucleotide sequence shown by SEQ ID NO: 3.
<Host Cell of the Present Invention>
The host cell of the present invention includes a host cell transformed with the nucleic acid of the present invention. In one embodiment, the host cell of the present invention is a host cell transformed with the expression vector of the present invention. In one embodiment, the host cell of the present invention is a host cell transformed with the expression vector of the present invention which is a plasmid vector.
The host cell transformed with the nucleic acid of the present invention is not particularly limited, and any cell known in the art can be selected as long as it is a cell that can be used for nucleic acid replication.
Examples of the host cell that can be used for nucleic acid replication include various cells such as natural cells or artificially established cells commonly used in the technical field of the present invention (for example, animal cells (for example, CHOK1SV cells), insect cells (for example, Sf9), bacteria (for example, E. coli), and yeasts (for example, Saccharomyces and Pichia)). In one embodiment, E. coli can be used as a host cell. Transformation itself can be carried out by known methods.
<Method for Producing the Nucleic Acid of the Present Invention and Nucleic Acid Which can be Produced by the Method>
Examples of the method for producing the nucleic acid of the present invention include a method for producing a nucleic acid or an expression vector, which comprises a step of culturing host cells transformed with the nucleic acid or the expression vector of the present invention. In one embodiment, the method for producing the nucleic acid of the present invention comprises a step of culturing the host cell transformed with the nucleic acid of the present invention, and replicating the nucleic acid of the present invention. In one embodiment, the method for producing the nucleic acid of the present invention comprises a step of culturing the host cell transformed with the expression vector of the present invention, and replicating the expression vector of the present invention.
In one embodiment, the host cell used in the method for producing the nucleic acid of the present invention is E. coli. For culture of E. coli, an appropriate culture medium such as LB medium, M9 medium, Terrific Broth medium, SOB medium, SOC medium, or 2× YT medium can be selected. In addition, the culturing of E. coli can be carried out in an environment where carbon (it is not particularly limited as long as it is an assimilable carbon compound; for example, polyols such as glycerin, or organic acids such as pyruvic acid, succinic acid, or citric acid), nitrogen (it is not particularly limited as long as it is a nitrogen compound that can be used by E. coli; for example, peptone, meat extract, yeast extract, casein hydrolysate, soybean meal alkaline extract, or ammonia or a salt thereof), inorganics and inorganic ions (it is not particularly limited, and examples thereof include phosphate, carbonate, sulfate, magnesium, calcium, potassium, iron, manganese and zinc), a vitamin source, and an antifoaming agent are controlled to an appropriate concentration. In addition, the control of culturing includes control of parameters such as pH, temperature, stir, air flow and dissolved oxygen. In one embodiment, the conditions of culturing include pH of 6.7 to 7.5, temperature of 20° C. to 37° C., and a stirring speed of 200 to 300 rpm.
The method for producing the nucleic acid of the present invention may comprise a step of obtaining lysate from collected culture solutions. The lysate can be obtained, for example, by treating the collected culture solutions with an alkaline lysis method or boiling method. Also, the step of obtaining the lysate may include a step of sterile filtration of a final lysate material.
The method for producing the nucleic acid of the present invention may further comprise a step of purifying nucleic acid or an expression vector from lysate. Ion exchange chromatography and/or hydrophobic interaction chromatography can be used to purify the nucleic acid or the expression vector from the lysate. The step of purifying the nucleic acid or the expression vector from the lysate may include a step of ultrafiltration and/or diafiltration. In addition, as a final treatment of the purification step, a sterile filtration step may be comprised.
In one embodiment, the nucleic acid of the present invention is a nucleic acid produced by the method for producing the nucleic acid of the present invention.
In one embodiment, the expression vector of the present invention is an expression vector produced by the method for producing the nucleic acid of the present invention.
<Pharmaceutical Composition of the Present Invention>
The pharmaceutical composition of the present invention includes a pharmaceutical composition comprising the nucleic acid of the present invention and a pharmaceutically acceptable excipient. In one embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition comprising the vector of the present invention and the pharmaceutically acceptable excipient. The pharmaceutical composition of the present invention can be prepared by a generally used method with an excipient generally used in the field, that is, a pharmaceutical excipient, a pharmaceutical carrier or the like. Examples of dosage forms of these pharmaceutical compositions include, for example, parenteral agents such as injections and drip agents, which can be administered by intravenous administration, subcutaneous administration, intradermal administration, and intramuscular administration. In formulating, excipients, carriers, additives, and the like can be used according to these dosage forms within the pharmaceutically acceptable range.
In one embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition comprising the nucleic acid or the expression vector of the present invention and the pharmaceutically acceptable excipient.
While the administration amount of the nucleic acid of the present invention or the expression vector varies depending on the degree of symptoms and age of the patient, and the dosage form of the preparation used, for example, the amount in a range of 0.001 mg/kg to 100 mg/kg can be used. Further, it is possible to prepare a formulation by adding the nucleic acid or the expression vector of the present invention in an amount corresponding to such administration amount.
The pharmaceutical composition of the present invention can be used as an agent for preventing or treating allergy caused by an allergen selected from Der p 1, Der p 2, Der p 23, and Der p 7. Further, the pharmaceutical composition of the present invention can be used as an agent for prevention or treating the mite allergy.
The present invention includes a pharmaceutical composition for preventing or treating allergy, comprising the nucleic acid of the present invention. In addition, the present invention includes a method for preventing or treating allergy, comprising administering a prophylactically effective or therapeutically effective amount of the nucleic acid of the present invention. The present invention also includes the nucleic acid of the present invention for use in preventing or treating allergy. In addition, the present invention includes use of the nucleic acid of the present invention for the manufacture of a pharmaceutical composition for preventing or treating allergy. In one embodiment, the above-described allergy is allergy caused by an allergen selected from the group consisting of Der p 1, Der p 2, Der p 23, and Der p 7. In addition, in one embodiment, the above-described allergy is allergy affecting an allergy patient having an antibody that responds to an allergen selected from the group consisting of Der p 1, Der p 2, Der p 23, and Der p 7. Further, in one embodiment, the above-described allergy is mite allergy.
In one embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition for preventing or treating allergy, comprising the following nucleic acid and a pharmaceutically acceptable excipient:
a nucleic acid comprising a nucleotide sequence encoding a chimeric protein, wherein the nucleotide sequence is a nucleotide sequence comprising the following nucleotide sequences in this order:
a nucleotide sequence encoding a signal peptide consisting of the amino acid sequence of amino acid numbers 1 to 27 of SEQ ID NO: 2,
a nucleotide sequence encoding an intra-organelle stabilizing domain of LAMP consisting of the amino acid sequence of amino acid numbers 28 to 380 of SEQ ID NO: 2,
a nucleotide sequence encoding an allergen domain comprising Der p 1 consisting of the amino acid sequence of amino acid numbers 383 to 594 of SEQ ID NO: 2, Der p 2 consisting of the amino acid sequence of amino acid numbers 599 to 727 of SEQ ID NO: 2, Der p 23 consisting of the amino acid sequence of amino acid numbers 732 to 800 of SEQ ID NO: 2, and Der p 7 consisting of the amino acid sequence of amino acid numbers 805 to 1002 of SEQ ID NO: 2,
a nucleotide sequence encoding a transmembrane domain consisting of the amino acid sequence of amino acid numbers 1006 to 1028 of SEQ ID NO: 2, and
a nucleotide sequence encoding an endosomal/lysosomal targeting domain of LAMP consisting of the amino acid sequence of amino acid numbers 1037 to 1040 of SEQ ID NO: 2.
In one embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition for preventing or treating allergy, comprising the following nucleic acid and a pharmaceutically acceptable excipient:
a nucleic acid comprising a nucleotide sequence encoding a chimeric protein, wherein the nucleotide sequence is a nucleotide sequence comprising the following nucleotide sequences in this order:
a nucleotide sequence encoding a signal peptide consisting of the amino acid sequence of amino acid numbers 1 to 27 of SEQ ID NO: 2,
a nucleotide sequence encoding an intra-organelle stabilizing domain of LAMP consisting of the amino acid sequence of amino acid numbers 28 to 380 of SEQ ID NO: 2,
a nucleotide sequence encoding an allergen domain comprising Der p 1 consisting of the amino acid sequence of amino acid numbers 383 to 594 of SEQ ID NO: 2, Der p 2 consisting of the amino acid sequence of amino acid numbers 599 to 727 of SEQ ID NO: 2, Der p 23 consisting of the amino acid sequence of amino acid numbers 732 to 800 of SEQ ID NO: 2, and Der p 7 consisting of the amino acid sequence of amino acid numbers 805 to 1002 of SEQ ID NO: 2 in this order,
a nucleotide sequence encoding a transmembrane domain consisting of the amino acid sequence of amino acid numbers 1006 to 1028 of SEQ ID NO: 2, and
a nucleotide sequence encoding an endosomal/lysosomal targeting domain of LAMP consisting of the amino acid sequence of amino acid numbers 1037 to 1040 of SEQ ID NO: 2.
In one embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition for preventing or treating allergy, comprising a nucleic acid comprising a nucleotide sequence encoding a chimeric protein consisting of the amino acid sequence shown by SEQ ID NO: 2 and a pharmaceutically acceptable excipient.
The present invention includes a pharmaceutical composition for preventing or treating allergy, comprising the expression vector of the present invention. In addition, the present invention includes a method for preventing or treating allergy, comprising administering a prophylactically effective or therapeutically effective amount of the expression vector of the present invention. The present invention also includes the expression vector of the present invention for use in preventing or treating allergy. In addition, the present invention includes use of the expression vector of the present invention for the manufacture of a pharmaceutical composition for preventing or treating allergy. In one embodiment, the above-described allergy is allergy caused by an allergen selected from the group consisting of Der p 1, Der p 2, Der p 23, and Der p 7. In addition, in one embodiment, the above-described allergy is allergy affecting an allergy patient having an antibody that responds to an allergen selected from the group consisting of Der p 1, Der p 2, Der p 23, and Der p 7. Further, in one embodiment, the above-described allergy is mite allergy.
In one embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition for preventing or treating allergy, comprising an expression vector comprising the following nucleic acid and a pharmaceutically acceptable excipient:
a nucleic acid comprising a nucleotide sequence encoding a chimeric protein, wherein the nucleotide sequence is a nucleotide sequence comprising the following nucleotide sequences in this order:
a nucleotide sequence encoding a signal peptide consisting of the amino acid sequence of amino acid numbers 1 to 27 of SEQ ID NO: 2,
a nucleotide sequence encoding an intra-organelle stabilizing domain of LAMP consisting of the amino acid sequence of amino acid numbers 28 to 380 of SEQ ID NO: 2,
a nucleotide sequence encoding an allergen domain comprising Der p 1 consisting of the amino acid sequence of amino acid numbers 383 to 594 of SEQ ID NO: 2, Der p 2 consisting of the amino acid sequence of amino acid numbers 599 to 727 of SEQ ID NO: 2, Der p 23 consisting of the amino acid sequence of amino acid numbers 732 to 800 of SEQ ID NO: 2, and Der p 7 consisting of the amino acid sequence of amino acid numbers 805 to 1002 of SEQ ID NO: 2,
a nucleotide sequence encoding a transmembrane domain consisting of the amino acid sequence of amino acid numbers 1006 to 1028 of SEQ ID NO: 2, and
a nucleotide sequence encoding an endosomal/lysosomal targeting domain of LAMP consisting of the amino acid sequence of amino acid numbers 1037 to 1040 of SEQ ID NO: 2.
In one embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition for preventing or treating allergy, comprising an expression vector comprising the following nucleic acid and a pharmaceutically acceptable excipient:
a nucleic acid comprising a nucleotide sequence encoding a chimeric protein, wherein the nucleotide sequence is a nucleotide sequence comprising the following nucleotide sequences in this order:
a nucleotide sequence encoding a signal peptide consisting of the amino acid sequence of amino acid numbers 1 to 27 of SEQ ID NO: 2,
a nucleotide sequence encoding an intra-organelle stabilizing domain of LAMP consisting of the amino acid sequence of amino acid numbers 28 to 380 of SEQ ID NO: 2,
a nucleotide sequence encoding an allergen domain comprising Der p 1 consisting of the amino acid sequence of amino acid numbers 383 to 594 of SEQ ID NO: 2, Der p 2 consisting of the amino acid sequence of amino acid numbers 599 to 727 of SEQ ID NO: 2, Der p 23 consisting of the amino acid sequence of amino acid numbers 732 to 800 of SEQ ID NO: 2, and Der p 7 consisting of the amino acid sequence of amino acid numbers 805 to 1002 of SEQ ID NO: 2 in this order,
a nucleotide sequence encoding a transmembrane domain consisting of the amino acid sequence of amino acid numbers 1006 to 1028 of SEQ ID NO: 2, and
a nucleotide sequence encoding an endosomal/lysosomal targeting domain of LAMP consisting of the amino acid sequence of amino acid numbers 1037 to 1040 of SEQ ID NO: 2.
In one embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition for preventing or treating allergy, comprising an expression vector comprising a nucleic acid comprising a nucleotide sequence encoding a chimeric protein consisting of the amino acid sequence shown by SEQ ID NO: 2 and a pharmaceutically acceptable excipient.
Specific examples are provided herein for reference in order to obtain further understanding of the present invention; however, these examples are for the purpose of illustration and the present invention is not limited thereto.
EXAMPLES
Example 1
Construction of LAMP-Der p 1-Der p 2-Der p 23-Der p 7 Plasmid
LAMP-Der p 1-Der p 2-Der p 23-Der p 7 plasmid consisting of the nucleotide sequence shown by SEQ ID NO: 3 (an expression vector comprising a nucleic acid comprising a nucleotide sequence comprising the following nucleotide sequences in this order (that is, a nucleotide sequence encoding a chimeric protein consisting of the amino acid sequence shown by SEQ ID NO: 2): a nucleotide sequence encoding a signal peptide of LAMP-1 (the amino acid sequence of 1 to 27 of SEQ ID NO: 2), a nucleotide sequence encoding an intra-organelle stabilizing domain of LAMP-1 (the amino acid sequence of 28 to 380 of SEQ ID NO: 2), a nucleotide sequence encoding an allergen domain comprising Der p 1, Der p 2, Der p 23, and Der p 7 in this order (the amino acid sequence of 383 to 1002 of SEQ ID NO: 2), a nucleotide sequence encoding a transmembrane domain of LAMP-1 (the amino acid sequence of 1006 to 1028 of SEQ ID NO: 2), and a nucleotide sequence encoding an endosomal/lysosomal targeting domain of LAMP-1 (the amino acid sequence of 1037 to 1040 of SEQ ID NO: 2)) was constructed. The plasmid can be constructed by inserting synthetic DNA, in which Xho I recognition sequence is added to 5′ end of the nucleotide sequence of 1147 to 3006 of SEQ ID NO: 1 (a nucleotide sequence encoding an allergen domain comprising Der p 1, Der p 2, Der p 23, and Der p 7 in this order) and Eco RI recognition sequence is added to the 3′ end of the nucleic acid sequence, into Eco RI-Xho I site of the plasmid shown by SEQ ID NO: 6 of Japanese Patent No. 5807994. E. coli was transformed with the constructed LAMP-Der p 1-Der p 2-Der p 23-Der p 7 plasmid and cultured in a liquid medium. The amplified LAMP-Der p 1-Der p 2-Der p 23-Der p 7 plasmid was obtained by a method of centrifuging the culture solution and collecting the cells based on a general plasmid extraction and purification method (miniprep method).
Example 2
Expression of LAMP-Der p 1-Der p 2-Der p 23-Der p 7 Chimeric Protein
In vitro expression of the LAMP-Der p 1-Der p 2-Der p 23-Der p 7 chimeric protein (a chimeric protein consisting of an amino acid sequence encoded by the nucleotide sequence shown by SEQ ID NO: 1 (that is, the amino acid sequence shown by SEQ ID NO: 2)) by using human fetal kidney-derived 293T cell line was evaluated.
(1) Cell Culture and Plasmid Introduction
Human fetal kidney-derived 293T cells (Thermo Fisher Scientific, Cat. HCL4517) were seeded in 6-well plates (Cat. 3810-006 manufactured by IWAKI) at 3×105 cells/well in D-MEM medium (Sigma-Aldrich, Cat. D5796) containing 10% fetal bovine serum (Hyclone, Cat. SH30070.03) and 100-fold diluted penicillin-streptomycin (Thermo Fisher Scientific, Cat. 15070063). After overnight culture of the seeded cells at 37° C. in the presence of 5% CO2, a mixed solution having a ratio of LAMP-Der p 1-Der p 2-Der p 23-Der p 7 plasmid: Lipofectamine 2000 (Thermo Fisher Scientific, Cat. 11668027)=2.5 (μg): 10 (μL) was added. After overnight culture of the seeded cells at 37° C. in the presence of 5% CO2 again, the medium was removed and washed once with PBS, and then western blotting was performed.
(2) Western Blotting
Pretreatment: Cells were lysed in RIPA buffer (Pierce, Cat. 89900) containing a protease inhibitor (Sigma-Aldrich, Cat. 1873580), and the protein concentration of the supernatant after centrifugation at 20,000×g for 5 minutes was measured. To 5 μL of the cell lysate diluted with PBS containing protease inhibitor, 5 μL of LDS sample buffer (Thermo Fisher Scientific, Cat. NP0007) containing 100 mM DTT was added so that the protein concentration would be 200 μg/mL, and heat-treated at 70° C. for 10 minutes.
SDS-PAGE: Using NuPAGE (Registered trademark) MOPS SDS Running buffer (Thermo Fisher Scientific, Cat. NP0001) and NuPAGE (Registered trademark) 4%-12% Bis-Tris Gel (Thermo Fisher Scientific, Cat. NP0323), the above-mentioned pretreated cell lysate was applied to the gel and electrophoresis was performed at a constant voltage of 200 V.
Blotting: Blotting was performed by bringing PVDF membrane (Thermo Fisher Scientific, Cat. LC2005) into contact with the gel after SDS-PAGE, and electrifying for 90 minutes at 180 mA in XCell II Blot Module (Thermo Fisher Scientific, Cat. EI9051) filled with NuPAGE (Registered trademark) Transfer buffer (Thermo Fisher Scientific, Cat. NP0006) containing 20% of methanol.
Blocking: The membrane after electrification was immersed in Blocking One (Nacalai Tesque, Cat. 03953-95) and shaken at room temperature for one hour.
Primary antibody: Anti-human LAMP-1 antibody (Sino biological, Cat. 11215-RP01) was added at 1000-fold dilution in TBS Tween-20 buffer (Thermo Fisher Scientific, Cat. 28360) containing 10% of Blocking One. The membrane was immersed in this buffer and shaken overnight at 4° C.
Secondary antibody: The membrane was washed with TBS Tween-20 buffer. Anti-rabbit IgG (H+L chain) pAb-HRP (MBL, Cat. 458) was added at 3000-fold dilution in TBS Tween-20 buffer containing 10% of Blocking One. The membrane was immersed in this buffer and shaken at room temperature for one hour.
Detection: The membrane was washed with TBS Tween-20 buffer. The membrane was immersed in ECL prime western blotting detection reagent (GE Healthcare, Cat. RPN2232), and an image was detected with LumiVision PRO 400EX (Aisin Seiki Co., Ltd.). In the image, a band responsive to the anti-human LAMP-1 antibody corresponding to the chimeric protein was detected.
As the result of the above-mentioned tests, it was confirmed that LAMP-Der p 1-Der p 2-Der p 23-Der p 7 chimeric protein in the cell was expressed by introducing the LAMP-Der p 1-Der p 2-Der p 23-Der p 7 plasmid to the human fetal kidney-derived 293T cell line.
Example 3
Induction of IgG2a Production by Administration of LAMP-Der p 1-Der p 2-Der p 23-Der p 7 Plasmid
Evaluation of induction of antibody production in vivo was performed. In eight examples in each group, 25 μL of a PBS solution containing 50 μg of LAMP-Der p 1-Der p 2-Der p 23-Der p 7 plasmid was administered in the ear of 7-week-old BALB/c female mice (Charles River Laboratories Japan, Inc.) at the start of administration intradermally three times every week (Day 0, 7 and 14). One week after the final administration, blood was collected and plasma samples were obtained (Day 21). As a control, LAMP-Der p 23-Der p 7-Der p 2-Der p 1 plasmid (an expression vector comprising a nucleic acid comprising a nucleotide sequence comprising the following nucleotide sequences in this order: a nucleotide sequence encoding the amino acid sequence of amino acid numbers 1 to 380 of SEQ ID NO: 2 (hereinafter, refer to as N-terminal of LAMP-1 in Examples 3 and 4), a nucleotide sequence encoding an allergen domain comprising Der p 23 consisting of the amino acid sequence of amino acid numbers 732 to 800 of SEQ ID NO: 2 (hereinafter, refer to as Der p 23 domain in Examples 3 and 4), Der p 7 consisting of the amino acid sequence of amino acid numbers 805 to 1002 of SEQ ID NO: 2 (hereinafter, refer to as Der p 7 domain in Examples 3 and 4), Der p 2 consisting of the amino acid sequence of amino acid numbers 599 to 727 of SEQ ID NO: 2 (hereinafter, refer to as Der p 2 domain in Examples 3 and 4), and Der p 1 consisting of the amino acid sequence of amino acid numbers 383 to 594 of SEQ ID NO: 2 (hereinafter, refer to as Der p 1 domain in Examples 3 and 4) in this order, and a nucleotide sequence encoding the amino acid sequence of amino acid numbers 1006 to 1040 of SEQ ID NO: 2 (hereinafter, refer to as C-terminal of LAMP-1 in Examples 3 and 4)); a mixture of LAMP-Der p 1-Der p 2 plasmid (an expression vector comprising a nucleic acid comprising a nucleotide sequence comprising the following nucleotide sequences in this order: a nucleotide sequence encoding N-terminal of LAMP-1, a nucleotide sequence encoding an allergen domain comprising Der p 1 domain and Der p 2 domain in this order, and a nucleotide sequence encoding C-terminal of LAMP-1) and LAMP-Der p 23-Der p 7 plasmid (an expression vector comprising a nucleic acid comprising a nucleotide sequence comprising the following nucleotide sequences in this order: a nucleotide sequence encoding N-terminal of LAMP-1, a nucleotide sequence encoding allergen domain containing Der p 23 domain and Der p 7 domain in this order, and a nucleotide sequence encoding C-terminal of LAMP-1); and a mixture of LAMP-Der p 1 plasmid (an expression vector comprising a nucleic acid comprising a nucleotide sequences comprising the following nucleotide sequences in this order: a nucleotide sequence encoding N-terminal of LAMP-1, a nucleotide sequence encoding an allergen domain comprising Der p 1 domain, and a nucleotide sequence encoding C-terminal of LAMP-1), LAMP-Der p 2 plasmid (an expression vector comprising a nucleic acid comprising a nucleotide sequence comprising the following nucleotide sequences in this order: a nucleotide sequence encoding N-terminal of LAMP-1, a nucleotide sequence encoding an allergen domain comprising Der p 2 domain, and a nucleotide sequence encoding C-terminal of LAMP-1), LAMP-Der p 7 plasmid (an expression vector comprising a nucleic acid comprising a nucleotide sequence comprising the following nucleotide sequences in this order: a nucleotide sequence encoding N-terminal of LAMP-1, a nucleotide sequence encoding an allergen domain comprising Der p 7 domain, and a nucleotide sequence encoding C-terminal of LAMP-1), and LAMP-Der p 23 plasmid (an expression vector comprising a nucleic acid comprising a nucleotide sequence comprising the following nucleotide sequences in this order: a nucleotide sequence encoding N-terminal of LAMP-1, a nucleotide sequence encoding an allergen domain comprising Der p 23 domain, and a nucleotide sequence encoding C-terminal of LAMP-1) were prepared. Each control plasmid can be prepared by the same method as the method described in Example 1. To mice, 25 μL of PBS solution containing 50 μg of the above plasmid or the above plasmid mixture or 25 μL of PBS was administered. An antibody titer was measured by ELISA using a 100-fold or 1000-fold diluted plasma sample, and the absorbance at 450 nm was measured. ELISA measurement was performed based on a general ELISA method using F96 MAXISORP NUNC-IMMUNO PLATE (Nunc, Cat. 439454) as a test plate. Der p 1 which is a purified protein (Indoor biotechnologies, NA-DP1-1, lot: 38052), Der p 2 which is a purified protein (Indoor biotechnologies, NA-DP2-1, lot: 36118), Der p 7 which is a recombinant purified protein (Indoor biotechnologies, RP-DP7-1, lot: 34033), or Der p 23 which is a recombinant purified protein (Sysmex, UniProtKB: A0A0K2DQU8) is prepared to 1 μg/mL with PBS, added at 50 μL/well and allowed to stand overnight at 4° C. After washing a test plate three times with a washing buffer (PBS Tween-20 buffer; Thermo Fisher Scientific, Cat. 28352), 100 μL/well of PBS containing 1% of BSA (Sigma-Aldrich, Cat. A8022) was added and allowed to stand at room temperature for one hour. After washing three times with the washing buffer, 50 μL/well of a 100-fold or 1000-fold diluted plasma sample in PBS containing 1% of BSA was added and allowed to stand at room temperature for one hour. After washing three times with the washing buffer, 50 μL/well of a 50000-fold diluted secondary antibody, Goat anti-mouse IgG2a HRP Conjugated (Bethyl Laboratories, Cat. A90-107P), in PBS containing 1% of BSA was added, and the test plate was allowed to stand at room temperature for one hour. After washing three times with the washing buffer, 50 μL/well of TMB Microwell Peroxidase Substrate System (SeraCare Life Sciences, Inc., Cat.50-76-03) which is a substrate solution was added and the plate was allowed to stand at room temperature for 15 minutes with blocking light. A reaction stop solution (2N H2SO4) was added at 50 μL/well and absorbance at 450 nm was measured.
As the result of the above-mentioned tests, the production of Der p 1, Der p 2, Der p 23 and Der p 7 specific IgG2a was detected by administering LAMP-Der p 1-Der p 2-Der p 23-Der p 7 plasmid (Der p1-p2-p23-p7) to mice (FIG. 1). On the other hand, even when LAMP-Der p 23-Der p 7-Der p 2-Der p 1 plasmid (Der p23-p7-p2-p1), a mixture of LAMP-Der p 1-Der p 2 plasmid and LAMP-Der p 23-Der p 7 plasmid (Der p1-p2+Der p23-p7), and a mixture of LAMP-Der p 1 plasmid, LAMP-Der p 2 plasmid, LAMP-Der p 7 plasmid and LAMP-Der p 23 plasmid (4 plasmid mix) were administered to the mice, the production of Der p 1-specific IgG2a was not detected. In addition, when a mixture of LAMP-Der p 1-Der p 2 plasmid and LAMP-Der p 23-Der p 7 plasmid (Der p1-p2+Der p23-p7) is administered to mice, the production of Der p 2 specific IgG 2a was not detected as well. That is, it was only the LAMP-Der p 1-Der p 2-Der p 23-Der p 7 plasmid that the production of IgG2a specific for all allergens encoded in the plasmid was detected. From the above results, it has been suggested that among the tested plasmids, only LAMP-Der p 1-Der p 2-Der p 23-Der p 7 plasmid induces Th1 immune responses for all the allergens and causes class switch of activated B cells to the IgG2a isotype.
Example 4
Induction of IFN-γ and IL-4 Production by LAMP-Der p 1-Der p 2-Der p 23-Der p 7 Plasmid
Evaluation of cytokine production induction upon stimulation with allergen was performed on splenocytes collected from mice administered with LAMP-Der p 1-Der p 2-Der p 23-Der p 7 plasmid. Splenocytes were prepared according to a general method from the mice used in Example 3 and the mice administered with a control plasmid (an expression vector comprising a nucleic acid comprising a nucleotide sequence comprising the following nucleotide sequences in this order: a nucleotide sequence encoding N-terminal of LAMP-1 and a nucleotide sequence encoding C-terminal of LAMP-1) with the same protocol as in Example 3 on Day 63. The control plasmid can be prepared by deleting Eco RI-Xho I site of the plasmid shown by SEQ ID NO: 6 of Japanese Patent No. 5807994. Splenocytes were seeded in 96-well plates (Cat. 3860-096 manufactured by IWAKI) at 8 x 105 cells/well in RPMI-1640 medium (Sigma-Aldrich, Cat. R8758) containing 10% fetal bovine serum (Hyclone, Cat. SH30070.03) and 100-fold diluted penicillin-streptomycin (ThermoFisher Scientific, Cat. 15070063). Der p 1 (Indoor biotechnologies, NA-DP1-1, lot: 38052), Der p 2 (Indoor biotechnologies, NA-DP2-1, lot: 36118), Der p 7 (Indoor biotechnologies, RP-DP7-1, lot: 34033), or Der p 23 (Sysmex, UniProtKB: A0A0K2DQU8) were added such that the final concentrations thereof were respectively 3, 3, 3, and 1.3 μg/mL. Culturing was performed at 37° C. under 5% of CO2 for 72 hours. The concentrations of IFN-γ and IL-4 in the culture supernatant were measured by ELISA method. A supernatant sample diluted 10-fold with TBS containing 0.1% BSA and 0.05% Tween 20 was used for the measurement of IFN-y, and a supernatant undiluted sample was used for the measurement of IL-4. As a test plate for ELISA measurement, F96 MAXISORP NUNC-IMMUNO PLATE (Nunc, Cat. 439454) was used. The measurement was carried out using mouse IFN-γ DuoSet ELISA (R&D Systems, Cat. DY485) and mouse IL-4 DuoSet ELISA (R&D Systems, Cat. DY 404) according to attached protocol. As the result of the above-mentioned test, a mite-derived allergen-specific IFN-γ production was induced by administering 50 μg of LAMP-Der p 1-Der p 2-Der p 23-Der p 7 plasmid (Der p 1-p2-p 23-p 7) to mice three times (FIG. 2). Also, even in a case where LAMP-Der p 23-Der p 7-Der p 2-Der p 1 plasmid (Der p23-p7-p2-p1), a mixture of LAMP-Der p 1-Der p 2 plasmid and LAMP-Der p 23-Der p 7 plasmid (Der p1-p2+Der p23-p7), and a mixture of LAMP-Der p 1 plasmid, LAMP-Der p 2 plasmid, LAMP-Der p 7 plasmid,and LAMP-Der p 23 plasmid (4 plasmid mix) were administered to the mice three times, comparable mite-derived allergen-specific IFN-γ production was induced. On the other hand, in the mice administered three times with 50 μg of LAMP-Der p 1-Der p 2-Der p 23-Der p 7 plasmid (Der p 1-p2-p23-p′7), the mite-derived allergen-specific IL-4 production was the lower limit of detection (FIG. 3). Even in a case where LAMP-Der p 23-Der p 7-Der p 2-Der p 1 plasmid (Der p23-p7-p2-p1), a mixture of LAMP-Der p 1-Der p 2 plasmid and LAMP-Der p 23-Der p 7 plasmid (Der p1-p2+Der p23-p′7), and a mixture of LAMP-Der p 1 plasmid, LAMP-Der p 2 plasmid, LAMP-Der p 7 plasmid, and LAMP-Der p 23 plasmid (4 plasmid mix) were administered to the mice three times, the mite-derived allergen-specific IL-4 production was below the lower limit of detection.
As the result of the above-mentioned tests, a mixture of LAMP-Der p 1-Der p 2-Der p 23-Der p 7 plasmid, LAMP-Der p 23-Der p 7-Der p 2-Der p 1 plasmid, LAMP-Der p 1-Der p 2 plasmid, and LAMP-Der p 23-Der p 7 plasmid, and a mixture of LAMP-Der p 1 plasmid, LAMP-Der p 2 plasmid, LAMP-Der p 7 plasmid, and LAMP-Der p 23 plasmid have been shown to induce Th1 cell dominant immune responses.
INDUSTRIAL APPLICABILITY
The nucleic acid of the present invention is expected to be useful for the prevention or treatment of mite allergy. In addition, the method for producing the nucleic acid of the present invention is useful for producing the nucleic acid.
Sequence Listing Free Text
The numerical heading <223> in the following sequence listing describes the description of “Artificial Sequence”. Specifically, the nucleotide sequence shown by SEQ ID NO: 1 in the sequence listing is a nucleotide sequence encoding LAMP-Der p 1-Der p 2-Der p 23-Der p 7 chimeric protein, and the amino acid sequence shown by SEQ ID NO: 2 in the sequence listing is the amino acid sequence encoded by SEQ ID NO: 1. In addition, the nucleotide sequence shown by SEQ ID NO: 3 is the nucleotide sequence of LAMP-Der p 1-Der p 2-Der p 23-Der p 7 plasmid.
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17377297
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astellas pharma inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Apr 27th, 2022 08:36AM
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Apr 27th, 2022 08:36AM
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Astellas Pharma
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tyo:4503
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Astellas Pharma
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Dec 20th, 2005 12:00AM
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Apr 2nd, 2002 12:00AM
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https://www.uspto.gov?id=US06977157-20051220
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Clock gene promoter
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The present invention discloses a Period2 gene promoter, a construct containing the promoter and a reporter gene, a cell containing the construct, a transgenic animal harboring the construct, and a method for screening a substance which controls expression or oscillatory expression of a biological clock gene. The screening method uses the above cell, a suprachiasmatic nucleus section or peripheral tissue of the above transgenic animal or the above transgenic animal.
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6977157
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1. An isolated DNA which maintains a basal promoter activity and has a promoter activity transcriptionally-activated by a BMAL1/CLOCK heterodimer, which comprises the nucleotide sequence described in the following (a), (b), (c) or (d):
(a) a sequence consisting of nucleotides at positions 7,463 to 7,931 in the nucleotide sequence represented by SEQ ID NO:1,
(b) a sequence consisting of nucleotides at positions 6,417 to 7,931 in the nucleotide sequence represented by SEQ ID NO:1,
(c) a sequence consisting of nucleotides at positions 5,249 to 7,931 in the nucleotide sequence represented by SEQ ID NO:1, and
(d) a sequence consisting of nucleotides at positions 4,415 to 7,931 in the nucleotide sequence represented by SEQ ID NO:1.
2. A DNA according to claim 1, which consists of the nucleotide sequence described in (a), (b), (c) or (d).
3. A construct which comprises the DNA according to claim 1 or 2 operably linked to a reporter gene.
4. A cell which comprises the construct according to claim 3.
5. A method for screening a substance which controls expression of Period2 gene, comprising the steps of:
allowing the cell according to claim 4 to contact with a substance to be tested, and measuring activity of the reporter gene.
6. A transgenic rat or mouse transfected with the construct according to claim 3, and wherein the suprachiasmatic nucleus and/or peripheral tissues of the rat or mouse exhibit the function of reporter gene expression.
7. The transgenic rat according to claim 6.
8. A method for screening a substance which controls expression and/or oscillatory expression of Period2 gene, comprising the steps of:
allowing the cell according to claim 4 to react with a substance to be tested, and
measuring activity of the reporter gene for oscillatory expression of the Period2 gene.
9. A method for screening a substance which controls expression and/or oscillatory expression of Period2 gene, comprising the steps of:
administering a substance to be tested to the transgenic rat or mouse according to claim 6, and
measuring activity of the reporter gene in the suprachiasmatic nucleus of the animal for oscillatory expression of the Period2 gene.
10. A method for screening a substance which controls expression and/or oscillatory expression of Period2 gene, comprising the steps of:
allowing a suprachiasmatic nucleus section or peripheral tissue of the transgenic rat or mouse according to claim 6 to react with a substance to be tested, and
measuring activity of the reporter gene for oscillatory expression of the Period2 gene.
11. The transgenic rat or mouse according to claim 6, wherein the reporter gene is one member selected from the group consisting of a gene encoding luciferase, a gene encoding secretion type alkaline phosphatase (SEAP), a gene encoding green fluorescent protein (GFP), a gene encoding chloramphenicol acetyltransferase (CAT), a gene encoding β-glucuronidase (GUS), a gene encoding β-D-galactosidase and a gene encoding aequorin.
12. The transgenic rat according to claim 7, wherein the reporter gene is one member selected from the group consisting of a gene encoding luciferase, a gene encoding secretion type alkaline phosphatase (SEAP), a gene encoding green fluorescent protein (GFP), a gene encoding chloramphenicol acetyltransferase (CAT), a gene encoding β-glucuronidase (GUS), a gene encoding β-D-galactosidase and a gene encoding aequorin.
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TECHNICAL FIELD
The present invention relates to a biological clock gene promoter, a construct containing the promoter and a reporter gene, a cell containing the construct, a transgenic animal harboring the construct, and a method for screening a substance which controls expression and/or oscillatory expression of a biological clock gene.
BACKGROUND OF THE INVENTION
It is known that a large number of organisms are possessed of a mechanism in their bodies, which generates circadian rhythm with ablout 24 hour cycle (Reference 1). In mammals, the mechanism generating circadian rhythm controls sleep-wake rhythm, blood pressure, body temperature and a part of hormone secretion rhythm (Reference 2, Reference 3). As diseases caused by the circadian rhythm disturbance, sleep-wake rhythm disorder (delayed sleep phase syndrome (DSPS), non-24 hour sleep-wake syndrome), seasonal depression, jet lag syndrome (JET-LAG), sleep disturbance in night and day shift workers, nocturnal poriomania and delirium found in patients with senile dementia and the like have been reported (References 4 to 7). In addition, there is a report stating that a part of children with school refusal or workers with refusal to attend firm as a social problem is caused by a circadian rhythm disorder (Reference 8). Increase in the number of patients with rhythm disorder is expected in the future by the increase in advanced aged and the progress of globalization of social structure, but it is the present situation that a secure rhythm disorder improving agent is not present. On the other hand, there are reports stating that bright light therapy, including staring at a light of about 5,000 luxes continuously for several hours in the early morning, shows excellent therapeutic effect on nocturnal poriomania and delirium in patients with senile dementia, rhythm disturbance in patients with delayed sleep phase syndrome (DSPS) or the like (References 6, 7 and 9 to 12). However, since the bright light therapy which requires looking at a high lux light source for a prolonged period of time is painful or burden for patients and their caretakers, agents as substitutes for this light therapy are highly expected.
Based on tissue destruction and tissue transplantation experiments, it has been found in 1972 that the rhythm center of mammals is present in suprachiasmatic nucleus (SCN) (References 13 and 14). However, molecular mechanism of the rhythm generation has been unclear until recent years (Reference 15). On the other hand, an arrhythmic mutant (Period mutant) of Drosophila melanogaster has been prepared by a genetic technique and then a Period gene of Drosophila melanogaster has been cloned (Reference 16, Reference 17). Since oscillatory expression of Period gene with about 24 hour cycle is achieved in Drosophila melanogaster through the migration of translated Period protein (PERIOD) into the nucleus and subsequent inhibition of its own transcription (negative feedback mechanism), it is considered that outputs of circadian rhythm (behavior, timing of eclosion) are finally developed by this (References 18 and 19). In mammals, on the other hand, human and mouse Period1 gene (Period1; Per1) has been cloned in 1997 as a homologue of the Drosophila Period gene (Reference 20, Reference 21). Thereafter, mouse Period2 gene (Period2; Per2) (References 22 and 23) and mouse Period3 gene (Period3; Per3) (References 24 and 25) have been cloned. In addition to these, mouse Clock gene (Clock) (Reference 26) and mouse Bmal1 gene (Bmal1) (Reference 27) have been reported as mammalian clock genes. Thus, it is possible for now to understand the rhythm generation mechanism at the molecular level. Actually, it has been found that the Clock gene and Bmal1 gene are important for the circadian oscillation of a clock gene (References 44 and 45) and that a protein encoded by the Clock gene and a protein encoded by the Bmal1 gene bind to a CACGTG type E-box and activate transcription of said clock gene, namely, the CACGTG type E-box sequence is essential for the transcriptional activation by the CLOCK and BMAL1 (Reference 27).
A transgenic rat (mPer1; luc transgenic rat) harboring a DNA prepared by ligating an upstream sequence of a mouse clock gene, Period1 gene (mPer 1), with a luciferase gene has been reported in 2000 (Reference 28). It has been reported that the Period1 shows oscillatory expressions in not only the suprachiasmatic nucleus but also in peripheral tissues of the living body by measuring the luciferase activity in real time using a photomultiplier tube detector (Photomal) (Reference 28). In addition, similar results have been reported also on a transgenic mouse (mPer1; luc transgenic mouse) harboring a DNA prepared by ligating an upstream sequence of the Period1 gene with a luciferase gene (Reference 43).
It has been suggested that the Period2 gene among the three Period gene homologues takes an important role in the rhythm generation, because its circadian rhythm disappears in mutant mice with artificial gene mutation (Reference 29). Also, it has been reported that the cause of a familial advanced sleep phase syndrome (ASPS) is a point mutation of the Period2 gene (Reference 30). Thus, the Period2 gene is a gene which not only shows abnormal rhythm in the mutant mice but also relates to rhythm disorders in human has been confirmed.
Only the one report regarding the upstream region of Period2 gene is WO 01/07654 on a mouse sequence, and said international publication describes a DNA sequence by defining it as a sequence which controls mouse Period2 transcription and describes about a method for identifying a Period2 transcription inhibitor, which comprises supplying a cell containing sequence (which controls Per2 transcription)-linked reporter gene, introducing a test compound and assaying transcription of the reporter gene. However, there is no specific example on actually obtaining the above DNA sequence described as a sequence which controls mouse Period2 transcription, and there is no description such that it can be obtained. Also, there is no specific example on the determination of transcription start site or measurement of transcriptional activity, too. In addition, there are positions in the disclosed DNA sequence where bases cannot be specified, and sequence information regarding upstream sequence of the Period2 gene is not specifically disclosed.
Also, there are many reports on the analysis of the upstream sequence of Period1 gene, but since Period1 and Period2 genes are located in different chromosomes and have no characteristic common sequence, it did not become information for deducing the Period2 promoter sequence.
Great concern has been directed toward the development of a tool for the screening of useful substances as rhythm disorder improving agents having a mechanism of function to control expression of biological clock genes and a method for screening substances capable of controlling expression of biological clock genes.
DISCLOSURE OF THE INVENTION
As a result of intensive studies, the present inventors have determined for the first time a human Period2 gene promoter sequence as a region which controls transcriptional activity of the human Period2 gene and also as a region that contributes to the oscillatory expression, thereby obtaining a construct containing the above promoter and a reporter gene and a cell containing the above construct. Also, a construct containing the above promoter and a reporter gene and a cell containing the above construct were obtained by determining a mouse Period2 gene promoter sequence. In addition, a transgenic animal harboring the above construct was prepared. Next, in spite of the absence of the CACGTG type E-box sequence in the mouse-derived Period2 promoter of the present invention, transcriptional activation of the Period2 gene by a heterodimer (BMAL1/CLOCK heterodimer) consisting of a protein encoded by a mouse Bmal1 gene and a protein encoded by a mouse Clock gene and oscillatory expression were unexpectedly found. Also, a system by which promoter activity of this gene can be easily detected was constructed. As a result, the present invention provides a Period2 gene promoter, a construct containing the above promoter and a reporter gene, a cell containing the above construct and a transgenic animal harboring the above construct, as tools useful for screening rhythm disorder improving agents as substances which control expression and/or oscillatory expression of biological clock genes, and also provides a convenient method for screening rhythm disorder improving agents, and thus, the present invention has been completed. Also, the term “construct” as used herein means a construct consisting of a DNA constructed by a combination of DNAs such that it shows the function of interest.
The CACGTG type E-box sequence is present in five positions of the upstream of the first exon of the mouse Period1 gene as one of the Period gene homologues, and the region which contributes to the basal activity of Per1 transcription is present in a region containing first exon, its environs and a human-mouse conserved segment of the first intron, but it does not contain these five CACGTG type E-box sequences. The BMAL1/CLOCK heterodimer activates transcription of the mouse Period1 gene by binding to the CACGTG type E-box sequence, but it hardly enhance the transcriptional activity derived from the region which contributes a basal transcriptional activity and contains no CACGTG type E-box sequence (Reference 27). Also, when the five CACGTG type E-boxes of the mouse Period1 gene were mutated, the transcriptional activity in the presence of the BMAL1/CLOCK heterodimer becomes a similar level of the basal transcriptional activity (Reference 34). Based on these facts, it is considered that the BMAL1/CLOCK heterodimer induces transcriptional activation of the mouse Period1 gene via the CACGTG type E-box. On the other hand, it has been reported that the circadian oscillation of Period1 and Period2 genes disappear under constant dark condition in Bmal1 knockout mice (reference 44), and it has been reported that the circadian oscillation of Period1 and Period2 genes attenuate under constant dark condition in Clock/Clock mutant mice (reference 45). That is, it has been found that the Bmal1 and Clock are important for the oscillations. When considered from these results, the DNA sequence defined as sequence, which controls mouse Period2 transcription, in the above WO/01/07654 (that is, it corresponds to a part (from positions 6,050 to 7,761) of a sequence consisting of nucleotides of positions 4,415 to 7,931 in the nucleotide sequence represented by SEQ ID NO:1 which is the DNA nucleotide sequence of the present invention, but is a sequence having inconsistent sequences in several positions) merely indicates a region having basal transcriptional activity because it does not contain the CACGTG type E-box. In the same manner, it was not considered that the DNA derived from Period2 gene of the present invention which does not contain the CACGTG type E-box will show transcriptional enhancement by a BMAL1/CLOCK heterodimer, and its contribution to the oscillatory expression could not be expected, too. However, it was unexpectedly found that the DNA of the present invention which does not contain the CACGTG type E-box shows transcriptional enhancement by a BMAL1/CLOCK heterodimer and also contributes to the oscillatory expression, namely that it is a region important for the oscillatory expression, so that a convenient system for measuring the oscillatory expression was constructed. Namely, since the Period2 promoter of the present invention showed higher activity than that of the mouse Period2 gene sequence containing the CACGTG type E-box (namely, pCH1 of this specification), it was able to construct a more easily detectable system by using the DNA of the present invention and to provide a method for obtaining more useful rhythm improving agents. Thus, the present invention has been accomplished.
Accordingly, the present invention relates to
[1] A DNA which maintains a basal promoter activity and has a promoter activity transcriptionally-activated by a BMAL1/CLOCK heterodimer, which comprises the nucleotide sequence described in the following (a), (b), (c), (d) or (e):
(a) a sequence consisting of nucleotides at positions 7,463 to 7,931 in the nucleotide sequence represented by SEQ ID NO:1,
(b) a sequence consisting of nucleotides at positions 6,417 to 7,931 in the nucleotide sequence represented by SEQ ID NO:1,
(c) a sequence consisting of nucleotides at positions 5,249 to 7,931 in the nucleotide sequence represented by SEQ ID NO:1,
(d) a sequence consisting of nucleotides at positions 4,415 to 7,931 in the nucleotide sequence represented by SEQ ID NO:1, and
(e) a sequence consisting of nucleotides at positions 3,820 to 6,068 in the nucleotide sequence represented by SEQ ID NO:2.
[2] A DNA which consists of the nucleotide sequence described in the (a), (b), (c), (d) or (e) according to [1].
[3] A construct which comprises the DNA according to [1] or [2] and a reporter gene.
[4] A cell which comprises the construct according to [3].
[5] A method for screening a substance which controls expression of Period2 gene, comprising the steps of:
allowing the cell according to [4] to contact with a substance to be tested, and
measuring a reporter activity.
[6] An transgenic animal transfected with the construct according to [3].
[7] The transgenic animal according to [6], wherein the animal is a rat.
[8] A method for screening a substance which controls expression and/or oscillatory expression of Period2 gene, comprising the steps of:
allowing the cell according to [4] or a suprachiasmatic nucleus section or peripheral tissue of the transgenic animal according to [6] or [7] to react with a substance to be tested, and
measuring oscillatory expression.
[9] A method for screening a substance which controls expression and/or oscillatory expression of Period2 gene, comprising the steps of:
administering a substance to be tested to the transgenic animal according to [6] or [7], and
measuring oscillatory expression of suprachiasmatic nucleus of the animal.
[10] The screening method according to any one of [5], [8] and [9], wherein the substance which controls expression and/or oscillatory expression of Period2 gene is a substance for improvement of rhythm disorders.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a result of the sequence comparison between upstream regions of mouse Period2 gene and human Period2 gene (hPer 2).
FIG. 2 shows nucleotide sequences of 7 segments highly conserved between mouse (SEQ ID NO. 1) and human (SEQ ID NO. 2).
FIG. 3 shows basal promoter activities of mouse Period2 gene upstream regions.
FIG. 4 shows a function of a heterodimer of transcription factors, BMAL1/CLOCK, upon a vector pCH3 containing mouse Period2 gene upstream region.
FIG. 5 shows a result of continuous measurement of luminescence from a suprachiasmatic nucleus section, carried out for 10 days after commencement of the measurement.
FIG. 6 shows a part of the graph shown in FIG. 5 expanded only in vertical direction.
FIG. 7 shows a result of continuous measurement of luminescence from a liver section, carried out for 10 days after commencement of the measurement.
FIG. 8 shows a result of continuous measurement of luminescence from a lung section, carried out for 10 days after commencement of the measurement.
FIG. 9 shows a result of a continuous measurement of luminescence from an eyeball, carried out for 10 days after commencement of the measurement.
FIG. 10 shows a result of continuous measurement of luminescence from a pCH3-transfected culture cell, carried out for 4 days after commencement of the measurement.
FIG. 11 shows a result of continuous measurement of luminescence from a pTM15-transfected culture cell, carried out for 5 days after commencement of the measurement.
FIG. 12 shows basal activities from various upstream regions of mouse Period2 gene and functions of a heterodimer of transcription factors, BMAL1/CLOCK, upon vectors containing the same regions.
FIG. 13 shows a result of continuous measurement of luminescence from a pCH3-D3-transfected culture cell, carried out for 6 days after commencement of the measurement.
BEST MODE FOR CARRYING OUT THE INVENTION
The present invention is explained below in detail.
[DNA of the Present Invention]
The DNA of the present invention contains (a) a sequence consisting of nucleotides at positions 7,463 to 7,931 in the nucleotide sequence represented by SEQ ID NO:1, (b) a sequence consisting of nucleotides at positions 6,417 to 7,931 in the nucleotide sequence represented by SEQ ID NO:1, (c) a sequence consisting of nucleotides at positions 5,429 to 7,931 in the nucleotide sequence represented by SEQ ID NO:1, (d) a sequence consisting of nucleotides at positions 4,415 to 7,931 in the nucleotide sequence represented by SEQ ID NO:1, or (e) a sequence consisting of nucleotides at positions 3,820 to 6,068 in the nucleotide sequence represented by SEQ ID NO:2, and shows a Period2 gene promoter activity.
The term “Period2 gene promoter activity” as used herein means a promoter activity which maintains at least a basal promoter activity as a DNA consisting of a sequence consisting of nucleotides at positions 4,415 to 7,931 in the nucleotide sequence represented by SEQ ID NO:1 (that is, having at least 50% of the activity of the basal promoter activity of a DNA consisting of a sequence consisting of nucleotides at positions 4,415 to 7,931 in the nucleotide sequence represented by SEQ ID NO:1, preferably having substantially the same level of basal promoter activity of the DNA or superior to that) and is also enhanced by BMAL1/CLOCK heterodimer on transcriptional activity (namely to contribute to oscillatory expression).
The term “basal promoter activity” as used herein means a promoter activity when a predetermined period of time (e.g., 48 hours) is passed under no stimulus condition, and the term “no stimulus condition” means specifically conditions in the absence of a BMAL1/CLOCK heterodimer as shown in Example 3. Also, the above “BMAL1/CLOCK heterodimer” is transcription factors which controls each transcription of the Period1 gene, Period2 gene and Period3 gene.
Although the method for judging whether or not a certain DNA shows “Period2 gene promoter activity” is not particularly limited, it can be judged, for example, by verifying that it is substantially the same as or superior to the promoter activity of the DNA consisting of a sequence consisting of nucleotides at positions 4,415 to 7,931 in the nucleotide sequence represented by SEQ ID NO:1, through the measurement of basal promoter activity in the absence of the BMAL1/CLOCK heterodimer as shown in Example 3, and further verifying whether or not it shows dose-dependent transcriptional activation by a BMAL1/CLOCK heterodimer.
The desirable DNA of the present invention includes a DNA consisting of a sequence consisting of nucleotides at positions 7,463 to 7,931 in the nucleotide sequence represented by SEQ ID NO:1, a sequence consisting of nucleotides at positions 6,417 to 7,931 in the nucleotide sequence represented by SEQ ID NO:1, a sequence consisting of nucleotides at positions 5,429 to 7,931 in the nucleotide sequence represented by SEQ ID NO:1 or a sequence consisting of nucleotides at positions 4,415 to 7,931 in the nucleotide sequence represented by SEQ ID NO:1, or a DNA consisting of a sequence consisting of nucleotides at positions 3,820 to 6,068 in the nucleotide sequence represented by SEQ ID NO:2. However, any DNA is included in the DNA of the present invention, so long as the DNA contains a DNA consisting of a sequence consisting of nucleotides at positions 7,463 to 7,931 in the nucleotide sequence represented by SEQ ID NO:1, a sequence consisting of nucleotides at positions 6,417 to 7,931 in the nucleotide sequence represented by SEQ ID NO:1, a sequence consisting of nucleotides at positions 5,429 to 7,931 in the nucleotide sequence represented by SEQ ID NO:1 or a sequence consisting of nucleotides at positions 4,415 to 7,931 in the nucleotide sequence represented by SEQ ID NO:1, or a sequence consisting of nucleotides at positions 3,820 to 6,068 in the nucleotide sequence represented by SEQ ID NO:2, and has a Period2 gene promoter activity.
As is shown later in Example 1, the sequence consisting of nucleotides at positions 4,415 to 7,931 in the nucleotide sequence represented by SEQ ID NO:1 is an upstream region of the mouse Period2 gene and, as is shown later in Example 3, has a Period2 gene promoter activity.
As is shown later in Example 1, the DNA consisting of a sequence consisting of nucleotides at positions 3,820 to 6,068 in the nucleotide sequence represented by SEQ ID NO:2 is an upstream region of the human Period2 gene. From the experimental results on mouse genes (Examples 1 and 3 which are described later), it was found that the mouse DNA showing promoter activity (namely the DNA consisting of a sequence consisting of nucleotides at positions 4,415 to 7,931 in the nucleotide sequence represented by SEQ ID NO:1) is a DNA resulting from the elimination of the region containing conserved segments (IV, V, VI and VII) in the first intron of Period2 gene from the DNA containing the human/mouse seven conserved segments. Accordingly, it is considered that a human DNA corresponding to the DNA resulting from the elimination of the region containing conserved segments (IV, V, VI and VII) in the first intron of Period2 gene from the DNA containing the human/mouse seven preserved regions, namely the DNA consisting of a sequence consisting of nucleotides at positions 3,820 to 6,068 in the nucleotide sequence represented by SEQ ID NO:2, also has a Period2 gene promoter activity.
Although not particularly limited, the DNA of the present invention can be prepared, for example, by (1) using polymerase chain reaction (PCR) method or by (2) screening a phage library.
(1) Polymerase Chain Reaction (PCR) Method
When the DNA of the present invention is prepared using the PCR method, a primer set capably of amplifying the DNA of the present invention is firstly designed based on the information on each nucleotide sequence represented by SEQ ID NO:1 or 2.
In the case of a DNA of the present invention containing a sequence consisting of nucleotides at positions 4,415 to 7,931 in the nucleotide sequence represented by SEQ ID NO:1, which is the mouse Period2 gene promoter, a primer set is designed based on the information on the nucleotide sequence represented by SEQ ID NO:1 in such a manner that the amplified product contains the sequence consisting of nucleotides at positions 4,415 to 7,931 in the nucleotide sequence represented by SEQ ID NO:1 but does not contain in and after the conserved segment IV (position 8,534). Also, in the case of a DNA of the present invention containing a sequence consisting of nucleotides at position 3,820 to 6,068 in the nucleotide sequence represented by SEQ ID NO:2, which is the human Period2 gene promoter, a primer set is designed based on the information on the nucleotide sequence represented by SEQ ID NO:2 in such a manner that the amplified product contains a sequence consisting of nucleotides at positions 3,820 to 6,068 in the nucleotide sequence represented by SEQ ID NO:2 but does not contain in and after the conserved segment IV (position 6,531).
The DNA of the present invention can be obtained by carrying out PCR using the thus designed respective primer set and a genomic DNA as the template.
(2) Phage Library Screening Method
When the DNA of the present invention is prepared by screening a phage library (e.g., Maniatis, T. et al., Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory, NY, 1982), a probe which can screen phage clones containing the DNA of the present invention is firstly designed base on the information on each nucleotide sequence of SEQ ID NO:1 or 2.
In the case of a DNA of the present invention containing a sequence consisting of nucleotides at positions 4,415 to 7,931 in the nucleotide sequence represented by SEQ ID NO:1, which is the mouse Period2 gene promoter, a probe is designed based on the information on the sequence consisting of nucleotides at positions 4,415 to 7,931 in the nucleotide sequence represented by SEQ ID NO:1. Also, in the case of a DNA of the present invention containing a sequence consisting of nucleotides at positions 3,820 to 6,068 in the nucleotide sequence represented by SEQ ID NO:2, which is the human Period2 gene promoter, a probe is designed based on the information on the sequence consisting of nucleotides at positions 3,820 to 6,068 in the nucleotide sequence represented by SEQ ID NO:2.
A phage clone containing the DNA of the present invention can be obtained by screening a phage library using the thus designed respective probe. The DNA of the present invention can be obtained by treating the thus obtained phage clone with appropriate restriction enzymes and then purifying a DNA fragment of interest using an appropriate purification means (e.g., agarose gel electrophoresis).
[Construct and Cell of the Present Invention]
The construct of the present invention contains the DNA of the present invention and a reporter gene. As the reporter gene, a gene encoding a known reporter protein which can be used as an index of gene expression in the cells can be used. The reporter protein includes luciferase, secretion type alkaline phosphatase (SEAP), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), β-glucuronidase (GUS), β-D-galactosidase, aequorin and the like. According to the construct of the present invention, it is preferable to use a luciferase gene as the reporter gene.
According to the construct of the present invention, arranging positions of the DNA of the present invention and a reporter gene are not limited, so long as the reporter gene is arranged in a downstream position of the DNA of the present invention and under control of the promoter activity of the DNA of the present invention. Also, the construct of the present invention is not particularly limited, so long as the construct contains at least the DNA of the present invention and a reporter gene, but it is preferable that it further contains a vector region.
Although the construct of the present invention is not particularly limited, a construct of the present invention further containing a vector region in addition to the DNA of the present invention and a reporter gene can be prepared, for example, by introducing the DNA of the present invention into a multi-cloning site of an appropriate reporter vector (namely, a vector containing a reporter gene). The reporter vector includes a vector pGL3-basic containing a gene encoding luciferase (Promega), a vector pSEAP2-basic containing a gene encoding SEAP (Clontech), and a vector pd1EGFP containing a gene encoding labile type GFP (Clontech).
More specifically, the construct of the present invention can be prepared by introducing the DNA of the present invention obtained by using the PCR method into a multi-cloning site of a reporter vector.
Also, the construct of the present invention can be prepared by introducing the DNA of the present invention, which has been prepared by treating a phage clone obtained by screening a phage library with appropriate restriction enzymes and then purifying it using an appropriate purification means (e.g., agarose gel electrophoresis), if necessary further subjecting to a blunt-ended treatment, into a multi-cloning site of a reporter vector. For example, in the case of a phage clone containing a sequence consisting of nucleotides at positions 3,820 to 6,068 in the nucleotide sequence represented by SEQ ID NO:2, which is the human Period2 gene promoter, the construct of the present invention containing the human Period2 gene promoter can be prepared by treating the phage clone with appropriate restriction enzymes (e.g., a combination of a restriction enzyme Aor51HI which cuts at position 3,513 position and a restriction enzyme PshBI which cuts at position 6,447), obtaining a 2,935 bp DNA fragment by purifying it using an appropriate purification means (e.g., an agarose gel electrophoresis), smooth-ending the DNA fragment and then introducing it into a multi-cloning site of a reporter vector. A deletion construct containing the DNA of the present invention can be prepared, for example, by treating a longer DNA among the DNAs of the present invention obtained by the above method with appropriate restriction enzymes and purifying the digest by an appropriate purification method and then subjecting the thus obtained DNA fragment of interest to self-ligation. More specifically, it can be obtained by the method described in Example 10.
The cell of the present invention contains the construct of the present invention. Although not particularly limited, the cell of the present invention can be prepared by transforming an appropriate host cell (preferably a eucaryote) with the construct of the present invention (preferably, a construct of the present invention further containing a vector region in addition to the DNA of the present invention and a reporter gene).
The host cell of eucaryote includes cells such as vertebrate, insect and yeast, and examples of the vertebrate cells include mouse NIH3T3 cell, monkey COS cell (Reference 37), Chinese hamster ovary cell (CHO) dihydrofolate reductase deficient strain (Reference 38), mouse L cell, mouse A9 cell, monkey BS-C-1 cell and the like, but it is preferable to use mouse NIH3T3 cell.
The construct of the present invention can be incorporated into a host cell by, for example, a DEAE-dextran method (Reference 39), a calcium phosphate-DNA coprecipitation method (Reference 40), a method using commercially available transfection reagents [e.g., Lipofectamine 2000 (GIBCO-BRL), FuGENE™6 Transfection Reagent (Roche Diagnostics)], electroporation (Reference 41) or the like.
The cell of the present invention can be cultured in accordance with a conventional method. As the medium which can be used in the culturing, various generally used media can be appropriately selected in response to the host cell employed. For example, in the case of the NIH3T3 cell, a medium prepared by supplementing DMEM (Dulbecco's modified Eagle's medium) with glucose (final concentration=4.5 g/l) and fetal bovine serum (final concentration=10%) can be used.
[Transgenic Animal of the Present Invention]
The transgenic animal of the present invention is not particularly limited, so long as the animal is transfected with the construct of the present invention, but it can be prepared based on a conventionally known method (e.g., Reference 35), except that the construct of the present invention is used as the DNA to be transfected. Specifically, it can be prepared based on the procedure described later in Example 4.
Also, the term “animal” as used herein means an animal excluding human (namely non-human animal), and examples include mammals excluding human (e.g., rat, mouse, dog, cat, monkey, pig, cattle, sheep, rabbit, goat, dolphin or horse), birds (e.g., domestic fowl or quail), amphibia (e.g., frog), reptiles, insects and the like, and rat and mouse are preferred, and rat is particularly preferred.
[Screening Method of the Present Invention]
The screening method of the present invention can be carried out using the cell of the present invention, a suprachiasmatic nucleus section or peripheral tissue of the transgenic animal of the present invention or the transgenic animal of the present invention itself. Although the test substance to be used in the screening is not particularly limited, examples include commercially available compounds (including peptides), various known compounds (including peptides) registered in chemical file, compounds obtained by combinatorial chemistry techniques (Reference 31), culture supernatants of microorganisms, natural components derived from plants and marine organisms, animal tissue extracts or compounds (including peptides) prepared by chemically or biologically modifying the compounds (including peptides) selected by the screening method of the present invention.
According to the screening method of the present invention which uses the cell of the present invention, a substance which controls expression of the Period2 gene, namely a substance which modifies its transcription activity, can be selected by allowing the cell of the present invention to contact with a substance to be tested and measuring the reporter activity (namely, a reporter assay).
When the cell of the present invention is allowed to contact with a substance to be tested, a cell into which the construct is temporarily or stably introduced is prepared and drug (namely substance to be tested) stimulation is carried out. A substance which can control expression of the Period2 gene can be screened by carrying out a reporter assay after a predetermined period of time of the drug (namely substance to be tested) stimulation.
The reporter assay can be carried out by a known assay method in response to the kinds of a reporter protein to be used. For example, when a firefly luciferase is used as the reporter protein, luciferin can be used as its chemical substrate for luciferin-luciferase luminescence, and when a Renilla luciferase derived from sea pansy is used, coelenteradin can be used as its chemical substrate for luciferin-luciferase luminescence. Also, when SEAP is used, CSPD [disodium 3-(methoxyspiro(1,2-dioxetane-3,2′-(5′-chloro)tricyclo[3.3.1.13.7]decan)-4-yl)phenyl phosphate] and MUP (4-methylunbellifery phosphate) can be used as its chemical substrates for the respective luminescent and fluorescent assay. A luciferase assay is preferred as the reporter assay, and the luciferase assay can be carried out preferably under the conditions described in Example 3 which is described later.
All of the substances which enhance expression of the Period2 gene and the substances which inhibit the same are useful as agents improving rhythm disorders, and when a substance which inhibits expression of the Period2 gene is screened, the screening can be effected by an assay in the coexistence of a known Period2 gene transcription activating factor such as BMAL1/CLOCK heterodimer. A synchronizing factor during daytime has a property to accelerate transcription or release the transcription inhibition (transcription acceleration as a consequence) of the Period2 gene, and it can synchronized the circadian rhythm when reacted in daytime and causes phase shift when reacted in night. Also, a synchronizing factor during night has a property to inhibit transcription or release the transcription acceleration (transcription inhibition as a consequence) of the Period gene, and it has been confirmed that it can synchronized the rhythm when reacted in night and causes phase shift when reacted in daytime. Accordingly, an agent which accelerates transcription of the Period2 gene can synchronize the rhythm when taken during the day, and on the contrary, an agent which inhibits transcription of the Period2 gene can synchronize the rhythm when taken during the night.
According to the screening method of the present invention which uses the cell of the present invention or a suprachiasmatic nucleus section or peripheral tissue of a transgenic animal, a substance which controls expression of the Period2 gene can be selected by allowing the cell of the present invention or a suprachiasmatic nucleus section or peripheral tissue of a transgenic animal to react with a substance to be tested and measuring the oscillatory expression.
There is a report stating that oscillatory curves with circadian cycle can be obtained by continuous measurement of luminescence level from a suprachiasmatic nucleus section or peripheral tissue, under culturing, of a mPer1:luc transgenic rat using a photomultiplier tube detector (Photomal) (Reference 28). When the oscillation of luminescence from a suprachiasmatic nucleus section or peripheral tissue of a transgenic animal harboring a construct containing a DNA showing Period2 gene promoter activity and a reporter gene is continuously measured using this method, and the oscillation of the luminescence becomes stable, a substance to be tested (e.g., a substance to be tested such as a candidate for an improving agent of biological rhythm screened by the screening method of the present invention which uses the cell of the present invention) is allowed to act upon the above suprachiasmatic nucleus section or peripheral tissue of a transgenic animal on one hand, and, as a control, a solvent [e.g., dimethyl sulfoxide (DMSO) or the like] alone of the substance to be tested is allowed to act upon the suprachiasmatic nucleus section or peripheral tissue on the other hand, and the measurement is continued. As the transgenic animal, a transgenic rat is desirable. Phase shift (positional change in oscillation peak or bottom), namely time delay or time advance of oscillatory expression, can be evaluated by comparing the oscillatory curve of luminescence obtained from the group treated with a substance to be tested (e.g., a biological rhythm disorder-improving candidate substance to be tested) with the oscillatory curve of luminescence obtained from the untreated control group. A substance which controls expression and/or oscillatory expression of the Period2 gene can be selected by screening substances showing ideal phase shift in this manner. In addition, the above screening can also be carried out using the cell of the present invention, that is, a cell into which a DNA prepared by ligating an upstream sequence of the mouse Period2 gene with a reporter gene is introduced, instead of a suprachiasmatic nucleus section or peripheral tissue, and synchronizing the cells. The cells can be synchronized by stimulation with high concentration serum or dexamethasone (DEX), but preferably can be synchronized by the method described in Example 6.
For example, a substance which controls expression and/or oscillatory expression of the Period2 gene can be selected by carrying out the continuous measurement of luminescence level from a suprachiasmatic nucleus section or peripheral tissue (e.g., liver, kidney, lung or eyeball) of a transgenic rat (mPer2:luc transgenic rat) prepared by introducing into a rat a DNA in which an upstream sequence of the mouse Period2 gene is ligated with a luciferase gene, by the method described in Example 5 which is described later, and evaluating the phase shift by comparing the oscillation curves of luminescence level obtained from the test substance-treated group with those obtained from the untreated group (control). When it is carried out by the method described in Example 5, it is possible to evaluate the function upon the rhythm of substances to be tested, by changing one suprachiasmatic nucleus section to a fresh medium to which a solvent (e.g., DMSO, etc.) alone is added as a control, and the other to a fresh medium to which a substance to be tested dissolved in a solvent (e.g., DMSO, etc.) is added, at a stage of from the 3rd to 9th day after commencement of the measurement during which the oscillatory expression is observed, and comparing the oscillation after addition of the substance to be tested with that of the control. Also, regarding peripheral tissues, it is possible to carry out screening of substances to be tested by changing medium of, for example liver, lung and eyeball, to a medium containing a substance to be tested (a solvent alone in the case of control) in the same manner, during the 4th and 5th days after commencement of the measurement, during the 1st to 5th days after commencement of the measurement and during the 1st to 3rd days after commencement of the measurement, respectively, during which the oscillatory expression is observed.
Also, as shown in the following Example 5, in peripheral tissues, oscillation rhythm occurred several times and then attenuated by intercellular de-synchronization (cf. FIGS. 7 to 9), and it is possible to obtain an agent showing a function to improve attenuated locomoter activity rhythm and/or sleep rhythm in the aged by screening an agent which inhibits this attenuation.
Also, when a large peripheral tissue (e.g., liver, lung, etc.) is used in the screening, it is desirable to a peripheral tissue section.
According to the screening method of the present invention which uses the transgenic animal of the present invention, a substance which controls expression and/or oscillatory expression of the Period2 gene can be selected by administering a substance to be tested to the transgenic animal of the present invention and measuring oscillatory expression in the suprachiasmatic nucleus of the animal.
Specifically, a substance to be tested (e.g., a substance to be tested such as a biological rhythm improving agent candidate screened by the screening method of the present invention which uses the cell of the present invention) is administered to a transgenic animal harboring a construct containing a DNA showing the Period2 promoter activity and a reporter gene. When luciferase is used as the reporter protein, it is possible to screen a compound by evaluating its function upon oscillation of the rhythm center suprachiasmatic nucleus, by the use of a method in which luciferin is supplied from the vertex into the vicinity of the suprachiasmatic nucleus through a capillary and the luminescence level is simultaneously and continuously measured through a micro-optical fiber inserted into just above the suprachiasmatic nucleus while keeping the individual alive (Reference 42).
EXAMPLES
The present invention is specifically described based on examples, but they do not limit the scope of the present invention. Also, unless otherwise indicated, these were carried out in accordance with known methods (e.g., Maniatis, T. et al., Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory, NY., 1982; and Hille, B., Ionic Channels of Excitable Membranes, 2nd Ed., Sinauer Associates Inc., MA, 1992).
Example 1
<Preparation of Mouse and Human Period2 Upstream DNA Sequences and Comparative Analysis>
Preparation of a mouse Period2 upstream sequence was carried out by a screening using a mouse genomic DNA phage library and using Genome Walker Kit (Clontech).
Firstly, polymerase chain reaction (PCR) was carried out by preparing a primer set which can amplify a fragment between a position 218 and a position 723 in mouse Period2 gene cDNA from the reported sequence of mouse Period2 gene (GenBank AFO36893), namely a forward primer consisting of the nucleotide sequence represented by SEQ ID NO:3 [a sequence at positions 218 to 239 in the mouse Period2 gene cDNA (GenBank AFO36893)] and a reverse primer consisting of the nucleotide sequence represented by SEQ ID NO:4 [a sequence complimentary to the sequence at positions 702 to 723 in the mouse Period2 gene cDNA (GenBank AFO36893)]. In this case, the above PCR was carried out using a Taq polymerase (AmpliTaq Gold; Applied Biosystems) as the enzyme and repeating 40 times of a cycle consisting of incubation at 95° C. (10 minutes) and subsequent incubation at 94° C. (15 seconds), 60° C. (30 seconds) and 72° C. (1 minute).
Using the thus obtained amplified fragment (505 bp) as the probe, screening from a mouse genomic DNA phage library was carried out. As a result, a clone containing the first intron of the mouse Period2 gene 6.0 kb upstream from the second exon was obtained. When a sequencing reaction was carried out on the thus obtained phage DNA using a DNA sequencing reagent (BigDye Terminator Cycle Sequencing FS Ready Reaction Kit; Applied Biosystems) in accordance with its manufacture's instructions and then the DNA nucleotide sequence was analyzed using a DNA sequencer (ABI PRISM 377; Applied Biosystems), a sequence consisting of nucleotides at positions 10,104 to 17,004 in the nucleotide sequence represented by SEQ ID NO:1 was obtained.
Next, in order to obtain a further upstream sequence from the phage DNA sequence, preparation of the upstream sequence was carried out using Genome Walker Kit (Clontech). Libraries MDL1 to MDL5 attached to the kit were used as the template of the Genome Walker Kit, and a Taq DNA polymerase (Advantage Genomic polymerase Mix; Clontech) was used as the enzyme for PCR.
Firstly, using AP1 attached to the kit and a first primer consisting of the nucleotide sequence represented by SEQ ID NO:5 (a sequence complimentary to a sequence consisting of nucleotides at positions 13,005 to 13,034 in the nucleotide sequence represented by SEQ ID NO:1) as a primer set, PCR was carried out in the presence of 5% dimethyl sulfoxide (DMSO) by repeating 7 times of a cycle consisting of 94° C. (2 seconds) and 72° C. (3 minutes) and subsequent 36 times of a cycle consisting of 94° C. (2 seconds) and 67° C. (3 minutes) and then finally incubating at 67° C. for 4 minutes. Next, using 1 μl of 50 times diluted solution of the thus obtained reaction product as the template and using AP2 attached to the kit and a second primer consisting of the nucleotide sequence represented by SEQ ID NO:6 (a sequence complimentary to a sequence consisting of nucleotides at positions 12,429 to 12,458 in the nucleotide sequence represented by SEQ ID NO:1) as a primer set, PCR was carried out by repeating 5 times of a cycle consisting of 94° C. (2 seconds) and 72° C. (3 minutes) and subsequent 24 times of a cycle consisting of 94° C. (2 seconds) and 67° C. (3 minutes) and then finally incubating at 67° C. for 4 minutes.
A sequence consisting of nucleotides at positions 9,870 to 12,458 in the nucleotide sequence represented by SEQ ID NO:1 was obtained by sequencing analysis of the amplified band obtained by the above two step PCR. Based on this sequence, a primer set for obtaining a further upstream sequence, namely a first primer consisting of the nucleotide sequence represented by SEQ ID NO:7 (a sequence complimentary to a sequence consisting of nucleotides at positions 10,103 to 10,132 in the nucleotide sequence represented by SEQ ID NO:1) and a second primer consisting of the nucleotide sequence represented by SEQ ID NO:8 (a sequence complimentary to a sequence consisting of nucleotides at positions 10,021 to 10,050 in the nucleotide sequence represented by SEQ ID NO:1), were prepared.
The above two step PCR was repeated except that a first primer consisting of the nucleotide sequence represented by SEQ ID NO:7 and a second primer consisting of the nucleotide sequence represented by SEQ ID NO:8 were used instead of the first primer consisting of the nucleotide sequence represented by SEQ ID NO:5 and the second primer consisting of the nucleotide sequence represented by SEQ ID NO:6, and sequencing analysis of the thus obtained amplified band was carried out to obtain a sequence consisting of nucleotides at positions 9,146 to 10,050 in the nucleotide sequence represented by SEQ ID NO:1.
Subsequently, in order to obtain further upstream sequences one by one, the above procedures, namely preparation of first primer and second primer, two step PCR and sequencing analysis of obtained amplified band, were repeated. Combination of the first primer and second primer used in each two step PCR was,
a combination of a first primer consisting of the nucleotide sequence represented by SEQ ID NO:9 (a sequence complimentary to a sequence consisting of nucleotides at positions 9,355 to 9,384 in the nucleotide sequence represented by SEQ ID NO:1), and
a second primer consisting of the nucleotide sequence represented by SEQ ID NO:10 (a sequence complimentary to a sequence consisting of nucleotides at positions 9,307 to 9,336 in the nucleotide sequence represented by SEQ ID NO:1);
a combination of a first primer consisting of the nucleotide sequence represented by SEQ ID NO:11 (a sequence complimentary to a sequence consisting of nucleotides at positions 7,838 to 7,857 in the nucleotide sequence represented by SEQ ID NO:1), and
a second primer consisting of the nucleotide sequence represented by SEQ ID NO:12 (a sequence complimentary to a sequence consisting of nucleotide sequences at positions 7,818 to 7,837 in the nucleotide sequence represented by SEQ ID NO:1); and
a combination of a first primer consisting of the nucleotide sequence represented by SEQ ID NO:13 (a sequence complimentary to a sequence consisting of nucleotides at positions 2,234 to 2,263 in the nucleotide sequence represented by SEQ ID NO:1), and
a second primer consisting of the nucleotide sequence represented by SEQ ID NO:14 (a sequence complimentary to a sequence consisting of at positions 2,134 to 2,163 in the nucleotide sequence represented by SEQ ID NO:1), and used in this order.
Also, a further upstream sequence was not able to be obtain by the two step PCR using primers designed based on a sequence consisting of nucleotides at positions 8,918 to 9,336 in the nucleotide sequence represented by SEQ ID NO:1, which was obtained as a result of the sequencing analysis of a DNA band amplified by the two step PCR using a combination of the first primer consisting of the nucleotide sequence represented by SEQ ID NO:9 and the second primer consisting of the nucleotide sequence represented by SEQ ID NO:10. Accordingly, in order to obtain a further upstream sequence, the two step PCR was carried out using a combination of a first primer consisting of the nucleotide sequence represented by SEQ ID NO:11 and a second primer consisting of the nucleotide sequence represented by SEQ ID NO:12, which had been designed based on the first exon sequence.
As a result of the analysis so far carried out, a sequence consisting of nucleotides at positions 1 to 17,004 in the nucleotide sequence represented by SEQ ID NO:1 (however, excluding a sequence consisting of nucleotides at positions 7,858 to 9,145) was obtained. In order to compensate the gap between the first exon and the first intron, which corresponds to the undetermined sequence consisting of nucleotides at positions 7,858 to 9,145, two step PCR (nested PCR) was carried out using a mouse genomic DNA as the template and using a Taq DNA polymerase (Advantage Genomic polymerase Mix; Clontech) as the enzyme. Using a forward primer consisting of the nucleotide sequence represented by SEQ ID NO:15 (a sequence consisting of nucleotides at positions 7,516 to 7,545 in the nucleotide sequence represented by SEQ ID NO:1) and a reverse primer consisting of the nucleotide sequence represented by SEQ ID NO:16 (a sequence complimentary to a sequence consisting of nucleotides at positions 9,063 to the 9,092 in the nucleotide sequence represented by SEQ ID NO:1) as a primer set, the first PCR was carried out in the presence of 5% DMSO by incubating at 95° C. for 1 minute, repeating 35 times of a cycle consisting of 94° C. (15 seconds), 60° C. (30 seconds) and 68° C. (5 minutes) and then finally incubating at 68° C. for 10 minutes. The second PCR was carried out under the same conditions as in the first PCR, except that a forward primer consisting of the nucleotide sequence represented by SEQ ID NO:17 (a sequence consisting of at positions 7,556 to 7,585 in the nucleotide sequence represented by SEQ ID NO:1) and a reverse primer consisting of the nucleotide sequence represented by SEQ ID NO:18 (a sequence complimentary to a sequence consisting of positions 8,977 to 9,006 in the nucleotide sequence represented by SEQ ID NO:1) were used as a primer set.
A 1.4 kbp DNA fragment was obtained as a result of the above two step PCR, and by its sequence analysis, it was able to compensate the gap between the first exon and the first intron.
The nucleotide sequence represented by SEQ ID NO:1 was obtained by summarizing all of the above analyzed results. It was found that the first exon (positions 7,736 to 7,857), the second exon (positions 16,158 to 16,396) and the first intron (positions 7,858 to 16,157) are included in the nucleotide sequence represented by SEQ ID NO:1, and that the translation start site of the mouse Period2 is present in the second exon (position 16,176). Also, the transcription start site is present at position 7,736 (cf. the following Example 2).
Since it is expected that a region important for the regulation of gene expression is conserved between species when upstream sequences of mouse and human genes are compared, an attempt was made to obtain an upstream sequence of the human Period2 gene (GenBank NM-00389). As a result of the retrieval of a draft sequence from a net [BLAST 2; National Center for Biotechnology Information (NCBI)], a clone containing an upstream region of the human Period2 gene (GenBank AC013400.5; 188.2 kbp; phase 1) was found.
Since there was a gap between the first intron and the second exon in this draft sequence clone AC013400.5, PCR was carried out using a human genomic DNA (Clontech) as the template. Using a forward primer consisting of the nucleotide sequence represented by SEQ ID NO:19 (a sequence consisting of nucleotides at positions 14,577 to 14,606 in the nucleotide sequence represented by SEQ ID NO:2) and a reverse primer consisting of the nucleotide sequence represented by SEQ ID NO:20 (a sequence complimentary to a sequence consisting of nucleotides at positions 15,855 to 15,884 in the nucleotide sequence represented by SEQ ID NO:2) as a primer set and using a Taq DNA polymerase (AmpliTaq Gold; Applied Biosystems) as the enzyme, the above PCR was carried out by incubating at 95° C. for 10 minutes and then repeating 40 times of a cycle consisting of 94° C. (15 seconds), 60° C. (30 seconds) and 72° C. (2 minutes), and the DNA sequence of a DNA fragment containing the gap (about 1.2 kbp) was analyzed.
On the other hand, since it was found thereafter that a new clone (GenBank AC012485.13; 168.7 kbp; phase 3 complete sequence) was registered on the net, a region of 17,112 bp (a sequence complimentary to positions 35,354 to 52,465 in the GenBank AC012485.13) corresponding to the 17,004 bp of the above mouse genomic sequence was extracted from the clone GenBank AC012485.13 and shown as SEQ ID NO:2. From the analysis of the thus obtained upstream sequence of the human Period2 gene, it was found that the human translation start site is also present in the second exon similar to the case of mouse. Thus, sequence information on the about 17 kbp upstream from the second exon extending about 17 kbp of the mouse Period2 gene and human Period2 gene was obtained.
The thus obtained sequence information on the upstream regions extending about 17 kbp of the mouse Period2 gene and human Period2 gene was subjected to homology comparison analysis (analyzing conditions are default values) using BLAST 2 SEQUENCES VERSION BLASTN2.2.1 Aug. 1, 2001 program of NCBI. A region having a high Expect accuracy of e-3 or less was defined as positive in preservation ability. Results of the analysis are shown in FIG. 1. As shown in FIG. 1, it was found that seven fragmentarily conserved segments (I to VII) are present in before and after the first exon between human and mouse.
Respective sequences in the conserved segments are shown in FIG. 2. In FIG. 2, the symbol “H” indicates human sequence, the symbol “M” indicates mouse sequence and the “|” between human sequence and mouse sequence indicates a position where the kind of base coincided between human and mouse.
By summarizing all results of the above analysis, it was found that the first intron (positions 6,069 to 16,461) and the second exon (positions 16,462 to 16,710) are contained in the nucleotide sequence represented by SEQ ID NO:2, and the human Period2 translation start site is present at position 16,481.
When an upstream region DNA fragment is obtained by PCR using a genomic DNA as the template, there is a possibility of causing a gene mutation. An attempt was made to clone a phage containing an upstream region DNA fragment having no mutation from 1×106 phage particles in accordance the manufacture's instructions of a mouse genomic phage library (Clontech).
In order to prepare a probe, a primer consisting of the nucleotide sequence represented by SEQ ID NO:17 and a primer consisting of the nucleotide sequence represented by SEQ ID NO:18 were designed based on the mouse Period2 gene upstream sequence information obtained in the above. Using a mouse genomic DNA (Clontech) as the template and using a Taq DNA polymerase (Advantage Genomic DNA polymerase Mix; Clontech) as the enzyme, PCR was carried out by incubating at 95° C. for 1 minute and then repeating 35 times of a cycle consisting of 95° C. (15 seconds), 60° C. (30 seconds) and 68° C. (2 minutes), thereby obtaining a DNA fragment of 1.4 kb.
The thus obtained DNA fragment was cloned into the EcoRV site of a plasmid (pBluescript; STRATAGENE) and amplified. This plasmid was subjected to restriction enzyme NotI/SalI treatment and then to an agarose gel electrophoresis to obtain a DNA fragment for probe. Next, the DNA fragment for probe was labeled using [α-32P]dCTP (Amersham Pharmacia Biotech) in accordance with the manufacture's instructions of a labeling kit (BcaBEST Labeling Kit; Takara Shuzo). As a result of the screening of the mouse genomic phage library using this probe, a clone containing six conserved segments between human and mouse was obtained. This phage clone in a large amount was prepared, and the phage DNA containing the upstream region was extracted using Phage DNA Extraction Kit (QIAGEN) in accordance with the manufacture's instructions.
In order to obtain a DNA fragment containing seven conserved regions, the thus obtained phage genome was digested with restriction enzymes EcoT221 and NheI and then subjected to an agarose gel electrophoresis to extract and purify a band of a 6.4 kb DNA fragment. The nucleotide sequence of the above DNA fragment is a sequence consisting of nucleotides at positions 4,415 to 10,877 in the nucleotide sequence represented by SEQ ID NO:1. Blunt-ending of the purified DNA fragment was carried out using DNA Blunting Kit (Takara Shuzo).
Since a luciferase vector pGL3-basic (Promega; to be referred to as pGL3-b hereinafter) is a reporter vector having no promoter sequence, it can evaluate the promoter activity of upstream fragments. A vector pCH1 was prepared by digesting this vector pGL3-b with a restriction enzyme SmaI and then inserting the mouse upstream fragment which had been blunt-ended in advance. Subsequently, a vector pCH3 in which the conserved segments (IV, V, VI, VII) in the first intron were removed from the vector pCH1 was prepared by subjecting the vector pCH1 to a treatment with restriction enzymes, XhoI and SnaBI, removing the DNA fragment including the conserved segments (IV, V, VI, VII) by an agarose gel electrophoresis and then carrying out self-ligation. The upstream region sequence of the mouse Period2 gene in the vector pCH3 corresponds to a sequence consisting of nucleotides at positions 4,415 to 7,931 in the nucleotide sequence represented by SEQ ID NO:1.
Example 2
<Identification of Transcription Start Site of Mouse Period2>
Whether or not the vector pCH1 and vector pCH3 obtained in Example 1 containing the mouse Period2 gene upstream region fragment contain a promoter region can be verified by examining the presence of the transcription start site. In order to identify the transcription start site, an attempt was made to identify a cap site using a mouse total brain cDNA library prepared by an oligo-cap method (Reference 32, Reference 33).
Since an adapter consisting of the nucleotide sequence represented by SEQ ID NO:21 is attached to the cap site, a primer consisting of the nucleotide sequence represented by SEQ ID NO:22 (a sequence consisting of nucleotides at positions 1 to 21 in the nucleotide sequence represented by SEQ ID NO:21) and a primer consisting of the nucleotide sequence represented by SEQ ID NO:23 (a sequence consisting of nucleotides at positions 11 to 30 in the nucleotide sequence represented by SEQ ID NO:21) were prepared for the adapter. For the mouse Period2 gene, on the other hand, a primer consisting of the nucleotide sequence represented by SEQ ID NO:24 [a sequence complimentary to a sequence consisting of nucleotides at positions 439 to 459 in the mouse Period2 gene cDNA (GenBank AFO36893)], a primer consisting the nucleotide sequence represented by SEQ ID NO:25 [a sequence complimentary to a sequence consisting of nucleotides at positions 377 to 398 in the mouse Period2 gene cDNA (GenBank AFO36893)] and a primer consisting of the nucleotide sequence represented by SEQ ID NO:26 [a sequence complimentary to a sequence consisting of nucleotides at positions 300 to 319 in the mouse Period2 gene cDNA (GenBank AFO36893)] were prepared.
When a nested PCR containing the first PCR which used the primer consisting of the nucleotide sequence represented by SEQ ID NO:22 and the primer consisting of the nucleotide sequence represented by SEQ ID NO:24 and the second PCR which used the primer consisting of the nucleotide sequence represented by SEQ ID NO:23 and the primer consisting of the nucleotide sequence represented by SEQ ID NO:25 was carried out, a DNA fragment of 414 bp containing the cap site was obtained. Also, when another nested PCR containing the first PCR which used the primer consisting of the nucleotide sequence represented by SEQ ID NO:22 and the primer consisting of the nucleotide sequence represented by SEQ ID NO:24 and the second PCR which used the primer consisting of the nucleotide sequence represented by SEQ ID NO:23 and the primer consisting of the nucleotide sequence represented by SEQ ID NO:26 was carried out, a DNA fragment of 335 bp containing the cap site was obtained.
As a result of the sequence analysis of these DNA fragments, it was found that the transcription start site is present in 8,440 bp upstream (nucleotide at position 7,736 in the nucleotide sequence represented by SEQ ID NO:1) from the translation start site (nucleotide at position 16,176 in the nucleotide sequence represented by SEQ ID NO:1). Based on this, it was confirmed that the transcription start site is contained in the constructed vector pCH1 and vector pCH3, the vector pCH1 has nucleotides of −3,321 to +3,142 (when the transcription start site is defined as “+1”) in the mouse Period2 gene, and the vector pCH3 has nucleotides of −3,321 to +196 in the mouse Period2 gene.
Example 3
<Analysis of Promoter Activity and Enhancer Activity of the Upstream Region of Mouse Period2 Gene>
A mouse cultured cell line NIH3T3 was inoculated at 1×105 cells per well into a 6 well plate on the day before a luciferase assay. Using Lipofectamine 2000 (GIBCO-BRL) as the transfection reagent and using 1 μg of each of various reporter vectors and 20 ng of an internal control vector (PRL-SV40; Promega), transfection was carried out in accordance with the manufacture's instructions. As the reporter vectors, the vector pCH1 and vector pCH3 obtained in the above Example 1, a vector pGL3-b having no promoter activity (Promega) as a negative control and a vector pGL3-promoter in which the SV40 promoter is connected to the upstream of luciferase gene (Promega; to be referred to as pGL3-p hereinafter) as a positive control were used.
Also, the luciferase gene contained in vectors other than the internal control vector (PRL-SV40) (namely vector pGL3-b, vector pCH1, vector pCH3 and vector pGL3-p) is a firefly origin, while the luciferase gene contained in the internal control vector (PRL-SV40) is a sea pansy origin.
After a lapse of 48 hours from the transfection, the cells were washed once with phosphate buffered saline (PBS) and then the assay was carried out using an assay kit (Picka Gene Dual Reporter Assay Kit; Nippon Gene) in accordance with the manufacture's instructions. The luminescence measurement was carried out using Microtiter Luminometer (Dynatech Laboratories).
The results are shown in FIG. 3. In FIG. 3, “BLK” means an amount of luminescence in the host cell not treated with transfection. Also, the “luciferase relative activity (normalized)” shown in FIG. 3 means a value normalized based on the expression level of luciferase originated from the internal control vector (PRL-SV40). As shown in FIG. 3, basal promoter activity of the vector pCH1 was a low activity (7% of the vector pGL3-p), while the vector pCH3 showed a promoter activity of 55% of the vector pGL3-p.
In order to evaluate whether or not the vector pCH3 which contains the first exon upstream three conserved segmentss (I, II and III) among the conserved seven sevens (from I to VII) is functional, whether or not the reporter activity of pCH3 is enhanced by a BMAL1/CLOCK heterodimer, which act as a trans-acting factor for Period, was examined.
That is, transfection was carried out using Lipofectamine 2000 (GIBCO-BRL) by adding 25 ng, 50 ng or 250 ng of each of pCl-neo-Bmal1 and pC1-neo-Clock, together with the vector pCH3 (10 ng). Also, the vector pCl-neo-Bmal1 and vector pCl-neo-Clock have been prepared by introducing mouse Bmal1 or mouse Clock, respectively, into a pCl-neo vector (Promega) (Reference 34), and the mouse Bmal1 and mouse Clock are expressed constitutionally in cultured cells. Also, in carrying out the above transfection, 0.5 ng of pRL-CMV (Promega) was added as an internal control, and the DNA to be transfected was adjusted with the pCl-neo vector (Promega) to a total amount of 1 μg.
After a lapse of 48 hours from the transfection, luminescence level was measured using an assay kit (Pica Gene Dual Reporter Assay Kit; Nippon Gene) in the same manner as the above procedure.
The results are shown in FIG. 4. The “BLK” in FIG. 4 has the same meaning as the “BLK” shown in FIG. 3. As shown in FIG. 4, dose-dependent transcriptional activation by the BMAL1/CLOCK heterodimer was confirmed. Based on this result, it was found that the thus constructed luciferase vector containing the upstream region of mouse Period2 gene is functional.
Example 4
Preparation of mPer2:luc Transgenic Rat>
Since it is considered, based on the results of in vitro experiments carried out in the above Example 3, that the vector pCH3 contains sufficient elements for showing an intrinsic activity of the Period2 gene expression, an attempt was made to prepare an mPer2:luc transgenic rat using this.
Firstly, in preparing a gene-introduced rat, sequences originating from the vector itself (e.g., replication origin ori, ampicillin resistance gene, etc.) were removed by treatment with restriction enzymes, SalI and MluI, and subsequent agarose gel electrophoresis. Purification of the fragment of interest was carried out using QIAquick Gel Extraction Kits (QIAquick; QUIAGEN), and after carrying out its phenol/chloroform treatment, a DNA solution (concentration=75 ng/μl) was prepared by dissolving it in 100 μl of sterile TE buffer [10 mmol/l Tris-HCl (pH 8.0), 1 mmol/l EDTA (pH 8.0)]. The above DNA fragment contains a sequence consisting of nucleotides at positions 4,415 to 7,931 in the nucleotide sequence represented by SEQ ID NO:1 and a gene encoding luciferase.
In order to prepare a transgenic rat, Wistar rats were purchased from Charles River Japan. The transgenic rat preparation operation was carried out basically based on a known method (Reference 35), and micro-injection of the DNA solution for injecting into rat pronucleus stage fertilized eggs was carried out in the following manner.
That is, sexually matured Wistar rats of 8-weeks-old were reared under conditions of 12 hour light-dark cycle (from 4:00 to 16:00 was used as the light period), 23±2° C. in temperature and 55±5% in humidity, and the hormone treating day was selected by observing sexual cycle of females by vaginal smear. Firstly, 150 IU/kg of a pregnant mare serum gonadotropic hormone [PMS Zen-yaku (pregnant mare serum gonadotropin; PMSG); Nippon Zen-yaku] was intraperitoneally administered to female rats to carry out superovulation treatment, 75 IU/kg of a human chorionic gonadotropic hormone [Puvelogen (human chorionic gonadotropin; hCG); Sankyo Zoki] was administered 48 hours thereafter, and then crossing was carried out by allowing them to lodge with males. After a lapse of 32 hours from the hCG administration, pronucleus stage fertilized eggs were collected by oviduct perfusion. The mKRB solution (Reference 36) was used for the oviduct perfusion and culturing of eggs. After removing cumulus cells by carrying out an enzyme treatment of the collected fertilized eggs at 37° C. for 5 minutes in mKRB solution containing 0.1% hyaluronidase (Hyaluronidase Type I-S; Sigma), the eggs were washed three times with mKRB solution to remove the enzyme and then stored in a CO2 incubator (5% CO2, 37° C., saturation humidity) until the DNA injection operation. The DNA solution prepared in the above was injected into male pronuclei of the thus prepared rat fertilized eggs. The injection operation was carried out for 525 embryos, and among survived 431 embryos, 420 morphologically normal embryos were transplanted into oviducts of pseudopregnancy-induced allomothers.
In order to examine whether or not exogenous DNA (including luciferase gene) was introduced into the rats, a primer consisting of the nucleotide sequence represented by SEQ ID NO:27 [a sequence consisting of nucleotides at positions 410 to 432 in the cloning vector pGL3-b (GenBank U47295)] and a primer consisting of the nucleotide sequence represented by SEQ ID NO:28 [a sequence complimentary to a sequence consisting of nucleotides at positions 980 to 1,000 in the cloning vector pGL3-b (GenBank U47295)] were designed as PCR primers for the luciferase gene. Introduction of exogenous DNA (including luciferase gene) can be verified by the presence or absence of a DNA product (591 bp) amplified by the PCR using these primers.
A total of 65 rats were born by the transplantation of 420 embryos. At the time of 3-weeks-old, about 1 cm of the tail tip of each born individual was cut out using a surgical knife and dissolved by adding 800 μl of a lysis solution and shaking overnight in a 55° C. constant temperature bath. In this case, the above lysis buffer was prepared by dissolving actinase E and protease K, both to a final concentration of 10 mg/ml, in a lysis buffer [a solution containing, as final concentrations, 50 mmol/l Tris-HCl (pH 8.0), 100 mmol/l EDTA (pH 8.0), 100 mmol/l NaCl and 1% SDS].
Subsequently, phenol treatment was carried out twice, and the upper water layer after centrifugation was collected and transferred into a tube containing isopropanol. After mixing, the thus formed filamentous genomic DNA was wound with the tip of a processed glass micro-pipette, soaked in 70% ethanol for 5 minutes and then in 100% ethanol for 5 minutes and finally dissolved by soaking in TE, thereby preparing genomic DNA of each individual.
The PCR which used the genomic DNA of each individual as the template was carried out using a Taq DNA polymerase (Roche Diagnostics) by incubating at 94° C. for 1 minute and then repeating 40 times of a cycle consisting of 94° C. (30 seconds), 57° C. (30 seconds) and 72° C. (60 seconds). As a result, it was found that 8 of the 65 born rats were mPer2:Luc transgenic rats.
In addition, at the time of 7-weeks-old, about 5 mm of the tail tip of each of the 8 mPer2:Luc transgenic rats was cut out using a surgical knife. A luciferin solution was applied to the cut surface, and, turning up the cut surface, the luminescence was measured using a photomultiplier tube detector [Photomal (model name LM-300Y2); Hamamatsu Photonics]. In this case, the above luciferin solution was prepared by adding HEPES (pH 7.2; final concentration=10 mmol/l), penicillin antibiotic (final concentration=25 U/ml), streptomycin antibiotic (final concentration=25 μg/ml), sodium bicarbonate (NaHCO3; final concentration=0.3 g/l) and luciferin [Cat. E1601 (Promega); final concentration=0.1 mmol/l] to a Phenol Red-free DMEM medium (cat. 13000-054; GIBCO-BRL).
As a result, tails of 4 of the 8 rats showed 923 cps, 4,392 cps, 1,122 cps and 865 cps, respectively. Since these luminescence levels were significantly high compared to the around 100 cps of the control (tail of wild type rat), it was confirmed that 4 animals of the mPer2:Luc transgenic rat in which the luciferase functions in the living body were obtained.
Among these 4 female transgenic rats (FO), the line showing the highest level of luminescence from tail (4,392 cps) was crossed with a wild type male, and 11 F1 rats (5 males and 6 females) were obtained.
Example 5
<Operation of Continuous Measurement of Luminescence Levels from Suprachiasmatic Nucleus Section and Peripheral Tissues (Liver Section, Lung Section and Eyeball) of mPer2:luc Transgenic Rat>
Since the F1 rats obtained in Example 4 are cross-breeds of wild type and hetero transgenic rat, wild type and hetero transgenic rats are included in their legitimate F1 children. In order to select transgenic rats to be subjected to the test in this Example from the above F1 rats, tails of the above F1 rats (namely, the F1 rats obtained by the crossing of the line having the most highest tail luminescence with a wild type, prepared in Example 4) were cut off at the time of 6-weeks-old, and whether or not they show luminescence was examined in the same manner as in Example 4. As a result, it was found that F1 transgenic rats showing significant luminescence were 8 animals (4 males and 4 females). Each of suprachiasmatic nucleus section as a rhythm center and peripheral tissues (liver section, lung section and eyeball) was prepared from one female (7-week-old) among these 8 transgenic rats in accordance with the following procedure, and real time oscillation measurement was carried out.
Firstly, preparation of suprachiasmatic nucleus sections was carried out in the following manner. That is, the transgenic rat was anesthetized under diethyl ether and sacrificed by cervical dislocation, both eyes were excised to block input by light and then excision of total brain was carried out. Unnecessary temporal part, frontal lobe and cerebellum among the total brain were excised, and the resulting brain was fixed on an ice-cooled slicer table with an adhesive (Alon Alfa 201; Toa Gosei). The brain fixed on the slicer table was filled with Hanks buffer [1×Hanks Buffer (GIBCO-BRL; cat. 14060-057) and 10 mmol/l HEPES (pH 7.2) (GIBCO-BRL; cat. 15630-080) in final concentrations], and preparation of sections (400 μm in thickness) was carried out using a slicer (Microslicer DTK-1000; Dohan E M) while ice-cooling. A section containing the suprachiasmatic nucleus was selected by observing under a stereoscopic microscope (SMZ645; Nikon). Preparation of the suprachiasmatic nucleus section was carried out by cutting out the suprachiasmatic nucleus alone located under the third cerebral ventricle from this section using a surgical knife under the stereoscopic microscope.
On the other hand, 1.2 ml of a luciferin-containing measuring medium [prepared by adding 10 mmol/l HEPES (pH 7.2) (GIBCO-BRL; cat. 15630-080), 0.1 mmol/l luciferin potassium salt (Promega), antibiotics (25 U/ml penicillin and 25 μg/ml streptomycin; GIBCO-BRL) and 0.3 g/l NaHCO3 (Wako Pure Chemical Industries), to respective final concentrations, to Phenol Red-free DMEM (Dulbecco's modified Eagle's medium; GIBCO-BRL; cat. 13000-054)] was added to a 35 mm dish, and a membrane filter (MiliCell CM; Millipore; cat. PICMORG50) was floated on the medium. The suprachiasmatic nucleus section prepared in the above was put on this membrane filter to which the medium was supplied from the bottom, and a slide glass cover (40 mm×50 mm; Matsunami Glass Industry) was put on the 35 mm dish and the space between them was sealed by applying a silicon grease compound (Toray Dow Coming) to prevent drying of the medium during the culture. Continuous measurement of luminescence level (real time monitoring) was carried out by setting the 35 mm dish containing suprachiasmatic nucleus section prepared in this manner on inside of an photomultiplier tube detector (LM-300Y2; Hamamatsu Photonics) connected to a computer. The continuous measurement of luminescence level was continued for 20 days, and the measuring medium was exchanged only once during the period (on the 10th day after commencement of the measurement).
Regarding the peripheral tissues, each of the liver, lung and eyeball was excised from the transgenic rat. Thereafter, regarding the liver and lung, tissue sections of about 1 mm square were prepared using a surgical knife and the following measurement was carried out. Regarding the eyeball on the other hand, the following measurement was carried out using the excised tissue as such. A medium was prepared by adding a growth enhancing agent B-27 additive (50×) [B-27 Supplement (50×); GIBCO-BRL] to a final concentration of 2% (1×) to the same above luciferin-containing measuring medium used for the culturing of suprachiasmatic nucleus section, each peripheral tissue (liver section, lung section or eyeball) was directly put into the medium and sealed with a cover glass, and then continuous luminescence measurement was carried out using the photomultiplier tube detector in the same manner as the case of the suprachiasmatic nucleus section. The luminescence measurement was continued for 20 days, and the measuring medium was exchanged only once during the period (on the 10th day after commencement of the measurement).
Results of the continuous luminescence measurement of the suprachiasmatic nucleus section during the first 10 days (namely from the commencement of the measurement until the lapse of 10 days) are shown in FIG. 5 and FIG. 6, and results of the continuous luminescence measurement of the peripheral tissues (liver section, lung section and eyeball) during the first 10 days are shown in FIG. 7, FIG. 8 and FIG. 9, respectively. In FIG. 5 to FIG. 9, the abscissa shows the number of days from the commencement of the measurement (the measurement starting day was expressed as day 0), and the ordinate shows luminescence (cpm). Also, FIG. 6 shows a part of the graph shown in FIG. 5 expanded only in vertical direction.
As shown in FIG. 5 and FIG. 6, luminescence quantity of the suprachiasmatic nucleus section showed an oscillation peak after about 9 hours on the 3rd day from the commencement of the measurement, and the oscillation rhythm was found thereafter at intervals of about 24 hours. Also, this oscillation continued until on the 20th day after the commencement of the measurement.
As shown in FIG. 7, luminescence quantity of the liver section showed oscillation peaks after about 18 hours on the 3rd day and after about 18 hours on the 4th day from the commencement of the measurement, but the oscillation was not observed thereafter due to its attenuation. In addition, since the oscillation rhythm recovered to 4 times by the medium exchange on the 10th day after commencement of the measurement, it is considered that the attenuation of oscillation rhythm is not due to death of cells but due to de-synchronization among cells.
As shown in FIG. 8, the luminescence quantity of the lung section showed a total of 5 oscillation rhythms after the commencement of the measurement, though accompanied by rapid changes.
As shown in FIG. 9, the luminescence quantity of the eyeball showed an oscillation rhythm having a peak after about 14 hours on the 1st day of the commencement of the measurement, and the oscillation rhythm was observed three times thereafter though the oscillation was small.
Thus, though there are differences in the oscillation phase and continuing frequency of oscillation among respective tissues of the suprachiasmatic nucleus and peripheral tissues (liver, lung and eyeball), the oscillation rhythm after about 24 hours was observed in all of the tissues. It is considered that screening of a substance capable of controlling expression of the Period2 gene becomes possible by the continuous Period2 gene expression monitoring system which uses the transgenic rat of the present invention or its suprachiasmatic nucleus sections or peripheral tissues.
Example 6
<Confirmation of Oscillation-inducing Ability of Mouse Period2 Promoter in Cultured Cell Line>
It has been reported that when a rat cultured cell Rat-1 is stimulated with high concentration horse serum or DEX, a group of cells are synchronized, and clock genes and a group of genes controlled by the clock genes start the 24 hour interval expression oscillation all at once, which continues for several days and then attenuates (Reference 2).
An attempt was made to introduce the above pCH3 construct prepared by connecting a mouse Period2 gene upstream region to a luciferase vector, transiently into the Rat-1 cell, to carry out DEX stimulation thereafter, to change the medium to a medium containing luciferase and then to measure luminescence therefrom real time using the above ultra-weak emission counter. Its illustrative method is described below. Firstly, the Rat-1 cell (purchased from ATCC: designation=Rat1-R12; ATCC number=CRL-2210) was cultured and maintained at 37° C. under an atmosphere of 5% CO2 in a 225 cm2 flask (Iwaki) charged with 2 ml of a medium [prepared by adding antibiotics in respective final concentrations (100 U/ml penicillin and 100 μg/ml streptomycin; GIBCO-BRL) to Phenol Red-containing DMEM (cat. 11965-092)] containing 10% FBS (fetal bovine serum; JRH Bioscience). The Rat-1 cell was inoculated at 1.2×106 cells per well into a 35 mm dish (Falcon) on the day before the transfection, and the culturing was continued using 2 ml of the above medium containing 5% FBS. On the next day, 1 μg of the pCH3 construct was subjected to transfection using a transfection reagent Lipofectamine 2000 (GIBCO-BRL) in accordance with the manufacture's instructions attached thereto. Three hours after the transfection, the medium was exchanged with 2 ml of fresh above medium containing 10% FBS to continue the culturing. Sixteen hours after the transfection, the stimulation was started by changing the medium to the above medium (containing 10% EBS) containing 0.1 μmol/l at final concentration of DEX (SIGMA; cat. D-8893). After 2 hours of the DEX stimulation, the medium was exchanged with 2 ml of a luciferin-containing measuring medium (the above medium (containing 10% FBS) containing 0.1 mmol/l at final concentration of luciferin potassium salt (Promega)). After addition of the luciferin-containing measuring medium, this was covered with a slide glass, sealed using a silicon grease compound and arranged in the ultra-weak emission counter connected to a computer, and then continuous luminescence measurement (real time monitoring) was carried out.
The results are shown in FIG. 10. The abscissa in FIG. 10 shows the number of days from the commencement of the measurement (the measurement starting day was expressed as day 0), and the ordinate shows luminescence quantity (cpm). As shown in FIG. 10, oscillation of the luminescence was observed at intervals of 24 hours. Based on this, it was confirmed that the mouse Period2 gene upstream region claimed by us (positions 4,415 to 7,931 in the nucleotide sequence represented by SEQ ID NO:1) has the oscillating ability also in cultured cells. Accordingly, it is considered that screening of a substance capable of changing the oscillation in cultured cells is possible by the use of this region.
Example 7
<Construction of Construct pTM15 in Which Human Period2 Upstream Sequence is Connected to Luciferase Vector>
From the results of mouse promoter activity analyses of Example 3 and Example 5, it was suggested that the regions I to III among the seven conserved segments are considered to be sufficient for the oscillation. Accordingly, in order to obtain a DNA fragment containing human I to III regions (nucleotides at positions 3,820 to 6,068 in the nucleotide sequence represented by SEQ ID NO:2), PCR was carried out using a human genomic DNA (Clontech) as the template and using a Taq polymerase (Advantage-GC Genomic polymerase Mix; Clontech) as the enzyme. Using a forward primer consisting of the nucleotide sequence represented by SEQ ID NO:29 (a sequence consisting of nucleotides at positions 3,644 to 3,671 in the nucleotide sequence represented by SEQ ID NO:2) and a reverse primer consisting of the nucleotide sequence represented by SEQ ID NO:30 (a sequence complimentary to a sequence consisting of nucleotides at positions 6,402 to 6,429 in the nucleotide sequence represented by SEQ ID NO:2), the PCR was carried out by incubating at 94° C. for 1 minute, repeating 35 times of a cycle consisting of 94° C. (15 seconds), 58° C. (30 seconds) and 68° C. (4 minutes) and then finally incubating at 68° C. for 10 minutes. As a result of the PCR, a DNA fragment of 2.8 kbp was obtained. In general, nucleotide A is added to the 3′-terminal of a PCR fragment amplified by Taq polymerase, so that, in order to carry out efficient cloning of a DNA fragment to which A is added, an attempt was made to prepare a cloning vector in which nucleotide T is added to the SmaI site of the luciferase vector pGL3-basic. That is, the pGL3-basic was digested by treating it with the restriction enzyme SmaI and then subjected to 1% agarose gel electrophoresis, and the digested vector was extracted from the gel, added to a PCR solution containing Taq polymerase (Roche Diagnostic) and incubated at 70° C. for 2 hours for addition of oligonucleotide T to the SmaI cut site. The PGL3-basic TA vector prepared in this manner can clone a PCR fragment efficiently into the SmaI site. A reporter vector pTM15 was prepared by inserting the 2.8 kbp PCR fragment obtained by the above PCR into the PGL3-basic TA vector. The sequence of the upstream region moiety of the human Period2 gene in the vector pTM15 corresponds to nucleotides at positions 3,644 to 6,429 in the nucleotide sequence represented by SEQ ID NO:2 and contains the conserved segments I to III (positions 3,820 to 6,068 in the sequence represented by SEQ ID NO:2).
Example 8
<Identification of Human Period2 Transcription Start Site>
Although it was found in Example 2 that the transcription start site of mouse Period2 is present inside the conserved segments I to III, there is no information regarding the transcription start site of human Period2. Accordingly, in order to identify the transcription start site of human Period2, an attempt was made to identify the cap region using a human cerebellum cDNA library prepared by oligo-cap method (total RNA to be used as the source was obtained from Clontech (cat. 64035-1); Reference 32 and Reference 33). That is, when a nested PCR containing the first PCR which used a primer consisting of the nucleotide sequence represented by SEQ ID NO:22 and a primer consisting of the nucleotide sequence represented by SEQ ID NO:31 [a sequence complimentary to a sequence consisting of nucleotides at positions 480 to 501 in a human Period2 gene cDNA (GenBank NM-003894.1)] and the second PCR which used a primer consisting of the nucleotide sequence represented by SEQ ID NO:23 and a primer consisting of the nucleotide sequence represented by SEQ ID NO:32 [a sequence complimentary to a sequence consisting of nucleotides at positions 122 to 143 in the human Period2 gene cDNA (GenBank NM-003894.1)] was carried out, two DNA fragments of 209 bp and 323 bp containing the cap region were obtained. As a result of the sequence analysis of these two DNA fragments, it was found that the first exon of the Period2 gene exists in two kinds (to be called exon 1A and exon 1B for convenience) which independently connect to the second exon (nucleotides at positions 16,462 to 16,710 in the nucleotide sequence represented by SEQ ID NO:2). The exon 1A is positions 5,625 to 5,773 in the nucleotide sequence represented by SEQ ID NO:2, and the exon 1B is positions 5,806 to 6,068 in the nucleotide sequence represented by SEQ ID NO:2. Thus, it was found that there are two human Period2 gene transcription start sites, and they are present at positions 5,625 (exon 1A) and 5,806 (exon 1B) in the nucleotide sequence represented by SEQ ID NO:2. Based on this, it was confirmed that transcription start sites are contained in the thus constructed vector pTM15.
Example 9
<Confirmation of Oscillation-inducing Ability of Human Period2 Promoter in Cultured Cell Line>
As a result of examination on the oscillation-inducing ability of the human Period2 promoter vector pTM15 in cultured cell by the same method of Example 6, significant oscillation shown in FIG. 11 was confirmed. The abscissa in FIG. 11 shows the number of days from the commencement of the measurement (the measurement starting day was expressed as day 0), and the ordinate shows luminescence quantity (cpm). Since DNA fragment containing the human Period2 gene upstream region claimed by us (positions 3,820 to 6,068 in the nucleotide sequence represented by SEQ ID NO:2) showed the oscillating ability also in cultured cells, it is considered that screening of a substance capable of changing the oscillation in cultured cells is possible by the use of this region.
Example 10
<Preparation of Deletion Construct in Which Mouse Period2 Upstream Sequence is Connected to Luciferase Vector>
It was revealed that the vector pCH3 obtained in Example 1 (contains a sequence of nucleotides at positions 4,415 to 7,931 in the nucleotide sequence represented by SEQ ID NO:1), containing a mouse Period2 gene upstream region fragment having three conserved segments (I, II and III) of upstream of the first exon, has strong promoter activity as shown in FIG. 3 of Example 3 and is transcription-activated by a Period transcription activation factor BMAL1/CLOCK heterodimer as shown in FIG. 4. In order to find that a region important for the basal promoter activity of the mouse Period2 gene and a region important for the transcriptional activation by BMAL1/CLOCK heterodimer are present in which of the three conserved segments, conserved segment deletion constructs were prepared and examined. Firstly, a deletion construct consisting of a shorter nucleotide sequence containing all of the conserved segments I, II and III (correspond to sequences consisting of nucleotides at positions 5,932 to 6,043, 6,087 to 6,179 and 7,518 to 7,735, respectively, in the nucleotide sequence represented by SEQ ID NO:1) was prepared. The above vector pCH3 was subjected to a treatment with restriction enzymes, MulI and BalI, and the digested DNA fragment was removed by an agarose gel electrophoresis. A vector pCH3-D1 was prepared from the vector pCH3 by self-ligation of the thus deletion DNA fragment-deleted product. In the vector pCH3-D1, the sequence of the upstream region moiety of the mouse Period2 gene corresponds to a sequence consisting of nucleotides at positions 5,249 to 7,931 in the nucleotide sequence represented by SEQ ID NO:1 (a nucleotide sequence of −2,487 to +196 when the transcription start site is defined as “+1”). Next, a deletion construct in which the preserved regions I and II were deleted was prepared. The vector pCH3 was subjected to a treatment with restriction enzymes, MulI and EcoRI, and the digested DNA fragment was removed by an agarose gel electrophoresis. A vector pCH3-D2 was prepared from the vector pCH3 by carrying out self-ligation of the thus deletion DNA fragment-deleted product. In the vector pCH3-D2, the sequence of the upstream region moiety of the mouse Period2 gene corresponds to a sequence consisting of nucleotides at positions 6,417 to 7,931 in the nucleotide sequence represented by SEQ ID NO:1 (a nucleotide sequence of −1,319 to +196 when the transcription start site is defined as “+1”). In addition, in order to construct a vector mainly containing the conserved segment III alone, the vector pCH3 was subjected to a treatment with MulI/BstXI restriction enzymes, MulI and BstXI, and the digested DNA fragment was removed by an agarose gel electrophoresis. A vector pCH3-D3 was prepared from the vector pCH3 by carrying out self-ligation of the thus deletion DNA fragment-deleted product. In the vector pCH3-D3, the sequence of the upstream region moiety of the mouse Period2 gene corresponds to a sequence consisting of nucleotides at positions 7,463 to 7,931 in the nucleotide sequence represented by SEQ ID NO:1 (a nucleotide sequence of −273 to +196 when the transcription start site is defined as “+1”).
Example 11
<Analysis of Promoter Activity and Enhancer Activity of Deletion Constructs of Mouse Period2 Upstream Region>
A mouse cultured cell line NIH3T3 was inoculated at 1×105 cells per well into a 6 well plate on the day before a luciferase assay. Using Lipofectamine 2000 (GIBCO-BRL) as the transfection reagent and using 1 μg of each of various reporter vectors and 20 ng of an internal control vector (PRL-SV40; Promega), transfection was carried out in accordance with the manufacture's instructions. As the reporter vectors described above, the vector pCH3-D1, vector pCH3-D2 and vector pCH3-D3 obtained in the above Example 10, a vector pGL3-b having no promoter activity (Promega) as a negative control and the vector pGL3 as a positive control were used.
Also, the luciferase gene contained in vectors other than the internal control vector (PRL-SV40) (namely vector pCH3-D1, vector pCH3-D2, vector pCH3-D3 and vector pCH3) is a firefly origin, while the luciferase gene contained in the internal control vector (PRL-SV40) is a sea pansy origin.
Forty-eight hours after the transfection, the cells were washed once with phosphate buffered saline (PBS) and then the assay was carried out using an assay kit (Pica Gene Dual Reporter Assay Kit; Nippon Gene) in accordance with the manufacture's instructions. The luminescence was measured using Microtiter Luminometer (Dynatech Laboratories).
The results are shown in FIG. 12. In FIG. 12, “BLK” means an amount of luminescence in the host cell not treated with transfection. Also, the “luciferase relative activity (standardized)” shown in FIG. 12 means a value standardized based on the luciferase expression quantity originated from the internal control vector (PRL-SV40). As shown in FIG. 12, the basal promoter activity of the vector pCH3-D1 and vector pCH3-D2 showed a promoter activity of about 60% of the vector pCH3. The basal promoter activity of the vector pCH3-D3 showed a promoter activity of about 80% of the vector pCH3. Based on the above, it was suggested that the region carrying out basal promoter activity of the mouse Period is present in a sequence consisting of nucleotides at positions 7,463 to 7,931 in the nucleotide sequence represented by SEQ ID NO:1. When the fact that the transcription start site and the conserved segment III are contained in this region is taken into consideration, it is considered that the conserved segment III is important for the basal promoter activity.
Next, a region necessary for the Period2 to undergo transcriptional activation by the trans-acting factor BMAL1/CLOCK was examined.
Transfection was carried out using Lipofectamine 2000 (GIBCO-BRL) by adding 250 ng of each of pCI-neo-Bmal1 and pCI-neo-Clock together with 10 ng of the above vector pCH3-D1, vector pCH3-D2, vector pCH3-D3 or vector pCH3. In carrying out the transfection, 0.5 ng of pRL-CMV (Promega) was added as an internal control, and the DNA to be transfected was adjusted with the pCl-neo vector (Promega) to a total amount of 1 μg.
Forty-eight hours after the transfection, the measurement of luminescence level was carried out using an assay kit (Pica Gene Dual Reporter Assay Kit; Nippon Gene) in the same manner as in the above procedure.
The results are shown in FIG. 12. The “BLK” in FIG. 12 has the same meaning as the “BLK” shown in FIG. 4. As shown in FIG. 12, transcriptional activity of all of the vector pCH3-D1, vector pCH3-D2, vector pCH3-D3 and vector pCH3 was enhanced 5.0 times, 5.6 times, 7.1 times and 5.6 times, respectively, by the co-transfection of Bmal1 gene and Clock gene, so that it was considered that they received transcriptional activation by the BMAL1/CLOCK heterodimer. Based on this result, it was suggested that the sequence important for the mouse Period2 to undergo transcription activation by the transcription activation factor BMAL1/CLOCK heterodimer is present in a sequence consisting of nucleotides at positions 7,463 to 7,931 in the nucleotide sequence represented by SEQ ID NO:1. Since the conserved segment III is contained in this region, it is considered that a responsive element, on which the BMAL1/CLOCK heterodimer functions, is present in the conserved segment III.
Example 12
<Analysis of Oscillation-inducing Ability of Mouse Period2 Upstream Region Deletion Construct>
As a result of the measurement of oscillation-inducing ability in cultured cells using the vector pCH3-D3 prepared in Example 10, carried out by a method similar to that in Example 6, significant oscillation shown in FIG. 13 was confirmed. The abscissa in FIG. 13 shows the number of days from the commencement of the measurement (the measurement starting day was expressed as day 0), and the ordinate shows luminescence quantity (cpm). Since a DNA fragment containing only the conserved segment III (corresponds to a sequence consisting of nucleotides at positions 7,463 to 7,931 in the nucleotide sequence represented by SEQ ID NO:1) among the mouse Period2 gene upstream region showed the oscillating ability in cultured cells, it is considered that a sequence important for the oscillatory expression is present in the conserved segment III of mouse Period2 and that screening of a substance capable of changing the oscillatory expression in cultured cells is possible by the use of this region.
INDUSTRIAL APPLICABILITY
A system for the screening of substances capable of controlling expression of biological clock genes can be constructed by using the Period2 gene promoter of the present invention, the construct of the present invention containing this promoter and a reporter gene, the cell of the present invention, the transgenic animal of the present invention or its suprachiasmatic nucleus sections or peripheral tissues. Substances selected by this screening system are useful as candidate substances of agents for improving circadian rhythm disorders (e.g., sleep disturbance, depression or abnormal behavior of patients with senile dementia, caused by abnormal circadian rhythm).
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Free Text in Sequence Listing
Explanation of “Artificial Sequence” is described in the numerical entry <223> in the following Sequence Listing. Specifically, the nucleotide sequence represented by the sequence of SEQ ID NO:21 in the Sequence Listing is an adapter sequence of oligo cap. The nucleotide sequence represented by the sequence of SEQ ID NO:22 in the Sequence Listing is a sequence consisting of nucleotides at positions 1 to 21 in the nucleotide sequence represented by the sequence of SEQ ID NO:21 in the Sequence Listing. The nucleotide sequence represented by the sequence of SEQ ID NO:23 in the Sequence Listing is a sequence consisting of nucleotides at positions 11 to 30 in the nucleotide sequence represented by the sequence of SEQ ID NO:21 in the Sequence Listing. The nucleotide sequence represented by the sequence of SEQ ID NO:27 in the Sequence Listing is a sequence consisting of nucleotides at positions 410 to 432 in the cloning vector pGL3-b (GenBank U47295). The nucleotide sequence represented by the sequence of SEQ ID NO:28 in the Sequence Listing is a sequence complimentary to a sequence consisting of nucleotides at positions 980 to 1,000 in the cloning vector pGL3-b (GenBank U47295).
Although the invention has been described in the above based on specified embodiments, modifications and improvements obvious to those skilled in the art are included in the scope of the present invention.
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10415489
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astellas pharma inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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435/29
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Apr 1st, 2022 05:13PM
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Apr 1st, 2022 05:13PM
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Astellas Pharma
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tyo:4503
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Astellas Pharma
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Nov 18th, 2008 12:00AM
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Dec 4th, 2003 12:00AM
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https://www.uspto.gov?id=US07452983-20081118
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Protein which binds to Akt2
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A novel polypeptide, a polynucleotide, an expression vector, a cell transfected with the expression vector, a method for screening an insulin resistance improving agent and a carbohydrate metabolism improving agent, and a method for producing a pharmaceutical composition are provided. The polypeptide is useful in screening an insulin resistance improving agent and a carbohydrate metabolism improving agent. When the polypeptide is overexpressed in a fat cell, the activity of Akt2 is reduced. Fat cells express the polypeptide. The polynucleotide encodes the polypeptide. The expression vector includes the polynucleotide. The screening method uses the polypeptide to screen for an insulin resistance improving agent and a carbohydrate metabolism improving agent. The production method uses the substance obtained by the screening method as the active ingredient of the pharmaceutical composition. The pharmaceutical composition is useful for insulin resistance improvement and carbohydrate metabolism improvement.
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7452983
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1. An isolated polypeptide which comprises the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4 and which binds to Akt-homolog-2 (“Akt2”).
2. An isolated polypeptide consisting of the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4.
3. An isolated polynucleotide encoding
(1) a polypeptide which comprises the amino acid sequence of SEQ ID NO:2 and which binds to Akt-2,
(2)a polypeptide which comprises the amino acid sequence of SEQ ID NO:4 and which binds to Akt-2,
(3) a polypeptide consisting of the amino acid sequence of SEQ ID NO:2, or
(4) a polypeptide consisting of the amino acid sequence of SEQ ID NO:4.
4. An expression vector comprising a polynucleotide encoding
(1) a polypeptide which comprises the amino acid sequence of SEQ ID NO:2 and which binds to Akt-2,
(2) a polypeptide which comprises the amino acid sequence of SEQ ID NO:4 and which binds to Akt-2,
(3) a polypeptide consisting of the amino acid sequence of SEQ ID NO:2, or
(4) a polypeptide consisting of the amino acid sequence of SEQ ID NO:4.
5. An isolated cell transformed with an expression vector comprising a polynucleotide encoding
(1) a polypeptide which comprises the amino acid sequence of SEQ ID NO:2 and which binds to Akt-2,
(2) a polypeptide which comprises the amino acid sequence of SEQ ID NO:4 and which binds to Akt-2,
(3) a polypeptide consisting of the amino acid sequence of SEQ ID NO:2, or
(4) a polypeptide consisting of the amino acid sequence of SEQ ID NO:4.
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5
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TECHNICAL FIELD
The present invention relates to a novel polypeptide which binds to Akt2, and a novel polynucleotide coding for said polypeptide, a vector comprising said polynucleotide, a transformed cell comprising said vector and a method for screening a substance which inhibits binding of the aforementioned polypeptide with Akt2.
BACKGROUND OF THE INVENTION
Insulin is secreted from β cells of the pancreatic islets of Langerhans and reduces blood sugar level by mainly acting upon muscle, the liver and adipose and thereby incorporating blood sugar into cells to effect its storage and consumption. Diabetes mellitus is induced by insufficient action of this insulin, and two types are present in its patients, namely type 1 having a disorder of the production or secretion of insulin and type 2 having a difficulty in accelerating carbohydrate metabolism by insulin. The blood sugar level in both of these patients becomes higher than that in healthy person, but while insulin in blood is absolutely insufficient in type 1, insulin resistance occurs in type 2 in which incorporation or consumption of blood sugar by cells is not accelerated in spite of the presence of insulin. The type 2 diabetes mellitus is a so-called lifestyle-related disease which is generated due to overeating, lack of exercise, stress and the like causes in addition to the hereditary predisposition. Nowadays, patients of this type 2 diabetes mellitus are rapidly increasing in advanced nations accompanied by the increase of ingestion calories, and this type occupies 95% of diabetes mellitus patients in Japan. Accordingly, necessity is increasing not only for a simple hypoglycemic drug as a therapeutic agent for diabetes mellitus but also on a study which aims at treating type 2 diabetes mellitus for the purpose of accelerating carbohydrate metabolism through the improvement of insulin resistance.
At present, insulin injections are prescribed for the treatment of type 1 diabetes mellitus patients. On the other hand, in addition to the insulin injections, sulfonylurea hypoglycemic agents (SU preparations) which prompts secretion of insulin by acting upon β cells of the pancreas and biguanide hypoglycemic agents which have actions to increase sugar usage and inhibit gluconeogenesis by anaerobic glycolysis action and to inhibit intestinal absorption of sugar, as well as α-glucosidase inhibitors which delay digestion and absorption of saccharides, are known as the hypoglycemic agents prescribed for type 2 patients. Though these indirectly improve insulin resistance, thiazolidine derivatives have been used in recent years as agents which directly improve insulin resistance. Its action is to accelerate incorporation of glucose into cells and use of glucose in cells. It is shown that the thiazolidine derivatives act as an agonist of peroxisome proliferator responding activated receptor gamma (PPARγ) (cf. Non-patent reference 1). However, it is known that the thiazolidinediones not only improve insulin resistance but also have a side effect to induce edema (cf. Non-patent reference 2, Non-patent reference 3). Since this induction of edema is a serious side effect which results in cardiac hypertrophy, more useful target molecule for drug development than the PPARγ is in demand for the improvement of insulin resistance.
The signal of insulin action is transferred into cells via an insulin receptor on the cell membrane. Two pathways of the first and second are present in this insulin action pathway (cf. Non-patent reference 4). In the first pathway, the signal is transferred in order from the activated insulin receptor to Akt1 (PKBα) or Akt2 (PKBβ), or PKCγ or PKCζ, via IRS-1, IRS-2, PI 3 kinase and PDK 1, and incorporation of sugar from the extracellular moiety is accelerated as the result by translocating a glucose transporter GLUT 4 existing inside the cell onto the cell membrane (cf. Non-patent reference 5). On the other hand, in the second pathway, the signal is transferred from the insulin receptor to CrK II, C3G and TC 10 in that order via c-Cbl and CAP, and incorporation of sugar by GLUT 4 is accelerated as the result (cf. Non-patent reference 6). However, there are portions still unclear regarding details of these insulin signal transduction pathways, and it is not clear particularly about the mechanism which finally mediates acceleration of sugar incorporation of cells by these signals via the glucose transporter.
Akt2 is present in the aforementioned insulin signal first pathway and activated by undergoing phosphorylation by insulin stimulation via PDK 1. The activated Akt2 transfers the signal as a kinase by phosphorylating a protein as its substrate. It has been reported that a homo-knockout mouse in which a gene coding for the Akt2 protein was artificially deleted shows a type 2 diabetes-like phenotype due to reduced insulin sensitivity mainly in muscle and the liver. Based on these facts, it has been considered that Akt2 is a signal mediating factor which functions in incorporating sugar into cells in response to the insulin signal, and its functional inhibition induces insulin resistance by partial interception of the insulin signal transduction (cf. Non-patent reference 7).
(Non-patent Reference 1)
“The Journal of Biological Chemistry”, (USA), 1995, vol. 270, pp. 12953-12956
(Non-patent Reference 2)
“Diabetes Frontier”, (USA), 1999, vol. 10, pp. 811-818
(Non-patent Reference 3)
“Diabetes Frontier”, (USA), 1999, vol. 10, pp. 819-824
(Non-patent Reference 4)
“The Journal of Clinical Investigation”, (USA), 2000, vol. 106, no. 2, pp. 165-169
(Non-patent Reference 5)
“The Journal of Biological Chemistry”, (USA), 1999, vol. 274, no. 4, pp. 1865-1868
(Non-patent Reference 6)
“Nature”, (England), 2001, vol. 410, no. 6831, pp. 944-948
(Non-patent Reference 7)
“Science”, (USA), 2002, vol. 292, no. 2, pp. 1728-1731
DISCLOSURE OF THE INVENTION
Based on the aforementioned information, the present inventors considered that insulin resistance may be improved when function of Akt2 can be increased. It was considered that this object may be achieved by increasing activity of the Akt2 itself, or by regulating the activity of a newly identified intracellular factor which binds to Akt2 and thereby controls its activity. However, since Akt2 is a kinase, it is difficult to regulate its enzyme activity toward increasing direction by a drug. Accordingly, a protein which binds to Akt2 was identified by a yeast two hybrid system. As a result, it was successful in cloning a mouse derived cDNA of a novel nucleotide sequence coding for a protein AKBP 2 (Akt2 Binding Protein 2) which binds to Akt2. Also, since expressed amount of this protein was considerably increased in muscle and adipose of model mice of diabetes mellitus in comparison with normal individuals, it was found that this protein is a causal factor of the morbid state of diabetes mellitus. In addition, by succeeding in cloning a human orthologue human AKBP 2 gene, it was found that said gene is expressed in fat cells as an insulin response tissue and that human AKBP 2 also binds to Akt2 as the case of mouse AKBP 2. In addition to this, by detecting that kinase activity of Akt2 is reduced by overexpression of mouse AKBP 2, it was found that insulin resistance is induced by the interception of insulin signal by AKBP 2, that is, insulin resistance is improved by-inhibiting binding of AKBP 2 with Akt2. Accordingly, a screening system for an insulin resistance improving agent and/or a carbohydrate metabolism improving agent was constructed making use of the interaction of AKBP 2 with Akt2.
As these results, the present invention was accomplished by providing a novel polypeptide useful in screening an insulin resistance improving agent and/or a carbohydrate metabolism improving agent, a polynucleotide coding for the aforementioned polypeptide, an expression vector comprising the aforementioned polynucleotide, a cell transformed with the aforementioned expression vector, a method for screening an insulin resistance improving agent and/or a carbohydrate metabolism improving agent, and a method for producing a pharmaceutical composition for insulin resistance improvement and/or carbohydrate metabolism improvement.
That is, the present invention relates to
[1] a polypeptide which comprises the amino acid sequence represented by SEQ ID NO:2 or SEQ ID NO:4, or an amino acid sequence in which from 1 to 10 amino acids are deleted, substituted and/or inserted in the amino acid sequence represented by SEQ ID NO:2 or SEQ ID NO:4, and which binds to Akt2,
[2] a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:2 or SEQ ID NO:4,
[3] a polynucleotide coding for the polypeptide described in [1] or [2],
[4] an expression vector comprising the polynucleotide described in [3],
[5] a cell transformed with the expression vector described in [4],
[6] a method for screening a substance which inhibits binding of a polypeptide described in claim 1 or claim 2 or a polypeptide consisting of an amino acid sequence having a homology of 90% or more with the amino acid sequence represented by SEQ ID NO:2 or SEQ ID NO:4 and which binds to Akt2, with Akt2, which comprises
allowing (1) the aforementioned polypeptide or a cell expressing the aforementioned polypeptide, to contact (2) a substance to be tested,
measuring binding of said polypeptide with Akt2, and
selecting a substance which inhibits the aforementioned binding,
[7] the screening method described in [6], wherein the binding inhibiting substance is an insulin resistance improving agent and/or a carbohydrate metabolism improving agent,
[8] the screening method described in [6] or [7], wherein the step of measuring binding of (1) the polypeptide described in [1] or [2] or a polypeptide consisting of an amino acid sequence having a homology of 90% or more with the amino acid sequence represented by SEQ ID NO:2 or SEQ ID NO:4, and which binds to Akt2, to (2) Akt2 is a step of measuring a change in Akt2 based on the change in the aforementioned binding, and
[9] a method for producing a pharmaceutical composition for insulin resistance improvement and/or carbohydrate metabolism improvement, which comprises
carrying out screening using the screening method described in [6] to [8], and
preparing a pharmaceutical preparation.
Virtually nothing is known about the sequences identical to the polypeptides and polynucleotides of the present invention described in SEQ ID NOs:1 to 4. Though sequences having homology with the polynucleotides of the present invention have been reported in a sequence data base GenBank as accession numbers AX714043, BC042155 and BC049110 after the priority date of this application, this is merely a disclosure of sequences and there is no description on their illustrative use. Also, a sequence data base GenPept carries, as an accession number AK056090, a polypeptide consisting of an amino acid sequence in which 68 amino acids of the amino acid sequence represented by SEQ ID NO:4 as one of the polypeptides of the present invention are deleted, and as an accession number AK019105, a polypeptide consisting of an amino acid sequence in which 228 amino acids of the amino acid sequence represented by SEQ ID NO:2 as one of the polypeptides of the present invention are deleted and 13 amino acids of the same are substituted. However, there is no information that these polypeptides were actually prepared, and there is no information on how to prepare them. In addition, illustrative use of said polypeptides is not described either. The present inventors have found the polypeptides and polynucleotides of the present invention for the first time and revealed for the first time that overexpression of the protein and increase of its binding to Akt2 are causal factors of the morbid state of diabetes mellitus. In addition, the screening methods of the present invention by making use of the binding of the polypeptide of the present invention with Akt2 are methods provided for the first time by the present inventors.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph showing expression of AKBP2 in cultured cells. Lanes 1 and 3 indicate molecular weight markers, and lane 2 an empty vector and lane 4 a case of introducing pcDNA-AKBP2.
FIG. 2 (1) is a graph showing comparison of AKBP2 expression level in adipose of normal mice C57BL/6J loaded with a normal feed or a high fat feed. Vertical axis of the drawing shows relative expression level in mouse fat. The white bar shows when the normal feed was loaded, and the black bar when the high fat feed was loaded.
The (2) is a graph showing comparison of AKBP2 expression level in muscle of the normal mice C57BL/6J loaded with a normal feed or a high fat feed. Vertical axis of the drawing shows relative expression level in mouse muscle. The white bar shows when the normal feed was loaded, and the black bar when the high fat feed was loaded.
The (3) is a graph showing comparison of AKBP2 expression level in adipose of the normal mice C57BL/6J and diabetes mellitus model mice KKAy/Ta. Vertical axis of the drawing shows relative expression level in mouse adipose. The white bar shows a result of the normal mice C57BL/6J, and the lined bar that of the diabetes mellitus model mice KKAy/Ta.
The (4) is a graph showing comparison of AKBP2 expression level in muscles of the normal mice C57BL/6J and the diabetes mellitus model mice KKAy/Ta. Vertical axis of the drawing shows relative expression level in mouse muscle. The white bar shows a result of the normal mice C57BL/6J, and the lined bar that of the diabetes mellitus model mice KKAy/Ta.
Expression level in normal feed C57BL/6J is shown as 1 in the comparison in (1) and (2), and expression level in C57BL/6J is shown as 1 in the comparison in (3) and (4), respectively.
FIG. 3 is a graph showing influence of mouse AKBP2 overexpression in NIH3T3 L1 fat cells upon Akt2 enzyme activity. Vertical axis of the drawing shows relative activity, and the value of the enzyme activity in the cells infected with a control virus under the condition of no insulin stimulus is shown as 1. Horizontal axis of the drawing shows insulin stimulation period (minute).
BEST MODE FOR CARRYING OUT THE INVENTION
The following describes the present invention in detail.
<Polypeptide of the Invention>
Included in the polypeptide of the present invention are
(1) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:2 or SEQ ID NO:4; and
(2) i) a polypeptide which comprises the amino acid sequence represented by SEQ ID NO:2 or SEQ ID NO:4 and which binds to Akt2, or ii) a polypeptide which comprises an amino acid sequence in which from 1 to 10 (preferably from 1 to 7, more preferably from 1 to 5, further preferably from 1 to 3) amino acids are deleted, substituted and/or inserted in the amino acid sequence represented by SEQ ID NO:2 or SEQ ID NO:4 and which binds to Akt2 (to be referred to as functionally equivalent variant hereinafter).
Those which reduce kinase activity of Akt2 by binding to Akt2 are particularly desirable as the polypeptides of the present invention.
Also, the polypeptides of the present invention are not particularly limited to the human and mouse derived polypeptides as long as they come under either of the aforementioned (1) and (2), and those which are derived from other vertebrates (e.g., rat, rabbit, horse, sheep, dog, monkey, cat, bear, pig, domestic fowl and the like) are also included therein. In addition, they are not limited to natural polypeptides and artificially produced mutants are also included therein, as long as they come under either of the aforementioned (1) and (2).
The term “binds to Akt2” means that a polypeptide binds to Akt2 (preferably human Akt2, more preferably the polypeptide encoded by the GenBank accession number M95936), and whether or not it “binds” may be verified by the following methods.
A partial or the entire length of a polypeptide to be examined for whether or not it binds, or a partial or the entire length of a polypeptide to be examined fused with a tag (e.g., GST, Flag, His or the like), is expressed in a cell. As the aforementioned cell, a cell which responds to insulin is desirable, and more illustratively, fat cell, hepatocyte or a skeletal muscle-derived cell is desirable. Akt2 protein and a protein which binds thereto may be concentrated from the aforementioned cell by immunoprecipitation using an anti-Akt2 antibody. Whether or not the polypeptide to be examined binds to Akt2 may be verified by separating concentrated solution of the thus obtained Akt2 and its binding protein by polyacrylamide gel electrophoresis through a conventionally known method and then carrying out western blotting using an antibody. Regarding the antibody to be used in this case, an antibody for the polypeptide to be examined or a polypeptide to be examined prepared based on its partial sequence, or an antibody which recognizes the aforementioned tag may be used.
In addition, binding of the polypeptide to be examined with a polypeptide can also be detected by combining a western blotting similar to the aforementioned one with an in vitro pull down method (H. Matsushime et al., Jikken Kogaku (Experimental Engineering), Vol. 113, No. 6, p. 528, 1994) which uses an extract of cells in which the polypeptide to be examined is expressed, or a mixed solution of proteins prepared by in vitro transcription and translation, and Akt2 protein purified by attaching a tag (e.g., GST or the like). Preferably, the binding may be detected using a mixed solution of proteins prepared by directly carrying out in vitro transcription and translation of the protein to be examined as shown in Example 6, from a plasmid for use in the expression of the protein to be examined (e.g., the plasmid for AKBP2 protein expression prepared in Example 1(5)) using an in vitro translation kit (e.g., TNT Kit (Promega)). More preferably, binding of the polypeptide to be examined to Akt2 may be detected by the method described in Example 6. The term “to reduce kinase activity of Akt2” means that the kinase activity possessed by Akt2 is reduced through the binding of the polypeptide to be examined to Akt2. Whether or not the “kinase activity is reduced” may be verified by the following method.
It is known that the kinase activity of Akt2 is accelerated when the 473rd serine (Ser 473) or the 308th threonine (Thr 308) in the molecule is phosphorylated (Biochem. J., 1998, 335 (1-13)). Making use of this, the presence or absence of Akt2 activity may be detected by detecting phosphorylated condition of the Ser 473 or Thr 308 of Akt2 by a western blotting which uses an antibody capable of specifically reacting with these phosphorylated residues (e.g. anti-phosphoSer antibody or the like). More illustratively, phosphorylation of Akt2, namely the presence or absence of Akt2 activity, may be detected by lysing cells (a cell which responds to insulin is desirable, and more illustratively, fat cell, hepatocyte or a skeletal muscle-derived cell is desirable) in which a part or the entire length of the polypeptide to be examined is expressed, and using this as the sample, carrying out western blotting, spot western blotting or the like method which uses the anti-phosphoSer antibody. Preferably, this may be detected by the method of Example 7. When reduction of the phosphorylation of Akt2 (namely activation of Akt2) was observed in this detection system by the use of a sample obtained from a cell in which the polypeptide to be examined was expressed, in comparison with a cell in which the polypeptide to be examined was not expressed, it may be judged that the polypeptide to be examined “reduces kinase activity of Akt2”.
In addition, whether or not it “reduces kinase activity of Akt2” can also be verified by an in vitro kinase assay method in which uptake of radioactive phosphoric acid based on a substrate is measured when a histone H2B, a GSK-3 fusion protein or the like is used as the substrate of Akt2 and allowed to react with an immune precipitate of Akt2. Illustratively, Akt2 protein may be concentrated from an extract of cells (a cell which respond to insulin is desirable, and more illustratively, fat cell, hepatocyte or a skeletal muscle-derived cell is desirable) in which a part or the entire length of the polypeptide to be examined is expressed, by immunoprecipitation using an anti-Akt2 antibody. By mixing a substrate of Akt2, such as GST-crosstide (GST fusion protein of GSK3-beta as a physiological substrate of Akt), with concentrated Akt2 protein, kinase activity of Akt2 may be measured-and determined using phosphorylation of the substrate as the index. Preferably, this may be measured by the method described in Example 7. When reduction of the phosphorylation of the substrate was observed in this measuring system by the use of a sample obtained from a cell in which the polypeptide to be examined was expressed, in comparison with a cell in which the polypeptide to be examined was not expressed, it may be judged that the polypeptide to be examined “reduces kinase activity of Akt2”.
<Polynucleotide of the Invention>
The polynucleotide of the present invention may be derived from any species, as long as it encodes the polypeptide of the present invention, namely a polypeptide represented by the amino acid sequence described in SEQ ID NO:2 or SEQ ID NO:4, or a functionally equivalent variant thereof. Preferred is a polynucleotide consisting of a nucleotide sequence coding for the amino acid sequence described in SEQ ID NO:2 or SEQ ID NO:4, and further preferred is the nucleotide sequence described in SEQ ID NO:1 or SEQ ID NO:3. In this connection, both of DNA and RNA are included in the “polynucleotide” according to this description.
All kinds of mutants may be included in the polynucleotide of the present invention, as long as they encode the polypeptide of the present invention. More illustratively, it can include allele mutants which are present in the natural world, mutants which are not present in the natural world, and mutants which have deletion, substitution, addition and insertion. The aforementioned mutation sometimes occur by natural mutation, but it can also be effected by carrying out artificial modification. All mutant genes coding for the aforementioned polypeptide of the present invention are included in the present invention, regardless of the cause and means of mutation of the aforementioned polypeptide. As the aforementioned artificial means leading to the preparation of mutants, for example, in addition to genetic engineering techniques such as base-specific substitution method (Methods in Enzymology, (1987), 154, 350, 367-382) and the like, chemical synthesis means such as phosphotriester method, phosphoamidide method and the like (Science, 150, 178, 1968) may be cited. By their combination, it is possible to obtain a DNA accompanied by the desired base substitution. Alternatively, it is possible to generate a substitution in a nonspecific base in the DNA molecule by the repetition of PCR or by allowing manganese ion or the like to be present in the reaction solution.
The polynucleotide and polypeptide of the present invention may be easily produced and obtained by general genetic-engineering techniques based on the sequence information disclosed by the present invention.
The polynucleotide coding for the polypeptide of the present invention may be prepared for example in the following manner, but it may be prepared not only by this method but also by conventionally known operations “Molecular Cloning, Sambrook, J. et al., Cold Spring Harbor Laboratory Press, 1989” and the like.
For example, (1) a method which uses PCR, (2) general genetic engineering techniques (namely a method in which a transformant comprising desired amino acids is selected from transformants transformed with a cDNA library), (3) a chemical synthesis method or the like may be cited. Each production method may be carried out in the same manner as described in WO 01/34785.
By the method which uses PCR, the polynucleotide described in this description may be produced for example by the procedure described in the “Mode for Carrying Out the Invention”, 1) Production method of protein gene, a) First production method, of the aforementioned patent reference. Regarding the “human cell or tissue having the ability to produce the protein of the present invention” in said description, adopose cells can for example be cited. Total mRNA is extracted from human or murine adipose cells. Next, a first-strand cDNA may be synthesized by subjecting this mRNA-to a reverse transcriptase reaction in the presence of random primers or oligo dT primers. The polynucleotide of the present invention or a part thereof may be obtained by subjecting the thus obtained first-strand cDNA to polymerase chain reaction (PCR) using two kinds of primers interposing a partial region of the gene of interest. More illustratively, the polynucleotide of the present invention may be produced for example by the methods described in Example 1 and Example 4.
By the method which uses general genetic engineering techniques, the polynucleotide coding for the polypeptide of the present invention may be produced for example by the procedure described in the “Mode for Carrying Out the Invention”, 1) Production method of protein gene, b) Second production method, of the aforementioned patent reference.
By the method which uses chemical synthesis, the polynucleotide coding for the polypeptide of the present invention may be produced for example by the procedure described in the “Mode for Carrying Out the Invention”, 1) Production method of protein gene, c) Third production method, d) Fourth production method, of the aforementioned patent reference.
By making use of partial or entire nucleotide sequence of the thus obtained polynucleotide of the present invention, expression level of the polynucleotide of the present invention in each individual or various tissues may be specifically detected.
Examples of such a detection method include RT-PCR (reverse transcribed-polymerase chain reaction), northern blotting analysis, in situ hybridization and the like methods.
<Production Methods of the Expression Vector, Cell and Polypeptide of the Invention>
Also included in the present invention is a method for producing the polypeptide of the present invention, which is characterized in that the transformed cell of the present invention is cultured.
The polynucleotide coding for the polypeptide of the present invention, obtained in the aforementioned manner, may be used for expressing the polypeptide of the present invention in a test tube or in a test cell by connecting it to the downstream of an appropriate promoter, by conventionally known methods described in “Molecular Cloning, Sambrook, J. et al., Cold Spring Harbor Laboratory Press, 1989” and the like.
Illustratively, by adding a polynucleotide containing a specified promoter sequence to upstream of the initiation codon for the polypeptide of the present invention obtained in the aforementioned manner, it is possible to effect expression of the polypeptide of the present invention by cell-free system transcription and translation of the gene using this as the template.
Alternatively, expression of the polypeptide of the present invention in a cell becomes possible when the aforementioned polynucleotide coding for the polypeptide of the present invention is integrated into an appropriate vector plasmid and introduced in the form of plasmid into a host cell. Alternatively, a cell in which such a construction is integrated into chromosomal DNA may be prepared and used. More illustratively, an isolated fragment containing a polynucleotide can transform host cells of eukaryotes and prokaryotes by again integrating into an appropriate vector plasmid. Furthermore, it is possible to effect expression of the polypeptide of the present invention in respective host cells by introducing an appropriate promoter and a sequence concerned in the gene expression into these vectors. The host cell is not particularly limited, as long as it can detect expression of the polypeptide of the present invention at the mRNA level or protein level. It is most desirable to use a fat-derived cell or muscle-derived cell as the host cell, in which endogenous Akt2 is abundantly present.
The method for expressing a gene by transforming a host cell may be carried out, for example, by the method described in the “Mode for Carrying Out the Invention”, 2) Method for producing the vector of the present invention, the host cell of the present invention and the recombinant protein of the present invention, of the aforementioned patent reference. The expression vector is not particularly limited, as long as it contains a desired polynucleotide. An example thereof is an expression vector obtained by inserting the desired polynucleotide into a conventionally known expression vector optionally selected in response to the host cell to be used. The cell of the present invention may be obtained, for example, by transfecting a desired host cell with the aforementioned expression vector. More illustratively, an expression vector for a desired protein may be obtained, for example, by integrating a desired polynucleotide into an expression vector for mammal cell, pcDNA3.1, as described in Example 2, and the transformed cell of the present invention may be produced by incorporating said expression vector into the 293 cell using the calcium phosphate method.
The desired transformed cell obtained in the above may be cultured in accordance with a general method, and the desired protein is produced by said culturing. Regarding the medium to be used in said culturing, various generally used kinds may be optionally selected in response to the employed host cell. For example, in the case of the aforementioned 293 cell, Dulbecco's modified Eagle's minimum essential medium (DMEM) supplemented with serum component (e.g., fetal bovine serum (FBS) or the like), to which G418 was further added, may be used. As the transformed cell of the present invention, a cell expressing the polypeptide of the present invention is desirable.
By culturing the cell of the present invention, the polypeptide of the present invention produced in the cell may be detected, determined and also purified. For example, it is possible to detect and purify the polypeptide of the present invention by western blotting or immunoprecipitation using an antibody which binds to the polypeptide of the present invention. Alternatively, by expressing the polypeptide of the present invention as a fusion protein with appropriate tag protein (e.g.,. glutathione-S-transferase (GST), protein A, β-galactosidase, maltose-binding protein (MBP) or the like), the polypeptide of the present invention may be detected by western blotting or immunoprecipitation using an antibody specific for such a tag proteins and purified making use of the tag protein. More illustratively, it may be purified making use of a tag protein in the following manner.
The polypeptide of the present invention (e.g., the polypeptide represented by SEQ ID NO:2 or SEQ ID NO:4) may be obtained by integrating the polynucleotide of the present invention (e.g., the polynucleotide represented by SEQ ID NO:1 or SEQ ID NO:3) for example into a His tag fusing vector, more illustratively for example into the pcDNA3.1/V5-His-TOPO (Invitrogen) or the like described in Example 1, to effect its expression in a cultured cell, purifying it using His tag, and then removing the tag moiety. For example, the mouse or human AKBP2 expression plasmid prepared in Example 1 or Example 5 using pcDNA3.1/V5-His-TOPO is designed in such a manner that V5 and His tag are added to the C-terminus of AKBP2 in both cases. Accordingly, the AKBP2 protein may be purified from the AKBP2-expressed cultured cells shown in Example 2 or Example 5, using the His tag. Illustratively, the AKBP2 protein fused with His tag may be isolated from an extract of disrupted cells by binding it to Ni2+-NTA-Agarose (Funakoshi) and centrifuging the product, in accordance with known methods (Jikken Igaku (Experimental Medicine) Supplement, Tanpakushitsu-no Bunshikan Sogosayo Jikkenhou (Experimentation on Intermolecular Interaction of Protein), page 32, 1996, Nakahara et al.). More illustratively, cells expressing the polypeptide of the present invention cultured using a culture flasks (e.g., Petri dish of 10 cm in diameter) are scratched off after adding an appropriate volume (e.g., 1 ml) of a buffer solution and then centrifuged at 1,5000 rpm for 5 minutes, and an appropriate amount (e.g., 50 μM) of Ni2+-NTA-Agarose substituted by an appropriate buffer solution is added to the thus separated supernatant and thoroughly mixed (e.g., 10 minutes or more of stirring using a rotator). Next, the supernatant is separated and removed by centrifugation (e.g., 2,000 rpm for 2 minutes) and again centrifuged by adding an appropriate amount (e.g., 0.5 ml) of a buffer solution adjusted to pH 6.8, thereby effecting washing. After repeating this 3 times, an appropriate amount (e.g., 50 μl) of 100 mM EDTA is added, the mixture is allowed to stand for 10 minutes and then the supernatant is recovered, thereby enabling purification of the released polypeptide of the present invention. As the aforementioned buffer solution, a buffer solution B (8 M urea, 0.1 M Na2HPO4, 0.1 M NaH2PO4, 0.01 M Tris-HCl pH 8.0) can for example be used. The His tag in the purified protein molecule may be removed from the molecule, for example, by designing in such a manner that His tag is fused to the N-terminal side and using TAGZyme System (Qiagen).
Alternatively, as occasion demands, it may be purified by a method which does not use a tag protein, for example, by various separation operations making use of the physical properties and chemical properties of the protein consisting of the polypeptide of the present invention. Illustratively, use of ultrafiltration, centrifugation, gel filtration, adsorption chromatography, ion exchange chromatography, affinity chromatography and high performance liquid chromatography may be exemplified.
The polypeptide of the present invention may be produced by general chemical synthesis in accordance-with the amino acid sequence information shown in SEQ ID NO:2 or SEQ ID NO:4. Illustratively, liquid phase and solid phase peptide synthesis methods are included. Its synthesis may be carried out by successively binding one amino acid after another, or by synthesizing a peptide fragment comprising several amino acids and the binding it. The polypeptide of the present invention obtained by these means may be purified in accordance with the aforementioned various methods.
<Inspection Method of Diabetes Mellitus>
By the use of a probe which hybridizes with the polynucleotide of the present invention under a stringent condition, expressed amount of a polynucleotide coding for the polypeptide of the present invention may be examined, and diagnosis of diabetes mellitus can be carried out using increase of the expressed amount (preferably the expressed amount in the fat tissue) as the index. In the inspection method of diabetes mellitus, the term “stringent condition” means a condition under which nonspecific binding does not occur, and illustratively, it means a condition in which 0.1× SSC (saline-sodium citrate buffer) solution containing 0.1% sodium lauryl sulfate (SDS) is used and the temperature is 65° C. As the probe, a DNA of at least 15 bp in chain length and having at least a part of or entire sequence (or a complementary sequence thereof) of the polynucleotide of the present invention is used.
According to the method for detecting diabetes mellitus, whether or not the subject is diabetes mellitus may be detected by allowing the aforementioned probes to contact a sample to be tested, and analyzing the bonded product of a polynucleotide coding for the polypeptide of the present invention (e.g., mRNA or cDNA derived therefrom) and the aforementioned probe by a conventionally known analyzing method (e.g., northern blotting). In addition, the expression quantity can also be analyzed by applying the aforementioned probe to a DNA tip. When the amount of the aforementioned bonded product, namely the amount of a polynucleotide coding for the polypeptide of the present invention, is increased in comparison with healthy parsons, it may be judged that the subject is diabetes mellitus.
As a method for measuring expressed level of the polynucleotide of the present invention, it is possible to employ methods in which the expressed level is measured by detecting the polypeptide of the present invention. Examples of the inspection method to be used include western blotting, immunoprecipitation, ELISA and the like methods, making use of an antibody which binds a sample to be tested to the polypeptide of the present invention, or an antibody which specifically binds to the polypeptide of the present invention. In determining the amount of the polypeptide of the present invention contained in the sample to be tested, the polypeptide of the present invention may be used as the standard amount. In addition, the polypeptide of the present invention is useful for preparing an antibody which binds to the polypeptide of the present invention. When the amount of the polypeptide of the present invention is increased in comparison with healthy parsons, it may be judged that the subject is diabetes mellitus.
<Screening Method of the Invention>
By using (1) the polypeptide of the present invention, (2) a polypeptide consisting of an amino acid sequence having a homology of 90% or more with the amino acid sequence represented by SEQ ID NO:2 or SEQ ID NO:4 and which binds to Akt2 (to be referred to as homologous polypeptide hereinafter), or (3) a polypeptide as a protein encoded by a polynucleotide which hybridizes with the polynucleotide having the nucleotide sequence represented by SEQ ID NO:1 or SEQ ID NO:3 under a stringent condition and which binds to Akt2 (to be referred to as hybridize polypeptide hereinafter), a method for screening a substance having an insulin resistance improving action and/or a substance having carbohydrate metabolism improving action (namely diabetes mellitus improving agents) may be constructed making use of the interaction of the aforementioned polypeptides (namely the polypeptide of the present invention, the homologous polypeptide and the hybridize polypeptide) with Akt2 kinase. The polypeptide of the present invention, the aforementioned homologous polypeptide and the aforementioned hybridize polypeptide are generally referred to as a polypeptide for the screening of the present invention.
The homologous polypeptide according to this description is not particularly limited, as long as it is a polypeptide consisting of an amino acid sequence having a homology of 90% or more with the amino acid sequence represented by SEQ ID NO:2 or SEQ ID NO:4 and which binds to Akt2, but regarding the amino acid sequence represented by SEQ ID NO:2 or SEQ ID NO:4, a polypeptide consisting of an amino acid sequence having a homology of preferably 95% or more, more preferably 98% or more, is desirable.
In this connection, the aforementioned term “homology” as used in this description means a value (Identities) obtained using parameters prepared as default by Clustal program (Higgins and Sharp, Gene, 73, 237-244, 1998; Thompson et al., Nucl. Acids Res., 22, 4673-4680, 1994) retrieval. The aforementioned parameters are as follows. As Multiple Alignment Parameters, Gap Penalty 15.00, Gap Length Penalty 6.66, Delay Divergent Seqs (%) 30 and DNA Transition Weight 0.50, and as Pairwise Alignment Parameters, Gap Penalty 15.00 and Gap Length Penalty 6.66, by Slow-Accurate.
Regarding the “stringent condition” for the hybridize polypeptide of this description under which a polynucleotide coding for the hybridize polypeptide of this description hybridizes with the polynucleotide having the nucleotide sequence represented by SEQ ID NO:1 or SEQ ID NO:3, it is a condition of “5× SSPE, 5× Denhard's solution, 0.5% SDS, 40% formamide, 200 μg/ml salmon sperm DNA, and 37° C. overnight” as the condition for hybridization, and a condition of “5× SSPE, 5× Denhard's solution, 0.5% SDS, 50% formamide, 200 μg/ml salmon sperm DNA, and 42° C. overnight” as more strict condition. Also, the condition for washing is “5× SSC, 1% SDS and 42° C.” as a mild condition, “0.5× SSC, 0.1% SDS and 42° C.” as a normal condition, and “0.2× SSC, 0.1% SDS and 65° C.” as a more strict condition.
Also included in the screening method of the present invention is a method for screening a substance which inhibits binding of the aforementioned polypeptide with Akt2, characterized in that it comprises a step of allowing the polypeptide for screening of the present invention or a cell expressing the polypeptide for screening of the present invention or to contact a substance to be tested, a step of measuring binding of said polypeptide with Akt2, and a step of selecting a substance which inhibits the aforementioned binding. The cells expressing the polypeptide for screening of the present invention may be either cells transformed with an expression vector containing a polynucleotide coding for the polypeptide for screening of the present invention or a naturally existing cells expressing the polypeptide of the present invention, but a transformed cells are desirable.
Since the AKBP2 as one of the polypeptides for screening of the present invention binds to Akt2, its expression is reduced in diabetes mellitus model mice, and the Akt2 activity is reduced in fat cells in which mouse AKBP2 was overexpressed, it was found that the polypeptide of the present invention negatively controls the insulin signal via its binding to Akt2. Thus, an insulin resistance improving agent and/or a carbohydrate metabolism improving agent may be screened by the aforementioned screening method.
In the aforementioned screening methods, the step of measuring binding of the polypeptide for screening of the present invention or cells expressing the polypeptide for screening of the present invention with Akt2 may be carried out by directly detecting binding of the polypeptide for screening of the present invention with Akt2, or can also be carried out by measuring a change of Akt2 caused by a change of the aforementioned binding.
Though not particularly limited, examples of the substance to be tested which may be used in the screening methods of the present invention include commercially available compounds (including peptides), various conventionally known compounds registered in chemical files (including peptides), a group of compounds obtained by the combinatorial chemistry techniques (N. K. Terrett, M. Gardner, D. W. Gordon, R. J. Kobylecki and J. Steele, Tetrahedron, 51, 8135-73, (1995)), culture supernatants of microorganisms, natural components derived from plants and marine organisms, animal tissue extracts, or compounds (including peptides) obtained by chemically or biologically modifying a compound selected by the screening methods of the present invention.
The aforementioned screening methods are not limited, the following screening methods may be exemplified.
1) Screening Method Which Uses Phosphorylation of Akt2
It is known that the kinase activity of Akt2 is accelerated when the 473rd serine (Ser 473) or the 308th threonine (Thr 308) in the molecule is phosphorylated (Biochem. J., 1998, 335 (1-13)). Making use of this, the presence or absence of Akt2 activity may be detected by detecting phosphorylated condition of the Ser 473 or Thr 308 of Akt2 by a western blotting which uses an antibody capable of specifically reacting with these phosphorylated residues (e.g., anti-phosphoSer antibody or the like).
A testing cell expressing a part or entire portion of the polypeptide for screening of the present invention is untreated or treated with a substance to be tested. As the testing cells, cells which respond to insulin are desirable, and more illustratively, fat cells, hepatocyte or skeletal muscle-derived cells are desirable. The cell untreated or treated with a substance to be tested is lysed, and using this as a sample, phosphorylation of Akt2, namely the presence or absence the Akt2 activity, may be detected making use of western blotting, spot western blotting or the like method which uses the anti-phosphoSer antibody. Preferably, this may be detected by the method of Example 7. In this detection system, a substance used for treating a sample in which acceleration of the phosphorylation of Akt2 (namely activation of Akt2) was observed in comparison with a sample untreated with the substance to be tested may be selected as a substance which inhibits binding of the polypeptide for screening of the present invention with Akt2, and based on this, an insulin resistance improving agent and/or carbohydrate metabolism improving agent, namely a substance having diabetes mellitus-treating effect, may be screened. As such a substance, it is desirable to select a substance which shows an ED50 value of the Akt2 phosphorylation acceleration action of 10 μM or less, preferably 1 μM or less, more preferably 0.1 μM or less, in said screening method.
2) Screening Method Which Uses In Vitro Kinase Method
The Akt2 activity can also be detected by an in vitro kinase assay method in which uptake of radioactive phosphoric acid based on a substrate is measured when a histone H2B, a GSK-3 fusion protein or the like is used as the substrate of Akt2 and allowed to react with an immune precipitate of Akt2. Illustratively, a testing cells expressing a part or entire portion of the polypeptide for screenings of the present invention are untreated or treated with a substance to be tested. As the testing cells, cells which responds to insulin are desirable, and more illustratively, fat cells, hepatocyte or a skeletal muscle-derived cells are desirable. Activated Akt2 protein may be concentrated from the aforementioned cells by immunoprecipitation using an anti-Akt2 antibody. By mixing a substrate of Akt2, such as GST-crosstide (GST fusion protein of GSK3-beta sequence as a physiological substrate of Akt), with concentrated Akt2 protein, kinase activity of Akt2 may be measured and determined using phosphorylation of the substrate as the index. Preferably, this may be measured by the method described in Example 7. It is possible to use the kinase measurements as screening methods of a large number of compound, by making use of the total kinase assay methods (Waga et al., J. immnunol. Methods, 190, pp. 71-77, 1996). In these measuring systems, substances used for treating samples in which acceleration of the kinase activity of Akt were observed in comparison with samples untreated with the substances to be tested may be selected as substances which inhibits binding of the polypeptide for screening of the present invention with Akt2, and based on this, insulin resistance improving agents and/or carbohydrate metabolism improving agents, namely substances having diabetes mellitus-treating effects, may be screened. As such substances, it is desirable to select a substance which shows an ED50 value of the Akt2 kinase acceleration action of 10 μM or less, preferably 1 μM or less , more preferably 0.1 μM or less, in said screening methods.
3) Screening Method Which Uses Binding of the Polypeptide for Screening of the Present Invention with Akt2
Since the polypeptide for screening of the present invention negatively controls the insulin signal via its binding to Akt2, the following screening method which uses binding of the polypeptide for screening of the present invention with Akt2 as the index may be exemplified. Illustratively, testing cells expressing a part or entire portion of the polypeptide for screenings of the present invention, or a part or entire portion of the polypeptide for screening of the present invention which is fused with a tag (e.g., GST, Flag, His or the like), is untreated or treated with a substance to be tested. As the testing cells, cells which respond to insulin is desirable, and more illustratively, fat cells, hepatocyte or skeletal muscle-derived cells are desirable. The Akt2 protein and a protein binding thereto may be concentrated from the aforementioned cells by immunoprecipitation using an anti-Akt2 antibody. In this concentration step, it is desirable that the same substance to be tested used in the aforementioned treatment of cells are contained in the reaction solution. A substance to be tested which inhibits binding of the polypeptide for screening of the present invention with Akt2 may be selected by separating the thus obtained concentrated solution of Akt2 and its binding protein by polyacrylamide gel electrophoresis using a conventionally known method and measuring the amount of the polypeptide for screening of the present invention by western blotting using an antibody. As such a substance, it is desirable to select a substance which shows an IC50 value, of the action to inhibit binding of the polypeptide of the present invention with Akt2, of 10 μM or less, preferably 1 μM or less, more preferably 0.1 μM or less, in the aforementioned screening method. Regarding the antibody to be used in this case, an antibody specific for the polypeptide for screening of the present invention or for the polypeptide for screening of the present invention prepared based on its partial sequence (e.g., anti-AKBP2 antibody), or an antibody which recognizes the aforementioned tag, may be used.
In the above screening methods of 1) to 3), the testing cells may be used by un-stimulating or stimulating with insulin, but preferably, the testing cells may be used by carrying out insulin stimulation.
In addition, substances to be tested which inhibits binding of the polypeptide for screening of the present invention with Akt2 can also be selected by combining a western blotting similar to the aforementioned one with an in vitro pull down method (H. Matsushime et al., Jikken Kogaku (Experimental Engineering), Vol. 13, No. 6, p. 528, 1994, ), using Akt2 protein purified by attaching a tag (e.g., GST or the like) from an extract of cells in which the polypeptide for screening of the present invention is expressed, or a mixed solution of proteins prepared by carrying out in vitro transcription and translation, to which a substance to be tested is added or not added. Preferably, substances to be tested which inhibits binding of Akt2 with the polypeptide for screening of the present invention can also be selected using a mixed solution of proteins prepared by directly carrying out in vitro transcription and translation of a protein consisting of the polypeptide of the present invention (e.g., AKBP2 protein) as shown in Example 6, from a plasmid which expresses the polypeptide for screening of the present invention (e.g., the AKBP2 expression plasmid prepared in Example 1(5)) using an in vitro translation kit (e.g., TNT Kit (Promega)). Each of these methods renders possible screening of a large number of substances to be tested by not carrying out polyacrylamide gel electrophoresis, but carrying out conventionally known spot western blotting. Also, it is possible to carry out a screening for selecting a substance to be tested capable of inhibiting binding of Akt2 with the polypeptide for screening of the present invention, in accordance with conventionally known ELISA methods, which comprises adding a substance to be tested to a lysate of cells in which the polypeptide for screening of the present invention expressed by fusing a tag similar to the aforementioned one and Akt2 are simultaneously expressed. In addition, it is possible to select a substance to be tested which inhibits binding of Akt2 with the polypeptide for screening of the present invention by screening it from the great majority of population through the detection of the existing CAT or luciferase activity, making use of the conventionally known two hybrid system for mammal cells (Clontech), and arranging Akt2 fused with the DNA binding region of GAL 4 as the bait, and the polypeptide for screening of the present invention fused with the transcription accelerating region of VP 16 to the pray side.
<Method for Producing a Pharmaceutical Composition for Insulin Resistance Improvement and/or Carbohydrate Metabolism Improvement>
Also included in the present invention is a method for producing a pharmaceutical composition for insulin resistance improvement, characterized in that it comprises a step of carrying out screening using the polypeptide for screening of the present invention, and a step of preparing a pharmaceutical preparation.
The pharmaceutical composition which contains a substance obtained by the screening method of the present invention as the active ingredient may be prepared using carriers, fillers and/or other additive agents generally used in making pharmaceutical preparations, in response to the type of the aforementioned active ingredient.
As its administration, oral administration by tablets, pills, capsules, granules, fine subtilaes, powders, solutions for oral use or the like, or parenteral administration by injections for intravenous injection, intramuscular injection, intraarticular injection or the like, suppositories, percutaneous administration preparations, transmucosal administration preparations or the like may be cited. Particularly, parenteral injection is desirable in the case of peptides which are digested in the stomach, intravenous injection or the like.
In the solid composition for use in the oral administration, one or more active substances may be mixed with at least one inert diluent such as lactose, mannitol, glucose, microcrystalline cellulose, hydroxypropylcellulose, starch, polyvinyl pyrrolidone, aluminum magnesium silicate or the like. In the usual way, the aforementioned composition can contain other additives than the inert diluent, such as a lubricant, a disintegrating agent, a stabilizing agent, a solubilizing or solubilization assisting agent or the like. As occasion demands, tablets or pills may be coated with a sugar coating or with a film of a gastric or enteric substance or the like.
The liquid composition for oral administration can contain, for example, emulsions, solutions, suspensions, syrups, elixirs or the like and can contain a generally used inert diluent such as purified water or ethyl alcohol. The aforementioned composition can contain additive agents other than the inert diluent, such as a moistening agent, a suspending agent, a sweetener, an aromatic, or an antiseptic.
The injections for parenteral administration can include aseptic aqueous or non-aqueous solutions, suspensions or emulsions. The aqueous solutions or suspensions can include, for example, distilled water for injection, physiological saline or the like as the diluent. As the diluent for use in the non-aqueous solutions or suspensions, propylene glycol, polyethylene glycol, plant oil (e.g., olive oil), alcohols (e.g., ethanol), polysorbate 80 or the like can for example be included. The aforementioned composition can further contain, a moistening agent, an emulsifying agent, a dispersing agent, a stabilizing agent, a solubilizing or solubilization assisting agent, an antiseptic or the like. The aforementioned composition may be sterilized, for example, by filtration through a bacteria retaining filter, blending of a germicide or irradiation. Alternatively, it may be used by producing a sterile solid composition and dissolving it in sterile water or other sterile solvent for injection prior to its use.
The dose may be optionally decided by taking into consideration strength of activity of the active ingredient, namely the substance obtained by the screening method of the present invention, symptoms, age, sex and the like of each patient to be treated.
For example, in the case of oral administration, the dose is generally approximately from 0.1 to 100 mg, preferably from 0.1 to 50 mg, per day per adult (as 60 kg body weight). In the case of parenteral administration in the form of injections, it is from 0.01 to 50 mg, preferably from 0.01 to 10 mg, per day.
EXAMPLES
The present invention is described in the following based on examples, but the invention is not restricted by said examples. In this connection, unless otherwise noted, these may be carried out in accordance with conventionally known methods (“Molecular Cloning, Sambrook et al., Cold Spring Harbor Laboratory Press, 1989” and the like). Also, when commercially available reagents and kits are used, these may be carried out in accordance with the instructions attached thereto.
Example 1
Cloning of Mouse AKBP2 Gene and Construction of Expression Vector
(1) Cloning of Akt2 Gene
Using the oligonucleotides represented by SEQ ID NO:5 and SEQ ID NO:6, designed with reference to the cDNA sequence described in the accession number M95936 of a gene data base GenBank, as primers, and a human skeletal muscle cDNA (Marathon-Ready™ cDNA; Clontech) as the template, PCR was carried out using a DNA polymerase (Pyrobest DNA Polymerase (Takara Shuzo)) under a condition of thermal denaturation at 95° C. for 3 minutes, 40 repetition of a cycle consisting of 98° C. for 10 seconds, 60° C. for 30 seconds and 74° C. for 1 minute and 30 seconds, and further heating at 74° C. for 7 minutes. Human Akt2 cDNA was cloned by inserting the resulting DNA fragment of about 1.5 kbp into the EcoRV recognition site of a plasmid pZErO™-2.1 (Invitrogen). Nucleotide sequence of the Akt2 cDNA cloned on the vector was determined by a sequencing kit (Applied Biosystems) and a sequencer (ABI 3700 DNA sequencer, Applied Biosystems), using the aforementioned oligonucleotides represented by SEQ ID NO:5 and SEQ ID NO:6, thereby confirming that its sequence coincided with the reported sequence.
(2) Preparation of Expression Plasmid for Yeast Two Hybrid
In order to insert the human Akt2 cDNA into an expression vector for yeast two hybrid, pDBtrp (Invitrogen), primers represented by SEQ ID NO:7 and SEQ ID NO:8 were designed by adding a region homologous with 40 nucleotides in front and in the rear of the pDBtrp vector multi-cloning site to the 5′-side and 3′-side of the human Akt2 gene sequence. PCR was carried out using the Akt2 plasmid cloned in the above as the template and using a DNA polymerase (Pyrobest DNA polymerase; Takara Shuzo), by heating at 98° C. (1 minute) and then repeating 35 times of a cycle consisting of 98° C. (5 seconds), 55° C. (30 seconds) and 72° C. (5 minutes) The DNA fragment obtained as the result has the complete code region of the human Akt2 gene.
The vector pDBtrp made into a linear chain by digesting with restriction enzymes SalI and NcoI and the PCR fragment containing Akt2 cDNA obtained in the above were simultaneously added to yeast strain MaV203 for two hybrid (Invitrogen) which was then transformed by a lithium acetate method (Guthrie C. and Fink R., Guide to Yeast Genetics and Molecular Biology, Academic, San Diego, 1991). As a result, homologous recombination occurred in the yeast cell, and a plasmid in which the Akt2 cDNA was inserted into the multi-cloning site of pDBtrp (to be referred to as pDB-Akt2 hereinafter) was formed. Yeast cell having the pDB-Akt2 plasmid were selected by culturing on a solid synthetic minimal medium (DISCO,20% agarose) from which tryptophan as a selection marker of the plasmid had been deleted, the yeast cells were treated with zymolyase (Seikagaku Kogyo) at 37° C. for 30 minutes, and then the plasmids were isolated and purified by the alkali method, and determination of their nucleotide sequences was carried out using a sequencing kit (Applied Biosystems) and a sequencer (ABI 3700 DNA sequencer, Applied Biosystems) to select those in which the Akt2 cDNA was inserted together with the code region and translation frame of GAL 4 DNA binding region of pDBtrp.
(3) Preparation of Mouse Fat Tissue Derived cDNA Library
By purchasing C57BL/6J male mice of 12 weeks of age and C57BL/KsJ-+m/+m male mice of 13 weeks of age from CLEA Japan, Poly(A)+ RNA was prepared from epididymis fat in accordance with the mRNA preparation method described in Experimental Medicine Supplement, Bio-manual Series 2, Gene Library Preparation Method (written in Japanese, edited by H. Nojima; published by Yohdosha on Feb. 20, 1994). Using ZAP-cDNA Synthesis Kit manufactured by Stratagene and in accordance with the protocol attached thereto, first-strand synthesis and second-strand synthesis were carried out using 5 μg of RNA, and the double-stranded cDNA was smooth-ended, ligated with the EcoRI adapter attached to the kit and then digested with restriction enzymes EcoRI and XhoI. Size fractionation was carried out using a spin column (CHROMA SPIN-400; Clontech), and shorter fragments were removed. A 100 μg portion of a vector pACT2 (Clontech) was digested with the restriction enzyme XhoI, treated with an alkaline phosphatase (Bacterial Alkaline Phosphatase; Takara Shuzo), and then digested with the restriction enzyme EcoRI and applied to a spin column (CHROMA SPIN-1000; Clontech). In accordance with the cDNA library preparation method described in Experimental Medicine Supplement, Bio-manual Series 2, Gene Library Preparation Method (edited by H. Nojima; published by Yohdosha on Feb. 20, 1994), the vector and cDNA were ligated, and the sample after ligation was treated with a filter cup (UFCP3TK50) manufactured by Millipore. Using the Escherichia coli for electroporation manufactured by GIBCO BRL (ElectroMAXX DH10B™ Cells), transformation was carried out by the electroporation method, and cultured overnight on a shaker using 1,000 ml of a culture medium. After confirming that 106 or more of independent colonies are present in the culture medium, plasmids were purified using a plasmid purification kit (Qiagen Plasmid Kit; Qiagen) and in accordance with the protocol attached to the kit.
(4) Yeast Two Hybrid Screening
The aforementioned yeast strain MaV203 for two hybrid transformed by pDB-Akt2 was suspended in 400 ml of YPD liquid medium (DIFCO), cultured at 30° C. for about 6 hours until absorbance at a wave length of 590 nanometer became from 0.1 to 0.4, and then made into competent cells by the lithium acetate method, and the final amount was suspended in 1.0 ml of 0.1 M lithium-tris buffer. The cells were transformed with 20 μg of the mouse fat tissue derived cDNA library prepared in the aforementioned (3), and the cells were selected by culturing on a solid synthetic minimal medium (DISCO, 20% agarose) from which tryptophan and leucine as respective selection markers of pDB-Akt2 and the library had been deleted, thereby obtaining a transformant into which both plasmids were introduced. At the same time, in order to select a cell having an activated reporter gene HIS3 which is expressed when a fusion protein of the GAL4 DNA binding domain artificially expressed in the two hybrid system is linked to a fusion protein of the GAL4 transcriptional activation domain, the transformed cells were cultured at 30° C. for 5 days on the solid minimal medium (20% agarose) from which histidine was removed together with tryptophan and leucine and to which 20 mM of 3AT (3-amino-1,2,4-triazole; Sigma) as an inhibitor of the enzyme encoded by HIS3 was added. The 3AT-resistant yeast colonies showing that a protein which binds to Akt2 is expressed under the same condition were obtained. These yeast cells were grown on the YPD solid medium for 24 hours, and then expression of the lacZ gene which is different from HIS3 but is a binding indicator reporter of the two hybrid system was examined using β-galactosidase activity as the index. Regarding the β-galactosidase activity, yeast cells on the medium were transferred on a nitrocellulose film, frozen in liquid nitrogen and then thawed at room temperature, and the filter was put on a filter paper soaked with 0.4% X-GAL (Sigma) solution and allowed to stand at 37° C. for 24 hours to measure change of color to blue caused by β-galactosidase. By selecting colonies in which contents of cells transferred on the filter changed from white to blue, yeast cells expressing a protein which binds to Akt2 were specified, and library derived plasmids were extracted from the cells in accordance with the method of Yeast Protocols Handbook of Clontech. Nucleotide sequences of the gene fragments contained therein were determined by a sequencing kit (Applied Biosystems) and a sequencer (ABI 3700 DNA sequencer, Applied Biosystems) using the nucleotide sequence represented by SEQ ID NO:9 (a sequence which binds to the GAL4 AD region; derived from GenBank accession number U29899 Cloning vector pACT2) as the primer, and it was confirmed as a result that the nucleotide sequence represented by SEQ ID NO:1 was contained therein.
(5) Determination of Initiation Codon of Mouse AKBP2 Gene
As a result of the aforementioned (4), a library derived plasmid having a gene fragment containing the nucleotide sequence represented by SEQ ID NO:1 was obtained. Accordingly, in order to determine initiation codon of the gene contained in said fragment, a primer of the nucleotide sequence represented by SEQ ID NO:10 which corresponds to a complementary chain of a nucleotide sequence of from the 1034th position to the 1011th position of the nucleotide sequence represented by SEQ ID NO:1 was synthesized (Proligo), and an attempt was made to amplify complete length cDNA derived from an expression product of said gene from the aforementioned fat tissue-derived cDNA library by PCR using said primer and the aforementioned primer of the nucleotide sequence represented by SEQ ID NO:9. The PCR was carried out using a DNA polymerase (TAKARA LA Taq; Takara Shuzo) and by heating at 94° C. (3 minutes) and then repeating 35 times of a cycle consisting of 94° C. (30 seconds), 58° C. (1.5 minutes) and 72° C. (4 minutes). The DNA fragments in the reaction solution were cloned into an expression vector (pcDNA3.1/V5-His-TOPO; Invitrogen) using TOPO TA Cloning System (Invitrogen). Nucleotide sequences of the inserted DNA fragments in the thus obtained plasmids were determined using a primer (TOPO TA Cloning Kit; Invitrogen; SEQ ID NO:11) which binds to the T7 promoter region on the vector, a sequencing kit (Applied Biosystems) and a sequencer (ABI 3700 DNA sequencer, Applied Biosystems). As a result, plasmids containing cDNA molecules of various lengths having the sequence of said gene were obtained, but chain lengths of the longest cDNA molecules were almost the same as that of the transcription product derived cDNA obtained in the aforementioned (4). Since several trials showed the same result, it was found that the chain length of sequence of the transcription product of said gene almost coincide with those of the cDNA obtained in (4). Based on this, it was found that the first ATG of the nucleotide sequence represented by SEQ ID NO:1 is the initiation codon of said gene, so that the open reading frame of said gene represented by SEQ ID NO:1 was confirmed. This gene was named mouse AKBP2 gene.
(6) Preparation of Mouse AKBP2 Expression Vector
As a result of the aforementioned (4), a library-derived plasmid having a gene fragment containing complete length of the nucleotide sequence represented by SEQ ID NO:1 was obtained, and the presence of a factor which binds to Akt2 was indicated. Also, its open reading frame was confirmed in the aforementioned (5). Accordingly, the primers represented by SEQ ID NO:12 and SEQ ID NO:13 were synthesized (Proligo) in accordance with the nucleotide sequence information shown in SEQ ID NO:1, and an AKBP2 cDNA coding for the net AKBP2 protein was amplified by PCR using said primers and the plasmid obtained in the aforementioned (4) as the template. These two kinds of DNA primers respectively have nucleotide sequences homologous with partial sequences of the 5′-side and 3′-side of the mouse AKBP2 gene represented by SEQ ID NO:1. PCR was carried out using a DNA polymerase (Pyrobest DNA Polymerase; Takara Shuzo), by heating at 98° C. (1 minute) and then repeating 35 times of a cycle consisting of 98° C. (5 seconds), 55° C. (30 seconds) and 72° C. (5 minutes) As a result of separating the PCR product by an agarose gel electrophoresis, it was confirmed that a DNA fragment of about 1.7 kbp was amplified. Accordingly, this DNA fragment in the reaction solution was subcloned into an expression vector (pcDNA3.1/V5-His-TOPO; Invitrogen) using TOPO TA Cloning System (Invitrogen). The primer represented by SEQ ID NO:13 used in this case was designed in such a manner that the stop codon sequence of AKBP2 was removed so that a vector-derived VS epitope (derived from V protein of paramyxovirus SV 5, Southern J. A., J. Gen. Virol., 72, 1551-1557, 1991) and His 6 tag (Lindner P., BioTechniques, 22, 140-149, 1997) were continued with the same frame of the triplet of mouse AKBP2 gene at the 3′-side after cloning. Nucleotide sequence of the inserted DNA fragment in the thus obtained plasmid was determined using a primer (TOPO TA Cloning kit; Invitrogen; SEQ ID NO:11) which binds to the T7 promoter region on the vector, a sequencing kit (Applied Biosystems) and a sequencer (ABI 3700 DNA sequencer, Applied Biosystems). As a result, it was confirmed that the 1719 base pair AKBP2 cDNA represented by SEQ ID NO:1 coding for the net AKBP2 protein was inserted into the aforementioned expression vector pcDNA3.1/V5-His-TOPO, as a DNA from which the 3′-side stop codon of the DNA sequence was removed. This expression plasmid is referred to as pcDNA-AKBP2 hereinafter.
Example 2
Preparation of Cultured Cell Which Expresses AKBP2 Protein
(1) Preparation of AKBP2 Expression Cell
The aforementioned expression plasmid pcDNA-AKBP2 prepared in Example 1(5) or an empty vector (pcDNA3.1/V5-His-TOPO) was introduced into the 293 cell (Cell Bank). The 293 cell was cultured in a culture dish of 6 well culture plate (well diameter 35 mm) until it became a state of 70% confluent, by adding 2 ml of a minimal essential medium DMEM (Gibco) containing 10% fetal bovine serum (Sigma) to each well. The pcDNA-AKBP2 (3.0 μg/well) was transiently introduced into this cell by the calcium phosphate-method (Graham et al., Virology, 52, 456, 1973; N. Arai, Gene Transfer and Expression/Analytical Method (written in Japanese), pp. 13-15, 1994). After 30 hours of culturing, the medium was removed and the cells were washed with phosphate buffered saline (to be referred to as PBS hereinafter), and then the cells were lysed by adding 0.1 ml per well of a cell lysis solution (100 mM potassium phosphate (pH 7.8), 0.2% Triton X-100).
(2) Detection of AKBP2 Protein
A 10 μl portion of 2× SDS sample buffer (125 mM Tris-HCl (pH 6.8), 3% sodium lauryl sulfate, 20% glycerol, 0.14 M β-mercaptoethanol, 0.02% Bromophenol Blue) was added to 10 μl of the aforementioned lysate of AKBP2 expression cell of Example 2(1), and this was treated at 100° C. for 2 minutes and then subjected to 10% SDS polyacrylamide gel electrophoresis to separate proteins contained in the sample. Proteins in the polyacrylamide gel were transferred on a nitrocellulose membrane using a semi-dry type blotting device (Bio-Rad), and then detection of the AKBP2 protein on said nitrocellulose membrane was carried out by western blotting in accordance with the usual way. A monoclonal antibody (Invitrogen) which recognizes V5 epitope fused to the C-terminus of AKBP2 was used as the primary antibody, and mouse IgG-HRP fusion antibody (Bio-Rad) was used as the secondary antibody. As a result, as shown in FIG. 1, it was confirmed that a protein of about 70 kDa which corresponds to an AKBP2-V5-His 6 fusion protein consisting of 618 amino acids containing the C-terminal side tag consisting of 45 amino acid is detected dependently on the gene transfer of the expression vector pcDNA-AKBP2. Based on this, it was revealed that complete length region of the aforementioned mouse AKBP2 gene cloned is certainly expressed and can form stable structure as the protein in the cultured cell.
Example 3
Measurement of AKBP2 Expression Level in Normal Mice, High Fat Diet-loaded Normal Mice and Diabetes Mellitus Model Mice
Based on the aforementioned information, it was found that the mouse AKBP2 protein of the present invention binds to Akt2, and is expressed in the insulin-responding tissues including both adipose and muscle. Since Akt2 protein is a factor which acts upon the insulin signal first pathway, it was considered that action of the AKBP2 of the present invention is related to the insulin resistance. Accordingly, measurement of messenger RNA (mRNA) expression level of the AKBP2 gene in muscle and fat was carried out using a type 2 diabetes mellitus model mice KKAy/Ta (Iwatsuka et al., Endocrinol. Japon.: 17, 23-35, 1970, Taketomi et al., Horm. Metab. Res., 7, 242-246, 1975) and a healthy mice C57BL/6J fed with a normal feed or a high fat diet.
Regarding the gene expression level, expression level of the mouse AKBP2 gene of the present invention was measured and corrected by the simultaneously measured expression level of glyceraldehyde 3-phosphate dehydrogenase (G3PDH) gene. As the measuring system, PRISM™ 7700 Sequence Detection System and SYBR Green PCR Master Mix (Applied Biosystems) were used. In this measuring system, expressed amount of the gene of interest is determined by detecting and monitoring fluorescence level of the SYBR Green I pigment incorporated by double-stranded DNA amplified by PCR in a real time manner.
Illustratively, the measurement was carried out by the following procedures.
(1) Preparation of Total RNA
C57BL/6J male mice of 14 weeks of age loaded with a usual feed or a high fat diet, and C57BL/6J male mice and KKAy/Ta mice of 15 weeks of age (all from CLEA Japan) were used. The high fat diet loading was carried out for 9 weeks from 5 weeks of age to 14 weeks of age. Composition of the high fat diet is as follows: casein 29.8%, sucrose 15.8%, vitamin mix 1.3%, mineral mix 8.8%, cellulose powder 5.0%, methionine 0.5%, safflower oil 28.9%, water 10%. On the other hand, CE-2 (CLEA Japan) was used as the normal feed. Muscle and fat of each of the aforementioned mice were extracted, and total RNA was prepared using a reagent for RNA extraction (Isogen; Nippon Gene) and in accordance with its instructions. Each total RNA thus prepared was then treated using deoxyribonuclease (Nippon Gene), subjected to phenol/chloroform treatment and ethanol precipitation, dissolved in sterile water and stored at −20° C.
(2) Synthesis of Single-stranded cDNA
Reverse transcription of total RNA into single-stranded cDNA was carried out in a system of 20 μl using 1 μg of RNA (fat), 1 μg of RNA (muscle of a mouse of 14 weeks of age) or 0.25 μg of RNA (muscle of a mouse of 15 weeks of age) prepared in (1), and using a kit for reverse transcription reaction (Advantage™ RT-for-PCR Kit; Clontech). After the reverse transcription, this was mixed with 180 μl of sterile water and stored at −20° C.
(3) Preparation of PCR Primers
Four oligonucleotides (SEQ ID NO:14 to SEQ ID NO:17) were designed as the PCR primers described in the following item (4). They were used as a combination of SEQ ID NO:14 with SEQ ID NO:15 for the mouse AKBP2 gene, and a combination of SEQ ID NO:16 with SEQ ID NO:17 for the G3PDH gene.
(4) Measurement of Gene Expression Quantity
Real time measurement of PCR amplification by PRISM™ 7700 Sequence Detection System was carried out in a system of 25 μl in accordance with the instructions. In each system, 5 μl of single-stranded cDNA, 12.5 μl of 2× SYBR Green reagent and 7.5 pmol of each primer were used. In this case, the single-stranded cDNA preserved in (2) was used by diluting 30 times regarding the G3PDH, or by diluting 10 times regarding the mouse AKBP2. Instead of the single-stranded cDNA, 0.1 μg/μl of a mouse genomic DNA (Clontech) was appropriately diluted and a 5 μl portion thereof was used for the preparation of calibration curve. PCR was carried out by heating at 50° C. for 10 minutes, subsequently heating at 95° C. for 10 minutes, and then repeating 45 cycles of a process consisting of 2 steps of 95° C. for 15 seconds and 60° C. for 60 seconds.
Expressed amount of the mouse AKBP2 gene in each sample was corrected by the expressed amount of G3PDH gene based on the following formula.
[Corrected amount of AKBP2 expression]=[expressed amount of AKBP2 (raw data)]/[expressed amount of G3PDH (raw data)]
FIG. 2 shows relative amounts in which the expressed amount in C57BL/6J mouse of usual feed was defined as 1 in comparing expressed amounts in fat, and the expressed amount in C57BL/6J also as 1 in comparing expressed amounts in muscle tissue.
As shown in FIG. 2, it was confirmed that expression of the mouse AKBP2 gene of the present invention is markedly increased in the fat and muscle at the time of high fat diet loading, or in the fat and muscle of the diabetes mellitus model mouse. Accordingly, it is considered that the mouse AKBP2 of the present invention induces insulin resistance by the acceleration of expression quantity in fat and muscle. Based on the above, it may be concluded that concern of the mouse AKBP2 of the present invention in the insulin resistance is large.
In addition, it was revealed from the results of this Example that diagnosis of diabetes mellitus morbid state may be made by measuring expression quantity of mouse AKBP2.
Example 4
Cloning of Human AKBP2 Gene, and Its Expression Distribution Analysis in Various Tissues
An attempt was made on the amplification of AKBP2 human orthologue gene complete length cDNA by the same PCR method shown in the aforementioned Example 1(5), using a human fat-derived cDNA library (Clontech) as the template and a pair of primers represented by SEQ ID NO:18 and SEQ ID NO:19. When nucleotide sequence of a DNA fragment of about 1.8 kbp obtained as a result thereof was determined in accordance with the same method shown in Example 1, it was confirmed that it contains complete length cDNA of the gene represented by SEQ ID NO:3. Said gene cDNA is a novel gene which encodes the polypeptide represented by SEQ ID NO:4. Said gene is a human orthologue gene of AKBP2 in which it has a homology of 76.8% with the. mouse AKBP2 gene represented by SEQ ID NO:1, and the encoded polypeptide has a homology of 71.3% with the mouse AKBP2 protein represented by SEQ ID NO:2, respectively.
Accordingly, an attempt was subsequently made to amplify a cDNA fragment of about 800 bases of the 3′-side of the human AKBP2 gene from cDNA samples derived from various human tissues, by PCR using the primer represented by SEQ ID NO:20 newly designed based on the sequence of said human AKBP2 gene and the aforementioned primer represented by SEQ ID NO:19, and the presence or absence of the expression of AKBP2 in respective tissues was examined. The PCR was carried out by DNA polymerase (Pyrobest DNA polymerase; Takara shuzo) using 2 μg of each of the various human tissue cDNA libraries (Clontech) as the template and, after heating at 98° C. (1 minute), repeating 35 cycles each cycle consisting of 98° C. (5 seconds), 55° C. (30 seconds) and 72° C. (5 minutes). When the thus obtained PCR products were separated by an agarose gel electrophoresis, a desired DNA fragment of about 800 base pair considered to be containing a 3′-terminal side partial sequence of the human AKBP2 gene was amplified from each of the skeletal muscle-, liver- and fat-derived cDNA libraries. When these DNA fragments were separated from respective agarose gels, and nucleotide sequences of said DNA fragments were respectively determined in accordance with the method described in the aforementioned Example 1(4) using the primer represented by SEQ ID NO:20, it was confirmed that they are the 3′-terminal side partial sequence of human AKBP2 gene represented by SEQ ID NO:3. Based on this, it was revealed that expression of the human AKBP2 gene is specifically controlled in fat, muscle, liver and the like limited organs which respond to the insulin signal.
As a result of this Example, since the human AKBP2 showed high homology with mouse AKBP2 and its expression was observed in insulin responding tissues, it was confirmed that it has the same functions of those of the mouse counterpart and therefore is useful for the diagnosis of diabetes mellitus and screening of a diabetes mellitus improving agent.
Example 5
Preparation of Cultured Cells Which Express Human AKBP2 Protein
The aforementioned AKBP2 gene complete length cDNA obtained in Example 4 was subcloned by the same method shown in the aforementioned Example 1(6). Thereafter, it was confirmed that the 1782 base pair human AKBP2 cDNA represented by SEQ ID NO:3 coding for the net human AKBP2 protein was inserted into the aforementioned expression vector pcDNA3.1/V5-His-TOPO, as a DNA from which the 3′-side stop codon of the DNA sequence was removed. This expression plasmid is referred to as pcDNA-human AKBP2 hereinafter. By introducing 5.1 μg per well of this expression plasmid pcDNA-human AKBP2 by the same method of Example 2(1), expression of human AKBP2 protein was detected in accordance with the method of Example 2(2). As a result, it was confirmed that a protein of about 70 kDa which corresponds to a human AKBP2-V5-His 6 fusion protein consisting of 638 amino acids containing the C-terminal side tag consisting of 45 amino acid is detected dependently on the gene transfer of the expression vector pcDNA-human AKBP2, so that it was revealed that the complete length region of the aforementioned cloned human AKBP2 gene is certainly expressed in the cultured cells and can form stable structure as the protein.
Example 6
Inspection of Interaction Between Human AKBP2 and Akt2
(1) Preparation of GST Fusion Akt2 Expression Plasmid
In order to insert human Akt2 cDNA into a GST fusion expression vector pGEX-3X (Amersham Bioscience), the human Akt2 cDNA obtained in Example 1(1) was digested with restriction enzymes HindIII and EcoRI, and the vector PGEX-3X with restriction enzymes BamHI and EcoRI, respectively, thereby making them into linear chains. In addition, in order to use the fragments represented by SEQ ID NO:21 and SEQ ID NO:22 as fragments for their ligation, they were separately treated at 60° C. for 30 minutes as a pretreatment and then mixed and allowed to stand at room temperature for 2 hours. A mixture of these treated human Akt2 cDNA fragment, vector pGEX-3X and fragment for ligation was mixed with a DNA ligase solution (DNA ligation kit II; Takara Shuzo) and treated at 16° C. for 3 hours, thereby preparing a plasmid in which Akt2 cDNA was inserted into the multi-cloning site of pGEX-3X (to be referred to as pGEX-Akt2 hereinafter). By carrying out determination of the nucleotide sequence using the oligonucleotide represented by SEQ ID NO:23 as a primer and using a sequencing kit (Applied Biosystems) and a sequencer (ABI 3700 DNA sequencer, Applied Biosystems), those in which the code region of Akt2 cDNA and the GST tag translation frame of pGEX vector were inserted in union were selected.
(2) Purification of GST Fusion Akt2 Protein
Using the plasmid pGEX-Akt2 obtained in the aforementioned (1), Escherichia coli BL21 was transformed by the heat shock method and cultured overnight on a shaker using 2.4 ml of a culture medium, total volume thereof was inoculated into 400 ml of a culture medium and cultured at 37° C. for 3 hours on a shaker, and then IPTG (SIGMA) was added thereto to a final concentration of 2.5 mM and the shaking culturing was further continued for 3 hours to induce expression of the GST fusion Akt2 protein (to be referred to as GST-Akt2 hereinafter). By recovering the cells, GST-Akt2 was purified on glutathione Sepharose beads (Glutathione Sepharose 4B; Amersham Pharmacia) in accordance with a conventionally known method (H. Matsushime et al., Jikken Kogaku (Experimental Engineering), Vol. 113, No. 6, p. 528, 1994). As a control, a protein of GST moiety alone (to be referred to as GST protein hereinafter) was expression-induced and purified from the Escherichia coli BL21 transformed with pGEX-3X, in the same manner as in the above. By carrying out separation by SDS polyacrylamide gel electrophoresis and Coomassie Brilliant Blue staining in accordance with conventionally known methods, it was confirmed that the proteins having expected molecular weights (GST-Akt2; 79 kDa, GST protein; 26 kDa) were purified.
(3) Verification of Biochemical Binding of Akt2 Protein with Human AKBP2 Protein
Using the GST fusion Akt2 protein (to be referred to as GST-Akt2 hereinafter) prepared in the aforementioned (2), the presence or absence of direct interaction between human AKBP2 protein and Akt2 protein was verified by the GST-pull down method (H. Matsushime et al., Jikken Kogaku, Vol. 113, No. 6, p. 528, 1994). Firstly, using 0.5 μg of the pcDNA-human AKBP2 prepared in the aforementioned Example 5 as the template, and using 40 μl of a TNT kit (TNTR Quick Coupled Transcription/Translation System; Promega) and 1.3 MBq of a radioisotope (redivue Pro-mix L-[35S]; Amersham), radioisotope-labeled human AKBP2 protein was prepared by in vitro transcription and translation in accordance with the attached protocols. A 15 μl portion of this human AKBP2 protein preparation solution was mixed with 1 μg of the GST protein or GST-Akt2 purified on glutathione beads in the aforementioned (2) and shaken at 4° C. for 1 hour after adding 0.3 ml of Buffer A (50 mM Tris-HCl (pH 7.5), 10% glycerol, 120 mM NaCl, 1 mM EDTA, 0.1 mM EGTA, 0.5 mM PMSF, 0.5% NP-40). Thereafter, the protein which binds to GST protein or GST-Akt2 on beads was co-precipitated by centrifugation. This was suspended in 0.5 ml of a buffer solution in which the NaCl concentration of the aforementioned Buffer A was changed to 100 mM, and again co-precipitated by centrifugation. After repeating this operation 4 times, proteins in the precipitate were separated by SDS polyacrylamide gel electrophoresis in accordance with a conventionally known method, and the human AKBP2 protein was detected by autoradiography. As a result, a band which is not detected when the GST protein is mixed was detected when GST-Akt2 was mixed. Based on this, it was revealed that the human AKBP2 as one of the polypeptides of the present invention interacts with Akt2 protein similar to the case of the mouse AKBP2 of the present invention, thus proving that these human and mouse AKBP2 molecules are counterparts which carry out the same function in both animal species. Accordingly, it was found that the human AKBP2 of the present invention is concerned in the induction of insulin resistance via its interaction with Akt2 protein similar to the case of the mouse AKBP2 of the present invention.
Example 7
Influence of Mouse AKBP2 Over-expression in NIH3T3 L1 Fat Cell Upon Akt2 Kinase Activity
From the results of the aforementioned yeast two hybrid and biochemical binding analyses, it was shown that Akt2 and AKBP2 interact with each other. Accordingly, influence of AKBP2 upon the enzyme (kinase) activity of Akt2 was examined by an in vitro kinase assay using a cultured cell NIH3T3 L1.
(1) Preparation of Substrate GST-crosstide for In Vitro Kinase Assay
The synthetic oligo DNA molecules represented by SEQ ID NO:24 and SEQ ID NO:25 coding for the phosphorylation region of GSK3p which is a physiological substrate of Akt2 were mixed and integrated into the EcoRI XhoI site of the pGEX-6P-1 vector. This was used as GST-crosstide. Using the plasmid GST-crosstide, Escherichia coli BL21 was transformed by the heat shock method and cultured overnight on a shaker using 2.4 ml of a culture medium, total volume thereof was inoculated into 400 ml of a culture medium and cultured at 37° C. for 3 hours on a shaker, and then IPTG (SIGMA) was added thereto to a final concentration of 2.5 mM and the shaking culturing was further continued for 3 hours to induce expression of a GST fusion protein (to be referred to as GST-crosstide hereinafter). By recovering the cells, GST-crosstide was purified on glutathione Sepharose beads (Glutathione Sepharose 4B; Amersham Pharmacia) in accordance with a conventionally known method (H. Matsushime et al., Jikken Kogaku (Experimental Engineering), Vol. 113, No. 6, p. 528, 1994). By carrying out separation of these proteins by SDS polyacrylamide gel electrophoresis and their Coomassie Brilliant Blue staining in accordance with conventionally known methods, it was confirmed that the GST-crosstide was purified.
(2) Preparation of AKBP2 High Expression Virus Making Use of Adenovirus Vector
A gene fragment coding for mouse AKBP2 was cut out from the pcDNA-AKBP2 vector using restriction enzymes BamHI and SacIl and inserted into the multi-cloning site (for BGlII and NotI) of an adenovirus vector pAdTrack-CMV (obtained from Johns Hopkins Cancer Center) using linker oligo SEQ ID NO:26 and SEQ ID NO:27 which form SacII and NotI digestion fragments, thereby obtaining a vector AKBP2/pAdTrack-CMV.
Thereafter, preparation of a solution of high titer adenovirus capable of expressing AKBP2 was carried out in accordance with a conventionally known protocol [“A Practical Guide for using the AdEasy System” www.coloncancer.org/adeasy.htm” “www.coloncancer.org/adeasy/protocol2.htm”)]. The adenovirus for control was prepared from pAdTrack-CMV.
In this connection, regarding the amount of virus, absorbance at 260 nm (A260) was measured and converted by the following formula.
1 A260=1.1×1012 virus particles=3.3×1011pfu/ml [Formula]
(3) Over-expression of Mouse AKBP2 in NIH3T3 L1 Fat Cells and Immunoprecipitation of Akt2
NIH3T3 L1 cells were suspended in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS) and inoculated in 8×105 cells/well potions into a collagen-coated 6 well plate (Asahi Techno Glass). On the next day, the medium was exchanged with the DMEM (10% FCS) further supplemented with 10 μg/ml of insulin, 250 nM of dexamethasone and 0.5 mM of 3-isobutyl-1-methylxanthine (IBMX) to induce differentiation of 3T3-L1 cells. Two days thereafter, the medium was returned to 0.4 ml of DMEM (10% FCS). Four days thereafter, an adenovirus which expresses AKBP2 was added to the medium at a concentration of 8×1010 pfu per well. As a control, an adenovirus which expresses GFP alone was used.
After 36 hours of the adenovirus infection, the cells were cultured for 16 hours using serum-free DMEM medium, stimulated with 100 nM of insulin for a predetermined period of time (0, 30 or 60 minutes) and then immediately dissolved in 500 μl of a cell lysis solution (50 mM Tris-HCl pH 7.5, 1 mM EDTA, 5 mM EGTA, 0.5 mM Na3VO4, 0.1% 2-mercaptoethanol, 50 mM NaF, 5 mM sodium pyrophosphate, 10 mM β-glycerophosphate, 1% Triton X-100, 0.1 mM PMSF). After centrifugation at 15,000 rpm for 20 minutes, the supernatant was mixed with an anti-Akt2 antibody (Upstate) and Protein G-Sepharose (Amersham) to effect immunoprecipitation. The immune precipitate was washed twice with the cell lysis solution, twice with a washing solution (50 mM Tris-HCl pH 7.5, 0.03% Brij 35, 0.1% 2-mercaptoethanol) and then twice with a reaction solution (20 mM MOPS pH 7.2, 10 mM MgCl2, 25 mM β-glycerophosphate, 5 mM EDTA, 1 mM DTT) and subjected to the kinase reaction.
(4) In Vitro Kinase Assay
The aforementioned immune precipitate was suspended in 20 μl of the reaction solution. The reaction solution further supplemented with 15 μM of ATP, 10 μCi of [γ32P]-ATP and 3 μg of GST-crosstide was added thereto and heated at 30° C. for 20 minutes. The reaction was stopped by adding 10 μl of 4× SDS sample buffer. After separation by SDS polyacrylamide gel electrophoresis, the radioactivity incorporated into GST-crosstide was determined by analyzing it by BAS 2000 Bioimaging Analyzer (Fuji Photo Film). As shown in FIG. 3, the kinase activity of Akt2 in the NIH3T3 L1 cell was reduced by over-expression with AKBP2 to 0.82 times as compared with the control (GFP) under insulin-un-stimulated state. In the same manner, when activity increase of Akt2 by the 100 nM insulin stimulation was observed, the GFP-infected cells showed 1.39 times (30 minutes) and 1.5 times (60 minutes) increase in the enzyme activity in comparison with the un-stimulated state, while the cells infected with the AKBP2 virus showed only 1.30 times (30 minutes) and 1.22 times (60 minutes) of the stimulation dependent increase in the activity. Also, since phosphorylation of the 473rd serine is important for the activation of Akt2, the aforementioned Akt2 immuno-precipitated after the insulin stimulation was analyzed by western blotting using an anti-phosphorylated serine473 antibody (New England Biolab) to find that the phosphorylated state at the time of no stimulation was reduced in the cells infected with the AKBP2 virus in comparison with the control virus-infected cells. In addition, a stimulation-dependent acceleration of phosphorylation was found by 30 minutes of insulin stimulation in both of the cells infected with the AKBP2 virus and the control virus-infected cells, but degree of the phosphorylation acceleration was weakened in the cells infected with the AKBP2 virus than in the control virus-infected cells, so that the results of the in vitro kinase assay was supported. Based on the above results, it is considered that the mouse AKBP2 of the present invention interacts with Akt2 and induces insulin resistance by reducing increase of the insulin-dependent enzyme activity, in addition to the insulin-independent enzyme activity.
INDUSTRIAL APPLICABILITY
The polypeptides and polynucleotides of the present invention which have the property to bind to Akt2, reduce the kinase activity of Akt2 and increase the expression level in the diabetes mellitus morbid state are useful for the diagnosis of diabetes mellitus. In addition, the polypeptides, polynucleotides, expression vectors and cells of the present invention are useful for the screening of a substance which inhibits binding of the polypeptides of the present invention with Akt2 (namely a substance which reinforces function of Akt2). The substance selected by said screening is useful as a candidate substance for an insulin resistance improving agent and a diabetes mellitus improving agent.
Sequence Listing Free Text
An explanation of “Artificial Sequence” is described the numerical entry <223> of the following Sequence Listing. Illustratively, each of the nucleotide sequences represented by SEQ ID NOs:5 to 8 and 10 to 27 in the Sequence Listing is an artificially synthesized primer sequence. The nucleotide sequence represented by the sequence of SEQ ID NO:9 is a sequence consisting of the bases of from the 5183rd (5′) to the 5162nd (3′) positions of a cloning vector pACT2 (GenBank U29899).
In the foregoing, the invention has been described based on the specified embodiments, but changes and modifications obvious to those skilled in the art are included in the scope of the invention.
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10537767
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astellas pharma inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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536/ 23.1
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Apr 1st, 2022 05:13PM
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Apr 1st, 2022 05:13PM
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Astellas Pharma
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tyo:4503
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Astellas Pharma
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Dec 13th, 2011 12:00AM
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Aug 7th, 2006 12:00AM
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https://www.uspto.gov?id=US08076348-20111213
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Acylguanidine derivative or salt thereof
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[Problem] To provide a compound which can be used in the prevention and/or treatment of diseases in which 5-HT2B receptor and 5-HT7 receptor are concerned, particularly in the treatment of irritable bowel syndrome (IBS) and/or prevention of migraine.
[Means for Resolution] It was found that an acylguanidine derivative having a tricyclic structure or a pharmaceutically acceptable salt thereof has a strong antagonism to 5-HT2B receptor and 5-HT7 receptor. In addition, the compound of the present invention having antagonism to both of the receptors showed superior pharmacological action in comparison with the case of the single use of an antagonist selective for either one of the receptors. Based on the above, the compound of the present invention is useful in preventing and/or treating diseases in which 5-HT2B receptor and 5-HT7 receptor are concerned, particularly in treating irritable bowel syndrome (IBS) and/or preventing migraine.
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8076348
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1. An acylguanidine derivative represented by the following general formula (I) or a pharmaceutically acceptable salt thereof
symbols in the formula represent the following meanings
R1 and R2: the same or different from each other, and each represents lower alkyl which may be substituted, lower alkenyl, halogen, —CN, —NO2, —OR0, —O-halogeno-lower alkyl, —OC(O)R0, —NR0R0a, —NR0—C(O)R0a, —NR0—S(O)2R0a, —SH, —S(O)p-lower alkyl, —S(O)2—NR0R0a, —C(O)R0, —CO2R0, —C(O)NR0R0a, cycloalkyl, aryl or a hetero ring group, wherein the aryl and a hetero ring group in R1 and R2 may respectively be substituted,
R0 and R0a: the same or different from each other, and each represents —H or lower alkyl,
m, n and p: the same or different from one another and each is 0, 1 or 2,
X: —N(R5)—,
Y: a single bond,
R5: —H, lower alkyl which may be substituted, —C(O)R0, —CO2R0, —C(O)NR0R0a, —S(O)p-lower alkyl, —S(O)p-aryl, cycloalkyl, a hetero ring group, lower alkylene-cycloalkyl, lower alkylene-aryl, lower alkylene-hetero ring group,
—C(O)-aryl or —C(O)-hetero ring group,
wherein the aryl and a hetero ring group in R5 may be respectively substituted, and
ring A: benzene ring.
2. The compound described in claim 1, wherein the substitution position of the guanidinocarbonyl group is the para position against Y.
3. The compound described in claim 2, wherein R5 is lower alkyl, cycloalkyl, lower alkylene-cycloalkyl, a hetero ring group, lower alkylene-(hetero ring group which may be substituted with lower alkyl), —C(O)-lower alkyl or —S(O)2-lower alkyl.
4. The compound described in claim 1 which is selected from the group consisting of
N-(diaminomethylene)-9-isopropyl-9H-carbazole-2-carboxamide, and
N-(diaminomethylene)-5-(hydroxymethyl)-9-isopropyl-9H-carbazole-2-carboxamide, or a pharmaceutically acceptable salt thereof.
5. A pharmaceutical composition which comprises the compound described in claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
6. The pharmaceutical composition described in claim 5, which is a 5-HT2B and 5-HT7 receptor antagonist.
7. The pharmaceutical composition described in claim 5, which is a migraine-preventing agent.
8. The pharmaceutical composition described in claim 5, which is an IBS-treating agent.
9. A medicament comprising the acylguanidine derivative of claim 1 or a pharmaceutically acceptable salt thereof, wherein said medicament is a 5-HT2B and 5-HT7 receptor antagonist, a migraine preventing agent and/or an IBS treating agent.
10. A method for preventing migraine and/or treating IBS, which comprises administering an effective amount of the compound described in claim 1 or a salt thereof to a patient.
11. The compound described in claim 3, wherein n is 0.
12. The compound described in claim 11, wherein R1 is chosen from halogen, lower alkyl, —O-lower alkyl, lower alkylene-OH, and —C(O)H.
13. The compound described in claim 12, wherein R5 is lower alkyl.
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TECHNICAL FIELD
The present invention relates to a pharmaceutical, particularly an acylguanidine derivative which is useful as an agent for treating irritable bowel syndrome and/or an agent for preventing migraine.
BACKGROUND OF THE INVENTION
Serotonin (5-HT) is a monoamine neurotransmitter and exerts various physiological actions via 5-HT receptors. The 5-HT receptors are classified into 7 families of from 5-HT1 to 5-HT7. Particularly, three subtypes, 5-HT2A, 5-HT2B, and 5-HT2C, are known for 5-HT2 receptor (Non-patent Reference 1).
Irritable bowel syndrome (IBS) is a disease in which abdominal pain or abdominal unpleasantness continues for a long period of time. Based on the symptoms, IBS is classified into a diarrhea-predominant-type, a constipation-predominant-type and a diarrhea-constipation alternating-type. In each case, it has been pointed out that there is a causal relation between morbid state and amount of 5-HT in blood. For example, there is a report pointing out that increase of blood 5-HT concentration after meal occurs in patients of diarrhea-predominant IBS and this deeply relates to the morbid state (Non-patent Reference 2).
Currently, a 5-HT receptor antagonist or a 5-HT receptor agonist is already used in Europe and U.S.A. as an agent for treating IBS, though it is at a clinical trial stage in Japan. Alosetron (5-HT3 receptor antagonist) is used in the clinical field as an agent for treating diarrhea-predominant-type, but side effects such as ischemic colitis, constipation and the like have been reported. In addition, tegaserod (5-HT4 receptor agonist) is used in the clinical field in Europe and U.S.A. as an agent for treating constipation-predominant-type, but its side effects have also been reported (Non-patent References 3 and 4).
In recent years, pharmacological studies on other 5-HT receptor subtypes have also been in progress (Non-patent Reference 5). Regarding the 5-HT2B receptor and 5-HT7 receptor, there are reports pointing out about said receptors and roles in the digestive tracts. For example, there are reports stating that the 5-HT2B receptor is localized in human ileum longitudinal muscle and a 5-HT2B receptor antagonistic compound suppresses contraction by 5-HT (Non-patent Reference 6) and that the 5-HT2B receptor localizing in human colon relates to the 5-HT-induced contraction at the time of electric stimulation and a 5-HT2B receptor antagonistic compound suppresses it (Non-patent Reference 7).
In addition, there are reports stating that the 5-HT7 receptor is present in guinea pig small intestines (Non-patent Reference 8) and rat intestines (Non-patent Reference 9) and concerned in the peristalsis of guinea pig ileum (Non-patent Reference 10). Based on the above, it is expected that a compound having antagonism to 5-HT2B and 5-HT7 receptors is useful as an agent for treating IBS.
On the other hand, migraine is a pulsating headache which is a disease in which a strong pain occurs in one side or both sides of the head and continues for several hours to about 3 days. It is suggested that morbid state of migraine progresses by the following onset mechanism. That is, dura mater blood vessel once contracts by the action of neurotransmitters (e.g., 5-HT and the like) and then dilates again and, at this time, releases vasoactive peptides (e.g., calcitonin gene-related peptide (CGRP) and the like) and serum protein to accelerate inflammation which leads to the onset of headache.
The pharmaceutical targeted at migraine is divided into a preventive agent and a treating agent. The former aims at reducing attack frequency by preventively administering it continuously before onset of the disease and the latter aims at suppressing the pain by taking it after expression of the attack. As the preventive agent for migraine, Ca antagonists (e.g., lomerizine, flunarizine and the like), 5-HT antagonists (e.g., pizotifen, methysergide and the like), β-adrenergic blocking agents (e.g., propranolol and the like), and the like are clinically used in certain countries, but many side effects have been reported on them and sufficient clinical effects have not been obtained.
Regarding the pizotifen as a 5-HT antagonist among the preventive agents described in the above, its efficacy is high in comparison with other agents, but there is a problem in that fatigued feeling, drowsiness, dizziness, weight gain and the side effects are observed at its effective dose (Non-patent Reference 11). It is known that said compound has affinity for all of the 5-HT receptor subtypes and also has high affinity for various receptors such as α1 adrenaline receptor (α1), muscarine 1 receptor (M1), dopamine 2 receptor (D2) and the like.
In recent years, pharmacological studies on 5-HT receptor subtypes have been conducted. It has been reported that a 5-HT2B receptor antagonist suppresses guinea pig m-chlorophenylpiperazine (mCPP)-induced dura mater extravascular protein leakage (Non-patent Reference 12) and that the 5-HT2B receptor localizing on the vascular smooth muscle induces release of nitrogen monoxide (NO) and the NO accelerates release of neuro-peptides such as CGRP, substance P and the like from the trigeminal nerve (Non-patent References 13 and 14). Also, a result which suggests a migraine preventive action has been obtained by an animal model using a 5-HT2B receptor selective antagonist (Non-patent Reference 15). Also, there are reports stating that 5-HT7 receptor is present in the trigeminal nerve (Non-patent Reference 16), concerned in the vasodilation by 5-HT in cerebrovascular smooth muscle (Non-patent Reference 17) or concerned in the dura mater extravascular protein leakage acceleration action (Non-patent Reference 18). In addition, it has been reported in Patent References 1 and 2, applied by the instant applicant and published after the priority date of this application, that a selective 5-HT2B and 5-HT7 receptor dual antagonist is effective for preventing migraine. Based on the above, it is expected that a compound which has the antagonistic activity for 5-HT2B and 5-HT7 receptors and is selective against other receptors is useful as an agent for preventing migraine with less side effects.
As the compound having antagonistic activity for 5-HT2B and 5-HT7 receptors, the compounds shown in Patent References 1 and 2 have been reported.
It has been reported in Patent References 1 and 2, applied by the instant applicant and published after the priority date of this application, that a fluorene compound represented by the following formula (A) has antagonistic activity for 5-HT2B and 5-HT7 receptors and is effective for preventing migraine.
As acylguanidine derivatives having a tricyclic structure, Patent References 3 and 4 are known.
Patent Reference 3 describes that the compounds represented by the following formula (B) is effective for the treatment of central diseases. However, in these compounds, a ring group is linked to the guanidine moiety via a linker X.
(In the formula, R is a cycloalkyl, an aryl, a mono- to tricyclic heteroaryl or the like, R1 and R2 are independently H, an alkyl, alkenyl or the like, X is a bond, an alkene, an alkenylene or the like, and R3 is a cycloalkyl, an aryl, an alkylaryl or the like. See said publication for details.)
In Patent Reference 4, it is reported that the compounds represented by the following formula (C) have an NO synthase inhibitory activity and/or an active oxygen species scavenging activity. However, there is no illustrative disclosure in this publication on a compound in which Φ is a bond or a compound which has —NH2 as —NR13R14.
(In the formula, Φ is a bond or phenylene group, B is —CH2—NO2, an alkyl group, an aryl group or NR13R14 or the like, wherein R13 and R14 are independently hydrogen atom, an alkyl group, cyano group or the like, X is a bond, —O—, —S— or CO— or the like, Y is a bond, —(CH2)m or the like, W is not present, or a bond, S atom or NR15, and R1 to R5 are hydrogen, a halogen or the like. See said publication for details.)
Non-patent Reference 1: Pharmacological Reviews, (USA), 1994, vol. 46, p. 157-203
Non-patent Reference 2: Gut, (England), 1998, vol. 42, p. 42-46
Non-patent Reference 3: The American Journal of Gastroenterology, (USA), 2000, vol. 95, p. 2698-2709
Non-patent Reference 4: The American Journal of Gastroenterology, (USA), 2003, vol. 98, p. 750-758
Non-patent Reference 5: Drugs, (New Zealand), 2001, vol. 61, no. 3, p. 317-332
Non-patent Reference 6: Brutish Journal of Pharmacology, (England), 1995, vol. 114, p. 1525-1527
Non-patent Reference 7: Brutish Journal of Pharmacology, (England), 2002, vol. 135, p. 1144-1151
Non-patent Reference 8: European Journal of Pharmacology, (Holland), 1995, vol. 280, p. 243-250
Non-patent Reference 9: Life Science, (Holland), 2001, vol. 69, p. 2467-2475
Non-patent Reference 10: Brutish Journal of Pharmacology, (England), 2003, vol. 138, p. 1210-1214
Non-patent Reference 11: Journal of Neurology, (Germany), 1991, vol. 238, p. S45-S52
Non-patent Reference 12: Cephalalgia, (England), 2003, vol. 23, p. 117-123
Non-patent Reference 13: The Journal of Biological Chemistry, (USA), 2000, vol. 275, p. 9324-9331
Non-patent Reference 14: Circulation Research, (USA), 1992, vol. 70, p. 1313-1319
Non-patent Reference 15: “Cluster Headache and Related Conditions”, edited by D. W. Bonhaus, vol. 9, (England), Oxford University Press, 1999, p. 278-286
Non-patent Reference 16: Neuroscience Letters, (Holland), 2001, vol. 302, p. 9-12
Non-patent Reference 17: European Journal of Pharmacology, (Holland), 2002, vol. 439, p. 1-11
Non-patent Reference 18: Regional Anesthesia, (England), 1996, vol. 21, p. 219-225
Patent Reference 1: International Publication No. 2005/79845
Patent Reference 2: International Publication No. 2005/80322
Patent Reference 3: International Publication No. 99/20599
Patent Reference 4: International Publication No. 00/17191
DISCLOSURE OF THE INVENTION
Problems that the Invention is to Solve
As has been described in the above, the existing agents for treating IBS and agents for preventing migraine are not satisfactory in view of the efficacy, safety and the like, so that it is highly demanded to provide an agent for treating IBS and an agent for preventing migraine, having excellent efficacy and safety.
Means for Solving the Problems
As has been described in the above, it is be expected that a compound having antagonistic activity to 5-HT2B and 5-HT7 receptors becomes an IBS treating agent and/or a migraine preventing agent having less side effects and excellent efficacy. Accordingly, with the aim of providing a compound useful as an IBS treating agent and/or a migraine preventing agent, the present inventors have conducted intensive studies on a compound having antagonistic activity to 5-HT2B and 5-HT7 receptors. As a result, it was found that novel acylguanidine derivatives represented by the following general formula (I), which are characterized by the possession of a tricyclic structure, show excellent antagonism to both of the 5-HT2B and 5-HT7 receptors. The present invention has been accomplished by further finding that these acylguanidine derivatives have excellent IBS treating effect and/or migraine preventing effect in comparison with the conventional compounds which have the antagonistic activity to only one of the 5-HT2B and 5-HT7 receptors.
That is, the present invention relates to an acylguanidine derivative represented by the following general formula (I) or a pharmaceutically acceptable salt thereof
[symbols in the formula represent the following meanings
R1 and R2: the same or different from each other, and each represents lower alkyl which may be substituted, lower alkenyl, halogen, —CN, —NO2, —OR0, —O-halogeno-lower alkyl, —OC(O)R0, —NR0R0a, —NR0—C(O)R0a, —NR0—S(O)2R0a, —SH, —S(O)p-lower alkyl, —S(O)2—NR0R0a, —C(O)R0, —CO2R0, —C(O)NR0R0a, cycloalkyl, aryl or a hetero ring group,
wherein the aryl and a hetero ring group in R1 and R2 may respectively be substituted,
R0 and R0a: the same or different from each other, and each represents —H or lower alkyl,
m, n and p: the same or different from one another and each is 0, 1 or 2,
X: —C(R3)(R4)— or —N(R5)—,
Y: (i) when X is —C(R3)(R4)—: a single bond,
(ii) when X is —N(R5)—: a single bond or —O—,
R3 and R4: the same or different from each other, and each represents —H, lower alkyl which may be substituted, halogen, —OR0, —NR0R0a, —NR0—C(O)R0a, —SH or —S(O)p-lower alkyl,
or R3 and R4 may together form oxo, lower alkylene-O—, —O-lower alkylene-O—, lower alkylene-S—, —S-lower alkylene-S—, or lower alkylene which may be interrupted by 1 or 2 groups selected from —O—, —NR0— and —S(O)p—,
R5: —H, lower alkyl which may be substituted, —C(O)R0, —CO2R0, —C(O)NR0R0a, —S(O)p-lower alkyl, —S(O)p-aryl, cycloalkyl, a hetero ring group, lower alkylene-cycloalkyl, lower alkylene-aryl, lower alkylene-hetero ring group,
—C(O)-aryl or —C(O)-hetero ring group,
wherein the aryl and hetero ring group in R5 may be respectively substituted,
ring A: (i) when X is —C(R3)(R4)—: cycloalkene ring or 5- to 8-membered monocyclic hetero ring,
(ii) when X is —N(R5)—: benzene ring, cycloalkene ring or 5- to 8-membered monocyclic hetero ring;
the same shall apply hereinafter].
In addition, the present invention also relates to a pharmaceutical composition which comprises the aforementioned acylguanidine derivative or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, particularly a pharmaceutical composition which is a 5-HT2B and 5-HT7 receptor antagonist, a migraine preventing agent and/or an IBS treating agent.
That is, (1) a pharmaceutical composition which comprises the compound described in the formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
(2) The pharmaceutical composition described in (1), which is a 5-HT2B and 5-HT7 receptor antagonist.
(3) The pharmaceutical composition described in (1), which is a migraine preventing agent.
(4) The pharmaceutical composition described in (1), which is an IBS treating agent.
(5) Use of the compound described in the formula (I) or a pharmaceutically acceptable salt thereof for the manufacture of a 5-HT2B and 5-HT7 receptor antagonist, a migraine preventing agent and/or an IBS treating agent.
(7) A method for preventing migraine and/or treating IBS, which comprises administering a therapeutically effective amount of the compound described in the formula (I) or a salt thereof to a patient.
Advantage of the Invention
The compound of the present invention showed excellent antagonistic activity to both of the 5-HT2B and 5-HT7 receptors. In addition, since the compound of the present invention showed excellent IBS treating effect and/or migraine preventing effect in comparison with the conventional compounds which have the antagonistic activity to only one of the 5-HT2B and 5-HT7 receptors, it is useful as an IBS treating agent and/or migraine preventing agent.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph showing a result of the measurement of the number of feces at the time of PS-127445 administration in the rat confined stress defecation model of the test method (4). Significant difference was not found in the respective 1, 3 and 10 mg/kg administration groups in comparison with the non-administration group (N=10).
FIG. 2 is a graph showing a result of the measurement of the number of feces at the time of SB-269970 administration in the rat confined stress defecation model of the test method (4). Significant difference was not found in the respective 1, 3 and 10 mg/kg administration groups in comparison with the non-administration group (N=10).
FIG. 3 is a graph showing a result of the measurement of the number of feces at the time of RS-127445 and SB-269970 simultaneous administration in the rat confined stress defecation model of the test method (4). Statistical test was carried out by the Dunnett's method, and * shows a significance level of 5%, and ** that of 1% and *** that of 0.1% (N=10).
FIG. 4 is a graph showing a result of the measurement of the number of feces at the time of the administration of Example 84 in the rat confined stress defecation model of the test method (4). Statistical test was carried out by the Dunnett's method, and ** shows 1% and *** shows 0.1% (N=10).
FIG. 5 is a graph showing a result of the measurement of the leaked amount of protein at the time of RS-127445 administration in the guinea pig migraine model of the test method (5). Statistical test was carried out by the Dunnett's method, and * shows a significance level of 5%, and ** that of 1%.
FIG. 6 is a graph showing a result of the measurement of the leaked amount of protein at the time of SB-269970 administration in the guinea pig migraine model of the test method (5). Statistical test was carried out by the Dunnett's method, and ** shows a significance level of 1%.
FIG. 7 is a graph showing a result of the measurement of the leaked amount of protein at the time of RS-127445 and SB-269970 simultaneous administration in the guinea pig migraine model of the test method (5). Statistical test was carried out by the T test, and * shows a significance level of 5%.
FIG. 8 is a graph showing a result of the measurement of the leaked amount of protein at the time of the administration of the compound of Example 1 in the guinea pig migraine model of the test method (5). Statistical test was carried out by the T test, and * shows a significance level of 5%.
BEST MODE FOR CARRYING OUT THE INVENTION
The following describes the present invention in detail.
In this description, the term “lower” means a straight or branched hydrocarbon chain having from 1 to 6 carbon atoms unless otherwise noted.
The “lower alkyl” means a C1-6 alkyl. Illustratively, methyl, ethyl, normal-propyl, isopropyl, normal-butyl, isobutyl, sec-butyl, tert-butyl, normal-pentyl, normal-hexyl and the like may be exemplified. Preferred is a C1-4 alkyl, and particularly preferred are methyl, ethyl, normal-propyl and isopropyl.
The “lower alkenyl” means a C2-6 alkenyl. The double bond may be at any optional position, and it may have two or more double bonds. Illustratively, for example, vinyl, 1-propenyl, allyl, isopropenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1,3-butadienyl, 1-pentenyl, 1-hexenyl and the like may be cited. Preferred is a C2-4 alkenyl, and particularly preferred are vinyl, 1-propenyl, allyl and isopropenyl.
The “lower alkylene” means a divalent group resulting from the removal of one hydrogen at an optional position of C1-6 alkyl. Illustratively, methylene, ethylene, methylmethylene, dimethylmethylene, trimethylene, propylene; butylene, pentylene, hexylene and the like may be exemplified. Preferred is a C1-4 alkylene, and particularly preferred are methylene and ethylene.
The “halogen” means a halogen atom. Illustratively, fluoro, chloro, bromo and iodo may be cited. Preferred are fluoro and chloro.
The “halogeno-lower alkyl” means a group in which 1 or more optional hydrogen atoms of the aforementioned “lower alkyl” are substituted by the aforementioned “halogen” which are the same or different from one another. Illustratively, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, pentafluoroethyl, hexafluoropropyl and the like may be exemplified. Preferred is a C1-2 alkyl substituted by 1 to 5 fluoro, and particularly preferred is trifluoromethyl.
The “cycloalkyl” means a C3-10 non-aromatic hydrocarbon ring group which may form a bridged ring or spiro ring or may be partially unsaturated. Illustratively, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, adamantyl, norbornyl, cyclopentenyl, cyclohexenyl and the like may be exemplified. Preferred is a C3-7 cycloalkyl, and particularly preferred are cyclobutyl, cyclopentyl and cyclohexyl.
The “cycloalkene ring” in the ring A means a C5-8 monocyclic non-aromatic hydrocarbon ring having a double bond at the fused site with the ring containing X and Y, which may further have a double bond. Illustratively, cyclopentene, cyclohexene, cycloheptene, cyclooctene and the like may be exemplified. Preferred are cyclopentene and cyclohexene.
The “aryl” means a monocyclic to tricyclic C6-14 aromatic hydrocarbon ring group. Illustratively, for example, phenyl, naphthyl, anthranyl and the like may be cited. Preferred are phenyl and naphthyl. In addition, a C5-8 non-aromatic hydrocarbon ring may be ring-fused and may form, for example, indanyl or tetrahydronaphthyl.
The “hetero ring group” means a saturated, unsaturated or partially unsaturated 3- to 8-membered monocyclic hetero ring group, 8- to 14-membered bicyclic hetero ring group or 11- to 20-membered tricyclic hetero ring group, which contains 1 to 4 hetero atoms selected from O, S and N. It may form oxide or dioxide through the oxidation of the S or N as the ring atom, or may form a bridged ring or spiro ring. As the monocyclic hetero ring group, illustratively, azetidinyl, oxiranyl, oxetanyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, pyranyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, furyl, dihydrofuryl, thienyl, pyrrolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, thiadiazolyl, imidazolyl, triazolyl, tetrazolyl, tetrahydrofuranyl, tetrahydropyranyl and the like may be exemplified. As the bicyclic hetero ring group, illustratively, indolyl, benzofuranyl, dihydrobenzofuranyl, benzothienyl, benzoxazolyl, benzimidazolyl, benzothiazolyl, quinolinyl, quinazolinyl, quinoxalinyl, cinnolinyl and the like may be exemplified. As the tricyclic hetero ring group, illustratively, carbazolyl, acridinyl and the like may be exemplified. Preferred are azetidinyl, oxiranyl, oxetanyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, pyranyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, furyl and thienyl, and particularly preferred are pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, pyranyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, furyl and thienyl.
The “5- to 8-membered monocyclic hetero ring” in the ring A means an unsaturated or partially unsaturated 5- to 8-membered monocyclic hetero ring which contains 1 to 4 hetero atoms selected from O, S and N and has a double bond at the fused site with the ring containing X and Y. Illustratively, pyridine, pyridazine, pyrimidine, imidazole, furan, thiophene, oxazole, thiazole, tetrahydropyridine and the like. Preferred is a 5- or 6-membered monocyclic hetero ring, and particularly preferred are thiophene, pyridine and tetrahydropyridine.
The “lower alkylene which may be interrupted by 1 or 2 groups selected from —O—, —NR0— and —S(O)p—” formed by R3 and R4 in combination means a group formed by inserting 1 or 2 groups selected from —O—, —NR0— and —S(O)p— into the carbon-carbon bond of lower alkylene. When 2 groups are inserted, the respective groups may be the same or different from each other. Illustratively,
—CH2—O—CH2CH2—, —CH2—S—CH2CH2—, —CH2—NH—CH2CH2—, —CH2—N(CH3)—CH2CH2—, —CH2CH2—O—CH2CH2—, —CH2CH2—S—CH2CH2—, —CH2CH2—NH—CH2CH2—, —CH2CH2—N(CH3)—CH2CH2—, —CH2—O—(CH2)3—, —CH2—S—(CH2)3—, —CH2—NH—(CH2)3—, —CH2—N(CH3)—(CH2)3—, —CH2—O—CH2—O—CH2— and the like may be exemplified.
The “which may be substituted” means that it is “not substituted” or “substituted with 1 to 5 substituents which may be the same or different from one another”.
According to the description, the acceptable substituent of the term “which may be substituted” may be any one as long as it is generally used in said technical field as a substituent of respective group. In addition, when two or more groups are present like the case, for example, of the lower alkyl of —C(O)—N(lower alkyl)2, the respective substituents may be the same or different from each other.
Preferred as the substituents of the “lower alkyl which may be substituted” in R1, R2, R3, R4 and R5 are halogen, —OR0, —O-lower alkylene-aryl, —OC(O)R0, —NR0R0a, —NR0—C(O)R0a, —NR0—S(O)2R0a, —SH, —S(O)p-lower alkyl, —S(O)2—NR0R0a, —C(O)R0, —CO2R0 or —C(O)NR0R0a. Particularly preferred is halogen, —OR0 or —NR0R0a.
Preferred as the substituent of the “aryl” and “hetero ring group” which may be respectively substituted in R1, R2 and R5 is halogen, lower alkyl which may be substituted with halogen or aryl, —OR0 or —O-halogeno-lower alkyl. Particularly preferred is halogen or lower alkyl.
Preferred embodiments of the present invention are shown below.
Preferred as R1 is halogen, lower alkyl, —O-lower alkyl, —NH2, lower alkylene-OR0 or —C(O)H, and more preferred is halogen, lower alkyl, —O-lower alkyl, lower alkylene-OH or —C(O)H.
Preferred as R2 is halogen or lower alkyl.
Preferred as m is 0 or 1, more preferably 0.
Preferred as n is 0.
Preferred as R3 and R4 are the case wherein they are the same or different from each other and each is lower alkyl or —OR0, or the case wherein they mean oxo, lower alkylene-O— or —S-lower alkylene-S— in combination. More preferred as R3 and R4 is lower alkyl or —OR0, or lower alkylene-O— or —S-lower alkylene-S— as R3 and R4 in combination, and further preferred is lower alkylene-O— as R3 and R4 in combination.
Preferred as R5, when Y is a single bond, is —H, lower alkyl, lower alkylene-OR0, cycloalkyl, lower alkylene-cycloalkyl, a hetero ring group, lower alkylene-(hetero ring group which may be substituted with lower alkyl), —C(O)-lower alkyl, —S(O)2-lower alkyl or —C(O)—N(lower alkyl)2, more preferred is lower alkyl, cycloalkyl, lower alkylene-cycloalkyl, a hetero ring group, lower alkylene-(hetero ring group which may be substituted with lower alkyl), —C(O)-lower alkyl or —S(O)2-lower alkyl, and further more preferred is lower alkyl. When Y is —O—, preferred as R5 is —H; lower alkyl, cycloalkyl, a hetero ring group or lower alkylene-(hetero ring group which may be substituted with lower alkyl), more preferred is lower alkyl, cycloalkyl, a hetero ring group or lower alkylene-(hetero ring group which may be substituted with lower alkyl), and further more preferred is lower alkyl.
Preferred as ring A, when X is —C(R3)(R4)—, is thiophene or pyridine, and more preferred is thiophene. When X is —N(R5)—, preferred as ring A is benzene, thiophene or pyridine, and more preferred is benzene.
Preferred as X is —C(R3)(R4)— or —N(R5)—.
Preferred as Y is a single bond.
Preferred as the tricyclic system constituted from ring A, a ring containing X and Y and benzene ring is carbazole, phenoxazine, indeno[2,1-b]thiophene or indeno[2,1-c]pyridine, more preferred is indeno[2,1-b]thiophene or indeno[2,1-c]pyridine, and further more preferred is indeno[2,1-b]thiophene.
In the aforementioned tricyclic system, preferred as the substitution position of the guanidinocarbonyl group is the para position against Y. Illustratively, it is the 2-position in the case of carbazole and phenoxazine, the 6-position in indeno[2,1-b]thiophene and the 7-position in indeno[2,1-c]pyridine.
A compound further consisting of the combination of the preferred groups described in the above is more desirable.
In addition, other preferred embodiments of the compound of the present invention represented by the general formula (I) are shown below.
(1) The compound described in the general formula (I), wherein X is —C(R3)(R4)—.
(2) The compound described in (1), wherein ring A is thiophene or pyridine.
(3) The compound described in (2), wherein the tricyclic system constituted from ring A, a ring containing X and Y and benzene ring is indeno[2,1-b]thiophene or indeno[2,1-c]pyridine.
(4) The compound described in (3), wherein the substitution position of the guanidinocarbonyl group is the para position against Y.
(5) The compound described in (4), wherein R3 and R4 are the same or different from each other and each is lower alkyl or —OR0, or oxo, lower alkylene-O— or —S-lower alkylene-S— as R3 and R4 in combination.
(6) The compound described in the general formula (I), wherein X is —N(R5)—.
(7) The compound described in (6), wherein Y is a single bond.
(8) The compound described in (7), wherein ring A is benzene.
(9) The compound described in (8), wherein the substitution position of the guanidinocarbonyl group is the para position against Y.
(10) The compound described in (9), wherein R5 is lower alkyl, cycloalkyl, lower alkylene-cycloalkyl, a hetero ring group, lower alkylene-(hetero ring group which may be substituted with lower alkyl), —C(O)-lower alkyl or —S(O)2-lower alkyl.
(11) The compound described in (6), wherein Y is —O—.
(12) The compound described in (11), wherein ring A is benzene.
(13) The compound described in (12), wherein the substitution position of the guanidinocarbonyl group is the para position against Y.
(14) The compound described in (13), wherein R5 is lower alkyl, cycloalkyl, a hetero ring group or lower alkylene-(hetero ring group which may be substituted with lower alkyl).
(15) A compound described in the general formula (I) selected from the group consisting of
N-(diaminomethylene)-4,5-dihydro-3H-spiro[furan-2,4′-indeno[1,2-b]thiophene]-6′-carboxamide,
N-(diaminomethylene)-9-isopropyl-9H-carbazole-2-carboxamide,
N-(diaminomethylene)-10-isopropyl-10H-phenoxazine-2-carboxamide,
N-(diaminomethylene)spiro[1,3-dithiolan-2,4′-indeno[1,2-b]thiophene]-6′-carboxamide, and
N-(diaminomethylene)-4-methoxy-4-methyl-4H-indeno[1,2-b]thiophene]-6-carboxamide, or a pharmaceutically acceptable salt thereof.
In addition, according to the description, the “binding affinity” means the ability to bind to a part of a receptor, and its evaluation is carried out by comparing the Ki value calculated by an in vitro receptor binding test as described in the test method or the IC50 value of a receptor binding test carried out under the same condition as occasion demands. In this connection, when the IC50 value cannot be calculated because sufficient inhibitory action is not shown at a certain concentration in the receptor binding test, IC50 value of the compound is regarded as said concentration or more in some cases.
When binding affinity of the compound of the present invention is “selective” for the 5-HT2B and 5-HT7 receptors, it means that binding affinity of said receptors is high in comparison with the binding affinity for “other receptors”. The “selective” according to the present invention indicates a case in which the Ki value or IC50 value showing binding affinity for said receptors is 1/10 or less in comparison with the value for the “other receptors”, and this value is preferably 1/50 or less, more preferably 1/100 or less, more further preferably 1/500 or less, particularly preferably 1/1000 or less.
The “other receptors” as used herein are other receptors which are reported regarding the existing nonselective 5-HT receptor antagonists and receptors particularly relating to undesirable actions. Thus, preferred as the compound of the present invention is a compound whose binding affinity for 5-HT2B and 5-HT7 receptors is selective in comparison with α1, M1 and D2 receptors, and more preferred is a compound whose binding affinity for 5-HT2B and 5-HT7 receptors is selective in comparison with α1, M1, D2, 5-HT1A, 5-HT1B, 5-HT2A, 5-HT2C, 5-HT3, 5-HT4 and 5-HT6 receptors.
There is a case in which geometrical isomers and tautomers are present in the compound (I) of the present invention. For example, the following tautomers are present.
The present invention includes one of such tautomers or a mixture thereof.
In addition, there is a case in which isomers based on the asymmetric carbon atom are present in the compound of the present invention. The present invention includes mixtures and isolated counterparts of these optical isomers.
In this connection, all of the compounds which are converted into the compound (I) or a salt thereof by undergoing metabolism in the living body, or so-called prodrugs, are included in the compound (I) of the present invention. Regarding the group which forms this prodrug, the groups described in “Progress in Medicine”, Lifescience Medica, 1985, vol. 5, p. 2157-2161 and the groups described in “Iyakuhin no Kaihatsu (Development of Medicines)”, published in 1990 by Hirokawa Shoten, vol. 7 Bunshi Sekkei (Molecular Design) 163-198 may be exemplified.
As the pharmaceutically acceptable salt of the compound (I) of the present invention, illustratively, acid addition salts with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid and the like), organic acids (e.g., formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, aspartic acid, glutamic acid and the like), and the like may be exemplified. Also, there is a case in which it forms a salt with a base depending on the kind of substituent, and for example, salts with inorganic bases containing metals such as sodium, potassium, magnesium, calcium, aluminum and the like or organic bases (e.g., methylamine, ethylamine, ethanolamine, lysine, ornithine and the like), ammonium salt, and the like may be cited.
In addition, the present invention also includes various hydrates, solvates and polymorphic substances of the compound (I) and salts thereof.
(Production Methods)
The compound (I) of the present invention and pharmaceutically acceptable salts thereof can be produced by employing various conventionally known synthetic methods by making use of the characteristics based on its basic backbone or kinds of its substituents. In that case, depending on the kinds of functional group, there is a case in which protection of said functional group with an appropriate protecting group or its replacement to a group which can be easily converted into said functional group, at the stages of the starting materials to intermediates, is effective in view of the production techniques. As such a functional group, it includes amino group, hydroxyl group, carboxyl group and the like, as their protecting groups, the protecting groups described for example in “Protective Groups in Organic Synthesis”, edited by T. W. Greene and 3rd edition, John Wiley & Sons, 1999 may be cited, and these may be optionally selected and used in response to the reaction conditions. By such a method, a desired compound can be obtained by carrying out the reaction by introducing said protecting group and then removing the protecting group or converting it into a desired group as occasion demands.
The following describes typical production methods of the compound of the present invention.
(L1 represents —OH or a leaving group.)
This production method is a method in which the compound (I) of the present invention is produced by subjecting a carboxylic acid or a reactive derivative thereof (1) and guanidine (2) or a salt thereof to amidation.
The reaction can be carried out using equivalent amounts of the carboxylic acid or a reactive derivative thereof (1) and guanidine (2), or guanidine in an excess amount. It can be carried out under cooling to under heating, preferably at from −20° C. to 60° C., in a solvent which is inert to the reaction, such as aromatic hydrocarbons (e.g., benzene, toluene or xylene or the like), halogenated hydrocarbons (e.g., dichloromethane, 1,2-dichloroethane or chloroform or the like), ethers (e.g., diethyl ether, tetrahydrofuran (THF), dioxane or dimethoxyethane (DME) or the like), N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), ethyl acetate, acetonitrile or water, or a mixed liquid thereof.
When a free carboxylic acid wherein L1 is OH is used as the starting compound (1), it is desirable to carry out the reaction in the presence of a condensing agent. In that case, it is desirable to use a condensing agent such as N,N′-dicyclohexylcarbodiimide (DCC), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (WSC), 1,1′-carbonyldiimidazole (CDI), 2-(1H-benzothiazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), diphenyl phosphoryl azide (DPPA), phosphorous oxychloride or the like, and further an additive agent (e.g., N-hydroxysuccinimide (HONSu), 1-hydroxybenzotriazole (HOBt) or the like). It can be carried out using equivalent amount or excess amount of the condensing agent based on the carboxylic acid.
As the reactive derivative of carboxylic acid wherein L1 is a leaving group regarding the starting compound (1), an acid halide (e.g., acid chloride, acid bromide or the like), an acid anhydride (e.g., a mixed acid anhydride with phenyl chlorocarbonate, p-toluenesulfonic acid, isovaleric acid or the like or symmetric acid anhydride), an active ester (e.g., an ester which can be prepared using phenol that may be substituted with an electron withdrawing group such as a nitro group, a fluorine atom or the like, HOBt, HONSu and the like), a lower alkyl ester and the like may be exemplified, and each of them can be produced from respective carboxylic acid using a reaction obvious to those skilled in the art. Depending on the kind of the reactive derivatives, it is sometimes advantageous for smooth progress of the reaction to carry out the reaction in the presence of a base (e.g., organic bases such as triethylamine, diisopropylethylamine, N-methylmorpholine, pyridine, 4-(N,N-dimethylamino)pyridine and the like or inorganic bases such as sodium bicarbonate). Pyridine can also serve as a solvent. In this connection, when a lower alkyl ester is used as the reactive derivative, it is desirable to carry out the reaction under room temperature to under heating under reflux.
Among the compound (I) of the present invention, a compound (I-b) in which —CR3R4— is represented by —CH(OH)— can be produced by subjecting a compound (I-a) of the present invention in which said moiety is a carbonyl group to reduction.
The reaction is carried out by treating the compound (I-a) with an equivalent amount or excess amount of a reducing agent. As the reducing agent, hydride reducing agents such as sodium borohydride or diisobutylaluminum hydride or the like or the reducing agent described in “Comprehensive Organic Transformation”, edited by Richard C. Larock, (USA), VCH Publishers, Inc., 1989, are used. The reaction is carried out under cooling to under heating, preferably at from −20° C. to room temperature with using as a solvent selected from aromatic hydrocarbons, ethers, DMF, DMSO, alcohols (e.g., methanol, ethanol, and the like), or water, or mixed solvents thereof.
(Production Method 3, Other Production Methods)
The compounds of the present invention having various functional groups such as an amino group, a carboxyl group, an amido group, a hydroxyl group, an alkylamino group and the like can be easily produced by those methods which are obvious to those skilled in the art or modified methods thereof using the compounds of the present invention having a corresponding nitro group, ester group, carboxyl group, amino group and the like as the starting materials. For example, these can be produced by the following reactions.
3-a: Reduction (1)
A compound having an amino group can be produced by reducing a compound having a nitro group. For example, the reaction can be carried out using a hydrogenation reaction which uses palladium-carbon, Raney nickel or the like as the catalyst.
3-b: Reduction (2)
A compound having a hydroxyalkyl group can be produced by reducing a compound having an ester group. For example, the reaction may be carried out using lithium aluminum hydride, sodium borohydride or the like as the reducing agent.
3-c: Hydrolysis
A compound having a carboxyl group can be produced by hydrolyzing a compound having an ester group. For example, this may be carried out in accordance with the deprotection reaction described in the aforementioned “Protective Groups in Organic Synthesis”.
3-d: Amidation
A compound having an amido group may be produced by the amidation of a compound having a carboxyl group or an amino group. This may be carried out in accordance with the aforementioned production method 1.
3-e: Alkylation
A compound having an alkylamino group can be produced by alkylating a compound having an amino group. As the alkylation reaction, the reaction can be carried out by a general method using various alkylating agents (e.g., an alkyl halide, an alkyl sulfonic acid ester or the like). In addition, a compound having an alkylamino group can be produced by carrying out reductive alkylation of a compound having an amino group with a carbonyl compound. The method described in “Jikken Kagaku Koza (Experimental Chemistry Course) (vol. 20) Yuki Gosei (Organic Synthesis) 2”, edited by The Chemical Society of Japan, 4th edition, Maruzen, 1992, p. 300, or the like can be applied to the reaction.
(Production of Starting Compounds)
The starting compound (I) in the production methods described in the above can be produced for example by the following method, a conventionally known method or a modified method thereof.
(Starting Material Synthesis 1)
(In the formulae, either one of Q and U is —Br, —Cl, —I or —O—SO2—CF3 and the other represents —B(OH)2 or B(O-lower alkyl)2. R10 represents a protective group such as lower alkyl or benzyl or the like. The same shall apply hereinafter.)
Among the starting compounds represented by the formula (I), a compound (1a) in which X is NH and Y is a single bond can be produced by the above reaction pathway.
In this case, the coupling reaction can be carried out by the method described in “Synthetic Communications”, (England), 1981, vol. 11, p. 513-519, “Synlett”, (Germany), 2000, vol. 6, p. 829-831, or “Chemistry Letters”, 1989, p. 1405-1408. The cyclization reaction can be carried out at room temperature to under heating in a solvent such as benzene, toluene or the like or without solvent, using triethyl phosphite, triphenylphosphine or the like.
(Starting Material Synthesis 2)
(L2 represents —OH or a leaving group such as halogen, —O-methanesulfonyl, —O-p-toluenesulfonyl or the like.)
Among the starting compounds (1), a compound (1b) in which X is NR5 and Y is a single bond can be produced from the compound (1a) by the reaction such as alkylation, acylation, sulfonylation or the like.
When a compound (6) in which L2 is a leaving group is used, the alkylation reaction can be carried out in the presence of a base such as sodium hydride, potassium hydride, potassium tert-butoxide or the like. Also, when a compound (6) in which L2 is —OH is used, it can be carried out using the usual method of Mitsunobu reaction, for example, in accordance with the method described in Tetrahedron Letters, (Netherlands), 2002, vol. 43, p. 2187.
Regarding the acylation or sulfonylation, the reaction can be carried out using an acid halide in which the leaving group of L2 is halogen, or the like, as the compound (6), in the presence of a base such as potassium hydride, potassium tert-butoxide or the like.
(Starting Material Synthesis 3)
Among the starting compounds represented by the formula (1), compounds (1c) and (1d) in which —CR3R4 is represented by —C(O)— can be produced by the above reaction pathway. The coupling reaction can be carried out in the same manner as the coupling reaction of the starting material synthesis 1. The hydrolysis and esterification can be carried out in accordance with the hydrolysis reaction and esterification reaction described in the aforementioned “Protective Groups in Organic Synthesis”. The usual method of intramolecular Friedel-Crafts' reaction can be used for the cyclization reaction and, for example, the method described in “Journal of the American Chemical Society”, (USA), 1941, vol. 63, p. 1948 may be cited.
(Starting Material Synthesis 4)
(In the formulae, R11 represents lower alkyl, R12 represents lower alkyl or two R12 together form lower alkylene, M represents the counter cation such as lithium ion, magnesium ion or the like of an organic metal reagent, E represents O or S, and R13 represents lower alkylene.)
Among the starting compounds (1), compounds (1e) to (1h) in which at least one of R3 and R4 has various types of substituent can be easily produced using each reaction of the alkylation, etherification, ketal formation and cyclization or a combination thereof.
The alkylation can be carried out using an organic metal reagent such as the Grignard reagent, an organic lithium reagent, an organic cerium reagent, or the like. The etherification is carried out using an alkylating agent in which L2 is a leaving group, in the presence of a base such as sodium hydride, potassium hydride, potassium tert-butoxide, silver oxide or the like. There is a case in which this is carried out under an acidic condition using a compound wherein L2 is —OH, which is carried out using an acid catalyst such as hydrochloric acid, sulfuric acid, p-toluenesulfonic acid or the like, a Lewis acid such as iron nitrate or iron perchlorate or the like, in a solvent such as methanol, ethanol, benzene, toluene, xylene or the like, and at room temperature to under heating.
The ketal formation can be carried out using an acid catalyst such as hydrochloric acid, sulfuric acid, p-toluenesulfonic acid or the like and a Lewis acid such as boron trifluoride diethyl ether or the like, at room temperature to under heating.
The cyclization is carried out using an acid catalyst such as hydrochloric acid, sulfuric acid, p-toluenesulfonic acid or the like, a Lewis acid such as iron nitrate or iron perchlorate or the like, in a solvent such as benzene, toluene, xylene or the like, and at from room temperature to under heating. Alternatively, this can be carried out in the presence of a base such as sodium hydride, potassium hydride, potassium tert-butoxide, silver oxide or the like, after converting the hydroxyl group of R3 into a leaving group such as halogen, sulfonic acid ester or the like.
Respective products of the above production methods can be introduced into corresponding carboxyl compounds by the deprotection of —CO2R10 group. For example, the deprotection reaction described in the aforementioned “Protective Groups in Organic Synthesis” can be used.
(Starting Material Synthesis 5)
The starting compound (1i) can be produced also by the hydrolysis of a compound (10) which has a cyano group. The compound (10) can be produced by a conventionally known method or by similar methods of the production methods of the starting material syntheses 1 to 4, using a corresponding starting material in which the —CO2R10 group is changed to a cyano group. The hydrolysis can be carried out at room temperature to under heating in a solvent which is inert to the reaction, such as acetic acid, water or the like, using an acid such as hydrochloric acid, sulfuric acid or the like. In addition, the reaction can also be carried out at room temperature to under heating in solvent such as an alcohol (e.g., ethanol or the like), water or the like, using a base such as potassium hydroxide or the like.
The compound (I) produced in this manner is isolated and purified as such or after making it into a salt by carrying out a salt formation treatment by a general method. The isolation and purification are carried out by employing general chemical operations such as extraction, concentration, evaporation, crystallization, filtration, recrystallization, various types of chromatography and the like.
Various isomers can be isolated by usual methods making use of the difference in a physicochemical property between isomers. For example, optical isomers can be respectively separated and purified by a method in which a racemic compound is derived into a diastereomer salt with an optically active organic acid (tartaric acid or the like) and then subjected to fractional recrystallization, or a technique such as a chiral filler-aided column chromatography or the like. In addition, an optically active compound can also be produced using an appropriate optically active compound as the starting material. In this connection, a diastereomer mixture can also be separated by a fractional crystallization, a chromatography or the like.
The reaction products obtained by the respective production methods described in the above can be isolated and purified as free compounds, salts thereof or their various solvates (e.g., hydrates and the like). The salts can be produced by subjecting to general salt formation treatment.
The isolation and purification are carried out by employing general chemical operations such as extraction, concentration, evaporation, crystallization, filtration, recrystallization, various types of chromatography and the like.
Various isomers can be isolated by usual methods making use of the difference in a physicochemical property between isomers. For example, optical isomers can be separated by a general optical resolution method such as a fractional crystallization, a chromatography or the like. In addition, an optically active isomer can also be produced from an appropriate optically active starting compound.
The pharmaceutical preparation which comprises one or two or more of the compound of the present invention or a salt thereof as the active ingredient can be prepared using carriers, fillers and other additive agents which are generally used in the preparation of medicines.
The administration may be either embodiment of oral administration by tablets, pills, capsules, granules, powders, solutions and the like, or parenteral administration by injections (e.g., intravenous, intramuscular and the like), suppositories, percutaneous preparations, transnasal preparations, inhalations and the like. The dose is optionally decided in response to each case by taking symptom, age, sex and the like of the object to be administered into consideration, but in the case of oral administration, it is generally approximately from 0.001 mg/kg to 100 mg/kg per day per adult, and this is administered once or by dividing into 2 to 4 times. Also, when intravenously administered, it is generally administered once to two or more times a day within the range of from 0.0001 mg/kg to 10 mg/kg per day per adult. Also, in the case of transnasal administration, it is generally administered once to two or more times a day within the range of from 0.0001 mg/kg to 10 mg/kg per day per adult. In addition, in the case of inhalation, it is generally administered once to two or more times a day within the range of from 0.0001 mg/kg to 1 mg/kg per day per adult.
As the solid composition for oral administration by the present invention, tablets, powders, granules and the like are used. In such a solid composition, one or more active substances are mixed with at least one inert filler such as lactose, mannitol, glucose, hydroxypropylcellulose, microcrystalline cellulose, starch, polyvinyl pyrrolidone, aluminum magnesium silicate or the like. In accordance with the usual way, the composition may contain inert additives such as lubricants (e.g., magnesium stearate and the like), disintegrators (e.g., carboxymethylstarch sodium and the like), solubilizing agents, and the like. As occasion demands, the tablets or pills may be coated with a sugar coating or a gastric or enteric coating.
As the liquid composition for oral administration, pharmaceutically acceptable emulsions, solutions, suspensions, syrups, elixirs and the like are included, which contain a generally used inert solvent such as purified water or ethanol. In addition to the inert solvent, this composition may contain auxiliary agents (e.g., solubilizing agents, moistening agents, suspending agents and the like), sweeteners, correctives, aromatics and antiseptics.
As the injections for parenteral administration, sterile aqueous or non-aqueous solutions, suspensions and emulsions are included. As the aqueous solvent, for example, distilled water for injection and physiological saline are included. Examples of the non-aqueous solvent include propylene glycol, polyethylene glycol, plant oils (e.g., olive oil or the like), alcohols (e.g., ethanol or the like), polysorbate 80 (the name in Pharmacopeia), and the like. Such a composition may further contain tonicity agents, antiseptics, moistening agents, emulsifying agents, dispersing agents, stabilizing agents and solubilizing agents. These are sterilized by, for example, filtration through a bacteria retaining filter, formulation of bactericides or irradiation. In addition, these can also be used by producing a sterile solid compositions and dissolving or suspending them in sterile water or a sterile solvent for injection prior to use.
Inhalations, transmucosal preparations transnasal preparations and the like are used in a solid, liquid or semisolid form and can be produced in accordance with conventionally known methods. For example, excipients such as lactose, starch or the like, as well as a pH adjusting agent, an antiseptic, a surfactant, a lubricant, a stabilizer, a thickener and the like, may be optionally added. An appropriate device for inhalation or blowing can be used for the administration. For example, using a conventionally known device such as a measured administration inhalation device or the like or a sprayer, a compound can be administered alone or as a powder of a prescribed mixture, or as a solution or suspension by a combination with a medicinally acceptable carrier. The dry powder inhaler or the like may be for single or multiple administration use, and a dry powder or a powder-containing capsule can be used. Alternatively, it may be in a form such as a pressurized aerosol spray or the like, which uses suitable gas such as chlorofluoroalkane, hydrofluoroalkane, carbon dioxide or the like.
(Test Methods)
Effects of the compound (I) of the present invention were confirmed by the following pharmacological tests.
Test Method (1) 5-HT2B Receptor Binding Test
(i) Membrane Sample Preparation
Cultured human 5-HT2B receptor-expressing HEK293-EBNA cells were washed with a phosphate buffer (PBS)(−). The cells were peeled off using a scraper in the presence of PBS(−), and the cells were recovered by a centrifugal treatment (1,000 rpm, 10 min, 4° C.). In the presence of 5 mM tris-hydrochloric acid (Tris-HCl) (pH 7.4) buffer, these were homogenized using a homogenizer (registered trademark: Polytron (PTA 10-TS)) and subjected to a centrifugal treatment (40,000×g, 10 min, 4° C.). In the presence of 50 mM Tris-HCl (pH 7.4) buffer, these were suspended using a glass-Teflon (registered trademark) homogenizer. By carrying out a centrifugal treatment (40,000×g, 10 min, 4° C.), these were suspended in 50 mM Tris-HCl (pH 7.4) and stored at −80° C.
(ii) Receptor Binding Test
A total volume of 500 μl containing 50 mM Tris-HCl, 4-mM-CaCl2 (pH 7.4) buffer, the human 5-HT2B receptor expressing HEK293-EBNA cell membrane sample and a radio ligand [3H] Mesulergine (3.1 TBq/mmol); was incubated at 25° C. for 1 hour. The compound was dissolved in 100% DMSO and diluted to respective concentrations. Nonspecific binding was regarded as the binding quantity in the presence of 1 μM ritanserin, and a result of subtracting the nonspecific binding quantity from the total binding quantity was regarded as the specific binding quantity. By adding 4 ml of 50 mM Tris-HCl buffer (pH 7.4), filtered under a reduced pressure trough a GF/B glass filter, and the filter was washed with the same buffer (4 ml×3). The glass filter was soaked in 5 ml of a liquid scintillator (trade name: Aquasol-2), and the radioactivity quantity was measured using a liquid scintillation counter. The compound concentration which inhibits 50% of the receptor binding, IC50 value, was calculated by non-linear regression analysis using a statistical analysis software (registered trademark: SAS (ver. 6.11)), and the Ki value which shows affinity for the receptor was calculated using the formula of Cheng & Prussoff; Ki=IC50/(1+[L]/[Kd]) ([L]: ligand concentration, [Kd]: dissociation constant). The results are shown in the following Table 1.
TABLE 1
Ex
Ki (nM)
1
4.6
66
0.26
81
0.83
83
1.5
84
1.3
Test Method (2) 5-HT7 Receptor Binding Test
(i) Membrane Sample Preparation
Cultured human 5-HT7 receptor expressing CHO cells were washed PBS(−). The cells were peeled off using a scraper in the presence of PBS(−), and the cells were recovered by a centrifugal treatment (1,000 rpm, 10 min, 4° C.). In the presence of 5 mM Tris-HCl (pH 7.4) buffer, these were homogenized using a homogenizer (registered trademark: Polytron (PTA 10-TS)) and subjected to a centrifugal treatment (40,000×g, 10 min, 4° C.). In the presence of 50 mM Tris-HCl (pH 7.4) buffer, these were suspended using a glass-Teflon (registered trademark) homogenizer. By carrying out a centrifugal treatment (40,000×g, 10 min, 4° C.), these were suspended in 50 mM Tris-HCl (pH 7.4) and stored at −80° C.
(ii) Receptor Binding Test
A total volume of 500 μl containing 50 mM Tris-HCl, 4 mM CaCl2 (pH 7.4) buffer, the human 5-HT7 receptor expressing CHO cell membrane sample and a radio ligand [3H] 5-HT (3.40 TBq/mmol) was incubated at 25° C. for 1 hour. The compound was dissolved in 100% DMSO and diluted to respective concentrations. Nonspecific binding was regarded as the binding quantity in the presence of 10 μM metergoline, and a result of subtracting the nonspecific binding quantity from the total binding quantity was regarded as the specific binding quantity. By adding 4 ml of 50 mM Tris-HCl buffer (pH 7.4), filtered under a reduced pressure trough a GF/B glass filter, and the filter was washed with the same buffer (4 ml×3). The glass filter was soaked in 5 ml of a liquid scintillator (trade name: Aquasol-2), and the radioactivity quantity was measured using a liquid scintillation counter. The compound concentration which inhibits 50% of the receptor binding, IC50 value, was calculated by non-linear regression analysis using SAS (ver. 6.11), and the Ki value which shows affinity for the receptor was calculated using the formula of Cheng & Prussoff; Ki=IC50/(1+[L]/[Kd]) ([L]: ligand concentration, [Kd]: dissociation constant). The results are shown in the following Table 2.
TABLE 2
Ex
Ki (nM)
1
0.64
66
2.6
81
0.78
83
0.99
84
2.5
Test Method (3) Affinity for Other Receptors
Affinities for 5-HT1A, 5-HT1B, 5-HT2A, 5-HT2C, 5-HT3, 5-HT4, 5-HT6, α1, M1 and D2 receptors can be confirmed using conventionally known methods (“Journal of Neurochemistry”, (England), 1986, vol. 47, p. 529-540; “Molecular Pharmacology”, (USA), 1982, vol. 21, p. 301-314; “European Journal of Pharmacology”, (Holland), 1985, vol. 106, p. 539-546; “The Journal of Pharmacology Experimental Therapeutics”, (USA), 1992, vol. 263, p. 1127-1132; “British Journal of Pharmacology”, (England), 1993, vol. 109, p. 618-624; “Molecular Pharmacology”, (USA), 1993, vol. 43, p. 320-327; “Molecular Pharmacology”, (USA), 1989, vol. 35, p. 324-330; “Cellular and Molecular Neurobiology”, (Germany), 1988, vol. 8, p. 181-191; “European Journal of Pharmacology”, (Holland), 1988, vol. 173, p. 177-182).
In this connection, affinities of the RS-127445 (2-amino-4-(4-fluoronaphth-1-yl)-6-isopropylpyrimidine); see WO 97/44326 for its production method) and SB-269970 ((R)-3-(2-(2-(4-methylpiperidine-1-yl)ethyl)pyrrolidine-1-sulfonyl)phenol; see WO 97/48681 for its production method), described in the following test method (4), are conventionally known, and regarding the RS-127445, it has been reported, for example in “British Journal of Pharmacology”, (England), 1999, vol. 127, p. 1075-1082, that its pKi for 5-HT2B receptor is 9.5, and it is 1000 times or more 5-HT2B receptor-selective against 5-HT1A, 5-HT1B, 5-HT2A, 5-HT2C, 5-HT3, 5-HT6, 5-HT7, α1, M1, D2 and the like receptors. Also, regarding the SB-269970, it has been reported, for example in “Journal of Medicinal Chemistry”, (USA), 2000, vol. 43, p. 342-345, that pKi of said compound for 5-HT7 receptor is 8.9, and it is 250 times or more 5-HT7 receptor-selective against 5-HT1A, 5-HT1B, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT4, 5-HT6, α1, D2 and the like receptors.
Test Method (4) Inhibitory Effect for Defecation at the Time of Confined Stress Loading
The IBS-treating effect of the compound (I) of the present invention was evaluated using a method in which a confined stress is applied to rats and the evacuation quantity is measured (cf. “The Journal of Pharmacology Experimental Therapeutics”, (USA), 1992, vol. 261, p. 297-303). This test is an animal model wherein it is known that the 5-HT3 receptor antagonist as a diarrhea-predominant IBS-treating agent shows its efficacy.
Test Method
An agent to be tested was administered to male Wistar rats (body weight 250 to 320 g, 10 animals for each group) and a confined stress was applied thereto 30 minutes thereafter. A confining cage (trade name: KN-468, 265 mm in width×95 mm in length×200 mm in height, Natsume Seisakusho, Tokyo) was used in the confined stress loading, and the number of feces during 1 hour after the stress loading was counted.
As shown in FIG. 1, the RS-127445 as a 5-HT2B receptor-selective antagonistic compound did not show the action to suppress defecation even when a dose of 10 mg/kg was orally administered (p.o.).
In addition, as shown in FIG. 2, the SB-269970 as a 5-HT7 receptor-selective antagonistic compound also did not show the action to suppress defecation even at a dose of 10 mg/kg (p.o.).
On the other hand, as shown in FIG. 3, it was found that a synergistic effect can be obtained when both of the compounds RS-127445 and SB-269970 are simultaneously administered. That is, as shown in FIG. 1 and FIG. 2, RS-127445 and SB-269970 did not show the action even at 10 mg/kg (p.o.) when used each independently, but it was revealed that significant suppressing action is shown starting at a dose of 1 mg/kg (p.o.) when both compounds are simultaneously administered.
This effect was the same when the compound of the present invention which selectively possesses both of the 5-HT2B receptor antagonism and 5-HT7 receptor antagonism was used. That is, as shown in FIG. 4, the compound of Example 84 which is described later showed an excellent suppressing action of 0.72 mg/kg (p.o.) in ED50 (50% effective dose).
Based on the above results, being possessed of both of the 5-HT2B receptor antagonism and 5-HT7 receptor antagonism, it can be expected that the compound of the present invention shows excellent effect to improve morbid state of IBS in comparison with one of the selective receptor antagonists.
Test Method (5) Preventive Effect in Guinea Pig Migraine Model
It has been suggested that an inflammatory protein leaked from dura mater blood vessel by 5-HT is concerned in the onset of migraine. This test system evaluates the migraine-preventive effect by measuring the amount of this leaked protein in the presence of a compound to be tested and was carried out by partially modifying the method described by Rachel A. Spokes and Vicki C. Middlefell in “European Journal of Pharmacology”, (Holland), 1995, vol. 281, p. 75-79.
Hartley male guinea pigs (250 to 350 g) were anesthetized by the intraperitoneal administration (i.p.) of urethane (1.5 g/kg). By applying a simple canulation to a latent vein, 50 mg/kg of a fluorescent protein (FITC-BSA) was intravenously administered (i.v.) and, 5 minutes thereafter, physiological saline or 1 μmol/ml/kg of 5-HT was intravenously administered. Perfusion was carried out with physiological saline 15 minutes thereafter and blood was washed out. RS-127445 and SB-269970 were administered intravenously, and the compound of Example 1 orally, respectively 30 minutes before the administration of fluorescent protein. By detaching the skull, the dura mater was extracted and incubated in an Eppendorf tube at 37° C. for 16 hours in the presence of physiological saline adjusted to pH 11. By carrying out a centrifugal operation, the supernatant was dispensed into a plate. The fluorescence intensity was measured using a fluorescence plate reader (excitation wavelength 485 nm, absorption wavelength 530 nm). By weighing the dura mater weight, the fluorescence intensity per mg dura mater protein was calculated.
The values of fluorescence intensity measured at the time of the administration and non-administration of each compound are shown in FIG. 4 to FIG. 7. In each of them, the axis of abscissa shows dose of the compounds, and the axis of ordinate fluorescence strength per 1 mg dura mater blood vessel. The control shows fluorescence strength at the time of not adding 5-HT, namely the standard value.
As shown in FIG. 5, a 5-HT2B-selective antagonistic compound RS-127445 showed the leaked protein quantity-reducing action at 3 mg/kg, but did not lower it to the standard value when the dose was increased from 3 mg/kg to 10 mg/kg.
In addition, as shown in FIG. 6, a 5-HT7-selective antagonistic compound SB-269970 also showed the action at from 10 mg/kg, but did not lower the leaked protein quantity to the standard value when the dose was increased from this to 30 mg/kg.
On the other hand, as shown in FIG. 7, it was found that a synergistic effect can be obtained when both of the compounds RS-127445 and SB-269970 are simultaneously administered. That is, as shown in FIG. 5 and FIG. 6, it was shown that the minimum amount by which the both compounds show the maximum drug efficacy is 3 mg/kg for RS-127445 and 10 mg/kg for SB-269970, and it was revealed that the leaked protein quantity is almost completely suppressed to the standard value when both compounds are simultaneously administered at the same doses. This result shows that, when both functions of the 5-HT2B receptor and 5-HT7 receptor are simultaneously inhibited, an excellent effect which cannot be obtained by the inhibition of one of the selective receptors can be obtained.
This effect was the same when the compound of the present invention which selectively possesses both of the 5-HT2B receptor antagonism and 5-HT7 receptor antagonism was used. That is, as shown in FIG. 8, the compound of Example 1 which is described later almost completely suppressed the leaked protein quantity by 30 mg/kg of oral administration.
It was shown based on the above results that, being possessed of both of the 5-HT2B receptor antagonism and 5-HT7 receptor antagonism, the compound of the present invention can completely suppress leaking amount the inflammatory protein. Accordingly, it was confirmed that the compound of the present invention has a possibility of effectively suppressing onset of migraine and has a superior effect to prevent migraine in comparison with one of the selective receptor antagonists.
EXAMPLES
The following illustratively describes production methods of the compounds of the present invention with reference to the production examples of the compounds of the present invention, though the present invention is not restricted by these examples. In this connection, since novel compounds are included in the starting compounds of the compounds of the present invention, production methods of these compounds are described as reference examples.
In this connection, the signs in the Reference Examples and Examples and in the tables which are described later have the following meanings (the same shall apply hereinafter).
REx: Reference Example number, Ex: Example number, No: compound number, Str: structural formula, Dat: physical data (EI: EI-MS; ESI: ESI-MS; APCI: APCI-MS; FAB: FAB-MS; NMR: δ (ppm) of characteristic peaks in DMSO-d6 by 1H NMR), Sal: salt (Blank space or no description indicates that it is a free form, and the numeral before the acid component shows molar ratio. For example, when 2HCl is described, it means that the compound is dihydrochloride), Me: methyl, Et: ethyl, nPr: normal-propyl, cPr: cyclopropyl, iPr: isopropyl, nBu: normal-butyl, cBu: cyclobutyl, nPen: normal-pentyl, cPen: cyclopentyl, cHex: cyclohexyl, Ph: phenyl, Bn: benzyl, Ac: acetyl, Boc: tert-butoxycarbonyl, null: no substitution. The numeral before substituent indicates the substitution position, for example, 5-F indicates 5-fluoro. RSyn and Syn: production method (The numeral indicates that it was produced using a corresponding starting material similar to the case of a compound having respective number as Reference Example number or Example number).
Reference Example 1
Methyl 2-nitrobiphenyl-4-carboxylate was obtained by allowing methyl 3-nitro-4-{[(trifluoromethyl)sulfonyl]oxy}benzoate and phenyl boronic acid, potassium phosphate and tetrakistriphenylphosphine palladium to undergo the reaction in DMF under heating. FAB: 258 (M+H)+.
Reference Example 2
Methyl 9H-carbazole-2-carboxylate was obtained by allowing methyl 2-nitrobiphenyl-4-carboxylate and triethyl phosphite to undergo the reaction under heating. FAB: 226 (M+H)+.
Reference Example 3
Methyl 9-isopropyl-9H-carbazole-2-carboxylate was obtained by allowing methyl 9H-carbazole-2-carboxylate, 2-propanol and (tributylphospholanylidene)acetonitrile to undergo the reaction in toluene under heating. ESI: 268 (M+H)+.
Reference Example 4
9-Isopropyl-9H-carbazole-2-carboxylic acid was obtained by allowing methyl 9-isopropyl-9H-carbazole-2-carboxylate and a 1 M sodium hydroxide aqueous solution to undergo the reaction in ethanol under heating. ESI: 252 (M−H)−.
Reference Example 5
Methyl 5-bromomethyl-9-isopropyl-9H-carbazole-2-carboxylate was obtained by allowing methyl 9-isopropyl-5-methyl-9H-carbazole-2-carboxylate, N-bromosuccinimide and 2,2′-azobisisobutyronitrile to undergo the reaction in carbon tetrachloride under heating. FAB: 360, 362 (M+H)+.
Reference Example 6
Methyl 5-dimethylaminomethyl-9-isopropyl-9H-carbazole-2-carboxylate was obtained by allowing methyl 5-bromomethyl-9-isopropyl-9H-carbazole-2-carboxylate, dimethylamine (2 M, methanol solution) and potassium carbonate to undergo the reaction at room temperature in THF. FAB: 325 (M+H)+.
Reference Example 7
Methyl 5-acetoxymethyl-9-isopropyl-9H-carbazole-2-carboxylate was obtained by allowing methyl 5-bromomethyl-9-isopropyl-9H-carbazole-2-carboxylate and potassium acetate to undergo the reaction at room temperature in DMF. EI: 339 (M)+.
Reference Example 8
Methyl 5-hydroxymethyl-9-isopropyl-9H-carbazole-2-carboxylate was obtained by allowing methyl 5-acetoxymethyl-9-isopropyl-9H-carbazole-2-carboxylate and potassium carbonate to undergo the reaction at room temperature in methanol-THF. FAB: 297 (M)+.
Reference Example 9
Methyl 9-isopropyl-5-methoxymethyl-9H-carbazole-2-carboxylate was obtained by allowing methyl 5-hydroxymethyl-9-isopropyl-9H-carbazole-2-carboxylate, methyl iodide and silver oxide to undergo the reaction under heating in acetonitrile. FAB: 311 (M)+.
Reference Example 10
Benzyl 9-isobutyryl-9H-carbazole-2-carboxylate was obtained by allowing benzyl 9H-carbazole-2-carboxylate and 2-methylpropionyl chloride to undergo the reaction at room temperature in DMF in the presence of sodium hydride. ESI: 372 (M+H)+.
Reference Example 11
9-Isobutyryl-9H-carbazole-2-carboxylic acid was obtained by allowing benzyl 9-isobutyryl-9H-carbazole-2-carboxylate, and palladium-carbon to undergo the reaction at room temperature in ethanol-DMF in an atmosphere of hydrogen gas. ESI: 282 (M+H)+.
Reference Example 12
Methyl 9-isopropyl-6-nitro-9H-carbazole-2-carboxylate was obtained by allowing methyl 9-isopropyl-9H-carbazole-2-carboxylate and concentrated nitric acid to undergo the reaction at room temperature in acetic acid. FAB: 313 (M+H)+.
Reference Example 13
Methyl 5-formyl-9-isopropyl-9H-carbazole-2-carboxylate was obtained by allowing methyl 5-hydroxymethyl-9-isopropyl-9H-carbazole-2-carboxylate and manganese dioxide to undergo the reaction at room temperature in chloroform. FAB: 296 (M+H)+.
Reference Example 14
9-Methyl-9H-carbazole-2-carboxylic acid was obtained by allowing methyl 9H-carbazole-2-carboxylate, methyl iodide and potassium hydroxide to undergo the reaction at room temperature in DMF. FAB: 226 (M+H)+.
Reference Example 15
Ethyl 9-ethyl-9H-carbazole-2-carboxylate was obtained by allowing methyl 9H-carbazole-2-carboxylate, ethyl iodide and potassium hydroxide to undergo the reaction under heating in DMF. ESI: 268 (M+H)+.
Reference Example 16a, Reference Example 16b
A mixture of 2,3,4,9-tetrahydro-1H-carbazole-7-carboxylic acid and 2,3,4,9-tetrahydro-1H-carbazole-5-carboxylic acid was obtained by allowing cyclohexanone and 3-hydrazinobenzoic acid to undergo the reaction under heating in acetic acid. By separating and purifying this mixture by a silica gel column chromatography, 2,3,4,9-tetrahydro-1H-carbazole-5-carboxylic acid [Reference Example 16a: FAB: 216 (M+H)+] and 2,3,4,9-tetrahydro-1H-carbazole-7-carboxylic acid [Reference Example 16b: FAB: 216 (M+H)+] were obtained.
Reference Example 17a, Reference Example 17b
By adding thionyl chloride to a methanol solution of a mixture of 2,3,4,9-tetrahydro-1H-carbazole-7-carboxylic acid and 2,3,4,9-tetrahydro-1H-carbazole-5-carboxylic acid at −10° C., followed by the reaction under heating and subsequent separation and purification by a column chromatography, methyl 2,3,4,9-tetrahydro-1H-carbazole-7-carboxylate [Reference Example 17a: ESI: 2230 (M+H)+] and methyl 2,3,4,9-tetrahydro-1H-carbazole-5-carboxylate [Reference Example 17b: ESI: 230 (M+H)+] were obtained.
Reference Example 18
3-{2-[1-(Ethoxycarbonyl)piperidin-4-ylidene]hydrazino}benzoic acid was obtained by allowing ethyl 4-oxopiperidine-1-carboxylate and 3-hydrazinobenzoic acid to undergo the reaction under heating in acetic acid. ESI: 306 (M+H)+.
Reference Example 19
A mixture of diethyl 1,3,4,5-tetrahydro-2H-pyrido[4,3-b]indole-2,7-dicarboxylate and diethyl 1,3,4,5-tetrahydro-2H-pyrido[4,3-b]indole-2,9-dicarboxylate was obtained by allowing 3-{2-[1-(ethoxycarbonyl)piperidin-4-ylidene]hydrazino}benzoic acid and concentrated hydrochloric acid to undergo the reaction under heating in ethanol. ESI: 317 (M+H)+.
Reference Example 20
A mixture of 2-(ethoxycarbonyl)-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole-7-carboxylic acid and 2-(ethoxycarbonyl)-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole-9-carboxylic acid was obtained by allowing a mixture of diethyl 1,3,4,5-tetrahydro-2H-pyrido[4,3-b]indole-2,7-dicarboxylate and diethyl 1,3,4,5-tetrahydro-2H-pyrido[4,3-b]indole-2,9-dicarboxylate and potassium hydroxide to undergo the reaction under heating in methanol-water. ESI: 287 (M−H)+.
Reference Example 21
10H-Phenoxazine-3-carboxylic acid was obtained by allowing 10H-phenoxazine-3-carbonitrile produced in accordance with “New Journal of Chemistry”, (England), 2001, vol. 25, no. 3, p. 385, and concentrated hydrochloric acid under heating in acetic acid. FAB: 227 (M)+.
Reference Example 22
10-Methyl-10H-phenoxazine-3-carbonitrile was obtained by allowing 10H-phenoxazine-3-carbonitrile produced in accordance with “New Journal of Chemistry”, (England), 2001, vol. 25, no. 3, p. 385, methyl iodide and sodium hydride at room temperature in THF. FAB: 223 (M+H)+.
Reference Example 23
10-Methyl-10H-phenoxazine-3-carboxylic acid was obtained by treating 10-methyl-10H-phenoxazine-3-carbonitrile with 8 M potassium hydroxide aqueous solution under heating in ethanol. FAB: 241 (M)±.
Reference Example 24
10H-Phenoxazine-2-carbonitrile was obtained by producing it in accordance with “New Journal of Chemistry”, (England), 2001, vol. 25, no. 3, p. 385.
Reference Example 25
10H-Phenoxazine-2-carboxylic acid was obtained by producing it in accordance with “Journal of Organic Chemistry”, 1960, vol. 25, p. 747.
Reference Example 26
Ethyl 10H-phenoxazine-2-carboxylate was obtained by producing it in accordance with “Journal of Organic Chemistry”, 1960, vol. 25, p. 747.
Reference Example 27
Diethyl 4-(2-thienyl)isophthalate was obtained by allowing 4-bromophthalic acid diethyl ester, 2-thienyl boronic acid, sodium carbonate and tetrakistriphenylphosphine palladium to undergo the reaction under heating in toluene-ethanol-water. FAB: 305 (M+H)+.
Reference Example 28
4-(2-Thienyl)isophthalic acid was obtained by treating an ethanol solution of diethyl 4-(2-thienyl)isophthalate with 5 M sodium hydroxide aqueous solution. FAB: 248 (M)+.
Reference Example 29
4-Oxo-4H-indeno[1,2-b]thiophene-6-carboxylic acid was obtained by allowing 4-(2-thienyl)isophthalic acid and trifluoroacetic anhydride to undergo the reaction under heating in trifluoroacetic acid. FAB: 230 (M)−.
Reference Example 30
Ethyl 4-oxo-4H-indeno[1,2-b]thiophene-6-carboxylate was obtained by allowing 4-oxo-4H-indeno[1,2-b]thiophene-6-carboxylic acid and concentrated sulfuric acid to undergo the reaction under heating in ethanol. EI: 258 (M)+.
Reference Example 31
Ethyl spiro[1,3-dithiolan-2,4′-indeno[1,2-b]thiophene]-6′-carboxylate was obtained by allowing ethyl 4-oxo-4H-indeno[1,2-b]thiophene-6-carboxylate, 1,2-ethanedithiol and boron trifluoride diethyl ether complex to undergo the reaction under heating in acetic acid. FAB: 334 (M)+.
Reference Example 32
4-Hydroxy-4-methyl-4H-indeno[1,2-b]thiophene-6-carboxylic acid was obtained by allowing 4-oxo-4H-indeno[1,2-b]thiophene-6-carboxylic acid and methylmagnesium bromide to undergo the reaction at 0° C. in THF. FAB: 245 (M−H)−
Reference Example 33
Methyl 4-methoxy-4-methyl-4H-indeno[1,2-b]thiophene-6-carboxylate was obtained by allowing 4-hydroxy-4-methyl-4H-indeno[1,2-b]thiophene-6-carboxylic acid, methyl iodide and sodium hydride to undergo the reaction at room temperature in DMF. FAB: 275 (M+H)+.
Reference Example 34
Methyl 4-allyl-4-hydroxy-4H-indeno[1,2-b]thiophene-6-carboxylate was obtained by allowing 4-allyl-4-hydroxy-4H-indeno[1,2-b]thiophene-6-carboxylic acid [ESI: 273 (M+H)+] synthesized by carrying out the same operation of Reference Example 32 using 4-oxo-4H-indeno[1,2-b]thiophene-6-carboxylic acid and allylmagnesium bromide to undergo the reaction with methyl iodide and sodium bicarbonate at 50° C. in DMF. ESI: 287 (M+H)+.
Reference Example 35
Methyl 4-hydroxy-4-(3-hydroxypropyl)-4H-indeno[1,2-b]thiophene-6-carboxylate was obtained by allowing methyl 4-allyl-4-hydroxy-4H-indeno[1,2-b]thiophene-6-carboxylate and BH3-THF complex to undergo the reaction at 0° C. in THF and then further carrying out the reaction at 60° C. by adding hydrogen peroxide aqueous solution and sodium hydroxide aqueous solution thereto. ESI: 305 (M+H)+.
Reference Example 36
4,5-Dihydro-3H-spiro[furan-2,4′-indeno[1,2-b]thiophene]-6′-carboxylic acid was obtained by treating, with 1 M sodium hydroxide aqueous solution, methyl 4-hydroxy-4-(3-{[(4-methylphenyl)sulfonyl]oxy}propyl)-4H-indeno[1,2-b]thiophene-6-carboxylate which had been obtained by allowing methyl 4-hydroxy-4-(3-hydroxypropyl)-4H-indeno[1,2-b]thiophene-6-carboxylate and 4-methylbenzenesulfonyl chloride to undergo the reaction at 0° C. in pyridine. ESI: 273 (M+H)+.
Reference Example 37
9-Oxo-9H-indeno[2,1-c]pyridine-7-carboxylic acid was obtained by allowing 4-pyridin-4-ylphthalic acid [FAB: 244 (M+H)+] which had been synthesized by carrying out the same operations of Reference Examples 27 and 28 using 4-bromoisophthalic acid diethyl ester and 4-pyridine boronic acid to undergo the reaction with lithium 2,2,6,6-tetramethylpiperidide at 0° C. in THF. By carrying out reaction of this compound by the same method of Reference Example 30, ethyl 9-oxo-9H-indeno[2,1-c]pyridine-7-carboxylate was obtained. FAB: 254 (M+H)+.
The reference example compounds shown in Tables 3 to 10 which are described later were produced using respectively corresponding starting materials in the same manner as in the methods of Reference Examples 1 to 37 described in the above.
Example 1
A 134 mg portion of CDI was added to 4 ml DMF solution of 140 mg 9-isopropyl-9H-carbazole-2-carboxylic acid, followed by stirring at 50° C. for 1 hour. After spontaneous cooling to room temperature, 238 mg of guanidine carbonate was added thereto, followed by stirring overnight at room temperature. After evaporation of the solvent, water was added thereto and the thus precipitated solid was purified by a silica gel column chromatography (Chromatorex (registered trademark), methanol/chloroform) to obtain 157 mg of N-(diaminomethylene)-9-isopropyl-9H-carbazole-2-carboxamide as a pale yellow solid.
Example 2
A 192 mg portion of sodium hydride (60%) was added to 6.5 ml DMF solution of 573 mg guanidine hydrochloride and stirred at room temperature for 1 hour. A 6.5 ml DMF solution of 270 mg methyl 9H-carbazole-2-carboxylate was added to this solution, followed by stirring at 70° C. for 2.5 hours. After spontaneous cooling to room temperature and subsequent evaporation of the solvent, water was added thereto and the thus precipitated solid was purified by Chromatorex (methanol/chloroform) to obtain 236 mg of N-(diaminomethylene)-9H-carbazole-2-carboxamide as a pale yellow solid.
Example 3
A 1.26 ml portion of 1 M hydrochloric acid and 30 mg of 20% palladium hydroxide were added to 9 ml ethanol solution of 300 mg N-(diaminomethylene)-9-[1-(diphenylmethyl)-azetidin-3-yl]-9H-carbazole-2-carboxamide, followed by stirring at room temperature for 4 days under an atmosphere of hydrogen gas. After carrying out celite filtration after adding 1 M sodium hydroxide aqueous solution, the solvent was evaporated, followed by purification by Chromatorex (methanol/chloroform) to obtain 89 mg of 9-azetidin-3-yl-N-(diaminomethylene)-9H-carbazole-2-carboxamide.
Example 4
A 1.0 ml portion of 1 M hydrochloric acid and 40 mg of 10% palladium-carbon were added to an ethanol 9 ml-THF 3 ml solution of 393 mg N-(diaminomethylene)-9-[2-(benzyloxy)ethyl]-9H-carbazole-2-carboxamide, followed by stirring at room temperature for 3 days under an atmosphere of hydrogen gas. After carrying out celite filtration by adding 1 M sodium hydroxide aqueous solution, the organic solvent was evaporated and the water layer was extracted with chloroform, followed by washing with saturated brine and drying with anhydrous magnesium sulfate. By evaporating the solvent, 140 mg of N-(diaminomethylene)-9-(2-hydroxyethyl)-9H-carbazole-2-carboxamide was obtained.
Example 5
A 20 mg portion of 10% palladium-carbon was added to an ethanol 5 ml-THF 3 ml solution of 106 mg N-(diaminomethylene)-9-isopropyl-6-nitro-9H-carbazole-2-carboxamide, followed by stirring at room temperature for 4 hours under an atmosphere of hydrogen gas. After carrying out Celite filtration, the solvent was evaporated to obtain 128 mg of 6-amino-N-(diaminomethylene)-9-isopropyl-9H-carbazole-2-carboxamide.
Example 6
A 0.6 ml portion of 4 M hydrogen chloride/ethyl acetate was added to 4.4 ml ethanol solution of 201 mg tert-butyl 4-(2-{[(diaminomethylene)amino]carbonyl}-9H-carbazol-9-yl)piperidine-1-carboxylate which had been synthesized in the same manner as in Example 1, followed by stirring overnight at room temperature. The thus precipitated-solid was collected by filtration and washed with EtOH to obtain 125 mg of N-(diaminomethylene)-9-piperidin-4-yl-9H-carbazole-2-carboxamide dihydrochloride as pale yellow solid.
Example 7
A 156 mg portion of sodium borohydride was added to a 10 ml methanol suspension of 280 mg N-(diaminomethylene)-8-oxo-8H-indeno[2,1-b]thiophene-6-carboxamide, followed by stirring at room temperature for 30 minutes. After evaporation of the solvent, the solid precipitated by adding water was purified by Chromatorex (DMF/chloroform) to obtain 284 mg of N-(diaminomethylene)-8-hydroxy-8H-indeno[2,1-b]thiophene-6-carboxamide as a pale green solid.
The Example compounds shown in Tables 11 to 19 which are described later were produced using respectively corresponding starting materials (except that Example 61 used a starting material in which the hydroxyl group was protected with acetyl group) in the same manner as in the methods of Examples 1 to 7 described in the above.
In addition, structures of other compounds of the present invention are shown in Tables 20 to 24. These can be easily synthesized using the aforementioned production methods, the methods described in examples and the methods obvious to those skilled in the art, or modified methods thereof.
TABLE 3
REx
RSyn
R1
Dat
38
1
2′-F
FAB: 275 (M)+
39
1
3′-F
FAB: 276 (M + H)+
40
1
4′-F
FAB: 276 (M + H)+
41
1
2′-Me
FAB: 272 (M + H)+
42
1
3′-Me
FAB: 272 (M + H)+
43
1
4′-Me
FAB: 272 (M + H)+
44
1
2′-OMe
FAB: 288 (M + H)+
45
1
3′-OMe
FAB: 288 (M + H)+
46
1
4′-OMe
FAB: 288 (M + H)+
47
1
2′-Cl
FAB: 292 (M + H)+
48
1
3′-Cl
FAB: 291 (M)+
49
1
4′-Cl
FAB: 292 (M + H)+
50
1
2′-CN
ESI: 283 (M + H)+
51
1
3′-CN
FAB: 283 (M + H)+
52
1
4′-CN
FAB: 283 (M + H)+
TABLE 4
REx
RSyn
R1
Dat
53
2
5-F
FAB: 244 (M + H)+
54
2
6-F
FAB: 244 (M + H)+
55
2
7-F
FAB: 244 (M + H)+
56
2
8-F
FAB: 244 (M + H)+
57
2
5-Me
FAB: 240 (M + H)+
58
2
6-Me
FAB: 240 (M + H)+
59
2
7-Me
FAB: 240 (M + H)+
60
2
8-Me
FAB: 240 (M + H)+
61
2
5-OMe
FAB: 256 (M + H)+
62
2
6-OMe
FAB: 255 (M)+
63
2
7-OMe
FAB: 256 (M + H)+
64
2
8-OMe
FAB: 256 (M + H)+
65
2
5-Cl
FAB: 259 (M)+
66
2
6-Cl
FAB: 260 (M + H)+
67
2
7-Cl
FAB: 260 (M + H)+
68
2
8-Cl
FAB: 260 (M + H)+
69
2
5-CN
FAB: 249 (M − H)−
70
2
6-CN
ESI: 249 (M − H)−
71
2
7-CN
ESI: 249 (M − H)−
72
2
8-CN
ESI: 249 (M − H)−
TABLE 5
REx
RSyn
R5
Dat
73
3
nPr
ESI: 268 (M + H)+
74
3
nBu
ESI: 282 (M + H)+
75
3
nPen
ESI: 296 (M + H)+
76
3
—(CH2)2OMe
ESI: 284 (M + H)+
77
3
—(CH2)2OBn
ESI: 360 (M + H)+
78
3
—(CH2)2NMe2
ESI: 297 (M + H)+
79
3
—(CH2)3OMe
FAB: 297 (M)+
80
3
—(CH2)2Ph
FAB: 330 (M + H)+
81
3
Bn
ESI: 316 (M + H)+
82
3
cBu
ESI: 280 (M + H)+
83
3
cPen
ESI: 294 (M + H)+
84
3
cHex
ESI: 308 (M + H)+
85
3
—CH(C2H5)2
ESI: 296 (M + H)+
86
3
FAB: 408 (M)+
87
3
EI: 309 (M)+
88
3
—CH2-cPr
ESI: 280 (M + H)+
89
3
APCI: 310 (M + H)+
90
3
ESI: 306 (M + H)+
91
3
ESI: 447 (M + H)+
92
3
ESI: 306 (M + H)+
93
3
FAB: 323 (M)+
94
10
FAB: 339 (M + H)+
TABLE 6
REx
RSyn
R5
R
Dat
95
3
Et
Et
ESI: 268 (M + H)+
96
3
Bn
EI: 357 (M)+
97
10
Ac
Bn
ESI: 344 (M + H)+
98
10
—S(O)2-Me
Bn
EI: 379 (M)+
99
10
—S(O)2-iPr
Bn
ESI: 408 (M + H)+
100
10
—C(O)NMe2
Bn
FAB: 373 (M + H)+
TABLE 7
REx
RSyn
R5
Dat
101
4
nPr
ESI: 252 (M − H)−
102
4
nBu
ESI: 266 (M − H)−
103
4
nPen
ESI: 280 (M − H)−
104
4
—(CH2)2OBn
ESI: 344 (M − H)−
105
4
—(CH2)3OMe
ESI: 282 (M − H)−
106
4
Bn
ESI: 300 (M − H)−
107
4
—(CH2)2Ph
ESI: 314 (M − H)−
108
4
cBu
FAB: 266 (M + H)+
109
4
cPen
ESI: 278 (M − H)−
110
4
cHex
ESI: 292 (M − H)−
111
4
—CH(C2H5)2
ESI: 280 (M − H)−
112
4
ESI: 294 (M − H)−
113
4
ESI: 393 (M − H)−
114
4
—CH2-cPr
ESI: 264 (M − H)−
115
4
ESI: 290 (M − H)−
116
4
ESI: 290 (M − H)−
117
4
ESI: 308 (M − H)−
118
4
FAB: 325 (M + H)+
119
11
ESI: 266 (M − H)−
120
11
Ac
ESI: 254 (M + H)−
121
11
—S(O)2-Me
FAB: 289 (M)+
122
11
—S(O)2-iPr
ESI: 316 (M − H)−
123
11
—C(O)NMe2
FAB: 283 (M + H)+
TABLE 8
REx
RSyn
R1
R
Dat
124
3
5-F
Me
FAB: 286 (M + H)+
125
3
6-F
Me
FAB: 286 (M + H)+
126
3
7-F
Me
FAB: 286 (M + H)+
127
3
8-F
Me
FAB: 286 (M + H)+
128
3
5-Me
Me
FAB: 282 (M + H)+
129
3
6-Me
Me
FAB: 282 (M + H)+
130
3
7-Me
Me
FAB: 282 (M + H)+
131
3
8-Me
Me
FAB: 282 (M + H)+
132
3
5-OMe
Me
FAB: 298 (M + H)+
133
3
6-OMe
Me
FAB: 297 (M)+
134
3
7-OMe
Me
FAB: 298 (M + H)+
135
3
8-OMe
Me
FAB: 298 (M + H)+
136
3
6-Cl
Me
FAB: 302 (M + H)+
137
3
5-Cl
Me
ESI: 302 (M + H)+
138
3
7-Cl
Me
ESI: 302 (M + H)+
139
3
8-Cl
Me
FAB: 302 (M + H)+
140
3
5-CN
Me
FAB: 293 (M + H)+
141
3
6-CN
Me
FAB: 293 (M + H)+
142
3
7-CN
Me
FAB: 293 (M + H)+
143
3
8-CN
Me
FAB: 293 (M + H)+
144
4
5-CN
H
FAB: 279 (M + H)+
145
4
6-CN
H
FAB: 277 (M − H)−
146
4
7-CN
H
FAB: 279 (M + H)+
147
4
8-CN
H
FAB: 279 (M + H)+
148
4
6-NO2
H
FAB: 297 (M − H)−
149
4
5-C(O)H
H
FAB: 282 (M + H)+
TABLE 9
REx
RSyn
Str
Dat
150
27
FAB: 305 (M + H)+
151
28
FAB: 247 (M − H)−
152
29
FAB: 231 (M + H)+
153
4
FAB: 305 (M − H)−
154
4
FAB: 259 (M − H)−
155
27
FAB: 300 (M + H)+
156
4
ESI: 226 (M + H)+
TABLE 10
REx
RSyn
R5
R
Dat
157
3
CO2Et
ESI: 340 (M + H)+
158
3
—(CH2)2—NMe2
CO2Et
ESI: 327 (M + H)+
159
3
CO2Et
ESI: 340 (M + H)+
160
3
Et
CO2Et
APCI: 283 (M)+
161
3
cBu
CO2Et
ESI: 310 (M + H)+
162
4
Et
CO2H
ESI: 254 (M − H)−
163
4
cBu
CO2H
ESI: 280 (M − H)−
164
22
Me
CN
FAB: 222 (M)+
165
23
Me
CO2H
FAB: 241 (M)+
TABLE 11
Ex
Syn
R5
Sal
Dat
1
1
iPr
HCl
NMR: 1.69 (6H, d, J = 6.9 Hz), 5.31 (1H, sept, J = 6.9 Hz),
8.65 (1H, s). ; FAB: 295 (M + H)+
2
2
H
HCl
NMR: 7.24 (1H, dt, J = 7.3, 1.0 Hz), 7.51 (1H, dt, J = 7.3,
1.0 Hz), 8.33 (1H, s). ; FAB: 253 (M + H)+
3
3
2HCl
NMR: 4.52 (2H, dd, J = 8.3, 8.3 Hz), 4.99 (2H, dd, J = 8.3, 8.3 Hz), 8.76 (1H, s). ; FAB: 308 (M + H)+
4
4
—(CH2)2OH
HCl
NMR: 3.84 (2H, t, J = 5.4 Hz), 4.58 (2H, t, J = 5.4 Hz),
8.67 (1H, s). ; FAB: 297 (M + H)+
8
1
Me
HCl
NMR: 4.01 (3H, s), 7.29 (1H, dt, J = 7.3, 1.0 Hz), 8.71 (1H,
d, J = 1.5 Hz). ; FAB: 267 (M + H)+
9
2
Et
HCl
NMR: 1.37 (3H, t, J = 7.3 Hz), 4.59 (2H, q, J = 7.3 Hz),
8.72 (1H, d, J = 1.5 Hz). ; FAB: 281 (M + H)+
10
1
nPr
HCl
NMR: 0.92 (3H, t, J = 7.3 Hz), 1.86 (2H, tq, J = 7.3, 7.3
Hz), 8.78 (1H, s). ; FAB: 295 (M + H)+
11
1
nBu
HCl
NMR: 0.89 (3H, t, J = 7.3 Hz), 1.35 (2H, tq, J = 7.4, 7.3
Hz), 8.74 (1H, d, J = 1.5 Hz). ; FAB: 309 (M + H)+
12
1
nPen
HCl
NMR: 0.81 (3H, t, J = 6.8 Hz), 1.82 (2H, tt, J = 7.4, 6.8
Hz), 8.70 (1H, d, J = 1.5 Hz). ; FAB: 323 (M + H)+
13
1
—CH(Et)2
HCl
NMR: 0.66 (6H, t, J = 6.4 Hz), 4.60-5.00 (1H, m), 8.89
(1H, s). ; ESI: 323 (M + H)+
14
2
—(CH2)2OMe
HCl
NMR: 3.18 (3H, s), 4.69 (2H, t, J = 5.2 Hz), 8.58 (1H,
s). ; ESI: 311 (M + H)+
15
1
—(CH2)2OBn
HCl
NMR: 3.90 (2H, t, J = 4.9 Hz), 4.45 (2H, s), 8.84 (1H,
s). ; FAB: 387 (M + H)+
16
1
—(CH2)3OMe
HCl
NMR: 2.07 (2H, tt, J = 6.9, 6.3 Hz), 3.20 (3H, s), 8.65 (1H,
d, J = 0.9 Hz). ; FAB: 325 (M + H)+
17
2
—(CH2)2N(Me)2
2HCl
NMR: 2.97 (6H, s), 4.96 (2H, brt, J = 7.8 Hz), 8.83 (1H,
s). ; FAB: 324 (M + H)+
18
1
cBu
HCl
NMR: 1.92-2.00 (1H, m), 5.49 (1H, quint, J = 8.8 Hz), 8.57
(1H, d, J = 1.6 Hz). ; ESI: 307 (M + H)+
19
1
cPen
HCl
NMR: 1.76-1.88 (2H, m), 5.47 (1H, quint, J = 9.0 Hz), 8.58
(1H, s). ; FAB: 321 (M + H)+
20
1
cHex
HCl
NMR: 1.64-1.77 (4H, m), 1.84-1.93 (4H, m), 8.80 (1H,
s). ; FAB: 335 (M + H)+
TABLE 12
21
1
HCl
NMR: 1.82 (2H, brd, J = 11.5 Hz), 4.09 (2H, brdd, J = 11.5, 2.0 Hz), 8.65-8.88 (3H, m). ; FAB: 337 (M + H)+
6
6
2HCl
NMR: 2.01 (2H, brd, J = 11.2 Hz), 5.33-5.43 (1H, m), 8.99 (1H, s). ; FAB: 336 (M + H)+
22
1
Ac
HCl
NMR: 3.00 (3H, s), 8.40 (1H, d, J = 8.3 Hz), 8.93 (1H, d,
J = 0.9 Hz). ; FAB: 295 (M + H)+
23
1
—C(O)-iPr
HCl
NMR: 1.35 (6H, d, J = 6.3 Hz), 3.88 (1H, sept, J = 6.3 Hz),
8.94 (1H, s). ; FAB: 323 (M + H)+
24
1
—S(O)2-iPr
HCl
NMR: 1.20 (6H, d, J = 6.8 Hz), 4.10 (1H, sept, J = 6.8 Hz),
8.69 (1H, d, J = 1.4 Hz). ; FAB: 359 (M + H)+
25
1
—C(O)—NMe2
HCl
NMR: 3.09 (6H, s), 7.38-7.43 (1H, m), 8.37 (1H, s). ;
FAB: 324 (M + H)+
26
1
HCl
NMR: 3.78 (1H, dd, J = 5.9, 11.2 Hz), 3.85 (1H, dd, J = 11.2, 3.9 Hz), 8.74 (1H, s). ; EI: 308 (M)+
27
2
NMR: 1.34 (3H, s), 4.54 (2H, s), 8.27 (1H, s). ; FAB: 337 (M + H)+
28
1
—CH2-cPr
HCl
NMR: 0.43-0.47 (2H, m), 4.48 (2H, d, J = 7.3 Hz), 8.79
(1H, d, J = 1.5 Hz). ; FAB: 307 (M + H)+
29
1
HCl
NMR: 5.80 (2H, s), 6.37 (1H, dd, J = 3.4, 2.0 Hz), 8.91 (1H, s). ; FAB: 333 (M + H)+
30
1
HCl
NMR: 5.62 (2H, s), 6.37 (1H, d, J = 1.5 Hz), 8.90 (1H, d, J = 1.4 Hz). ; FAB: 333 (M + H)+
31
1
—CH2CH2Ph
HCl
NMR: 3.13 (2H, t, J = 7.3 Hz), 4.74 (2H, t, J = 7.3 Hz),
8.67 (1H, d, J = 1.0 Hz). ; FAB: 357 (M + H)+
32
1
HCl
NMR: 3.18 (2H, dd, J = 11.7, 11.7 Hz), 3.79 (2H, dd, J = 11.7, 2.4 Hz), 8.84 (1H, d, J = 0.9 Hz). ; FAB: 351 (M + H)+
33
1
Bn
HCl
NMR: 5.83 (2H, s), 8.40 (1H, d, J = 8.3 Hz), 8.77 (1H,
s). ; FAB: 343 (M + H)+
34
1
—S(O)2-Me
HCl
NMR: 3.29 (3H, s), 7.48 (1H, t, J = 7.8 Hz), 8.79 (1H,
s). ; FAB: 331 (M + H)+
35
2
NMR: 4.76 (1H, s), 5.45-5.48 (1H, m), 8.73 (1H, s).; ESI: 474 (M + H)+
36
1
HCl
NMR: 3.53-3.63 (4H, m), 7.42 (1H, t, J = 7.8 Hz), 8.45 (1H, d, J = 1.0 Hz). ; FAB: 366 (M + H)+
TABLE 13
Ex
Syn
R1
Sal
Dat
37
2
5-F
HCl
NMR: 7.04 (1H, dd, J = 10.3, 7.8 Hz), 7.51 (1H,
dt, J = 7.8, 5.6 Hz), 8.35 (1H, s). ; FAB: 271
(M + H)+
38
2
7-F
HCl
NMR: 7.09 (1H, ddd, J = 9.5, 8.8, 2.5 Hz), 7.35
(1H, dd, J = 9.8, 2.5 Hz), 8.32 (1H, s). ; FAB:
271 (M + H)+
TABLE 14
Ex
Syn
R1
Sal
Dat
5
5
6-NH2
2HCl
NMR: 1.69 (6H, d, J = 6.8 Hz), 7.55 (1H, d,
J = 8.8 Hz), 8.74 (1H, s). ; FAB: 310
(M + H)+
39
2
5-F
HCl
NMR: 1.70 (6H, d, J = 6.8 Hz), 7.08 (1H,
dd, J = 10.3, 7.9 Hz), 8.74 (1H, s). ; FAB:
313 (M + H)+
40
2
6-F
HCl
NMR: 1.68 (6H, d, J = 7.4 Hz), 7.40 (1H,
dt, J = 9.3, 2.8 Hz), 8.64 (1H, s). ; FAB: 313
(M + H)+
41
2
7-F
HCl
NMR: 1.68 (6H, d, J = 6.9 Hz), 7.12 (1H,
dt, J = 9.1, 2.0 Hz), 8.64 (1H, s). ; FAB:
313 (M + H)+
42
2
8-F
HCl
NMR: 1.68 (6H, d, J = 6.9 Hz), 7.12 (1H,
dt, J = 9.1, 2.0 Hz), 8.64 (1H, s). ; FAB:
313 (M + H)+
43
2
5-Me
HCl
NMR: 1.69 (6H, d, J = 7.3 Hz), 2.85 (3H, s),
8.64 (1H, s). ; FAB: 309 (M + H)+
44
2
6-Me
HCl
NMR: 1.67 (6H, d, J = 7.3 Hz), 2.49 (3H, s),
8.62 (1H, s). ; FAB: 309 (M + H)+
45
2
7-Me
HCl
NMR: 1.69 (6H, d, J = 6.8 Hz), 2.54 (3H, s),
8.55 (1H, s). ; FAB: 309 (M + H)+
46
2
8-Me
HCl
NMR: 1.75 (6H, d, J = 6.8 Hz), 2.82 (3H, s),
8.47 (1H, s). ; FAB: 309 (M + H)+
47
2
5-OMe
HCl
NMR: 1.68 (6H, d, J = 6.8 Hz), 4.06 (3H, s),
8.61 (1H, s). ; FAB: 325 (M + H)+
48
2
6-OMe
HCl
NMR: 1.66 (6H, d, J = 6.9 Hz), 3.87 (3H, s),
8.53 (1H, s). ; ESI: 325 (M + H)+
49
2
7-OMe
HCl
NMR: 1.69 (6H, d, J = 6.8 Hz), 3.92 (3H, s),
8.56 (1H, s). ; FAB: 325 (M + H)+
50
2
8-OMe
HCl
NMR: 1.68 (6H, d, J = 6.8 Hz), 4.01 (3H, s),
8.59 (1H, s). ; ESI: 325 (M + H)+
51
1
5-CN
HCl
NMR: 1.72 (6H, d, J = 6.8 Hz), 7.72 (1H, t,
J = 7.3 Hz), 8.79 (1H, s). ; FAB: 320
(M + H)+
52
1
6-CN
HCl
NMR: 1.71 (6H, d, J = 6.8 Hz), 7.89 (1H,
dd, J = 8.8, 1.5 Hz), 8.90 (1H, d, J = 1.5
Hz). ; FAB: 320 (M + H)+
53
1
7-CN
HCl
NMR: 1.72 (6H, d, J = 7.4 Hz), 7.65 (1H,
dd, J = 8.3, 1.0 Hz), 8.71 (1H, s). ; FAB:
320 (M + H)+
54
1
8-CN
HCl
NMR: 1.83 (6H, d, J = 7.3 Hz), 7.43 (1H, t,
J = 7.8 Hz), 8.64 (1H, s). ; FAB: 320
(M + H)+
TABLE 15
55
2
5-Cl
HCl
NMR: 1.70 (6H, d, J = 6.9 Hz), 7.34 (1H, t, J = 7.8 Hz),
8.72 (1H, s).; FAB: 329 (M + H)+
56
2
6-Cl
HCl
NMR: 1.68 (6H, d, J = 6.9 Hz), 7.54 (1H, dd, J = 8.7, 1.9 Hz),
8.65 (1H, s).; FAB: 329 (M + H)+
57
2
7-Cl
HCl
NMR: 1.69 (6H, d, J = 6.8 Hz), 7.30 (1H, dd, J = 8.3, 2.0 Hz),
8.65 (1H, s).; FAB: 329 (M + H)+
58
2
8-Cl
HCl
NMR: 1.76 (6H, d, J = 7.3 Hz), 7.27 (1H, t, J = 7.8 Hz),
8.54 (1H, s).; FAB: 329 (M + H)+
59
1
6-NO2
HCl
NMR: 1.73 (6H, d, J = 6.8 Hz), 8.02 (1H, d, J = 9.2 Hz),
8.77 (1H, s).; FAB: 340 (M + H)+
60
2
5-CH2NMe2
2HCl
NMR: 1.71 (6H, d, J = 6.8 Hz), 2.89 (6H, s), 8.81 (1H, s).;
FAB: 352 (M + H)+
61
2
5-CH2OH
HCl
NMR: 1.69 (6H, d, J = 7.3 Hz), 5.10 (2H, s), 8.66 (1H, s).;
FAB: 325 (M + H)+
62
2
5-CH2OMe
HCl
NMR: 1.70 (6H, d, J = 6.8 Hz), 3.42 (3H, s), 8.65 (1H, s).;
FAB: 339 (M + H)+
63
1
5-C(O)H
HCl
NMR: 1.73 (6H, d, J = 6.8 Hz), 8.71 (1H, s), 10.44 (1H,
s).; FAB: 323 (M + H)+
TABLE 16
Ex
Syn
R5
Sal
Dat
64
1
H
HCl
NMR: 6.49 (1H, dd, J = 7.9, 1.5
Hz), 6.58-6.66 (2H, m), 7.04 (1H,
d, J = 2.4 Hz).; FAB: 269 (M +
H)+
65
1
Me
HCl
NMR: 3.14 (3H, s), 6.73-6.80 (3H,
m), 7.40 (1H, d, J = 2.0 Hz). ;
FAB: 283 (M + H)+
66
1
iPr
HCl
NMR: 1.48 (6H, d, J = 7.0 Hz),
4.37 (1H, sept, J = 7.0 Hz), 7.47
(1H, d, J = 2.0 Hz). ; ESI: 311
(M + H)+
67
1
Et
HCl
NMR: 1.15 (3H, t, J = 6.9 Hz),
3.80 (2H, q, J = 6.9 Hz), 7.40 (1H,
d, J = 1.5 Hz). ; FAB: 297
(M + H)+
68
1
cBu
HCl
NMR: 1.68-1.87 (2H, m), 4.38
(1H, tt, J = 7.3, 7.3 Hz), 7.30 (1H,
d, J = 1.4 Hz). ; FAB: 323
(M + H)+
69
2
HCl
NMR: 1.86 (2H, dd, J = 12.2, 2.5 Hz), 2.22 (1H, dd, J = 12.2, 4.4 Hz), 7.61 (1H, s). ; FAB: 353 (M + H)+
70
2
—(CH2)2—NMe2
2HCl
NMR: 2.94 (6H, s), 4.19 (2H, t,
J = 7.8 Hz), 7.49 (1H, d, J = 1.4
Hz). ; EI: 339 (M)+
71
2
NMR: 4.11 (2H, d, J = 5.9 Hz), 4.56 (2H, d, J = 5.9 Hz), 7.28 (1H, d, J = 1.4 Hz). ; FAB: 353 (M + H)+
TABLE 17
Ex
Syn
R5
Sal
Dat
72
1
H
HCl
NMR: 6.51-6.54 (2H, m), 6.64-6.69 (2H, m),
7.29 (1H, d, J = 1.9 Hz). ; FAB: 269 (M + H)+
73
1
Me
HCl
NMR: 3.12 (3H, s), 6.76 (1H, dt, J = 7.3, 1.5
Hz), 7.37 (1H, d, J = 2.0 Hz). ; FAB: 283
(M + H)+
TABLE 18
Ex
Syn
Str
Sal
Dat
74
1
HCl
NMR: 1.76-1.89 (4H, m), 2.63-2.68 (2H, m), 8.15 (1H, d, J = 1.5 Hz). ; FAB: 257 (M + H)+
75
1
HCl
NMR: 1.71-1.79 (2H, m), 1.79-1.86 (2H, m), 7.11 (1H, t, J = 7.9 Hz). ; FAB: 257 (M + H)+
76
1
HCl
NMR: 1.20 (3H, t, J = 6.9 Hz), 2.86 (2H, brt, J = 5.3 Hz), 7.18 (1H, t, J = 7.8 Hz). ; FAB: 330 (M + H)+
77
1
HCl
NMR: 1.22 (3H, t, J = 6.9 Hz), 2.87 (2H, brt, J = 5.6 Hz), 8.18 (1H, s). ; FAB: 330 (M + H)+
TABLE 19
Ex
Syn
Str
Sal
Dat
78
1
HCl
NMR: 7.54 (1H, d, J = 4.4 Hz), 8.05 (1H, s), 8.28 (1H, d, J = 4.4 Hz). ; FAB: 272 (M + H)+
7
7
HCl
NMR: 5.63 (1H, brs), 7.45 (1H, d, J = 4.9 Hz), 8.12 (1H, s). ; FAB: 274 (M + H)+
79
1
NMR: 7.22 (1H, d, J = 4.9 Hz), 7.64 (1H, d, J = 4.9 Hz), 8.10 (1H, s). ; ESI: 272 (M + H)+
80
7
HCl
NMR: 5.45 (1H, s), 7.28 (1H, d, J = 4.9 Hz), 8.14 (1H, s). ; ESI: 274 (M + H)+
81
1
HCl
NMR: 3.78-3.85 (2H, m), 7.33 (1H, d, J = 5.2 Hz), 8.44 (1H, d, J = 1.6 Hz). ; FAB: 348 (M + H)+
82
1
HCl
NMR: 1.63 (3H, s), 7.24 (1H, d, J = 5.2 Hz), 8.16 (1H, d, J = 1.2 Hz). ; FAB: 288 (M + H)+
83
1
HCl
NMR: 1.66 (3H, s), 2.75 (3H, s), 8.17 (1H, s). ; FAB: 302 (M + H)+
84
1
HCl
NMR: 4.11-4.16 (1H, m), 7.26 (1H, d, J = 4.9 Hz), 8.27 (1H, d, J = 1.3 Hz). ; FAB: 314 (M + H)+
85
1
NMR: 7.92 (1H, d, J = 4.4 Hz), 8.40 (1H, s), 8.80 (1H, s). ; FAB: 267 (M + H)+
86
7
2HCl
FAB: 269 (M + H)+
TABLE 20
No
R5
1
—C(O)Ph
2
—S(O)2Ph
TABLE 21
No
R5
R1
3
Ac
null
4
—S(O)2Me
null
5
—C(O)iPr
null
6
—S(O)2iPr
null
7
iPr
6-F
8
iPr
8-F
9
iPr
7-Me
10
iPr
9-Me
TABLE 22
No
11
12
13
14
TABLE 23
No
15
16
17
18
TABLE 24
No
Str
19
20
21
22
INDUSTRIAL APPLICABILITY
Since the compound of the present invention has excellent antagonism to both of 5-HT2B and 5-HT7 receptors, it is useful as a pharmaceutical, particularly as an agent for treating IBS and/or an agent for preventing migraine.
|
11997956
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astellas pharma inc.
|
USA
|
B2
|
Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
|
Open
|
514/277
|
|
Apr 1st, 2022 05:13PM
|
Apr 1st, 2022 05:13PM
|
Astellas Pharma
|
|
|
|
tyo:4503
|
Astellas Pharma
|
Nov 4th, 2014 12:00AM
|
Mar 28th, 2011 12:00AM
|
https://www.uspto.gov?id=US08877214-20141104
|
Pharmaceutical composition for modified release
|
A pharmaceutical composition for modified release comprising (R)-2-(2-aminothiazol-4-yl)-4′-[2-[(2-hydroxy-2-phenylethyl)amino]ethyl]acetic acid anilide or a pharmaceutically acceptable salt thereof, and a carrier for a sustained release pharmaceutical composition, wherein a dissolution rate of the drug from the composition is less than 85% after 30 minutes from the beginning of a dissolution test, is disclosed.
|
8877214
|
1. A pharmaceutical composition for modified release comprising a drug, which is (R)-2-(2-aminothiazol-4-yl)-4′-[(2-[2-hydroxy-2-phenylethyl)amino]ethyl]acetic acid anilide or a pharmaceutically acceptable salt thereof, and a carrier for a sustained release pharmaceutical composition, wherein a dissolution rate of the drug from the composition is less than 85% after 30 minutes from the beginning of a dissolution test, and said pharmaceutical composition is selected from the group consisting of a gel formulation in which a plurality of gums is combined, an osmotic pump type formulation, a formulation utilizing a swelling polymer, a matrix formulation utilizing a water-soluble polymer and a matrix formulation utilizing an insoluble polymer.
2. The pharmaceutical composition for modified release according to claim 1, wherein a dissolution rate is 75% or less after 1.5 hours from the beginning of the dissolution test.
3. The pharmaceutical composition for modified release according to claim 1, wherein the dissolution rate is 75% or less after 1.5 hours from the beginning the dissolution test, and a dissolution rate is 75% to 100% after 7 hours from the beginning of the dissolution test.
4. A method of reducing an effect of food intake, comprising the step of administering a pharmaceutical composition comprising a drug, which is (R)-2-(2-aminothiazol-4-yl)-4′-[(2-hydroxy-2-phenylethyl)amino]ethyl]acetic acid anilide or a pharmaceutically acceptable salt thereof, and a carrier for a sustained release pharmaceutical composition, wherein a dissolution rate of the drug from the composition is less than 85% after 30 minutes from the beginning of a dissolution test, and said pharmaceutical composition is selected from the group consisting of a gel formulation in which a plurality of gums is combined, an osmotic pump type formulation, a formulation utilizing a swelling polymer, a matrix formulation utilizing a water-soluble polymer and a matrix formulation utilizing an insoluble polymer.
5. The method of reducing an effect of food intake according to claim 4, wherein a dissolution rate is 75% or less after 1.5 hours form the beginning of the dissolution test.
6. The method of reducing an effect of food intake according to claim 4, wherein the dissolution rate is 75% or less after 1.5 hours from the beginning the dissolution test, and a dissolution rate is 75% to 100% after 7 hours from the beginning of the dissolution test.
7. The pharmaceutical composition for modified release according to claim 1, which is the gel formulation in which a plurality of gums is combined, wherein the carrier for a sustained release pharmaceutical composition is a gum base which is a sustained release filler comprising a homopolysaccharide which can form a crosslinkage with a heteropolysaccharide gum when exposed to the heteropolysaccharide gum and environmental fluids.
8. The pharmaceutical composition for modified release according to claim 7, wherein the sustained release filler further comprises calcium sulfate and/or a water-soluble base.
9. The pharmaceutical composition for modified release according to claim 1, which is the osmotic pump type formulation, wherein a bilayered compressed core consisting of a drug layer and a push layer is coated with a semipermeable membrane.
10. The pharmaceutical composition for modified release according to claim 9, wherein the push layer contains one or more osmotic active components.
11. The pharmaceutical composition for modified release according to claim 1, which is the formulation utilizing a swelling polymer, wherein the swelling polymer is a water-soluble high molecular weight polymer which swells upon imbibition of water.
12. The pharmaceutical composition for modified release according to claim 11, wherein the swelling polymer has a weight average molecular weight of approximately 4,500,000 or more.
13. The pharmaceutical composition for modified release according to claim 1, which is the matrix formulation utilizing a water-soluble polymer, wherein the drug is homogenously dispersed in one or more water-soluble polymers.
14. The pharmaceutical composition for modified release according to claim 13, wherein the water-soluble polymer is gradually gelled, eroded, dissolved, and/or disintegrated when exposed to an environmental fluid.
15. The pharmaceutical composition for modified release according to claim 1, which is the modified release formulation with a coating membrane, wherein a coating liquid contains a membrane forming agent, a plasticizer, a water-soluble base, or a dispersing agent.
16. The pharmaceutical composition for modified release according to claim 1, which is the matrix formulation utilizing an insoluble polymer, wherein the drug is uniformly dispersed in a water-insoluble polymer.
17. The pharmaceutical composition for modified release according to claim 16, wherein the water-insoluble polymer is selected from the group consisting of dibenzyl phthalate, dihexyl phthalate, butyl octyl phthalate, beeswax, carnauba wax, cetyl alcohol, cetyl stearyl alcohol, glyceryl behenate, lipids, fats, resins, cellulose derivatives, polyacrylate derivatives, polymethacrylate derivatives, hydroxypropylmethyl cellulose acetate succinate, polylactic acid, and polyglycolic acid.
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17
|
TECHNICAL FIELD
The present invention relates to a pharmaceutical composition for modified release capable of reducing food effects observed in conventional tablets, by combining an active ingredient with one or more excipients and controlling a releasing rate of the active ingredient.
More particularly, the present invention relates to a pharmaceutical composition for modified release comprising (R)-2-(2-aminothiazol-4-yl)-4′-[2-[(2-hydroxy-2-phenylethyl)amino]ethyl]acetic acid anilide or a pharmaceutically acceptable salt thereof, and a carrier for a sustained release pharmaceutical composition, in which the changes of an area under a blood drug concentration versus time curve (AUC) and a maximum blood drug concentration (Cmax) by the intake of food are reduced by controlling a releasing rate of the active ingredient.
BACKGROUND ART
(R)-2-(2-aminothiazol-4-yl)-4′-[2-[(2-hydroxy-2-phenylethyl)amino]ethyl]acetic acid anilide has been created by Astellas Pharma Inc., and it has been reported that this compound has not only both an activity of promoting insulin secretion and an activity of enhancing insulin sensitivity, but also an antiobestic activity and an antihyperlipemic activity based on an activity of selectively stimulating a β3 receptor, and is useful in treating diabetes (see, for example, patent literature 1).
Further, it has been reported that the compound can be used as a therapeutic agent for overactive bladder, such as overactive bladder accompanied by prostatic hyperplasia, or overactive bladder accompanied by urinary urgency, urinary incontinence, and urinary frequency (see, for example, patent literature 2).
A clinical trial of (R)-2-(2-aminothiazol-4-yl)-4′-[2-[(2-hydroxy-2-phenylethyl)amino]ethyl]acetic acid anilide in, the form of conventional formulations revealed that pharmacokinetic data unexpectedly varied according to the presence or absence of the intake of food. For example, the rate of decrease of Cmax in a fed state was 67%, and the rate of decrease of AUC in the fed state was 47%, in comparison with those in a fasted state. In this case, Cmax in the fasted state was three times higher than that in the fed state. These problems are considered to be raised by, for example, the changes in pharmacokinetics caused by food, and therefore, the development of a formulation capable of avoiding the effects by food intake is desired.
As a technique of preparing a formulation for modified release, a hydrogel sustained release tablet containing an additive which ensures penetration of water into the tablet, and a hydrogel-forming polymer is disclosed (see, for example, patent literature 3).
However, patent literature 3 does not refer to (R)-2-(2-aminothiazol-4-yl)-4′-[2-[(2-hydroxy-2-phenylethyl)amino]ethyl]acetic acid anilide, and further improvements are needed to produce a pharmaceutical composition.
CITATION LIST
Patent Literature
[patent literature 1] International Publication No. WO 99/20607 (Example 41)
[patent literature 2] International Publication No. WO 2004/041276
[patent literature 3] International Publication No. WO 94/06414
SUMMARY OF INVENTION
Technical Problem
An object of the present invention is to provide a pharmaceutical composition for modified release comprising (R)-2-(2-aminothiazol-4-yl)-4′-[2-[(2-hydroxy-2-phenylethyl)amino]ethyl]acetic acid anilide or a pharmaceutically acceptable salt thereof, in which the pharmaceutical composition has efficacy the same as or higher than those of conventional formulations and has no limitations on food intake.
Solution to Problem
The elimination half-life (T1/2) of (R)-2-(2-aminothiazol-4-yl)-4′-[2-[(2-hydroxy-2-phenylethyl)amino]ethyl]acetic acid anilide is long (approximately 18 to 24 hours), and thus, a formulation thereof for modified release is not necessarily needed to maintain its blood level. Taking into consideration the results of the clinical trial described above, the present inventors conducted intensive studies to design the formulation by paying attention to the control of a release rate of the drug from a formulation to the extent that the release is not affected by food intake or the like, rather than the addition of release control.
On the basis of blood concentration profiles (in a fasted state/after the intake of food) after administration of a conventional formulation (immediate release formulation), the absorption rate of the drug in a fed state was calculated by a deconvolution method to predict continuous absorption for about 4 hours. The present inventors considered from this result that a formulation capable of continuous drug release for 4 hours or more would be able to reduce the effects by food, because the drug release from the formulation would become the rate-limiting step for absorption.
The present inventors carried out a clinical trial in human using three types of formulations in which the release rate of the drug was controlled, and found that all formulations could reduce the effects by food, to complete the present invention.
It is generally known that the retention time in the stomach and the release rate of formulations for modified release vary according to the presence or absence of food intake, and as a result, there is a possibility that blood concentration profiles is changed. However, surprisingly, when using this formulation, the change of the blood concentration profiles was small in the presence or absence of food intake.
The present invention is characterized by providing a pharmaceutical composition for modified release which is not affected by the effects of food intake and exhibits a decreased change in AUC or Cmax, by controlling the releasing rate of the active ingredient.
The present invention provides:
[1] a pharmaceutical composition for modified release comprising (R)-2-(2-aminothiazol-4-yl)-4′-[2-[(2-hydroxy-2-phenylethyl)amino]ethyl]acetic acid anilide or a pharmaceutically acceptable salt thereof, and a carrier for a sustained release pharmaceutical composition, wherein a dissolution rate of the drug from the composition is less than 85% after 30 minutes from the beginning of a dissolution test,
[2] the pharmaceutical composition for modified release of [1], wherein a dissolution rate is 75% or less after 1.5 hours from the beginning of the dissolution test,
[3] the pharmaceutical composition for modified release of [1], wherein the dissolution rate is 75% or less after 1.5 hours from the beginning the dissolution test, and a dissolution rate is 75% to 100% after 7 hours from the beginning of the dissolution test,
[4] the pharmaceutical composition for modified release of any one of [1] to [3], which is selected from the group consisting of a sustained release hydrogel-forming formulation, a multi-layered formulation consisting of a drug core and a release-controlling layer which are geometrically arranged, a gel formulation in which a plurality of gums is combined, an osmotic pump type formulation, a formulation utilizing a swelling polymer, a matrix formulation utilizing a water-soluble polymer, a modified release formulation with a coating membrane, and a matrix formulation utilizing an insoluble polymer,
[5] a method of reducing an effect of food intake, comprising the step of administering a pharmaceutical composition comprising (R)-2-(2-aminothiazol-4-yl)-4′-[2-[(2-hydroxy-2-phenylethyl)amino]ethyl]acetic acid anilide or a pharmaceutically acceptable salt thereof, and a carrier for a sustained release pharmaceutical composition, wherein a dissolution rate of the drug from the composition is less than 85% after 30 minutes from the beginning of a dissolution test,
[6] the method of reducing an effect of food intake of [5], wherein a dissolution rate is 75% or less after 1.5 hours from the beginning of the dissolution test,
[7] the method of reducing an effect of food intake of [5], wherein the dissolution rate is 75% or less after 1.5 hours from the beginning the dissolution test, and a dissolution rate is 75% to 100% after 7 hours from the beginning of the dissolution test, and
[8] the method of reducing an effect of food intake of any one of [5] to [7], wherein the pharmaceutical composition is selected from the group consisting of a sustained release hydrogel-forming formulation, a multi-layered formulation consisting of a drug core and a release-controlling layer which are geometrically arranged, a gel formulation in which a plurality of gums is combined, an osmotic pump type formulation, a formulation utilizing a swelling polymer, a matrix formulation utilizing a water-soluble polymer, a modified release formulation with a coating membrane, and a matrix formulation utilizing an insoluble polymer.
As formulation techniques for reducing or avoiding the changes in pharmacokinetics such as AUC or Cmax accompanied by food intake, a formulation technique concerning a sustained-release pharmaceutical composition containing tamsulosin hydrochloride is disclosed (see Japanese Unexamined Patent Publication (Kokai) No. 2005-162736 and Japanese Unexamined Patent Publication (Kokai) No. 2005-162737). This formulation technique is limited to tamsulosin, and applied to a formulation containing the drug at a low dose (0.4 mg per unit formulation). This formulation enables to control the release of tamsulosin therefrom by being mainly composed of a sustained-release base. By contrast, the pharmaceutical composition contains the drug at a high dose (i.e., high content per unit formulation), and it is considered difficult to control the release rate of the drug from a formulation containing the sustained-release base at a low content, and therefore, the present invention is technically quite different from the formulation disclosed in these references.
Advantageous Effects of Invention
According to the present invention, a pharmaceutical composition for modified release which has no limitations on food intake and which is capable of reducing the changes in Cmax and AUC can be provided.
With respect to a conventional formulation, the rate of decrease of Cmax in a fed state was 67% in comparison with that in a fasted state. By contrast, with respect to the pharmaceutical composition for modified release of the present invention, the rate of decrease of Cmax in a fed state was 4% or 10% in comparison with that in a fasted state, and this result showed that reduction of Cmax caused by food intake could be significantly alleviated by forming its formulation into the pharmaceutical formulation for modified release.
With respect to a conventional formulation, the rate of decrease of AUC in a fed state was 47% in comparison with that in a fasted state. By contrast, with respect to the pharmaceutical composition for modified release of the present invention, the rate of decrease of AUC in a fed state was 10% or −4% in comparison with that in a fasted state, and this result showed that reduction of AUC caused by food intake could be significantly alleviated by forming its formulation into the pharmaceutical formulation for modified release.
BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 is a graph showing the drug release property from each of the formulations prepared in Examples 1A to 1C in Experimental Example 1.
FIG. 2 is a graph showing the drug release property from each of the formulations prepared in Examples 2A to 2D in Experimental Example 2.
FIG. 3 is a graph showing the drug release property from the formulation prepared in Example 3 in Experimental Example 3.
FIG. 4 is a graph showing the drug release property from the formulation prepared in Example 4 in Experimental Example 4.
FIG. 5 is a graph showing the drug release property from each of the formulations prepared in Examples 5A to 5C in Experimental Example 5.
FIG. 6 is a graph showing the drug release property from each of the formulations prepared in Examples 6A to 6G in Experimental Example 6.
FIG. 7 is a graph showing the drug release property from each of the formulations prepared in Examples 6H to 6N in Experimental Example 6.
FIG. 8 is a graph showing the drug release property from each of the formulations prepared in Examples 7A to 7E in Experimental Example 7.
FIG. 9 is a graph showing the drug release property from each of the formulations prepared in Examples 8A to 8C in Experimental Example 8.
FIG. 10 is a graph showing the drug release property from each of the formulations prepared in Examples 8D to 8G in Experimental Example 8.
FIG. 11 is a graph showing the relation between Cmax and the increase in heart rate from the base line in Experimental Example 9 (a dotted line shows 95% confidence interval).
FIG. 12 is a graph showing blood concentration profiles after the administration of the formulation of Example 1A in a fasted state or after 30 minutes from the intake of food in Experimental Example 10.
FIG. 13 is a graph showing the blood concentration profiles after the administration of the formulation of Example 1B in a fasted state or after 30 minutes from the intake of food in Experimental Example 10.
DESCRIPTION OF EMBODIMENTS
The pharmaceutical composition for modified release of the present invention will be explained hereinafter.
The term “immediate release formulation (conventional formulation)” as used herein means a formulation in which the dissolution rate of the drug from the formulation is 85% or more after 30 minutes from the beginning a dissolution test, which is carried out in accordance with a dissolution test (paddle method) described in the United States Pharmacopoeia under the conditions that 900 mL of an appropriate test fluid (such as a USP buffer, pH 6.8) is used and the paddle rotation speed is 100 rpm. Alternatively, the term means a formulation in which the dissolution rate of the drug from the formulation is 85% or more after 30 minutes from the beginning a dissolution test, which is carried out in accordance with a dissolution test, method 2 described in the Japanese Pharmacopoeia under the conditions that 900 mL of an appropriate test fluid (such as a Mc. Ilvain buffer, pH 6.8) is used and the paddle rotation speed is 50 rpm. Alternatively, the term means a formulation in which the dissolution rate of the drug from the formulation is 85% or more after 30 minutes from the beginning a dissolution test, which is carried out in accordance with a dissolution test, method 2 (paddle method) described in the Japanese Pharmacopoeia under the conditions that 900 mL of a USP phosphate buffer (pH 6.8) is used as a test fluid and the paddle rotation speed is 200 rpm.
The term “pharmaceutical composition for modified release” as used herein means a formulation in which the dissolution rate of the drug from the formulation is less than 85% after 30 minutes from the beginning a dissolution test carried out under the above conditions, and the drug release is controlled to the extent that the effects by food are reduced.
The wording “the effects by food are reduced” as used herein means, for example, a reduction by 10% or more, a reduction by 20% or more in another embodiment, and a reduction by 30% or more in still another embodiment, in comparison with Cmax of a conventional formulation. Alternatively, the term means, for example, a reduction by 10% or more with respect to the rates of decrease of Cmax and AUC in administration after food intake, in comparison with Cmax and AUC in administration in the fasted state, a reduction by 20% or more in another embodiment, and a reduction by 30% or more in still another embodiment.
The rates of decrease of Cmax and AUC are calculated by the following equations:
Rd(Cmax)=[Cmax(FS)−Cmax(FI)]×100/Cmax(FS)
Rd(AUC)=[AUC(FS)−AUC(FI)]×100/AUC(FS)
Rd(Cmax): Rate of decrease of Cmax (%)
Cmax(FS): Cmax in administration in the fasted state
Cmax(FI): Cmax in administration after food intake
Rd(AUC): Rate of decrease of AUC (%)
AUC(FS): AUC in administration in the fasted state
AUC(FI): AUC in administration after food intake
The term “formulation in which the effects by food are reduced” as used herein means a formulation in which the dissolution rate of the drug from the formulation is less than 85% after 30 minutes from the beginning a dissolution test, which is carried out under the above conditions. In another embodiment, it means a formulation in which the dissolution rate of the drug from the formulation is 75% or less after 1.5 hours from the beginning a dissolution test. In still another embodiment, it means a formulation in which the dissolution rate of the drug from the formulation is 75% or less after 1.5 hours and 75% to 100% after 7 hours from the beginning a dissolution test.
(R)-2-(2-aminothiazol-4-yl)-4′-[2-[(2-hydroxy-2-phenylethyl)amino]ethyl]acetic acid anilide (hereinafter sometimes referred to as compound A) is represented by the following structural formula.
Compound A may be used in a free form which is not a salt, and may form a salt with an acid in other embodiments. Examples of such a salt include an acid addition salt with a mineral acid such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid, or the like; and an acid addition salt with an organic acid such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, citric acid, tartaric acid, carbonic acid, picric acid, methanesulfonic acid, ethanesulfonic acid, glutamic acid, or the like.
The dose of compound A may be appropriately selected in accordance with symptom, age, sex, and the like of the patient to be treated. The daily dose of compound A for oral administration to an adult is generally 0.01 to 100 mg/kg, which is administered once or divided into two to four doses per day.
The content of compound A per formulation is, for example, 1% by weight to 70% by weight, 5% by weight to 70% by weight in another embodiment, and 5% by weight to 50% by weight in still another embodiment. The content of compound A per formulation is 1 mg to 500 mg, and 10 mg to 200 mg in another embodiment.
A carrier for a sustained release pharmaceutical composition, which is contained in the pharmaceutical composition for modified release of the present invention together with compound A or a pharmaceutically acceptable salt thereof, is not particularly limited, so long as it is a carrier, a pharmaceutical formulation, or a technique for manufacturing pharmaceutical preparations capable of achieving a specific release rate.
Examples of such a carrier (or a pharmaceutical formulation, or a technique for manufacturing pharmaceutical preparations) which forms the composition or components in the present invention include, for example,
(1) a sustained release hydrogel-forming formulation in which the formulation is almost completely gelled during the retention in the stomach and the small intestine of the upper digestive tract and the drug can be released in the colon of the lower digestive tract,
(2) a multi-layered formulation consisting of a drug core and a release-controlling layer which are geometrically arranged,
(3) a gel formulation in which a plurality of gums is combined,
(4) an osmotic pump type formulation,
(5) a formulation utilizing a swelling polymer,
(6) a matrix formulation utilizing a water-soluble polymer,
(7) a modified release formulation with a coating membrane,
(8) a matrix formation utilizing an insoluble polymer, and the like, as described in detail below. The compositions relating to these techniques for manufacturing pharmaceutical preparations, and the techniques per se are incorporated herein by reference.
Hereinafter, each embodiment of the pharmaceutical composition for modified release of the present invention will be explained in detail. Each embodiment described below is mainly explained with reference to cases using compound A as the active ingredient, but instead of compound A, a pharmaceutically acceptable salt thereof may be used.
(1) Sustained Release Hydrogel-Forming Formulation
The sustained release hydrogel-forming formulation contains, as the carrier for a sustained release pharmaceutical composition, an additive that allows water to penetrate into the formulation (designated as a gelling agent, a promoting agent for gelling, and a hydrophilic base, but hereinafter referred to as hydrophilic base), and a polymer which forms a hydrogel (hereinafter referred to as hydrogel-forming polymer).
It is necessary that the hydrogel-forming polymer used in the present invention can control the release rate of the drug, to the extent that the blood concentration profile of the drug is not affected by the presence or absence of food intake.
The molecular weight of the hydrogel-forming polymer is, for example, 100,000 or more, 100,000 to 8,000,000 in another embodiment, 100,000 to 5,000,000 in still another embodiment, and 100,000 to 2,000,000 in still another embodiment. The viscosity of the hydrogel-forming polymer is, for example, 12 mPa·s or more in a 5% aqueous solution at 25° C.; 12 mPa·s or more in a 5% aqueous solution at 25° C., and 40,000 mPa·s or less in a 1% aqueous solution at 25° C. in another embodiment; 400 mPa·s or more in a 2% aqueous solution at 25° C., and 7,500 mPa·s or less in a 1% aqueous solution at 25° C. in still another embodiment; and 400 mPa·s or more in a 2% aqueous solution at 25° C., and 5,500 mPa·s or less in a 1% aqueous solution at 25° C. in still another embodiment.
In the pharmaceutical composition for modified release of the present invention, the release period of time of the drug from the formulation can be arbitrarily controlled by adjusting the viscosity of the polymer which is used as the hydrogel-forming polymer.
The hydrogel-forming polymer used in the present invention is not particularly limited, so long as the release of the drug can be controlled to the extend that the effects of food on compound A may be reduced. Examples of the hydrogel-forming polymer include polyethylene oxide, hypromellose, hydroxypropyl cellulose, carboxymethyl cellulose sodium, hydroxyethyl cellulose, and carboxyvinyl polymers. Examples of the hydrogel-forming polymer in another embodiment include polyethylene oxide, hypromellose, and hydroxypropyl cellulose.
Examples of polyethylene oxide (hereinafter sometimes referred to as PEO) include product names, Polyox WSR-308 [average molecular weight: 8,000,000, viscosity: 10,000-15,000 mPa·s (1% aqueous solution at 25° C.)], Polyox WSR-303 [average molecular weight: 7,000,000, viscosity: 7,500-10,000 mPa·s (1% aqueous solution at 25° C.)], Polyox WSR Coagulant [average molecular weight: 5,000,000, viscosity: 5,500-7,500 mPa·s (1% aqueous solution at 25° C.)], Polyox WSR-301 [average molecular weight: 4,000,000, viscosity: 1,650-5,500 mPa·s (1% aqueous solution at 25° C.)], Polyox WSR-N-60K [average molecular weight: 2,000,000, viscosity: 2,000-4,000 mPa·s (2% aqueous solution at 25° C.)], Polyox WSR-N-12K [average molecular weight: 1,000,000, viscosity: 400-800 mPa·s (2% aqueous solution at 25° C.)], Polyox WSR-1105 [average molecular weight: 900,000, viscosity: 8,800-17,600 mPa·s (5% aqueous solution at 25° C.)], Polyox WSR-205 [average molecular weight: 600,000, viscosity: 4,500-8,800 mPa·s (5% aqueous solution at 25° C.)], Polyox WSR-N-750 [average molecular weight: 300,000, viscosity: 600-1200 mPa·s (5% aqueous solution at 25° C.)], Polyox WSR-N-80 [average molecular weight: 200,000, viscosity: 55-90 mPa·s (5% aqueous solution at 25° C.)], and Polyox WSR-N-10 [average molecular weight: 100,000, viscosity: 12-50 mPa·s (5% aqueous solution at 25° C.)] (DOW).
Examples of hypromellose (hereinafter sometimes referred to as HPMC) include product name Metolose 90SH50000 [viscosity in a 2% aqueous solution at 20° C.: 2,900-3,900 mPa·s], Metolose SB-4 (product name, Shin-Etsu Chemical Co., Ltd.) (viscosity in a 2% aqueous solution at 20° C.: approximately 4 mPa·S), TC-5RW (product name, Shin-Etsu Chemical Co., Ltd.) (viscosity in a 2% aqueous solution at 20° C.: approximately 6 mPa·S), TC-5S (product name, Shin-Etsu Chemical Co., Ltd.) (viscosity in a 2% aqueous solution at 20° C.: approximately 15 mPa·S), TC-5R (product name, Shin-Etsu Chemical Co., Ltd.) (viscosity in a 2% aqueous solution at 20° C.: approximately 6 mPa·S), TC-5M (product name, Shin-Etsu Chemical Co., Ltd.) (viscosity in a 2% aqueous solution at 20° C.: approximately 4.5 mPa·S), TC-5E (product name, Shin-Etsu Chemical Co., Ltd.) (viscosity in a 2% aqueous solution at 20° C.: approximately 3 mPa·S), Metolose 60SH-50 (product name, Shin-Etsu Chemical Co., Ltd.) (viscosity in a 2% aqueous solution at 20° C.: approximately 50 mPa·s), Metolose 65SH-50 (product name, Shin-Etsu Chemical Co., Ltd.) (viscosity in a 2% aqueous solution at 20° C.: approximately 50 mPa·s), Metolose 90SH-100 (product name, Shin-Etsu Chemical Co., Ltd.) (viscosity in a 2% aqueous solution at 20° C.: approximately 100 mPa·s), Metolose 90SH-100SR (product name, Shin-Etsu Chemical Co., Ltd.) (viscosity in a 2% aqueous solution at 20° C.: approximately 100 mPa·s), Metolose 65SH-400 (product name, Shin-Etsu Chemical Co., Ltd.) (viscosity in a 2% aqueous solution at 20° C.: approximately 400 mPa·s), Metolose 90SH-400 (product name, Shin-Etsu Chemical Co., Ltd.) (viscosity in a 2% aqueous solution at 20° C.: approximately 400 mPa·s), Metolose 65SH-1500 (product name, Shin-Etsu Chemical Co., Ltd.) (viscosity in a 2% aqueous solution at 20° C.: approximately 1,500 mPa·s), Metolose 60SH-4000 (product name, Shin-Etsu Chemical Co., Ltd.) (viscosity in a 2% aqueous solution at 20° C.: approximately 4,000 mPa·s), Metolose 65SH-4000 (product name, Shin-Etsu Chemical Co., Ltd.) (viscosity in a 2% aqueous solution at 20° C.: approximately 4,000 mPa·s), Metolose 90SH-4000 (product name, Shin-Etsu Chemical Co., Ltd.) (viscosity in a 2% aqueous solution at 20° C.: approximately 4,000 mPa·s), Metolose 90SH-4000SR (product name, Shin-Etsu Chemical Co., Ltd.) (viscosity in a 2% aqueous solution at 20° C.: approximately 4,000 mPa·s), Metolose 90SH-15000 (product name, Shin-Etsu Chemical Co., Ltd.) (viscosity in a 2% aqueous solution at 20° C.: approximately 15,000 mPa·s), Metolose 90SH-15000SR (product name, Shin-Etsu Chemical Co., Ltd.) (viscosity in a 2% aqueous solution at 20° C.: approximately 15,000 mPa·s), and Metolose 90SH-30000 (product name, Shin-Etsu Chemical Co., Ltd.) (viscosity in a 2% aqueous solution at 20° C.: approximately 30,000 mPa·s).
Examples of hydroxypropyl cellulose (hereinafter sometimes referred to as HPC) include HPC-SSL (product name, Nippon Soda Co., Ltd.) (viscosity in a 2% aqueous solution at 20° C.: 2.0-2.9 mPa·S), HPC-SL (product name, Nippon Soda Co., Ltd.) (viscosity in a 2% aqueous solution at 20° C.: 3.0-5.9 mPa·S), HPC-L (product name, Nippon Soda Co., Ltd.) (viscosity in a 2% aqueous solution at 20° C.: 6.0-10.0 mPa·S), HPC-M (product name, Nippon Soda Co., Ltd.) (viscosity in a 2% aqueous solution at 20° C.: 150-400 mPa·S), and HPC-H (product name, Nippon Soda Co., Ltd.) (viscosity in a 2% aqueous solution at 20° C.: 1,000-4,000 mPa·S).
Examples of methylcellulose (hereinafter sometimes referred to as MC) include Metolose SM15 (product name, Shin-Etsu Chemical Co., Ltd.) (viscosity in a 2% aqueous solution at 20° C.: approximately 15 mPa·S), Metolose SM25 (product name, Shin-Etsu Chemical Co., Ltd.) (viscosity in a 2% aqueous solution at 20° C.: approximately 25 mPa·S), Metolose SM100 (product name, Shin-Etsu Chemical Co., Ltd.) (viscosity in a 2% aqueous solution at 20° C.: approximately 100 mPa·S), Metolose SM400 (product name, Shin-Etsu Chemical Co., Ltd.) (viscosity in a 2% aqueous solution at 20° C.: approximately 400 mPa·S), Metolose SM1500 (product name, Shin-Etsu Chemical Co., Ltd.) (viscosity in a 2% aqueous solution at 20° C.: approximately 1,500 mPa·S), and Metolose SM4000 (product name, Shin-Etsu Chemical Co., Ltd.) (viscosity in a 2% aqueous solution at 20° C.: approximately 4,000 mPa·S).
Examples of carboxymethyl cellulose sodium (hereinafter sometimes referred to as CMCNa) include product names, Sunrose F-30MC [viscosity: 250-350 mPa·s (1% aqueous solution at 25° C.)], Sunrose F-150MC [average molecular weight: 200,000, viscosity: 1,200-1,800 mPa·s (1% aqueous solution at 25° C.)], Sunrose F-600MC [viscosity: 6,000-8,000 mPa·s (1% aqueous solution at 25° C.)], Sunrose F-1000MC [average molecular weight: 420,000, viscosity: 8,000-12,000 mPa·s (the same)], Sunrose F-1400MC [viscosity: 12,000-15,000 mPa·s (1% aqueous solution at 25° C.)], and Sunrose F-300MC [average molecular weight: 300,000, viscosity: 2,500-3,000 mPa·s (the same)](Nippon Paper Chemicals Co., Ltd.).
Examples of hydroxyethyl cellulose (hereinafter sometimes referred to as HEC) include product names, HEC DAICEL SE850 [average molecular weight: 1,480,000, viscosity: 2,400-3,000 mPa·s (1% aqueous solution at 25° C.)], and HEC DAICEL SE900 [average molecular weight: 1,560,000, viscosity: 4,000-5,000 mPa·s (1% aqueous solution at 25° C.)](Daicel chemical Industries, Ltd.).
Examples of carboxyvinyl polymers include Carbopol 71G (viscosity: 4,000-11,000 mPa·s), Carbopol 971P (viscosity: 4,000-11,000 mPa·s), Carbopol 981 (viscosity: 4,000-10,000 mPa·s), Carbopol 941 (viscosity: 4,000-10,000 mPa·s), Carbopol 934 (viscosity: 30,500-39,400 mPa·s), and Carbopol 934P (viscosity: 29,400-39,400 mPa·s) (B.F. Goodrich Chemical).
These hydrogel-forming polymers may be used alone, or as an appropriate combination of two or more thereof. A combination of different lots may be used.
The content of the hydrogel-forming polymer is not particularly limited, so long as it is an amount to the extent that the blood concentration profile of the drug is not affected by the presence or absence of food intake. The content of the hydrogel-forming polymer is, for example, 1% by weight to 70% by weight with respect to the total weight of the formulation, and 3% by weight to 70% by weight in another embodiment. The content of the hydrogel-forming polymer is 5% by weight to 70% by weight with respect to the total weight of the formulation, 10% by weight to 60% by weight in another embodiment, and 10% by weight to 40% by weight in still another embodiment. The content of the hydrogel-forming polymer is 0.1% by weight to 1,000% by weight with respect to the weight of the drug, 1% by weight to 500% by weight in another embodiment, and 5% by weight to 300% by weight in still another embodiment.
A polymer of which the viscosity (before mixing) is beyond the specific range can be used as an appropriate combination with one or more other polymers, in case that the mixture obtained by mixing these plural polymers has a viscosity (as measured before the use) within the specific range.
In the additive which ensures penetration of water into the pharmaceutical composition of the present invention (hydrophilic base), the amount of water necessary to dissolve 1 g of the hydrophilic base at 20±5° C. is 10 mL or less, 6 mL or less in another embodiment, 5 mL or less in still another embodiment, and 4 mL or less in still another embodiment. When the hydrophilic base has a high solubility to water, the effect that allows water to penetrate into the formulation is high.
Examples of the hydrophilic base include water-soluble polymers, such as polyethylene glycol [PEG: for example, product names PEG 400, PEG 1500, PEG 4000, PEG 6000, and PEG 20000 (NOF Corporation)], polyvinyl pyrrolidone (PVP: for example, product name PVP K30 (BASF), and the like; sugar alcohols, such as D-mannitol, D-sorbitol, xylitol, and the like; saccharides, such as lactose, sucrose, anhydrous maltose, D-fructose, dextran (for example, Dextran 40), glucose, and the like; surfactants, such as polyoxyethylene hydrogenated castor oil [HCO: for example, Cremophor RH40 (BASF), HCO-40, HCO-60 (Nikko Chemicals)], polyoxyethylene polyoxypropylene glycol [for example, Pluronic F68 (ADEKA Corporation and the like)], polyoxyethylene sorbitan higher fatty acid esters [Tween: for example, Tween 80 (Kanto Chemical)], and the like; salts, such as sodium chloride, magnesium chloride, and the like; organic acids, such as citric acid, tartaric acid, and the like; amino acids, such as glycine, β-alanine, lysine hydrochloride, and the like; and aminosaccharides, such as meglumine and the like.
As another embodiment, PEG, PVP, D-mannitol, D-sorbitol, xylitol, lactose, sucrose, anhydrous maltose, D-fructose, dextran, glucose, polyoxyethylene polyoxypropylene glycol, sodium chloride, magnesium chloride, citric acid, tartaric acid, glycine, β-alanine, lysine hydrochloride, or meglumine may be used. As still another embodiment, PEG, PVP, D-mannitol, lactose, sucrose, sodium chloride, polyoxyethylene polyoxypropylene glycol, or the like may be used.
These hydrophilic bases may be used alone, or as an appropriate combination of two or more thereof.
The content of the hydrophilic base is not particularly limited, so long as it is an amount capable of controlling the release of the drug to the extent that the release of the drug is not affected by food. The content of the hydrophilic base is, for example, 5% by weight to 75% by weight, 5% by weight to 70% by weight in another embodiment, and 20% by weight to 60% by weight in still another embodiment.
The sustained release hydrogel-forming formulation, as an embodiment of the pharmaceutical composition for modified release of the present invention, may be prepared as various dosage forms, which include, for example, formulations for oral administration such as tablets, capsules (including microcapsules), granules, and powder, and formulations for parenteral administration such as suppositories (for example, rectal suppositories or vaginal suppositories). These formulations may be safely administered orally or parenterally. Formulations for oral administration such as tablets, capsules, and granules may be selected in another embodiment.
Hereinafter, various pharmaceutical additives which may be used in the sustained release hydrogel-forming formulation, as an embodiment of the pharmaceutical composition for modified release of the present invention, and various methods for preparing the sustained release hydrogel-forming formulation will be explained, but these explanations are not particularly limited to the sustained release hydrogel-forming formulation, and can be applied to formulations other than the sustained release hydrogel-forming formulation.
The pharmaceutical composition for modified release of the present invention may be prepared by mixing the drug, the hydrogel-forming polymers, and the hydrophilic base, and forming the mixture into a predetermined shape. The mixing and forming may be carried out in accordance with conventional methods widely used in the technical field for formulation. A pharmaceutically acceptable carrier may be used in the mixing and/or forming, if desired.
In the preparation of the pharmaceutical composition for modified release of the present invention, further various pharmaceutical additives may be used, if desired. Such pharmaceutical additives are not particularly limited, so long as they are pharmaceutically acceptable. Examples of the pharmaceutical additives include various organic or inorganic carrier substances which are widely used as formulation materials, such as fillers, lubricants, binders, and disintegrating agents. Other formulation additives such as preservatives, antioxidants, stabilizers, film coating agents, coloring agents, and sweeteners may be used, if desired.
Examples of the fillers include lactose, sucrose, D-mannitol, D-sorbitol, starch, gelatinized starch, dextrin, crystalline cellulose, low substituted hydroxypropyl cellulose, carboxymethyl cellulose sodium, gum arabic, dextrin, pullulan, light anhydrous silicic acid, synthetic aluminum silicate, magnesium aluminate metasilicate, and the like.
Examples of the lubricants include magnesium stearate, calcium stearate, talc, colloidal silica, and the like.
Examples of the binders include gelatinized starch, sucrose, gelatin, gum arabic, methylcellulose, carboxymethyl cellulose, carboxymethyl cellulose sodium, crystalline cellulose, sucrose, D-mannitol, trehalose, dextrin, pullulan, hydroxypropyl cellulose, hypromellose, polyvinylpyrrolidone, and the like.
Examples of the disintegrating agents include lactose, sucrose, starch, carboxymethyl cellulose, carboxymethyl cellulose calcium, croscarmellose sodium, carboxymethyl starch sodium, light anhydrous silicic acid, low substituted hydroxypropylcellulose, and the like.
Examples of the preservatives include p-hydroxybenzoate esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid, and the like.
Examples of the antioxidants include butylated hydroxytoluene (BHT), propyl gallate (PG), butylhydroxyanisol (BHA), ascorbic acid, sodium ascorbate, erythorbic acid, sodium nitrite, sodium bisulfite, sodium pyrosulfite, citric acid, and edetate sodium; BHT, PG, and sodium ascorbate in another embodiment; and BHT in still another embodiment.
Examples of the stabilizers include yellow ferric oxide, red ferric oxide, black iron oxide, and the like.
Examples of the film coating agents include pharmaceutically commonly-used bases, such as water-soluble polymers, plasticizers, and inorganic substances, or a combination thereof.
Examples of the coloring agents include water-soluble edible tar pigments (examples: edible pigments such as food red No. 2, food red No. 3, food yellow No: 4, food yellow No. 5, food blue No. 1, and food blue No. 2), water-insoluble lake pigments (examples: aluminum salts of the above water-soluble edible tar pigments), natural pigments (examples: β-carotene, chlorophyll, and colcothar), and the like.
Examples of the sweeteners include saccharin sodium, dipotassium glycyrrhizinate, aspartame, stevia, and the like.
These carriers or formulation additives may be used alone, or as an appropriate combination of two or more thereof. With respect to the contents thereof, they may be used in appropriate amounts.
Hereinafter, the process of manufacturing the pharmaceutical composition for modified release of the present invention will be explained, the present invention is not limited to the following particular embodiments.
The pharmaceutical composition for modified release of the present invention may be prepared by known methods per se, such as dry granulation, wet granulation, fluidized bed granulation, intermittent granulation, agitation granulation, or the like.
As a method of de-lumping or pulverizing the drug, conventional crushing or pulverizing methods may be applied, for example, using an impact mill (Hosokawa Micron Corporation; Fine Impact Mill), a dry & wet mill (Powrex Corporation: Comil), or a cutting mill granulator (Dalton Corporation; Power Mill).
As a method of pulverizing the hydrophilic base, the hydrogel-forming polymer, or the formulation additives, conventional pulverizing methods may be applied, for example, using an impact mill (Hosokawa Micron Corporation; Fine Impact Mill or Sample Mill) or a jet mill (Horkos Corp; Jet Mill).
As a method of granulating the drug, conventional granulation methods may be used. Examples of such methods include a fluidized bed granulation method, an intermittent granulation method, an agitation granulation method, a high-speed agitation granulation method, a tumbling fluidized bed granulation method, an extrusion granulation method, a pulverization granulation method, a dry granulation method, and the like. In another embodiment, examples thereof include a fluidized bed granulation method, an intermittent granulation method, an agitation granulation method, a high-speed agitation granulation method, a tumbling fluidized bed granulation method, and a dry granulation method, and any method capable of granulating the drug may be used. Examples of a granulator include a fluidized bed granulator (for example, Flow Coater; Freund Corporation, or GPCG; Glatt GmbH), a granulation and coating apparatus equipped with a horizontal rotating disc having a flat powder contact portion [for example, a centrifugal fluidizing granulator (for example, CF granulator; Freund Corporation)], a granulation and coating apparatus having a rotating disk with a flat surface placed at the bottom of a fluidized bed and having an aeration portion (for example, Spiralflow, or Flowcoater with a rotor container; Freund Corporation), and a dry granulator in which material powder is directly compressed, molded, crushed, and sieved (for example, Roller Compactor; Freund Corporation).
In the dry granulation, for example, the drug, the hydrogel-forming polymer, the hydrophilic base, and additives such as a filler may be compression-molded using a dry granulator, and then, may be crushed and sieved to obtain granulated products having a desired size.
In the wet granulation, for example, while the drug, the hydrogel-forming polymer, the hydrophilic base, and additives such as a filler is fluidized, an appropriate amount of water or a liquid containing the hydrophilic base and the binder may be sprayed. The liquid containing the hydrophilic base may be prepared by dissolving or dispersing the essential component in a solvent such as water, ethanol, methanol, or the like. These solvents may be used as an appropriate mixture thereof.
The amount of water used in the granulation is not particularly limited, so long as the binder or formulation additives may be uniformly dissolved and/or suspended (dispersed) in the water. When the hydrophilic base is used in the solid form, the amount of water is not particularly limited, so long as the hydrogel-forming polymer can be granulated.
When the hydrophilic base is used in the liquid form, the amount of water to the hydrogel-forming polymer is generally 10% by weight or less, 8% by weight or less in another embodiment, and 5% by weight or less in still another embodiment. A method of adding water in the granulation is not particularly limited, so long as a nonuniform mixture consisting of untreated powder and aggregates, which are generally powdery, is not generated. Examples thereof include a continuous spray method in which water is continuously added, an intermittent spray method in which a dry step (and a shaking step, if desired) is carried out during the granulation step, and the like.
The addition rate of water in the granulation is not particularly limited, so long as a nonuniform mixture consisting of untreated powder and aggregates, which are generally powdery, is not generated. In the fluidized bed granulation, the addition rate of water to the hydrogel-forming polymer is generally 0.1% by weight/min. to 1% by weight/min., 0.2% by weight/min. to 0.8% by weight/min. in another embodiment, and 0.4% by weight/min. to 0.6% by weight/min. in still another embodiment.
The temperature of the powder in the granulation is not particularly limited, so long as it does not induce thermal denaturation of the hydrogel-forming polymer. The temperature is, for example, 20° C. to the melting point (62° C. to 67° C.) of the hydrogel-forming polymer, 20° C. to 50° C. in another embodiment, 20° C. to 35° C. in still another embodiment, and 25° C. to 30° C. in still another embodiment.
The concentration of the binder liquid as a solid content which may be used in the granulation is, for example, 1% to 20% as a formulation amount. The binder is not particularly limited, so long as it is pharmaceutically acceptable.
The binder may be added in the solid form to a granulator, and then, water may be sprayed as the binder liquid. Alternatively, the binder may be dissolved in water, and then, the resulting binder liquid may be sprayed.
An appropriate spray rate of the binder liquid varies according to a production method to be applied or its production scale. In a 1-kg scale production by the fluidized bed granulation, the spray rate is 2 g/min. to 20 g/min., and 5 g/min. to 15 g/min. in another embodiment.
An appropriate temperature of the product in the granulation is 15° C. to 50° C., and 15° C. to 40° C. in another embodiment.
The resulting granulated products may be, for example, dried or heated.
In the drying step, an apparatus and a method are not particularly limited, so long as the granulated products can be dried. Examples of an apparatus for drying include a fluidized bed granulator (for example, Flow Coater; Freund Corporation, or GPCG; Glatt GmbH), a granulation and coating apparatus equipped with a horizontal rotating disc having a flat powder contact portion [for example, a centrifugal fluidizing granulator (for example, CF granulator; Freund Corporation)], a granulation and coating apparatus having a rotating disk with a flat surface placed at the bottom of a fluidized bed and having an aeration portion (for example, Spiralflow, or Flowcoater with a rotor container; Freund Corporation), and the like. The conditions for drying are not particularly limited, so long as the granulated products may be generally dried in the fluidized bed. The drying of the granulated products will be almost completed, for example, under the conditions in which the dry inlet air temperature is 50° C. and the drying is carried out until the temperature of the granulated products becomes 40° C. and, in another embodiment, under the conditions in which the dry inlet air temperature is 40° C. and the drying is carried out until the temperature of the granulated products becomes 30° C. As the drying method, forced-air drying or drying under reduced pressure may be used.
The granulated products may be sieved.
In the sieving step, an apparatus and a method are not particularly limited, so long as the granulated products can be sieved. Examples of an apparatus for sieving include a screen, a dry & wet mill (Powrex Corporation: Comil), a cutting mill granulator (Dalton Corporation; Power Mill), and the like. The conditions for sieving are not particularly limited, so long as the granulated products may be generally sieved to obtain particles having a desired size.
Examples of tabletting include a direct tabletting method in which the drug, the hydrophilic base, and the hydrogel-forming polymer are mixed with an appropriate additive(s), and the mixture is compression-molded to obtain tablets; a method in which a composition obtained by a wet granulation (the granulation is carried out by spraying a mixture of the drug, the hydrophilic base, the hydrogel-forming polymer, and additives with a binder liquid) or a melting granulation (the granulation is carried out by heating a mixture of the drug, the hydrophilic base, the hydrogel-forming polymer, and an appropriate low-melting substance) is formed into tablets; and the like.
A rotary tabletting machine, a single punch tabletting machine, and the like may be used as a tabletting machine. A method as well as an apparatus is not particularly limited, so long as a compression-molded product (preferably tablets) can be pharmaceutically produced.
After the tabletting, the obtained tablets may be dried. The initial water content of the tablet is, for example, 2% by weight/tablet or less, 1.5% by weight/tablet or less in another embodiment, and 0.9% by weight/tablet or less in still another embodiment.
After the tabletting, the obtained tablets may be film coated using a pan coating machine at an amount of 1% by weight to 5% by weight per tablet.
(2) Multi-Layered Formulation Consisting of Drug Core and Release-Controlling Layer which are Geometrically Arranged
A multilayered formulation, an embodiment of the pharmaceutical composition for modified release according to the present invention, may be a two-layered or three-layered formulation for modified release, characterized by consisting of a drug-containing layer and a release-controlling layer, and consisting of:
a) the first layer (layer 1) which is prepared by compressing a mixture or granules containing 5 to 90 W/W % (preferably 10 to 85 W/W %) of a water-soluble polymer in this layer, and has a property of being swollen by contact with environmental fluids,
b) the second layer (layer 2) comprising compound A, a water-soluble polymer, and other filler(s), which is adjacent to the first layer, has a property suitable to compression-molding, and is designed to release the physiologically active substance within a predetermined period of time, and
c) the third layer (layer 3) (which may be optionally adjacent to the layer 2) which contains a water-soluble polymer capable of being generally gelled and/or swollen followed by optionally being disintegrated, and has a property of controlling the release of compound A from the layer 2. The “environmental fluids” include, for example, an aqueous solution as used in a dissolution test, as well as body fluids such as blood or gastrointestinal fluids.
Techniques for such a multilayered formulation which may be used in the pharmaceutical composition for modified release according to the present invention are disclosed in, for example, U.S. Pat. No. 4,839,177, U.S. Pat. No. 5,422,123, U.S. Pat. No. 5,780,057, U.S. Pat. No. 6,149,940, Japanese Patent Publication (Kokai) No. 2005-162736, and Japanese Patent Publication (Kokai) No. 2005-162737, the contents of which are incorporated herein by reference. As disclosed in U.S. Pat. No. 4,839,177 and U.S. Pat. No. 5,422,123, the multilayered formulation is characterized in that a release rate of the drug from the pharmaceutical formulation is controlled by sandwiching the layer 2 containing the drug between the layer 1 and the layer 3 in which the drug is not contained or is optionally contained. Further, as disclosed in U.S. Pat. No. 5,780,057 and U.S. Pat. No. 6,149,940, it is known that when the multilayered formulation is brought into contact with body fluids, at least one of the layer 1 and the layer 3 are rapidly swollen followed by the layer 2 is swollen, that is, the volume of the formulation is significantly increased, and as a result, the formulation remains in the stomach for a longer period of time, and almost all of the active substance contained therein is released and absorbed at the upper gastrointestinal tract in a controlled manner.
The layer 1 and the layer 3 may have the same composition and the same functional properties, or may have different compositions and different functional properties. When the layer 1 and the layer 3 have the same composition and functional properties, the amounts and thicknesses of the layers 1 and 3 which sandwich the layer 2 may be changed. At least one of the layers 1 and 3 acts as a barrier for the release of the active substance, that is, it is impermeable enough for compound A contained in the layer 2 not to be released or diffused therefrom. Further, at least one of the layers 1 and 3 can be rapidly swollen, that is, the volume thereof is rapidly increased. The layer 3 may optionally contain the drug so that a drug release which is different from that released from the layer 2 can be supplementally added to the pharmaceutical formulation.
The water-soluble polymers used in the layer 1, the layer 3, and the layer 2 are not particularly limited, so long as they are pharmaceutically acceptable and biocompatible. Such water-soluble polymers may be gradually dissolved and/or gelled in an aqueous liquid, and/or may be gelled rapidly or at a different rate and then optionally disintegrated. Examples of the water-soluble polymers include, for example, hydroxymethyl cellulose, hydroxyethyl cellulose, hypromellose having a molecular weight of 1,000 to 4,000,000, hydroxypropyl cellulose having a molecular weight of 2,000 to 2,000,000, carboxyvinyl polymers, chitosans, mannans, galactomannans, xanthans, carageenans, amylose, alginic acid, salts and derivatives thereof, pectin, acrylates, methacrylates, acrylate/methacrylate copolymers, polyacid anhydrides, polyamino acids, poly(methylvinyl ether/maleic anhydride) polymers, polyvinyl alcohols, glucans, scleroglucans, carboxymethyl cellulose and derivatives thereof, ethyl cellulose, methyl cellulose, or conventional water-soluble cellulose derivatives. Hypromellose having a molecular weight of 3,000 to 2,000,000 is preferable. The content of the water-soluble polymer in the layer 1 or the layer 3 is generally 5 to 90 W/W %, preferably 10 to 85 W/W %, more preferably 20 to 80 W/W %, with respect to the weight of each layer. The content of the water-soluble polymer in the layer 2 is generally 5 to 90 W/W %, preferably 10 to 85 W/W %, to the weight of the layer.
In the process for preparing the layer 1 and the layer 3, a water-soluble filler which promotes the degree of wetness of the layers may be used, to rapidly increase the volume of the multilayerd formulation containing the above water-soluble polymer. The water-soluble filler may be preferably selected from a group of fillers having an extremely rapid disintegrability, such as crosslinked polyvinylpyrrolidone, hydroxypropyl cellulose or hypromellose having a low or medium molecular weight, crosslinked carboxymethyl cellulose sodium, carboxymethyl starch or salts thereof, divinylbenzene/potassium methacrylate copolymers, or the like.
The content of the filler is 1 to 90 W/W % or less, preferably 5 to 50 W/W % of each layer.
If desired, a surfactant (anionic, cationic, or nonionic surfactants) may be further used to improve the degree of wetness. As a result, tablets and environmental fluids may conform with each other more rapidly, and the tablets, particularly the gel-forming layer, may be gelled more rapidly. Examples of the surfactant include, for example, sodium laurylsulfate, sodium ricinolate, sodium tetradecylsulfonate, sodium dioctylsulfosuccinate, cetomagrogol, poloxamer, glycerol monostearate, polysorbate, sorbitan monolaurate, lecithins, or other pharmaceutically acceptable surfactants.
If desired, another substance which modifies hydration may be further used. Such a substance may be selected from, for example, mannitol, lactose, starches derived from various organs, sorbitol, xylitol, microcrystalline cellulose, and/or a diluent capable of generally promoting a penetration of water or an aqueous liquid into a pharmaceutical composition; or a hydrophobic diluent to retard a penetration of water or an aqueous liquid into a pharmaceutical formulation, such as ethyl cellulose, glycerol monostearate, palmitate, or hydrogenated or non-hydrogenated vegetable oils (for example, hydrogenated castor oil, wax, monoglyceride, diglyceride, or triglyceride). It is preferable to select ethyl cellulose or hydrogenated vegetable oils as the hydrophobic diluent.
The content of the hydrophobic diluent in the layer 1 or the layer 3 is generally 1 to 60 W/W %, preferably 5 to 40 W/W %, more preferably 10 to 30 W/W %, with respect to the weight of each layer.
To control the release rate of compound A from the pharmaceutical formulation, microcrystalline or a water-soluble base, such as dextrose, sucrose, fructose, maltose, xylitol, citric acid, lactose, mannitol, or the like, may be used in the layer 2, if desired.
The content of microcrystalline and/or the water-soluble base in the layer 2 is generally 5 to 90 W/W %, preferably 10 to 80 W/W %, more preferably 20 to 70 W/W %, with respect to the weight of the layer.
The multilayered formulation of the present invention may contain, for example, a lubricant, such as magnesium stearate, talc, stearic acid, glycerol monostearate, polyoxyethylene glycol having a molecular weight of 400 to 7,000,000, hydrogenated castor oil, glycerol behenate, monoglyceride, diglyceride, triglyceride, or the like, a fluidizing agent such as colloidal silica or other silica, a binder, a buffer, an absorbing agent, or other pharmaceutically acceptable additives.
The multilayered formulation of the present invention may be manufactured, for example, by mixing powder and/or granules by a known manufacturing technique per se, and forming the mixture into tablets by compression. A two-layered or three-layered pharmaceutical formulation, such as a tablet, may be manufactured by a known method per se. The multilayered formulation of the present invention may be manufactured, for example, by using a rotary press capable of manufacturing multilayered tablets. It is preferable that a tabletting pressure is generally 7 to 50 kN. When the tablets are manufactured on a small scale, a mortar and pestle may be used to prepare powder and/or granules, and then, an oil press tabletting machine may be used to manufacture two-layered or three-layered tablets. The thickness of each layer of the formulation may vary according to the content of the active substance, and is preferably 0.2 to 8 mm, more preferably 1 to 4 mm. In the formulation of the present invention, for example, a coating layer with a macromolecular material may be applied to the pharmaceutical composition. Such a coating may be applied by using an organic or aqueous solution, in accordance with a known method per se.
When the multilayered formulation of the present invention is brought into contact with gastric juices in the gastrointestinal tract and/or liquids, the volume thereof is rapidly increased. This increase in volume may be limited in a single layer or several layers of the formulation. Such a formulation may be in a form of a tablet, small tablets, or a gelatin capsule consisting of small tablets. Further, at least two small tablets may be combined in the same formulation, and may be packed in, for example, a wafer capsule or a gelatin capsule. When the formulation consists of small tablets, each small tablet may have a different composition or the same composition.
(3) Gel Formulation in which a Plurality of Gums is Combined
A gel formulation in which a plurality of gums is combined, an embodiment of the pharmaceutical composition for modified release according to the present invention, is characterized by comprising at least compound A and a gum base. The gum base as used herein means a sustained release filler comprising a homopolysaccharide which can form a crosslinkage with a heteropolysaccharide gum when exposed to the heteropolysaccharide gum and environmental fluids (such as body fluids, an aqueous solution for an in vitro dissolution test, or the like). The sustained release filler may further comprise calcium sulfate and/or a water-soluble base. The gel formulation may further contain a commonly used filler.
Techniques for obtaining the gel formulation in which a plurality of gums is combined, which may be used in the pharmaceutical composition for modified release according to the present invention, are disclosed in, for example, U.S. Pat. No. 4,994,276, U.S. Pat. No. 5,128,143, U.S. Pat. No. 5,135,757, and Japanese Patent No. 2832248. As disclosed therein, it is known that a heterogeneously dispersed filler comprising a combination of a heteropolysaccharide and a homopolysaccharide exhibiting a synergistic effect, such as a combination of two or more polysaccharide gums, has a viscosity higher than that of any single gum, and can cause a rapid hydration, and thus a harder gel is generated more rapidly. The contents of the above patent references are incorporated herein by reference.
The heteropolysaccharide as used herein is defined as a water-soluble polysaccharide containing two or more sugar units. The heteropolysaccharide is not particularly limited, so long as it has a branched-chain or spiral configuration, and has an excellent water absorbing property and a high viscosity improving property. As the heteropolysaccharide, for example, xanthan gum or derivatives thereof (such as deacylated xanthan gum), carboxymethyl ether, or propylene glycol ester are preferable, and xanthan gum having a high molecular weight (>106) is more preferable.
The homopolysaccharide as used herein is not particularly limited, so long as it is a polysaccharide consisting of mannose and galactose, and can form a crosslinkage with a heteropolysaccharide. Locust bean gum having a high ratio of mannose to galactose is more preferable than other galactomannans such as guar or hydroxypropyl guar.
Other naturally-occurring polysaccharide gums may be used in the present invention. Examples of such polysaccharides include, for example, alginic acid derivatives, carrageenan, tragacanth gum, gum arabic, karaya gum, polyethylene glycol esters of these gums, chitin, chitosan, mucopolysaccharide, konjak, starch, substituted starch, starch fragment, dextrin, British gum having a molecular weight of approximately 10,000 Da, dextran, or the like. The starch may be used in an unmodified form, for example, an ungelled starch such as potato, rice, banana, or the like, or a semisynthetic or gelled starch.
As a combination of the heteropolysaccharide and the homopolysaccharide, the combination of xanthan gum and locust bean gum is particularly preferable. The content ratio of the heteropolysaccharide and the homopolysaccharide is not particularly limited, so long as it is an amount effective in enhancing a desired gel strength. Such a ratio (heteropolysaccharide gum: homopolysaccharide gum) is approximately 3:1 to approximately 1:3, preferably approximately 1:1.
The water-soluble cationic crosslinking agent as used herein is not particularly limited, so long as it is a pharmaceutically acceptable monovalent or polyvalent metal cation. As the binder, for example, calcium sulfate or the like may be used.
The water-soluble base as used herein is not particularly limited, so long as it is pharmaceutically acceptable. Examples of the water-soluble base include, for example, dextrose, sucrose, fructose, maltose, xylitol, citric acid, or the like.
The gel formulation in which a plurality of gums is combined of the present invention may be manufactured, for example, in a pharmaceutically acceptable form for oral administration such as a tablet or the like. In an embodiment, (1) a heteropolysaccharide gum, and a homopolysaccharide which can form a crosslinkage with the heteropolysaccharide gum when exposed to environmental fluids are mixed together under the dry condition with a pharmaceutically acceptable water-soluble base in a desired ratio, (2) the resulting mixture is subject to a wet granulation, (3) the granules are dried, (4) the dried granules are pulverized to obtain a sustained release filler having a desired particle size, (5) the resulting sustained release filler is granulated together with compound A, (6) the resulting granules are dried, (7) a conventional filler, such as a lubricant or the like, is added thereto, and (8) the resulting mixture is formed by compression into, for example, tablets. In another embodiment, a mixture of the sustained release filler and compound A may be granulated, together with an a solution of a hydrophobic substance (such as ethyl cellulose or the like) in an amount sufficient to retard the hydration of the filler (i.e., gums) without the destruction thereof, and then a conventional filler such as a lubricant is added thereto, and the resulting mixture is formed by compression into, for example, tablets.
In the wet granulation, predetermined amounts of the heteropolysaccharide gum, the homopolysaccharide gum, the cationic crosslinking agent, and the water-soluble base are homogeneously mixed; and then, a wetting agent, such as water, propylene glycol, glycerol, alcohol, or the like, is added thereto to prepare a wet aggregate; and the resulting wet aggregate is dried, and pulverized using a conventional apparatus to prepare granules having a predetermined particle size.
As the lubricant, for example, stearic acid or the like may be used. The mixing of the hydrophobic substance with the sustained release filler may be carried out, for example, by using a liquid in which the hydrophobic substance is dissolved and/or dispersed in an organic solvent, and further granulating the above-mentioned granules together with the liquid.
Examples of the hydrophobic substance include, for example, a pharmaceutical acceptable hydrophobic cellulose, such as ethyl cellulose or the like.
A combination and a mixing ratio of each component are not particularly limited. In a preferred embodiment, approximately 5 to 60 W/W % of xanthan gum (as the heteropolysaccharide) and locust bean gum (as the homopolysaccharide) (xanthan gum: locust bean gum=approximately 1:1) with respect to the total weight of the pharmaceutical formulation may be contained, and approximately 10 W/W % or less of calcium sulfate (as the water-soluble cationic crosslinking agent) and approximately 30 to 70 W/W % of dextrose (as an inert diluent) may be further contained. To control the release rate, the hydrophobic substance may be added, and, for example, approximately 5 to 10 W/W % of ethyl cellulose may be contained.
(4) Osmotic Pump Type Formulation
Osmotic pump type formulations utilize osmotic pressure to generate a driving force for imbibing fluid into a formulation, by a semipermeable membrane that permits free diffusion of fluid but not a drug or an osmoagent. The osmotic systems are characterized in that the action thereof is pH-independent, and a drug can be sustainedly released at a constant rate for a long time, even as the formulation transits the gastrointestinal tract and encounters environments having different pH values.
Such osmotic pump type formulations are reported in Santus and Baker, “Osmotic drug delivery: a review of the patent literature”, Journal of Controlled Release, 35, p. 1-21, (1995). Further, osmotic pump type formulations are described in U.S. Pat. Nos. 3,845,770, 3,916,899, 3,995,631, 4,008,719, 4,111,202, 4,160,020, 4,327,725, 4,519,801, 4,578,075, 4,681,583, 5,019,397, and 5,156,850, the contents of which are incorporated herein by reference.
In the osmotic pump type formulation of the present invention, a bilayered compressed core consisting of a drug layer containing compound A, and a push layer, is coated with a semipermeable membrane that permits water or outer fluid but not a drug, an osmoagent, an osmopolymer, or the like. The semipermeable membrane is provided with at least one drug delivery orifice for connecting the inside of the formulation with the exterior environment. Therefore, after the osmotic pump type formulation is orally administered, fluid such as water transits the semipermeable membrane, and penetrates into the inside of the formulation. As a result, an osmotic action is generated, and compound A is sustainedly released through the drug delivery orifice(s) at a constant rate for a long time.
The drug layer contains compound A, as a mixture with a pharmaceutically acceptable additive(s).
The push layer contains one or more osmotic active components, but does not contain compound A, as described in detail below. Typical osmotic active component(s) contained in the push layer may be composed of an osmoagent and one or more osmopolymers. The osmopolymer as used herein means a polymer which has relatively a large molecular weight and swells when fluid is imbibed, to release compound A through the drug delivery orifice(s).
The semipermeable membrane used is not particularly limited, so long as it is permeable to the passage of an external fluid, such as water and biological fluids, and substantially impermeable to the passage of compound A, an osmoagent, an osmopolymer, and the like. Such a semipermeable membrane is essentially nonerodible, and insoluble in a living body.
As polymers for forming the semipermeable membrane, for example, semipermeable homopolymers, semipermeable copolymers, and the like may be used. As materials for such polymers, cellulosic polymers, such as cellulose esters, cellulose ethers, cellulose ester-ethers, and the like, may be used. The cellulosic polymers have a degree of substitution (DS) of anhydroglucose units of more than 0 and 3 or less. The degree of substitution (DS) means the average number of hydroxyl groups originally present on the anhydroglucose units that are replaced by a substituting group or converted into another group. The anhydroglucose unit can be partially or completely substituted with groups, such as acyl, alkanol, alkenoyl, aroyl, alkyl, alkoxy, halogen, carboalkyl, alkylcarbamate, alkylcarbonate, alkylsulfonate, alkylsulfamate, semipermeable polymer forming groups, and the like, wherein the organic moieties contain 1 to 12 carbon atoms, preferably 1 to 8 carbon atoms.
As the typical semipermeable compositions, one member, or two or more members selected from the group consisting of cellulose acylate, cellulose diacylate, cellulose triacylate, cellulose acetate, cellulose diacetate, cellulose triacetate, mono-, di-, and tri-cellulose alkanylates, mono-, di-, and tri-alkenylates, mono-, di-, and tri-aroylates, and the like, may be used. Representative polymers include cellulose acetate having a DS of 1.8 to 2.3 and an acetyl content of 32 to 39.9%; cellulose diacetate having a DS of 1 to 2 and an acetyl content of 21 to 35%; cellulose triacetate having a DS of 2 to 3 and an acetyl content of 34 to 44.8%; and the like.
More specific cellulosic polymers include cellulose propionate having a DS of 1.8 and a propionyl content of 38.5%; cellulose acetate propionate having an acetyl content of 1.5 to 7% and an acetyl content of 39 to 42%; cellulose acetate propionate having an acetyl content of 2.5 to 3%, an average propionyl content of 39.2 to 45%, and a hydroxyl content of 2.8 to 5.4%; cellulose acetate butyrate having a DS of 1.8, an acetyl content of 13 to 15%, and a butyryl content of 34 to 39%; cellulose acetate butyrate having an acetyl content of 2 to 29%, a butyryl content of 17 to 53%, and a hydroxyl content of 0.5 to 47%; cellulose triacylates having a DS of 2.6 to 3, such as cellulose trivalerate, cellulose trilamate, cellulose tripalmitate, cellulose trioctanoate, and cellulose tripropionate; cellulose diesters having a DS of 2.2 to 2.6, such as cellulose disuccinate, cellulosedipalmitate, cellulose dioctanoate, cellulose dicaprylate, and the like; mixed cellulose esters, such as cellulose acetate valerate, cellulose acetate succinate, cellulose propionate succinate, cellulose acetate octanoate, cellulose valerate palmitate, cellulose acetate heptanoate, and the like. Semipermeable polymers are disclosed in U.S. Pat. No. 4,077,407, and can be synthesized and obtained by procedures described in Encyclopedia of Polymer Science and Technology, Vol. 3, pp. 325-354 (1964), Interscience Publishers Inc., New York, N.Y. The content of the polymers is not particularly limited, so long as it is an amount permeable to the passage of an external fluid, such as water and biological fluids, and substantially impermeable to the passage of compound A, an osmoagent, an osmopolymer, and the like. The content of the polymers is preferably 6 to 20 W/W %, more preferably 8 to 18 W/W %, with respect to the weight of a dilayered compressed core consisting of a drug layer and a push layer.
Semipermeable polymers for forming the semipermeable membrane further include cellulose acetaldehyde dimethyl acetate; cellulose acetate ethylcarbamate; cellulose acetate methyl carbamate; cellulose dimethylaminoacetate; semipermeable polyurethanes; semipermeable sulfonate polystyrenes; cross-linked selectively semipermeable polymers formed by the coprecipitation of an anion and a cation, as disclosed in U.S. Pat. Nos. 3,173,876, 3,276,586, 3,541,005, 3,541,006, and 3,546,142; semipermeable polymers, as disclosed in U.S. Pat. No. 3,133,132; semipermeable polystyrene derivatives; semipermeable poly (sodium styrenesulfonate); semipermeable poly (vinylbenzyltrimethylammonium chloride); and semipermeable polymers exhibiting a fluid permeability of 10−5 to 10−2 (cc mL/cm hr atm), expressed as hydrostatic or osmotic pressure differences per atmosphere across a semipermeable membrane. These polymers are described in U.S. Pat. Nos. 3,845,770, 3,916,899, and 4,160,020, and in Handbook of Common Polymers, Scott and Roff (1971) CRC Press, Cleveland, Ohio.
The semipermeable membrane may contain a flux-regulating agent. The flux-regulating agent means a substance added to assist in regulating the fluid permeability or flux through the semipermeable membrane. The flux-regulating agents include a substance which enhances the flux (hereinafter referred to as flux-enhancing agent) and a substance which decreases the flux (hereinafter referred to as flux-decreasing agent). The flux-enhancing agents are essentially hydrophilic, while the flux-decreasing agents are essentially hydrophobic. The flux-regulating agents include, for example, polyhydric alcohols, polyalkylene glycols, polyalkylenediols, polyesters of alkylen glycols, and the like.
Typical flux-enhancing agents include polyethylene glycols 300, 400, 600, 1500, 4000, 6000 and the like; low molecular weight glycols, such as polypropylene glycol, polybutylene glycol, and polyamylene glycol: polyalkylenediols, such as poly(1,3-propanediol), poly(1,4-butanediol), poly(1,6-hexanediol), and the like; fatty acids, such as 1,3-butylen glycol, 1,4-pentamethylene glycol, 1,4-hexamethylene glycol, and the like; alkylen triols, such as glycerine, 1,2,3-butanetriol, 1,2,4-hexanetriol, 1,3,6-hexanetriol, and the like; esters, such as ethylene glycol dipropionate, ethylene glycol butyrate, butylene glycol dipropionate, glycerol acetate esters, and the like. Preferred flux-enhancing agents include difunctional block-copolymers of propylene glycol, polyoxyalkylene or derivatives thereof, known as pluronics (trademark, BASF).
Typical flux-decreasing agents include phthalates substituted with an alkyl or alkoxy or with both an alkyl and alkoxy group such as diethyl phthalate, dimethoxyethyl phthalate, dimethyl phthalate, and [di(2-ethylhexyl) phthalate], and aryl phthalates such as triphenyl phthalate and butyl benzyl phthalate; insoluble salts such as calcium sulfate, barium sulfate, calcium phosphate, and the like; insoluble oxides such as titanium oxide; polymers in the form of powder, granules, and the like, such as polystyrene, polymethylmethacrylate, polycarbonate, and polysulfone; esters such as citric acid esters esterified with long chain alkyl groups; inert and water impermeable fillers; resins compatible with cellulose based semipermeable membrane forming materials; and the like.
The content of the flux-regulating agent contained in the semipermeable membrane is approximately 0.01 to approximately 20 W/W % or more.
Other substances may be contained in the semipermeable membrane to impart plasticity, flexibility, and elongation properties, to make the membrane less brittle, and to render tear strength. Such substances include phthalate plasticizers such as dibenzyl phthalate, dihexyl phthalate, butyl octyl phthalate, straight chain phthalates having 6 to 11 carbon atoms, di-isononyl phthalte, di-isodecyl phthalate, and the like. Other plasticizers include nonphthalates such as triacetin, dioctylazelate, epoxidized tallate, tri-isoctyl trimellitate, tri-isononyl trimellitate, sucrose acetate isobutyrate, epoxidized soybean oil, and the like.
The content of the plasticizer contained in the semipermeable membrane is approximately 0.01 to 20 W/W % or more.
The push layer is in contacting layered arrangement with the drug layer. The push layer contains an osmopolymer that imbibes an aqueous or biological fluid and swells to push compound A through the exit means of the formulation. The osmopolymer as used herein means a polymer that interacts with water or aqueous biological fluids and swells or expands to a high degree. Preferred osmopolymers are swellable and hydrophilic polymers exhibiting a 2 to 50-fold volume increase. The osmopolymer can be non-crosslinked or crosslinked, but is preferably at least lightly crosslinked in a preferred embodiment, to create an extended polymer network that is too large to exit the formulation. The content of the osmopolymer can be appropriately selected in accordance with various factors such as properties, content, and the like of a drug contained in the drug layer, but is not particularly limited, so long as it is an amount capable of releasing the drug from the drug layer at a desired dissolution rate by swelling. The amount is preferably 30 mg or more, more preferably 50 mg or more. The content is 40 to 80 W/W % with respect to the weight of the push layer.
The osmopolymers include one or more members selected from the group consisting of poly(alkylen oxide) having a number average molecular weight of 1,000,000 to 15,000,000, as represented by polyethylene oxide, and poly(alkali carboxymethylcellulose) having a number average molecular weight of 500,000 to 3,500,000, wherein the alkali is sodium, potassium, or lithium. The osmopolymers further include osmopolymers comprising polymers that form hydrogels, such as Carbopole (registered trademark), acidic carboxypolymers, polymers of acrylic cross-linked with polyallyl sucrose (known as carboxypolymethylene), and carboxyvinyl polymers having a molecular weight of 250,000 to 4,000,000; Cyanamer (registered trademark) polyacrylamides; cross-linked water swellable indenemaleic anhydride polymers; Good-rite (registered trademark) polyacrylic acid having a molecular weight of 80,000 to 200,000; Aqua-Keeps (registered trademark), acrylate polymer polysaccharides composed of condensed glucose units, such as diester cross-linked polygluran; and the like. Polymers that form hydrogels are described in U.S. Pat. Nos. 3,865,108, 4,002,173, and 4,207,893, and in Handbook of Common Polymers, Scott and Roff, Chemical Rubber Co., Cleveland, Ohio.
The osmoagent (sometimes referred to as an osmotic solute or an osmotically effective agent) may be contained in both of the drug layer containing compound A and the push layer, and is not particularly limited, so long as it exhibits an osmotic activity gradient across the semipermeable membrane. Suitable osmagents include a member or two or more members selected from the group consisting of sodium chloride, potassium chloride, lithium chloride, magnesium sulfate, magnesium chloride, potassium sulfate, sodium sulfate, lithium sulfate, potassium acid phosphate, mannitol, glucose, lactose, sorbitol, inorganic salts, organic salts, and carbohydrates. The content of the osmoagent used is 15 to 40 W/W % with respect to the weight of the push layer.
Solvents suitable for manufacturing the formulation components include aqueous or inert organic solvents that do not adversely harm the substances used in the system. Such solvents broadly include one or more members selected from the group consisting of aqueous solvents, alcohols, ketones, esters, ethers, aliphatic hydrocarbons, halogenated solvents, cycloaliphatic solvents, aromatic solvents, heterocyclic solvents, and mixtures thereof. Typical solvents include acetone, diacetone alcohol, methanol, ethanol, isopropyl alcohol, butyl alcohol, methyl acetate, ethyl acetate, isopropyl acetate, n-butyl acetate, methyl isobutyl ketone, methyl propyl ketone, n-hexane, n-heptane, ethylene glycol monoethyl ether, ethylene glycol monoethyl acetate, methylene dichloride, ethylene dichloride, propylene dichloride, carbon tetrachloride, nitroethane, nitropropane, tetrachloroethane, ethyl ether, isopropyl ether, cyclohexane, cyclooctane, benzene, toluene, naphtha, 1,4-dioxane, tetrahydrofuran, diglyme, water, aqueous solvents containing inorganic salts (such as sodium chloride, calcium chloride, and the like), and mixtures thereof (such as acetone and water, acetone and methanol, acetone and ethyl alcohol, methylene dichloride and methanol, and ethylene dichloride and methanol).
The drug layer is formed from a pharmaceutical composition consisting of compound A in an amount pharmacologically effective in treatment or prevention, and a carrier for a sustained release pharmaceutical composition. The carrier for a sustained release pharmaceutical composition may include hydrophilic polymers.
The hydrophilic polymers impart an action of releasing compound A at a constant releasing rate. Suitable hydrophilic polymers include poly(alkylene oxide) having a number average molecular weight of 100,000 to 750,000, such as poly(ethylene oxide), poly(methylene oxide), poly(buthylene oxide, and poly(hexylene oxide); and poly(carboxymethyl cellulose) having a number average molecular weight of 40,000 to 400,000, typically poly(alkali carboxymethyl cellulose), poly(sodium carboxymethyl cellulose), poly(potassium carboxymethyl cellulose), and poly(lithium carboxymethyl cellulose). The drug composition may contain hydroxypropylalkyl cellulose having a number average molecular weight of 9,200 to 125,000, typically hydroxypropylethyl cellulose, hypromellose, hydroxypropylbutyl cellulose, and hydroxypropylpentyl cellulose, to improve delivery properties of the formulation; and polyvinylpyrrolidone having a number average molecular weight of 7,000 to 75,000, to improve flow properties of the formulation. Among these polymers, poly(ethylene oxide) having a number average molecular weight of 100,000 to 300,000 is most preferable. The content of the hydrophilic polymer can be appropriately selected in accordance with various factors such as physicochemical properties, content, and the like of a drug contained, but is 40 to 90 W/W % with respect to the drug layer.
The drug layer may further contain surfactants and disintegrants, if desired. Suitable surfactants are those having an HLB value of approximately 10 to 25, such as polyethylene glycol 400 monostearate, polyoxyethylene-4-sorbitan monolaurate, polyoxyethylene-20-sorbitan monooleate, polyoxyethylene-20-sorbitan monopalmitate, polyoxyethylene-20-monolaurate, polyoxyethylene-40-stearate, sodium oleate, and the like. Disintegrants may be selected from starches, clays, celluloses, algins and gums and crosslinked starches, celluloses and polymers. Representative disintegrants include corn starch, potato starch, croscarmelose, crospovidone, sodium starch glycolate, Veegum HV, methylcellulose, agar, bentonite, carboxymethylcellulose, alginic acid, guar gum, and the like.
Pan coating may be used to prepare the completed formulation, except for the exit orifice for releasing a drug from the surface of the formulation. In the pan coating system, the composition for forming the semipermeable membrane is deposited by spraying the composition onto the surface of the bilayered compressed core formed from the drug layer and the push layer, accompanied by tumbling in a rotating pan. Alternatively, the compressed core may be coated with the semipermeable membrane by well-known techniques in the art. After the coating, the semipermeable membrane may be dried in a forced-air oven or in a temperature and humidity controlled oven to remove the solvent(s) used in the coating from the formulation. Drying conditions may be appropriately selected on the basis of an available equipment, ambient conditions, solvents, a coating agent, a coating thickness, and the like.
The osmotic pump type formulation, an embodiment of the pharmaceutical composition for modified release of the present invention, can be prepared by known conventional methods, such as wet granulation techniques. In the wet granulation, a drug and a carrier for a sustained release pharmaceutical composition are blended using an organic solvent, such as denatured absolute alcohol and the like, as a granulation solution. The remaining components may be dissolved in a portion of the granulation solution such as the above solvent, and a wet mixture separately prepared is gradually added to the drug mixture, accompanied by the continuous mixing in a blender. The granulation solution is added until a wet aggregate is generated, and the wet aggregate are sifted through a screen arranged on an oven tray. The mixture is dried at a temperature of approximately 24 to 35° C. in a forced-air oven for approximately 18 to 24 hours. The dried granules are sized. A lubricant such as magnesium stearate or the like is added to the drug granules, and the whole is put into a milling jar and mixed on a jar mill for approximately 10 minutes. The composition is pressed into a layer, for example, in a Manestye (registered trademark) press or a Korsch LCT press. For a bilayered core, the drug-containing layer is pressed, and a composition for the push layer, prepared in a similar fashion by wet granulation techniques, is pressed against the drug-containing layer. One exit orifice, or two more exit orifices, are drilled in the drug layer end of the formulation. Optional water soluble overcoats, which may be colored (for example, Opadry colored coatings) or clear (for example, Opadry Clear), may be coated on the formulation to provide the completed formulation.
The osmotic pump type formulation, an embodiment of the pharmaceutical composition for modified release of the present invention, has at least one exit orifice. A drug is constantly released from the formulation through the exit orifice(s) by the compressed core. The exit orifice may be provided during the manufacture of the formulation, or during the drug delivery by the formulation in a fluid environment of use. The terms “exit orifice”, “delivery exit”, “drug delivery exit”, and similar terms as used herein include terms selected from the group consisting of pass, opening, orifice, and bore. Further, these expressions include an orifice that is formed from a substance or polymer that erodes, dissolves or is leached from the outer wall.
This substance or polymer may include, for example, erodible poly(glycolic acid) or poly(lactic acid) in the semipermeable membrane; gelatinous filaments; water-removable polyvinyl alcohol); a leachable compound, such as a fluid removable pore-forming substance selected from the group consisting of inorganic and organic salts, oxides, and carbohydrates. The exit(s) are formed by leaching one or two or more members selected from the group consisting of sorbitol, lactose, fructose, glucose, mannose, galactose, talose, sodium chloride, potassium chloride, sodium citrate and mannitol to provide a uniform-release dimensioned pore-exit orifice(s). The exit can have any shape, such as round, rectangle, square, elliptical, and the like, for the uniform release of a drug from the formulation. The formulation can be constructed with one or two or more exits in spaced-apart relation or on one or more surfaces of the formulation. The pore size of the exit is not particularly limited, so long as it can cooperate with the compressed core to control the release of the drug, but is preferably 0.3 to 0.6 mm. Drilling, including mechanical and laser drilling, through the semipermeable membrane can be used to form the exit orifice. Such exits and equipments for forming such exits are disclosed in U.S. Pat. No. 3,916,899, by Theeuwes and Higuchi and in U.S. Pat. No. 4,088,864, by Theeuwes, et al., each of which is incorporated herein by reference.
(5) Formulation Utilizing Swelling Polymer
The formulation utilizing a swelling polymer, as an embodiment of the pharmaceutical composition for modified release of the present invention, is a formulation for modified release containing a water-soluble high molecular weight polymer which swells upon imbibition of water.
Formulation techniques using a swelling polymer which may be used in the formulation for modified release of the present invention are described in U.S. Pat. Nos. 6,340,475, 5,972,389, 5,582,837, and 5,007,790, the contents of which are incorporated herein by reference.
The “water-soluble high molecular weight polymer which swells upon imbibition of water” used is not particularly limited, so long as it is a pharmaceutically acceptable polymer that swells in a dimensionally unrestricted manner upon imbibition of water, and that releases a drug continuously. Suitable polymers are those having a weight average molecular weight of preferably approximately 4,500,000 or more, more preferably approximately 4,500,000 to approximately 10,000,000, most preferably approximately 5,000,000 to approximately 8,000,000.
Such polymers include cellulose polymers and derivatives thereof, polysaccharides and derivatives thereof, polyalkylene oxides, and crosslinked polyacrylic acids and derivatives thereof. The term “cellulose” as used herein means a linear polymer of anhydroglucose. Preferred cellulose polymers are alkyl-substituted cellulose polymers that dissolve in the gastrointestinal tract. Preferred alkyl-substituted cellulose derivatives are those substituted with alkyl groups having 1 to 3 carbon atoms each. Examples thereof include, for example, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hypromellose, and carboxymethylcellulose. A preferred viscosity ranges between approximately 100 and approximately 110,000 cps, as measured in a 2% aqueous solution at 20° C. A viscosity in other embodiments ranges between approximately 1,000 and approximately 4,000 cps, as measured in a 2% aqueous solution at 20° C. More preferred alkyl-substituted celluloses are hydroxyethylcellulose and hypromellose. Preferred hydroxyethylcellulose is NATRASOL (product name) 250H×NF.
Further, most preferred polymers are polyalkylene oxide derivatives, particularly polyethylene oxide, i.e., an unsubstituted linear polymer of ethylene oxide. Preferred polyethylene oxide has a weight average molecular weight of approximately 900,000 to approximately 8,000,000. A preferred viscosity ranges between approximately 50 to approximately 2,000,000 cps, as measured in a 2% aqueous solution at 20° C. Preferred polyethylene oxide is POLYOX (product name), such as grade WSR Coagulant and grade WSR 303
Other examples of such polymers include both naturally-occurring and modified (semi-synthetic) polysaccharide gums, such as dextran, xanthan gum, gellan gum, welan gum, and rhamsan gum. Xanthan gum is preferred. Crosslinked polyacrylic acids of greatest utility are those whose properties are the same as those described above for alkyl-substituted celluloses and polyalkylene oxide polymers. Preferred crosslinked polyacrylic acids are those with a viscosity ranging from approximately 4,000 to approximately 40,000 cps, for a 1% aqueous solution at 25° C. Preferred examples are CARBOPOL (product name) NF grades 971P, 974P, and 934P, and WATER LOCK (product name) which are starch/acrylates/acrylamide copolymers.
The content of the “water-soluble high molecular weight polymer which swells upon imbibition of water” with respect to the weight of the formulation is not particularly limited, but is preferably approximately 1 to approximately 95 W/W %.
The formulation utilizing a swelling polymer, an embodiment of the pharmaceutical composition for modified release of the present invention, can be prepared as a pharmaceutically acceptable solid dosage form for oral administration such as tablets, particles, and particles retained in tablets or capsules. A presently preferred dosage form is a size 0 gelatin capsule containing two or three polymer particles (pellets) containing a drug. For the two-pellet capsules, the pellets are cylindrically shaped, 6.6 or 6.7 mm (or more generally, 6.5 to 7 mm) in diameter and 9.5 or 10.25 mm (or more generally, 9 to 12 mm) in length. For the three-pellet capsules, the pellets are cylindrically shaped, 6.6 mm in diameter and 7 mm in length. For a size 00 gelatin capsule with two pellets, the pellets are cylindrical, 7.5 mm in diameter and 11.25 mm in length. For a size 00 gelatin capsule with three pellets, the pellets are cylindrical, 7.5 mm in diameter and 7.5 mm in length. Another presently preferred dosage form is a tablet, with dimensions 18 to 22 mm in length, 6.5 to 7.8 mm in width, and 6.2 to 7.5 mm in height, more preferably with dimensions 20 mm in length, 6.7 mm in width, and 6.4 mm in height. These are merely examples, and the shapes and sizes can be varied considerably.
A particulate drug/polymer mixture or a drug-impregnated polymer matrix can be prepared by various known conventional methods, such as mixing, comminution, and fabrication techniques. These methods include, for example, direct compression using appropriate punches and dies, injection, and compression molding. When compression molding is carried out, lubricants may be optionally added. Examples of lubricants include stearic acid, magnesium stearate, calcium stearate, sodium stearyl fumarate, and the like, and magnesium stearate is preferred. The content of the lubricant is 0.25 to 3 W/W %, preferably less than 1 W/W %, with respect to the weight of the formulation. As other lubricants, hydrogenated vegetable oils, and hydrogenated and refined triglycerides of stearic and palmitic acids are preferable, and the content is approximately 1 to 5 W/W %, preferably approximately 2 W/W %, with respect to the weight of the formulation.
Most preferable sets of various components described above include a combination of approximately 90 to approximately 97 W/W % (with respect to the weight of the formulation) of polyethylene oxide having a weight average molecular weight of approximately 2,000,000 to approximately 7,000,000 as the “water-soluble high molecular weight polymer which swells upon imbibition of water” and less than approximately 2 W/W % (with respect to the weight of the formulation) of magnesium stearate as the lubricant. Examples of a combination of, for example, two water-soluble polymers include a combination of approximately 48 W/W % of polyethylene oxide having a weight average molecular weight of approximately 900,000 to approximately 7,000,000 and approximately 48 W/W % of hypromellose having a viscosity of approximately 3 to approximately 10,000 cps, as measured in a 2% aqueous solution at 20° C. (weight ratio=about 1:1).
It is expected that the formulation utilizing a swelling polymer is retained in the stomach by swelling.
(6) Matrix Formulation Utilizing Water-Soluble Polymer
The matrix formulation utilizing water-soluble polymer, an embodiment of the pharmaceutical composition for modified release of the present invention, is a formulation for modified release in which the drug is homogenously dispersed in one or more water-soluble polymers, such as hypromellose (HPMC).
Techniques for obtaining such a matrix formulation which may be used in the formulation for modified release according to the present invention are disclosed, for example, in WO 93/16686, the contents of which are incorporated herein by reference.
When hypromellose, a water-soluble polymer, is brought into contact with water, hydration thereof is caused, and a hydrogel layer is formed on the surface of a matrix. This gel layer containing a drug formed on the matrix surface is gradually dissolved and eroded, to release the drug from the layer. The matrix formulation of the present invention is characterized in that a drug may be controllably released by repeating the contact with water, the formation of the gel layer containing the drug, and the dissolution and erosion of the gel layer.
The matrix formulation of the present invention is characterized in that a sustained release filler consisting of a water-soluble polymer, an inactive diluent, and a physiologically active substance are homogenously dispersed. The water-soluble polymer is not particularly limited, so long as it is gradually gelled, eroded, dissolved, and/or disintegrated when exposed to an environmental fluid. Examples of the water-soluble polymers include, for example, hypromellose having a molecular weight of 1,000 to 4,000,000, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose having a molecular weight of 2,000 to 2,000,000, hypromellose phthalate having a labeled viscosity of 30 to 200 mm2/s [at 20° C.; a 10% solution prepared by dissolving hypromellose phthalate in a methanol/dichloromethane mixture (1:1)], carboxyvinyl polymers, chitosans, mannans, galactomannans, xanthans, carageenans, amylose, alginic acid, salts and derivatives thereof, pectin, acrylates, aminoalkylmethacrylate copolymers, methacrylate copolymers, polyacid anhydrides, polyamino acids, poly(methylvinyl ether/maleic anhydride) polymers, polyvinyl alcohols, polyvinylpyrrolidone, glucans, scleroglucans, carboxymethyl cellulose and derivatives thereof, methyl cellulose, or conventional water-soluble cellulose derivatives. Hypromellose having a molecular weight of 1,000 to 2,000,000, or carboxyvinyl polymers of 3,000 to 45,000 cps (at 25° C.; a 0.5% aqueous solution) is preferable, and hypromellose having a molecular weight of 10,000 to 1,000,000, or carboxyvinyl polymers of 4,000 to 40,000 cps (at 25° C.; a 0.5% aqueous solution) is more preferable. The content of the water-soluble polymer is 10 W/W % or more per formulation unit, preferably 30 W/W % or more, more preferably 70 W/W % or more. These water-soluble polymers may be contained alone or as a combination thereof in an appropriate amount(s).
Various fillers for medicaments may be appropriately used to prepare the matrix formulation of the present invention. The fillers for medicaments are not particularly limited, so long as they are pharmaceutically acceptable and may be used as additives for medicament. As the fillers, for example, a diluent, a binder, a disintegrator, an acidulant, an effervescent agent, an artificial sweetener, a flavor, a lubricant, a coloring agent, or the like may be used. The diluent may be selected from mannitol, lactose, starches derived from various organs, sorbitol, xylitol, citric acid, microcrystalline cellulose, and/or a diluent capable of generally promoting a penetration of water or an aqueous liquid into a pharmaceutical preparation. The binders include, for example, hypromellose, hydroxypropyl cellulose, polyvinyl alcohol, methyl cellulose, gum arabic, and the like. The disintegrators include, for example, a corn starch, a starch, carmellose calcium, carmellose sodium, low-substituted hydroxypropyl cellulose, and the like. The acidulants include, for example, citric acid, tartaric acid, malic acid, and the like. The effervescent agents include, for example, sodium bicarbonate and the like. The artificial sweeteners include, for example, saccharin sodium, dipotassium glycyrrhizinate, aspartame, stevia, thaumatin, and the like. The flavors include, for example, lemon, lemon-lime, orange, menthol, and the like. The lubricants include, for example, magnesium stearate, calcium stearate, sucrose fatty acid esters, polyethylene glycol, talc, stearic acid, and the like. These fillers for medicaments may be contained alone or as a combination thereof in an appropriate amount(s).
The matrix formulation of the present invention may be manufactured by a known method per se. In particular, tablets may be manufactured by a tablet forming method which is commonly used and known to those skilled in the art. The tabletting pressure is generally within a range of 3 to 20 kN. In a small scale, tablets may be prepared, in accordance with methods explained in detail in the following Examples, by preparing powder and/or granules with a mortar and a pestle, and forming the powder and/or granules into tablets by using an oil press tabletting machine.
(7) Modified Release Formulation with Coating Membrane
As a method for controlling the release (i.e., modified release) of a drug from a pharmaceutical preparation, a coating membrane is applied to the surface of a pharmaceutical preparation by coating. The kind of coating membrane is not particularly limited. The coating may be applied to not only a shaped preparation such as a tablet or the like, but also various preparations such as powder, granules, pellets, or the like.
A coating liquid may contain, for example, a membrane forming agent (mainly a polymer), a plasticizer (which provides plasticity, flexibility, and extensibility to a coating membrane), a water-soluble base (such as lactose, sodium chloride, or the like), a dispersing agent (which prevents particles or tablets from adhering and aggregating after the coating), or the like. These components may be dissolved or dispersed in an appropriate solvent, such as water, alcohol, or the like, to prepare the coating liquid.
The release of a drug from the formulation can be controlled by appropriately adjusting, for example, the kinds and the mixing ratio of components contained in the coating liquid, the amount of coating, or the like. For example, a preferable ratio of the membrane forming agent to the water-soluble base is 99:1 to 50:50 (membrane forming agent: water-soluble base). The content of the coating membrane is preferably approximately 2 to 30 parts by weight, with respect to 100 parts by weight of an uncoated tablet.
Examples of a coating method include, for example, a method in which a coating liquid, such as an organic solvent solution, or a mixing solution or suspension of an organic solvent and water, is sprayed while being rotated, by using a coating pan, or a method in which a coating liquid is sprayed while being fluidized by air blown from the bottom of a fluidized bed. Further, a coating liquid prepared by dissolving or dispersing a membrane forming agent in a solvent may be sprayed, and then the solvent may be removed by drying to form a coating membrane on the surface of a pharmaceutical preparation. As a simple method, a coating membrane may be formed by immersing shaped preparations or the like in a coating liquid.
Examples of the membrane forming agent as used herein include, for example, a water-insoluble polymer or a water-soluble polymer. The membrane forming agent is not particularly limited, so long as it is pharmaceutically acceptable and biocompatible. These membrane forming agents may be added alone or as a combination thereof in an appropriate amount(s).
Examples of the water-insoluble polymer include, for example, dibenzyl phthalate, dihexyl phthalate, butyl octyl phthalate, beeswax, carnauba wax, cetyl alcohol, cetyl stearyl alcohol, glyceryl behenate, lipids, fats, resins such as shellac or the like, cellulose derivatives such as ethyl cellulose, cellulose acetate, or the like, polyacrylate derivatives such as aminoalkylmethacryl copolymer (product name: Eudragit RS) or the like, polymethacrylate derivatives such as methacrylate copolymer (product name: Eudragit L) or the like, hydroxypropylmethyl cellulose acetate succinate, polylactic acid, polyglycolic acid, or the like.
Examples of the water-soluble polymer include, for example, hypromellose, hydroxypropyl cellulose, hydroxyethyl cellulose, carmellose sodium, methyl cellulose, polyvinylpyrrolidone, polyethylene glycol, polyvinyl alcohol, or the like.
To enhance the hydrophilic property of the coating membrane, a water-soluble base may be added. Examples of the water-soluble base include, for example, maltose, sucrose, lactose, sodium chloride, citric acid, polyethylene glycol 400, dextrose, fructose, xylitol, polyoxyethylene sorbitan monooleate, or the like.
The coating liquid which may be used in the present invention preferably contains one or more of the above-mentioned water-insoluble polymers, and more preferably further contains one or more of the water-soluble polymers and/or one or more of the water-soluble bases.
Further, the coating liquid may contain a plasticizer to provide plasticity, flexibility, and extensibility to the coating membrane. Examples of the plasticizer include, for example, triacetin, dioctyl azelate, epoxidized tallate, triisooctyl trimellitate, triisononyl trimellitate, sucrose acetate isobutyrate, soybean oil, propylene glycol, glycerol, polyethylene glycol, glyceryl triacetate (triacetin), triethyl citrate, acetyl triethyl citrate, diethyl phthalate, diethyl sebacate, dibutyl sebacate, acetylated monoglyceride, castor oil, liquid paraffin, or the like.
If desired, a surfactant and/or a disintegrator may be added. As such a surfactant which may be used in the coating membrane, a surfactant having an HLB value of approximately 10 to 25, such as polyethylene glycol 400 monostearate, polyoxyethylene-4-sorbitan monolaurate, polyoxyethylene-20-sorbitan monooleate, polyoxyethylene-20-sorbitan monopalmitate, polyoxyethylene-20-monolaurate, polyoxyethylene-40-stearate, sodium oleate, or the like, may be used.
Examples of the disintegrator include, for example, starches, clay, cellulose, algin, gums, crosslinked starches, crosslinked cellulose, or crosslinked polymers. Typically, for example, corn starch, potato starch, croscarmellose, crospovidone, sodium starch glycorate, Veegum HV, methyl cellulose, agar, bentonite, carboxyl methyl cellulose, alginic acid, guar gum, or the like, may be used.
As a solvent suitable for manufacturing the formulation of the present invention, an aqueous or inert organic solvent which does not adversely affect substances used in the system may be used. Examples of the solvent include, for example, aqueous solvents, alcohols, ketones, esters, ethers, aliphatic hydrocarbons, halogenated solvents, cycloaliphatic, aromatic, or heterocyclic solvents, or a mixture thereof. Typical solvents may be, for example, acetone, diacetone alcohol, methanol, ethanol, isopropanol, butanol, methyl acetate, ethyl acetate, isopropyl acetate, n-butyl acetate, methyl isobutyl ketone, methyl propyl ketone, n-hexane, n-heptane, ethylene glycol monoethyl ether, ethylene glycol monoethyl acetate, methylene dichloride, ethylene dichloride, propylene dichloride, carbon tetrachloride, nitroethane, nitropropane, tetrachloroethane, ethyl ether, isopropyl ether, cyclohexane, cyclooctane, benzene, toluene, naphtha, 1,4-dioxane, tetrahydrofuran, diglyme, water, an aqueous solvent containing an inorganic salt such as sodium chloride, calcium chloride, or the like, or a mixture thereof, such as a mixture of acetone and water, a mixture of acetone and methanol, a mixture of acetone and ethanol, a mixture of methylene dichloride and methanol, or a mixture of ethylene dichloride and methanol.
(8) Matrix Formulation Utilizing Insoluble Polymer
A matrix formulation utilizing an insoluble polymer, an embodiment of the present invention, is a pharmaceutical composition for modified release in which the drug is uniformly dispersed in a water-insoluble polymer. Because the matrix consisting of the water-insoluble polymer can control the penetration of water into the formulation, the matrix formulation can modify the release of the drug from the formulation by controlling the dissolution rate of the drug in the matrix and the dispersion rate of the dissolved drug in the matrix.
The water-insoluble polymer used in the present invention is not particularly limited, so long as it is pharmaceutically acceptable. Examples of the water-insoluble polymer include, for example, dibenzyl phthalate, dihexyl phthalate, butyl octyl phthalate, beeswax, carnauba wax, cetyl alcohol, cetyl stearyl alcohol, glyceryl behenate, lipids, fats, resins such as shellac or the like, cellulose derivatives such as ethyl cellulose, cellulose acetate, or the like, polyacrylate derivatives such as aminoalkylmethacryl copolymer or the like, polymethacrylate derivatives such as methacrylate copolymer, ethyl acrylate methyl methacrylate copolymer or the like, hydroxypropylmethyl cellulose acetate succinate, polylactic acid, polyglycolic acid, or the like.
The content of the insoluble polymer is 1 W/W % or more per formulation unit, preferably 2 W/W % or more, more preferably 5 W/W % or more. These insoluble polymers may be contained alone or as a combination thereof in an appropriate amount(s).
Various fillers for medicaments may be appropriately used to prepare the matrix formulation of the present invention. The fillers for medicaments are not particularly limited, so long as they are pharmaceutically acceptable and may be used as additives for medicament. As the fillers, for example, a diluent, a binder, a disintegrator, an acidulant, an effervescent agent, an artificial sweetener, a flavor, a lubricant, a coloring agent, or the like may be used. The diluent may be selected from mannitol, lactose, starches derived from various organs, sorbitol, xylitol, citric acid, microcrystalline cellulose, and/or a diluent capable of generally promoting a penetration of water or an aqueous liquid into a pharmaceutical preparation. The binders include, for example, hypromellose, hydroxypropyl cellulose, polyvinyl alcohol, methyl cellulose, gum arabic, and the like. The disintegrators include, for example, a corn starch, a starch, carmellose calcium, carmellose sodium, low-substituted hydroxypropyl cellulose, and the like. The acidulants include, for example, citric acid, tartaric acid, malic acid, and the like. The effervescent agents include, for example, sodium bicarbonate and the like. The artificial sweeteners include, for example, saccharin sodium, dipotassium glycyrrhizinate, aspartame, stevia, thaumatin, and the like. The flavors include, for example, lemon, lemon-lime, orange, menthol, and the like. The lubricants include, for example, magnesium stearate, calcium stearate, sucrose fatty acid esters, polyethylene glycol, talc, stearic acid, and the like. These fillers for medicaments may be contained alone or as a combination thereof in an appropriate amount(s).
The matrix formulation of the present invention may be manufactured by a known method per se. In particular, tablets may be manufactured by a tablet forming method which is commonly used and known to those skilled in the art. The tabletting pressure is generally within a range of 3 to 20 kN. In a small scale, tablets may be prepared, in accordance with methods explained in detail in the following Examples, by preparing powder and/or granules with a mortar and a pestle, and forming the powder and/or granules into tablets by using an oil press tabletting machine.
EXAMPLES
The present invention will now be further illustrated by, but is by no means limited to, the following Examples.
In the following Examples, unless otherwise noted, a compound produced according to Example 41 of WO 99/20607 was used as compound A.
Example 1
Preparation of Sustained Release Hydrogel-Forming Formulation
In this Example, as the pharmaceutical composition for modified release of the present invention, sustained release hydrogel-forming formulations 1A to 1C were prepared.
Example 1A
400 g of the compound A, 100 g of polyethylene oxide, 291.2 g of polyethylene glycol, 0.8 g of finely ground dibutyl hydroxytoluene (BHT) (manufactured by Merck, the same was used hereinafter), and 8 g of magnesium stearate were weighed out, and mixed by using a mixer. The mixture was compression-molded by using Roller Compactor Mini (manufactured by Freund Corporation; the same apparatus was used hereinafter) and sieved to obtain a pharmaceutical composition for modified release (granules) of the present invention. The obtained granules were formed into tablets by using a rotary tabletting machine (manufactured by HATA IRON WORKS CO., LTD.; the same apparatus was used hereinafter) to obtain 400 mg/tablet of a pharmaceutical composition for modified release (tablet) of the present invention. The obtained tablet was coated with a film coating agent dispersed in water (Opadry, manufactured by Colorcon, Inc., the same was used hereinafter) by using High coater (HCT-30, manufactured by Freund Corporation, the same apparatus was used hereinafter) to obtain a pharmaceutical composition for modified release (tablet) of the present invention.
Example 1B
400 g of the compound A, 250 g of polyethylene oxide, 190.7 g of polyethylene glycol, 0.8 g of finely ground BHT, and 8.5 g of magnesium stearate were weighed out, and mixed by using a mixer. The mixture was compression-molded by using Roller Compactor Mini, and then sieved to obtain a pharmaceutical composition for modified release (granules) of the present invention. The obtained granules were formed into tablets by using a rotary tabletting machine to obtain 425 mg/tablet of a pharmaceutical composition for modified release (tablet) of the present invention. The obtained tablet was coated with a film coating agent dispersed in water by using High coater to obtain a pharmaceutical composition for modified release (tablet) of the present invention.
Example 1C
Into a fluidized bed granulating apparatus GPCG-5 (manufactured by Freund Corporation; the same apparatus was used hereinafter), 800 g of de-lumped compound A, 1120 g of polyethylene oxide, 1913.6 g of polyethylene glycol, and 120 g of hydroxypropylcellulose (HPC-SL, manufactured by Nippon Soda Co., Ltd.) were loaded, and granulated with purified water to obtain a pharmaceutical composition for modified release (granules) of the present invention. The pharmaceutical composition for modified release (granules) of the present invention was sieved, and mixed with 6.4 g of finely ground BHT and 40 g of magnesium stearate, and the obtained mixture was formed into tablets by using the rotary tabletting machine to obtain a pharmaceutical composition for modified release (tablet) of the present invention having a weight per tablet of 250 mg. The obtained tablets were spray-coated with an aqueous dispersion of the film coating agent using HiCoater to obtain a pharmaceutical composition for modified release (tablet) of the present invention having a weight per tablet of 257.5 mg.
Comparative Example 1
After 400 g of de-lumped compound A was mixed with 1200 g of D-mannitol, 320 g of purified water was added thereto, and the mixture was kneaded by using an agitation granulator (VG-25, manufactured by Powrex Corporation). The resulting product was sieved through a screen (opening: 850 μm), and dried by using a fluidized bed granulating apparatus (FLO-1, manufactured by Freund Corporation). The dried product was sieved through a screen (opening: 500 μm), and then filled into No. 1 capsules at a content of 320 mg per capsule to obtain a pharmaceutical composition of Comparative Example containing 80 mg of compound A.
Example 2
Preparation of Multi-Layered Formulation Consisting of Drug Core and Release-Controlling Layer which are Geometrically Arranged
In the Examples (Examples 2A to 2D), as the pharmaceutical composition for modified release of the present invention, multi-layered formulations 2A to 2D were prepared.
Step 1: Production of Mixed Powder Constituting Layer 2 Containing Active Substance
A mixed powder containing 50.0 mg of compound A and the composition unit shown in Table 1 was produced, and used in producing layer 2 as the intermediate layer of the three-layered tablet. The powder composed of the composition unit was prepared by weighing out necessary amounts of the active substance (compound A), mannitol, Hypromellose (90SH-15000, manufactured by Shin-Etsu Chemical Co. Ltd), polyvinyl pyrrolidone, microcrystalline cellulose, and magnesium stearate, and mixing them with a mortar and a pestle so that they were homogenized.
TABLE 1
Compound A
50.0
mg
Mannitol
15.0
mg
Hypromellose (90SH-15000)
15.0
mg
Polyvinyl pyrrolidone
4.8
mg
Microcrystalline cellulose
63.7
mg
Magnesium stearate
1.5
mg
Total
150.0
mg
Step 2: Production of Granules Constituting Layers 1 and 3 (Layers 1 and 3 Containing No Drug) Used for Modified Release of Drug
Granules made with the composition ratio shown in Table 2 were produced and used in producing layer 1 as the top layer and layer 3 as the bottom layer of the three-layered tablet. Specifically, the granules were prepared by weighing out necessary amounts of hypromellose, hydrogenated castor oil, yellow iron oxide, and magnesium stearate; mixing them by the use of a mortar and a pestle so that they were homogenized; further moistening them with a solution of ethylcellulose in alcohol (10% w/w); and drying the homogeneously wet aggregate.
TABLE 2
Hypromellose (90SH-15000)
80.25%
Hydrogenated castor oil
13.50%
Yellow iron oxide
0.25%
Ethylcellulose
5.00%
Magnesium stearate
1.00%
Total
100.00%
Step 3: Production of Three-Layered Tablet (Compression Molding)
Three-layered tablet were prepared by an oil press tabletting machine with a tabletting pressure of 1000 kg/punch. The granules of layer 3 prepared in Step 2 were put into a die, and subjected to light tapping so that the upper surface became flat. On the surface, the mixed powder of layer 2 containing the active substance prepared in Step 1 was loaded, which was subjected to light tapping so that the upper surface became flat. Furthermore, on the surface, the granules of the layer 1 prepared in the Step 2 were loaded into the die, and subjected to compression molding. Thus, three-layered tablets containing 50 mg of compound A (2A to 2D) were produced.
The weight and the punch diameter of each of layers 1, 2, and 3 in each of multi-layered formulations 2A to 2D are shown in Table 3.
TABLE 3
Examples
2 A
2 B
2 C
2 D
Layer 1
100.0
100.0
100.0
100.0
Layer 2
150.0
300.0
150.0
150.0
Layer 3
150.0
150.0
150.0
150.0
Total (mg)
400.0
550.0
400.0
400.0
Punch
8
8
7
6.5
diameter (mm)
Example 3
Preparation of Gel Formulation in which a Plurality of Gums is Combined
In this Example, as the pharmaceutical composition for modified release of the present invention, a gel formulation composed of the composition unit shown in Table 4 was prepared.
Specifically, necessary amounts of locust bean gum (GENUGUM type RL-200-J, manufactured by Sansho Co., Ltd.), xanthan gum (VS-900, manufactured by Nitta Gelatin Inc.), dextrose, and calcium sulfate were weighed out, and mixed sufficiently by using a mortar and a pestle so that the mixture was homogenized. Furthermore, an appropriate amount of purified water was added thereto, and the mixture was stirred and mixed. The mixture was sieved through a screen, and the obtained product was dried. To the dried product, a necessary amount of compound A was added. To the mixture, a solution of ethylcellulose in alcohol (100 mg/mL) was gradually added. The mixture was dried, and the dried product was put into a die, and subjected to compression molding by an oil press tabletting machine with a tabletting pressure of 1000 kg/punch by using a punch having a diameter of 8 mm.
TABLE 4
Compound A
50.0 mg
Locust bean gum (GENUGUM type RL-200-J)
50.0 mg
Xanthan gum (VS-900)
50.0 mg
Dextrose
70.0 mg
Calcium sulfate
10.0 mg
Ethylcellulose
14.0 mg
Total
244.0 mg
Example 4
Preparation of Osmotic Pump Type Formulation
In this Example, as the pharmaceutical composition for modified release of the present invention, an osmotic pump type formulation was prepared.
Step 1: Production of Mixed Powder Constituting Drug Layer Containing Active Substance
Mixed powder containing 50.0 mg of compound A and the composition unit shown in Table 5 was produced, and it was used in producing a bilayered compressed core.
The powder composed of the composition unit was prepared by weighing out necessary amounts of active substance (compound A), polyethylene oxide (Polyox WSR N-80, manufactured by DOW), hypromellose (TC-5 R, manufactured by Shin-Etsu Chemical Co. Ltd.), and magnesium stearate, and mixing them sufficiently by using a mortar and a pestle so that they were homogenized.
TABLE 5
Compound A
50.0
mg
Polyethylene oxide (Polyox WSR N-80)
100.0
mg
Hypromellose (TC-5 R)
6.0
mg
Magnesium stearate
1.0
mg
Total
157.0
mg
Step 2: Production of Mixed Powder Constituting Push Layer
A mixed powder composed of the composition unit shown in Table 6 was produced, and it was used in producing the bilayered compressed core.
Specifically, the mixed powder was produced by weighing out necessary amounts of polyethylene oxide (Polyox WSR Coagulant, manufactured by DOW), sodium chloride, hypromellose, red ferric oxide, and magnesium stearate, and mixing them sufficiently by using a mortar and a pestle so that they were homogenized.
TABLE 6
Polyethylene oxide (Polyox WSR Coagulant)
60.0
mg
Sodium chloride
30.0
mg
Hypromellose (TC-5 R)
4.0
mg
Red ferric oxide
1.0
mg
Magnesium stearate
0.5
mg
Total
95.5
mg
Step 3: Production of Bilayered Compressed Core Composed of Drug Layer and Push Layer
A bilayered compressed core was prepared by an oil press tabletting machine with a tabletting pressure of 1000 kg/punch. The mixed powder for a push layer prepared in Step 2 was put into a die, the mixed powder for a drug layer prepared in Step 1 was loaded thereon, and the both layers were subjected to compression molding to produce a bilayered compressed core containing 50 mg of compound A.
Step 4: Production of Semi-Permeable Membrane and Membrane Coating
Necessary amounts of polyethylene glycol 4000 and cellulose acetate (mass ratio of 6:94) were dissolved in a mixed solvent of dichloromethane and methanol (mass ratio of 9:1) to prepare a coating solution having a solid concentration of 2% w/w. By using this solution, a film coating was formed so that the coating component was 5% w/w with respect to the bilayered compressed core.
Step 5: Punching
A needle (27G) having a diameter of 0.4 mm was used to form orifices at the drug layer side of the semi-permeable-membrane-coated tablets prepared in Step 4, to prepare an osmotic pump type formulation as the pharmaceutical composition for modified release of the present invention.
Example 5
Preparation of Formulation Using Swelling Polymer
In the Examples (Examples 5A to 5C), as the pharmaceutical composition for modified release of the present invention, formulations 5A to 5C using a swelling polymer composed of the composition unit shown in Table 7 were prepared.
Specifically, necessary amounts of compound A and polyethylene oxide (various types of Polyox, manufactured by DOW) were weighed out, and mixed sufficiently by using a mortar and a pestle so that they were homogenized. The mixture was put into a die and subjected to compression molding by an oil press tabletting machine with a tabletting pressure of 1000 kg/punch by using a punch having a diameter of 7 mm.
TABLE 7
Examples
5 A
5 B
5 C
Compound A
50.0
50.0
50.0
Polyethylene oxide
200.0
—
—
(Polyox WSR N-60K)
Polyethylene oxide
—
200.0
—
(Polyox WSR N-12K)
Polyethylene oxide
—
—
200.0
(Polyox WSR N-205)
Total (mg)
250.0
250.0
250.0
Example 6
Preparation of Matrix Formulation Using Water-Soluble Polymer
In the Examples (Examples 6A to 6N), as the pharmaceutical composition for modified release of the present invention, matrix formulations 6A to 6N composed of the composition units shown in Tables 8 and 9 were prepared.
Specifically, necessary amounts of compound A and various additives [hypromellose (manufactured by Shin-Etsu Chemical Co. Ltd) or hydroxypropylcellulose (manufactured by Nippon Soda Co., Ltd.)] were weighed out, and mixed sufficiently by using a mortar and a pestle so that they were homogenized. The mixture was put into a die and subjected to compression molding by an oil press tabletting machine with a tabletting pressure of 1000 kg/punch.
TABLE 8
Examples
6 A
6 B
6 C
6 D
6 E
6 F
Compound A
100.0
100.0
100.0
100.0
100.0
100.0
Hypromellose
200.0
—
—
50.0
—
—
(METLOSE SR 90SH-100SR)
Hypromellose
—
200.0
—
—
25.0
—
(METLOSE SR 90SH-
4000SR)
Hypromellose
—
—
200.0
—
—
25.0
(METLOSE SR 90SH-
15000SR)
Total (mg)
300.0
300.0
300.0
150.0
125.0
125.0
TABLE 9
Examples
6 G
6 H
6 I
6 J
6 K
6 L
6 M
6 N
Compound A
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
Hydroxypropyl
—
—
—
—
50.0
—
—
—
cellulose
(HPC-L)
Hydroxypropyl
200.0
—
100.0
50.0
—
25.0
—
—
cellulose
(HPC-M)
Hydroxypropyl
—
200.0
—
—
—
—
25.0
10.0
cellulose
(HPC-H)
Total (mg)
300.0
300.0
200.0
150.0
150.0
125.0
125.0
110.0
Example 7
Preparation of Modified Release Formulation with Coating Membrane
In the Examples (Examples 7A to 7E), as the pharmaceutical composition for modified release of the present invention, modified release formulations 7A to 7E with a coating membrane composed of the composition unit shown in Table 10 were prepared.
Specifically, firstly, necessary amounts of the compound A and additives of a core tablet were weighed out, and mixed sufficiently by using a mortar and a pestle so that they were homogenized. The mixture was put into a die and subjected to compression molding by an oil press tabletting machine with a tabletting pressure of 1000 kg/punch to produce each core tablet. Additionally, necessary amounts of film coating components [as aminoalkyl methacrylate copolymer RS, various types of Eudragit, manufactured by Degussa corporation; and as Hypromellose, TC-5 E manufactured by Shin-Etsu Chemical Co. Ltd] were dissolved/dispersed in ethanol to prepare each coating solution having a solid concentration of 10% w/w. By using each solution, film coating was conducted so that the coating component had a prescribed amount with respect to the core tablet.
TABLE 10
Examples
7 A
7 B
7 C
7 D
7 E
Core tablet
Compound A
100.0
100.0
100.0
100.0
100.0
Mannitol
100.0
—
—
—
—
Polyethylene glycol 8000
—
200.0
200.0
200.0
200.0
Subtotal (mg)
200.0
300.0
300.0
300.0
300.0
Film coat
Aminoalkyl methacrylate
—
—
15.0
30.0
22.5
Copolymer RS (Eudragit RL PO)
Aminoalkyl methacrylate
10.0
12.0
—
—
—
Copolymer RS (Eudragit RS PO)
Hypromellose (TC-5 E)
—
—
—
—
7.5
Subtotal (mg)
10.0
12.0
15.0
30.0
30.0
Total (mg)
210.0
312.0
315.0
330.0
330.0
Example 8
Preparation of Matrix Formulation Using Insoluble Polymer
Examples 8A to 8C
As the pharmaceutical composition for modified release of the present invention, matrix formulations (tablets) 8A to 8C composed of the composition unit shown in Table 11 were prepared.
Specifically, necessary amounts of the compound A and ethylcellulose (Ethocel 10 premium, manufactured by DOW) were weighed out, and mixed sufficiently by using a mortar and a pestle so that they were homogenized. The mixture was put into a die and subjected to compression molding by an oil press tabletting machine with a tabletting pressure of 1000 kg/punch.
TABLE 11
Examples
8 A
8 B
8 C
Compound A
100.0
100.0
100.0
Ethylcellulose
200.0
25.0
10.0
(Ethocel 10 premium)
Total (mg)
300.0
125.0
110.0
Examples 8D and 8E
As the pharmaceutical composition for modified release of the present invention, matrix formulations (granules) 8D to 8E composed of the composition unit shown in Table 12 were prepared.
Specifically, necessary amounts of the compound A and ethylcellulose shown in Table 12 were weighed out, and mixed sufficiently by using a mortar and a pestle so that they were homogenized. Furthermore, an appropriate amount of ethanol was added thereto, and the mixture was stirred and mixed. The mixture was dried, and screened by using a sieve so as to remove coarse particles to prepare granule formulations 8D and 8E for modified release as the pharmaceutical composition for modified release of the present invention.
TABLE 12
Examples
8 D
8 E
Compound A
25.0
50.0
Ethylcellulose
75.0
100.0
Total (mg)
100.0
150.0
Examples 8F and 8G
Necessary amounts of the compound A and microcrystalline cellulose shown in Table 13 were weighed out, and mixed sufficiently by using a mortar and a pestle so that they were homogenized. Furthermore, a prescribed amount of solution of ethylcellulose in ethanol was added thereto, and the mixed solution was stirred and mixed. The mixture was dried and screened by using a sieve so as to remove coarse particles to prepare granule formulations 8F and 8G for modified release as the pharmaceutical composition for modified release of the present invention.
TABLE 13
Examples
8 F
8 G
Compound A
50.0
50.0
Microcrystalline cellulose
—
50.0
Ethylcellulose
8.0
10.0
Total (mg)
58.0
110.0
Experimental Example 1
Dissolution Test of Sustained Release Hydrogel-Forming Formulation
A drug release property from each of the formulations prepared in Examples 1A to 1C was evaluated by the dissolution test, method 2 (paddle method) described in the Japanese Pharmacopoeia. The test was carried out using 900 mL of a USP phosphate buffer (pH 6.8) as a test solution at a paddle rotating speed of 200 rpm. The drug concentration in the test solution was measured every hour, and the drug release property was evaluated. The results are shown in Table 14 and FIG. 1.
TABLE 14
Examples
1 A
1 B
1 C
Dissolution rate
54
15
27
1.5 hours
Dissolution rate
80
31
52
2.5 hours
Dissolution rate
99
66
95
7 hours
Experimental Example 2
Dissolution Test of Multi-Layered Formulation Consisting of Drug Core and Release-Controlling Layer which are Geometrically Arranged
A drug release property from each of the formulations prepared in Examples 2A to 2D was evaluated by the method described in Experimental Example 1. The results are shown in Table 15 and FIG. 2.
TABLE 15
Examples
2 A
2 B
2 C
2 D
Dissolution rate
19
19
18
21
1.5 hours
Dissolution rate
26
32
25
30
2.5 hours
Dissolution rate
57
75
73
88
7 hours
Experimental Example 3
Dissolution Test of Gel Formulation in which a Plurality of Gums is Combined
A drug release property from the formulation prepared in Example 3 was evaluated by the method described in Experimental Example 1. The results are shown in FIG. 3. As a result, the dissolution rates after 1.5 hours, 2.5 hours, and 7 hours were 11%, 18%, and 78%, respectively.
Experimental Example 4
Dissolution Test of Osmotic Pump Type Formulation
A drug release property from the formulation prepared in Example 4 was evaluated by the method described in Experimental Example 1. The results are shown in FIG. 4. As a result, the dissolution rates after 1.5 hours, 2.5 hours, and 7 hours were 21%, 43%, and 90%, respectively.
Experimental Example 5
Dissolution Test of Formulation Using Swelling Polymer
A drug release property from each of the formulations prepared in Examples 5A to 5C was evaluated by the method described in Experimental Example 1. The results are shown in Table 16 and FIG. 5.
TABLE 16
Examples
5 A
5 B
5 C
Dissolution rate
13
20
26
1.5 hours
Dissolution rate
23
38
47
2.5 hours
Dissolution rate
67
91
96
7 hours
Experimental Example 6
Dissolution Test of Matrix Formulation Using Water-Soluble Polymer
A drug release property from each of the formulations prepared in Examples 6A to 6N was evaluated by the method described in Experimental Example 1. The results are shown in Tables 17 and 18 and FIGS. 6 and 7.
TABLE 17
Examples
6 A
6 B
6 C
6 D
6 E
6 F
6 G
Dissolution rate
18
7
6
38
40
34
7
1.5 hours
Dissolution rate
29
11
10
59
51
43
12
2.5 hours
Dissolution rate
76
35
26
98
85
76
34
7 hours
TABLE 18
Examples
6 H
6 I
6 J
6 K
6 L
6 M
6 N
Dissolution rate
6
10
19
38
24
15
27
1.5 hours
Dissolution rate
8
17
29
59
39
25
43
2.5 hours
Dissolution rate
20
52
73
96
86
62
89
7 hours
Experimental Example 7
Dissolution Test of Modified Release Formulation with Coating Membrane
A drug release property from each of the formulations, prepared in Examples 7A to 7E was evaluated by the method described in Experimental Example 1. The results are shown in Table 19 and FIG. 8.
TABLE 19
Examples
7 A
7 B
7 C
7 D
7 E
Dissolution rate
1
3
5
5
42
1.5 hours
Dissolution rate
2
3
14
7
67
2.5 hours
Dissolution rate
8
6
99
54
104
7 hours
Experimental Example 8
Dissolution Test of Matrix Formulation Using Insoluble Polymer
A drug release property from each of the formulations prepared in Examples 8A to 8C was evaluated by the method described in Experimental Example 1. The results are shown in Table 20 and FIG. 9.
TABLE 20
Examples
8 A
8 B
8 C
Dissolution rate
12
22
28
1.5 hours
Dissolution rate
17
32
42
2.5 hours
Dissolution rate
38
69
86
7 hours
A drug release property from each of the formulations prepared in Examples 8D to 8G was evaluated by the method described in Experimental Example 1. The results are shown in Table 21 and FIG. 10.
TABLE 21
Examples
8 D
8 E
8 F
8 G
Dissolution rate
51
45
67
66
1.5 hours
Dissolution rate
57
50
77
75
2.5 hours
Dissolution rate
69
60
92
91
7 hours
Experimental Example 9
Pharmacokinetics (PK) Test of Immediate Release Formulation (Capsule Formulation) in Human
An immediate release formulation (capsule formulation) containing compound A was administered to healthy subjects in a fasted state, before 30 min from the intake of a meal, or after 30 min from the meal, and the drug concentration in the plasma was measured. The immediate release formulations (capsule formulations) containing 0.1 mg, 1 mg, 5 mg, 20 mg, and 80 mg of the compound A were used in combinations as needed so that the dose of compound A became 0.1 mg, 1 mg, 3 mg, 10 mg, 30 mg, 100 mg, 160 mg, 240 mg, and 340 mg.
Results are shown in FIG. 11. When the maximum plasma concentration (Cmax) of compound A and the increase in heart rate from the base line were analyzed, a positive correlation was observed.
Experimental Example 10
Pharmacokinetics (PK) Test of Sustained Release Hydrogel-Forming Formulation in Human
The pharmaceutical composition for modified release of the present invention prepared in Example 1A or 1B (containing compound A in an amount corresponding to 200 mg) was administered to healthy subjects in a fasted state (Fasted) or after 30 min from the intake of a meal (Fed), and the drug concentration in the plasma was measured. On the other hand, two capsules of the pharmaceutical composition (conventional formulation) (containing compound A in an amount corresponding to 160 mg) of Comparative Example 1 was administered to healthy subjects in a fasted state or after 30 min from the intake of a meal, and the drug concentration in the plasma was measured.
The results in the pharmaceutical composition for modified release of the present invention prepared in Example 1A are shown in FIG. 12, and the results in the pharmaceutical composition for modified release of the present invention prepared in Example 1B are shown in FIG. 13, respectively.
With respect to the conventional formulation, the rate of decrease of Cmax in the fed state was 67%, in comparison with that in the fasted state, and the rate of decrease of AUC was 47% (Cmax in the fasted state was approximately three times higher than that in the fed state). With respect to the pharmaceutical compositions for modified release (1A and 1B) of the present invention, the rates of decrease of Cmax in the fed state were 4% and 10%, in comparison with those in the fasted state, and the rates of decrease of AUC were 10% and −4%. These results indicated that the reductions of Cmax and AUC caused by food intake could be significantly alleviated by the pharmaceutical composition for modified release of the present invention.
Furthermore, the maximum plasma concentration after the administration of the pharmaceutical composition for modified release prepared in Example 1A of the present invention was 274 ng/mL and 264 ng/mL in the fasted state and in the fed state, respectively. Similarly, in Example 1B, they were 155 ng/mL and 140 ng/mL, respectively. Furthermore, the increase in heart rate is 13 bpm or less in both.
INDUSTRIAL APPLICABILITY
According to the present invention, a pharmaceutical composition for modified release which is not affected by the effects of food intake and exhibits a decreased change in AUC or Cmax by controlling the releasing rate of the active ingredient can be provided.
As above, the present invention was explained with reference to particular embodiments, but modifications and improvements obvious to those skilled in the art are included in the scope of the present invention.
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13073677
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astellas pharma inc.
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USA
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B2
|
Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
|
Open
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424/400
|
|
Apr 1st, 2022 05:13PM
|
Apr 1st, 2022 05:13PM
|
Astellas Pharma
|
|
|
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tyo:4503
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Astellas Pharma
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Feb 27th, 2007 12:00AM
|
Nov 19th, 2004 12:00AM
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https://www.uspto.gov?id=US07183412-20070227
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Ester or amide derivatives
|
An ester or amide derivative represented by the general formula (I) or a pharmaceutically acceptable salt thereof. Particularly, an ester or amide derivative of 4-oxo-1,4-dihydroqunoline-2-carboxylic acid represented by the general formula (I′) or (I″), or a pharmaceutically acceptable salt thereof.
|
7183412
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1. An ester compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein formula (I) is:
wherein:
X1 to X4 each independently represents C or N, provided that at least three of X1 to X4 represents C;
Y represents O;
R1 represents a C1 to C6 alkyl group;
R2 represents (a) a C3 to C10 alkyl group, or (b) an aryl-C1 to C10 alkyl group and that the aryl may be substituted with from one to three of halogen, C1 to C6 alkyl, C1 to C6 alkyl-O—, nitro, cyano, amino, hydroxycarbonyl, C1 to C6 alkyl-NH—, or di-C1 to C6 alkyl=N—;
R3 represents H, a C1 to C6 alkyl group, an aryl-C1 to C6 alkyl group, a heteroaryl-C1 to C6 alkyl group, a cycloalkyl-C1 to C6 alkyl group, an aryl group, a heteroaryl group, or a cycloalkyl group, provided that the aryl, heteroaryl, and cycloalkyl group may each be substituted with from one to three of halogen, C1 to C6 alkyl, C1 to C6 alkyl-O—, nitro, cyano, amino, hydroxycarbonyl, C1 to C6 alkyl-NH—, or di-C1 to C6 alkyl=N—; and
R4 to R7 may be the same or different and each represents H, a halogen, a nitro group, a cyano group, an amino group, a hydroxycarbonyl group, a C1 to C6 alkyl group, a C1 to C6 alkyl-O— group, a C1 to C6 alkyl-NH group, or a di-C1 to C6 alkyl=N— group,
provided that in the case where X1 to X4 each represents N, R4 to R7 to be bound thereto are not present and that R3 and R4 may be taken together to form a linear or branched C1 to C8 alkylene which may be inserted by N, O or S.
2. The ester compound or its pharmaceutically acceptable salt according to claim 1, wherein X1 to X4 are all C.
3. The ester compound or its pharmaceutically acceptable salt according to claim 1, wherein R3 is H.
4. The ester compound or its pharmaceutically acceptable salt according to claim 1, wherein R2 represents (a) C5 to C10 alkyl group, or (b) a phenyl-C1 to C6 alkyl group, provided that the phenyl may be substituted with from one to three of halogen, C1 to C6 alkyl, C1 to C6 alkyl-O—, nitro, amino, C1 to C6 alkyl-NH—, or di-C1 to C6 alkyl=N—.
5. The ester compound or its pharmaceutically acceptable salt according to claim 4, wherein R2 represents (a) a C6 to C8 alkyl group or (b) a benzyl group which may be substituted with from one to three of halogen, C1 to C6 alkyl, C1 to C6 alkyl-O—, nitro, amino, C1 to C6 alkyl-NH—, or di-C1 to C6 alkyl=N—.
6. The ester compound or its pharmaceutically acceptable salt according to claim 1, wherein R1 represents a methyl group.
7. The ester compound or its pharmaceutically acceptable salt according to claim 1, wherein R4 to R7 are all H.
8. The ester compound or its pharmaceutically acceptable salt according to claim 1, which is Benzyl 3-methyl-4-oxo-1,4-dihydroquinoline-2-carboxylate, or a salt thereof.
9. A pharmaceutical composition comprising an ester compound according to claim 1 or its pharmaceutically acceptable salt and a pharmaceutically acceptable carrier.
10. The ester compound or its pharmaceutically acceptable salt according to claim 2, wherein R3 is H.
11. The ester compound or its pharmaceutically acceptable salt according to claim 10, wherein R2 represents (a) a C5 to C10 alkyl group, or (b) a phenyl-C1 to C6 alkyl group, provided that the phenyl may be substituted with from one to three of halogen, C1 to C6 alkyl, C1 to C6 alkyl-O—, nitro, amino, C1 to C6alkyl-NH—, or di-C1 to C6alkyl=N—.
12. The ester compound or its pharmaceutically acceptable salt according to claim 11, wherein R2 represents (a) a C6 to C8 alkyl group or (b) a benzyl group which may be substituted with from one to three of halogen, C1 to C6 alkyl, C1 to C6 alkyl-O—, nitro, amino, C1 to C6 alkyl-NH—, or di-C1 to C6 alkyl=N—.
13. The ester compound or its pharmaceutically acceptable salt according to claim 11, wherein R1 represents a methyl group.
14. The ester compound or its pharmaceutically acceptable salt according to claim 13, wherein R4 to R7 are all H.
15. The ester compound or its pharmaceutically acceptable salt according to claim 2, wherein R2 represents (a) a C5 to C10 alkyl group, or (b) a phenyl-C1 to C6 alkyl group, provided that the phenyl may be substituted with from one to three of halogen, C1 to C6 alkyl, C1 to C6 alkyl-O—, nitro, amino, C1 to C6 alkyl-NH—, or di-C1 to C6 alkyl=N—.
16. The ester compound or its pharmaceutically acceptable salt according to claim 15, wherein R2 represents (a) a C6 to C8 alkyl group or (b) a benzyl group which may be substituted with from one to three of halogen, C1 to C6 alkyl, C1 to C6 alkyl-O—, nitro, amino, C1 to C6 alkyl-NH—, or di-C1 to C6 alkyl=N—.
17. The ester compound or its pharmaceutically acceptable salt according to claim 2, wherein R1 represents a methyl group.
18. The ester compound or its pharmaceutically acceptable salt according to claim 2, wherein R4 to R7 are all H.
19. The ester compound or its pharmaceutically acceptable salt according to claim 3, wherein R2 represents (a) a C5 to C10 alkyl group, or (b) a phenyl-C1 to C6 alkyl group, provided that the phenyl may be substituted with from one to three of halogen, C1 to C6 alkyl, C1 to C6 alkyl-O—, nitro, amino, C1 to C6 alkyl-NH—, or di-C1 to C6 alkyl=N—.
20. The ester compound or its pharmaceutically acceptable salt according to claim 19, wherein R2 represents (a) a C6 to C8 alkyl group or (b) a benzyl group which may be substituted with from one to three of halogen, C1 to C6 alkyl, C1 to C6 alkyl-O—, nitro, amino, C1 to C6 alkyl-NH—, or di-C1 to C6 alkyl=N—.
21. The ester compound or its pharmaceutically acceptable salt according to claim 3, wherein R1 represents a methyl group.
22. The ester compound or its pharmaceutically acceptable salt according to claim 3, wherein R4 to R7 are all H.
23. The ester compound or its pharmaceutically acceptable salt according to claim 4, wherein R1 represents a methyl group.
24. The ester compound or its pharmaceutically acceptable salt according to claim 4, wherein R4 to R7 are all H.
25. The ester compound or its pharmaceutically acceptable salt according to claim 5, wherein R1 represents a methyl group.
26. The ester compound or its pharmaceutically acceptable salt according to claim 5, wherein R4 to R7 are all H.
27. The ester compound or its pharmaceutically acceptable salt according to claim 6, wherein R4 to R7 are all H.
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27
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RELATED APPLICATIONS
This application is a divisional of application Ser. No. 10/363,033, filed Feb. 28, 2003, now U.S. Pat. 6,822,098; which is a national stage application under 35 U.S.C. § 371 of international application PCT/JP01/07307, filed Aug. 27, 2001, each of which are incorporated by reference.
TECHNICAL FIELD
The present invention relates to a novel ester or amide derivative, particularly an ester or amide derivative of 4-oxo-1,4-dihydroqunoline-2-carboxylic acid, or a pharmaceutically acceptable salt thereof, and a drug composition containing it as an active ingredient, particularly a preventive or remedy for Helicobacter pylori infectious diseases.
BACKGROUND ART
Helicobacter pylori is a pathogenic bacterium discovered in 1983 and is called a background pathogenic factor of peptic ulcers (such as gastric ulcer and duodenal ulcer), inflammations (such as gastritis), diseases of upper digestive tracts such as gastric cancer, MALT (mucosa-associated lymphoid tissue) lymphoma, or chronic heart disease. At present, studies on the therapy of Helicobacter pylori infectious diseases are being actively made. As the therapy, there are many reports for the purposes of bacterial elimination and prevention of recurrence, as described below. For example, there is numerated administration of a single drug of, e.g., bismuth, antibiotic, proton pump inhibitor (PPI), or anti-ulcer agent, or polypharmacy (such as two-drug therapy and three-drug therapy) comprising a combination of the foregoing drugs (see Internal Medicine, Special Issue, Vol. 78 (1), 1996, by Nankodo). However, these therapies still involve many problems to be solved, such as high frequency of administration of the drug(s), necessity of administration of a lager amount of the drug(s) than the regular dose, crisis of diarrhea or constipation by administration of the drug(s), and generation of resistant bacteria.
As an anti-helicobacter pylori agent, EP811613 discloses derivatives of 4-oxo-1,4-dihydroquinoline or naphthyridine in terms of the following general formula. In the general formula, the substituent (R2 group) at the 2-position of the ring is a C1 to C10 alkyl group, a (C1 to C10 alkyl)phenyl group, a C2 to C10 alkenyl group, a (C2 to C10 alkenyl)phenyl group, a C2 to C10 alkynyl group, a (C2 to C10 alkynyl)phenyl group, a phenyl group, a naphthyl group, a furyl group, a thiophenyl group, or a pyridyl group.
Further, JP-A-10-132784 discloses the following. 3-methyl-4-oxo-1,4-dihydroquinone derivative as an anti-helicobacter pylori agent, in which, however, the substituent at the 2-position is a nonenyl group.
However, creation of a compound having a stronger anti-helicobacter pylori action by single and oral administration is being demanded.
On the other hand, as derivatives of a 3-alkyl-4-oxo-1,4-dihydroquinoline-2-carboxylic acid, in DE1913466, Pol. J. Pharmacol. Pharm., 33(5), 539–544, 1981, Indian. J. Pharm., 39(1), 13–15, 1977, J. Indian Chemical Society, 50(3), 217–218, 1973, and J. Indian Chemical Society, 51(11), 967–969, 1974, there are disclosed compounds having an ethoxycarbonyl group or a (substituted or unsubstituted) NH2—NH—CO— group at the 2-position of the quinoline ring. However, any of these compounds are merely a synthesis intermediate or are merely reported that they have an anti-amoeba activity. These documents neither disclose nor suggest their anti-helicobacter pylori activity.
DISCLOSURE OF THE INVENTION
We, the present inventors made extensive and intensive investigations with respect to compounds having an anti-helicobacter pylori activity. As a result, it has been found that novel ester or amide derivatives of 4-oxo-1,4-dihydroquinoline-2-carboxylic acid, which are different in terms of structure from the conventional compounds in the point that on the 1,4-dihydroquinoline ring or 1,4-dihydronaphthyridine ring, not only a substituent at the 2-position is an ester residue or a substituted amide group, but also a substituent at the 3-position is an alkyl group, (1) have a strong and selective anti-bacterial action against Helicobacter pylori, (2) have a strong anti-bacterial action against Helicobacter pylori within digestive tracts by oral administration to mammals, and (3) are useful in the bacterial elimination therapy against patients infected with Helicobacter pylori.
Specifically, the invention relates to a novel ester or amide derivative represented by the following general formula (I) or a salt thereof:
wherein:
X1 to X4 each independently represents C or N, provided that at least one of X1 to X4 represents C;
Y represents O or NH;
R1 represents a C1 to C6 alkyl group;
R2 represents (a) a C1 to C10 alkyl group, provided that when X represents O, R2 represents a C3 to C10 alkyl group, or (b) an aryl-C1 to C10 alkyl group, a heteroaryl-C1 to C10 alkyl group, or a cycloalkyl-C1 to C10 alkyl group, provided that any carbon-carbon bond of the C1 to C10 alkyl group may be inserted by —O—, —NR8—, or —S(O)n—, wherein R8 represents H or a C1 to C6 alkyl group, and n is 0, 1 or 2, and that the aryl, heteroaryl and cycloalkyl may each be substituted with from one to three of halogen, C1 to C6 alkyl, C1 to C6 alkyl-O—, nitro, cyano, amino, hydroxycarbonyl, C1 to C6 alkyl-NH—, or di-C1 to C6 alkyl=N—;
R3 represents H, a C1 to C6 alkyl group, an aryl-C1 to C6 alkyl group, a heteroaryl-C1 to C6 alkyl group, a cycloalkyl-C1 to C6 alkyl group, an aryl group, a heteroaryl group, or a cycloalkyl group, provided that the aryl, heteroaryl and cycloalkyl may each be substituted with from one to three of halogen, C1 to C6 alkyl, C1 to C6 alkyl-O—, nitro, cyano, amino, hydroxycarbonyl, C1 to C6 alkyl-NH—, or di-C1 to C6 alkyl=N—; and
R4 to R7 may be the same or different and each represents H, a halogen, a nitro group, a cyano group, an amino group, a hydroxycarbonyl group, a C1 to C6 alkyl group, a C1 to C6 alkyl-O— group, a C1 to C6 alkyl-NH group, or a di-C1 to C6 alkyl=N— group,
provided that in the case where X1 to X4 each represents N, R4 to R7 to be bound thereto are not present and that R3 and R4 may be taken together to form a linear or branched C1 to C8 alkylene which may be inserted by N, O or S.
Also, the invention relates to a drug composition comprising the ester or amide derivative represented by the foregoing general formula (I) or its pharmaceutically acceptable salt and a pharmaceutically acceptable carrier, a particularly a drug composition as a preventive or remedy for Helicobacter pylori infectious diseases.
The compound (I) of the invention is structurally characterized in that a substituent at the 2-position of the 1,4-dihydroquinoline ring or 1,4-dihydronaphthyridine ring is an ester residue or an substituted amide group and is superior to the known compounds having a hydrocarbon group as the substituent at the 2-position in the point that it undergoes strong bacterial elimination against Helicobacter pylori within digestive tracts by oral administration to mammals.
The compound (I) of the invention will be hereunder described in detail.
Examples of the “halogen” include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.
The term “alkyl” means a linear or branched saturated hydrocarbon group. Specific examples of the C1 to C10 alkyl include a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group, and structural isomers thereof (such as an isopropyl group). Specific examples of the C1 to C6 alkyl include a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, and structural isomers thereof (such as an isopropyl group). Specific examples of the C6 to C8 alkyl include a hexyl group, a heptyl group, an octyl group, and structural isomers thereof (such as a methylhexyl group).
The term “alkylene” means a divalent group resulted from removal of hydrogen from the foregoing alkyl. Specific examples of the C1 to C8 alkylene include methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, and structural isomers thereof (such as 2-methylpropylene).
The term “aryl” means an aromatic hydrocarbon group, and preferably a C6 to C14 aryl. Specific examples include phenyl, naphthyl, and biphenyl, and particularly preferably phenyl.
The term “heteroaryl” means a 5-membered or 6-membered monocyclic heteroaryl having from 1 to 4 hetero atoms selected from N, S and O, or a bicyclic heteroaryl fused with a benzene ring, which may be partially saturated. Examples of the monocyclic heteroaryl include furyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, pyridyl, pyrazinyl, pyrimidyl, triazolyl, thiazolyl, pyridazinyl, triazinyl, oxazolyl, and pyrimidyl. Examples of the bicyclic heteroaryl include benzofuranyl, benzothienyl, benzothiadiazoyl, benzothiazolyl, benzoimidazolyl, indolyl, quinolyl, isoquinolyl, and quinoxalinyl. Of these are preferable 5-membered or 6-membered monocyclic heteroaryls, with furyl, thienyl, imidazolyl, thiazolyl and pyridyl being more preferable.
The term “cycloalkyl” means a saturated hydrocarbon group having from 3 to 8 carbon atoms. Specific examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.
In the general formula of the invention, specific examples of the ring represented by the formula:
In the invention, a quinoline ring is particularly preferable.
In the invention, is preferable a compound represented by the foregoing general formula (I) wherein X1 to X4 are all C, and specifically an ester or amide derivative of 3-alkyl-4-oxo-1,4-dihydroquinoline-2-carboxylic acid represented by the following general formula (I′) or a salt thereof.
In the general formula, X1 to X4, Y, and R1 to R7 have the same meanings as defined above.
In the invention, is more preferable a compound represented by the foregoing general formula (I) wherein X1 to X4 are all C, and R3 is H, and specifically an ester or amide derivative of 3-alkyl-4-oxo-1,4-dihydroquinoline-2-carboxylic acid represented by the following general formula (I″) or a salt thereof.
In the general formula, X1 to X4, Y, and R4 to R7 have the same meanings as defined above.
In the invention, is further preferable a compound represented by the foregoing general formula (I″) wherein R2 represents (a) a C1 to C10 alkyl group (provided that when X is O, R2 represents a C5 to C10 alkyl group), or (b) a phenyl-C1 to C6 alkyl group (provided that any carbon-carbon bond of the C1 to C10 alkyl group may be inserted by —O—, —NR8—, or —S(O)n— (wherein R8 represents H or a C1 to C6 alkyl, and n is 0, 1 or 2), and that the phenyl may be substituted with from one to three of halogen, C1 to C6 alkyl, C1 to C6 alkyl-O—, nitro, amino, C1 to C6 alkyl-NH—, or di-C1 to C6 alkyl=N—); and more preferably, R2 represents a C6 to C8 alkyl group or a benzyl group which may be substituted with one to three of halogen, C1 to C6 alkyl, C1 to C6 alkyl-O—, nitro, amino, C1 to C6 alkyl-N—, or di-C1 to C6 alkyl=N—.
Also, a compound wherein R1 represents a methyl group is preferable. A compound wherein R4 to R7 are all H is preferable.
In the invention, the following compounds are particularly preferable.
N-Heptyl-3-methyl-4-oxo-1,4-dihydroquinoline-2-carboxamide
N-(4-Methylbenzyl)-3-methyl-4-oxo-1,4-dihydroquinoline-2-carboxamide
N-(3-Methoxybenzyl)-3-methyl-4-oxo-1,4-dihydroquinoline-2-carboxamide
Benzyl 3-methyl-4-oxo-1,4-dihydroquinoline-2-carboxylate
With respect to the compound of the invention, there are optical isomers (such as optically active compounds and diastereomers) depending on the kinds of the groups. Further, the compound of the invention includes a compound having an amide bond. There may be tautomers based on the amide bond. Especially, among the compounds of the invention, the ester or amide derivatives of 3-alkyl-4-oxo-1,4-dihydroquinoline-2-carboxylic acid represented by the general formula (I″) include the following tautomers.
The invention includes isolated isomers and mixed isomers thereof.
The compound (I) of the invention may form a salt with an acid or a base depending on the kinds of the substituents. Such a salt is a pharmaceutically acceptable salt. Specific examples include acid addition salts with an inorganic acid such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, and phosphoric acid, or with an organic acid such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, aspartic acid, and glutamic acid; salts with an inorganic base such as sodium, potassium, magnesium, calcium, and aluminum, or with an organic base such as methylamine, ethylamine, ethanolamine, lysine, and ornithine; and ammonium salts.
In addition, the invention includes various hydrates, solvates and crystal polymorphisms of the compound (I) of the invention and its salt. Moreover, the invention includes prodrugs of the substance of the formula (I) as obtained in the customary means. The prodrugs as used herein mean a compound having a substituent(s) capable of converting into the substituent(s) of the substance of the formula (I) by solvolysis or under physiological conditions, especially a compound that will be converted into the substance of the formula (I) within a living body. As the substituent to form the prodrug are enumerated those groups described in Prog. Med., 5, 2157–2161 (1985) and Iyakuhin No Kaihatsu (Development of Drugs), Vol. 7, “Molecular Design”, 163–198 (1990), by Hirokawa-shoten.
For example, there is enumerated a compound having a hydroxyl group substituted at the 1-position of the quinoline ring of the ester or amide derivative of 3-alkyl-4-oxo-1,4-dihydroquinoline-2-carboxylic acid (I″).
(Production Process)
The compound (I) and its salt of the invention can be produced through application of various known synthesis processes by utilizing the characteristic features based on the basic skeleton thereof or kinds of the substituents.
First Process:
Among the compounds of the invention, an amide derivative (Ia) having a substituted amino group as the substituent at the 2-position of the 1,4-dihydroquinoline ring is produced by reacting a carboxylic acid derivative represented by the general formula (II) with an amine represented by the general formula (III) to form an amide bond.
In the formulae, X1 to X4 and R1 to R7 have the same meanings as defined above.
The reaction is usually carried out in a usual solvent such as acetone, dioxane, tetrahydrofuran, diethyl ether, diisopropyl ether, acetonitrile, chloroform, dichloromethane, 1,2-dichloroethane, ethyl acetate, N,N-dimethylformamide (DMF), toluene, and pyridine, or a mixture thereof. The reaction may also be carried out in other arbitrary organic solvent so far as it does not adversely affect the reaction. Although the reaction temperature and reaction time are not particularly limited, the reaction is usually carried out at room temperature overnight. The reaction can be carried out in the presence of a catalyst such as 1-hydroxybenzotriazole (HOBt) and 4-dimethylaminopyridine (DMAP), and/or a condensing agent such as dicyclohexylcarbodiimide (DCC), 1-[3-(dimethylamino)propyl-3-ethylcarbodiimide hydrochloride (WSCD-HCl), and carbonyldiimidazole (CDI). Further, the reaction can also be carried out in the presence of N,N-dimethylformamide (DMF) via an acid chloride using thionyl chloride or oxalyl chloride. Alternatively, the reaction can be carried out via an active ester using an acid anhydride such as acetic anhydride, or an acid chloride such as mesyl chloride.
Second Process:
Among the compounds of the invention, an ester derivative (Ib) having an ester residue as the substituent at the 2-position of the 1,4-dihydroquinoline ring is produced by reacting the carboxylic acid derivative represented by the general formula (II) with an alcohol represented by the general formula (IV) to form an ester bond.
In the formulae, X1 to X4 and R1 to R7 have the same meanings as defined above.
The reaction is usually carried out in the foregoing usual solvent or a mixture thereof. The reaction may also be carried out in other arbitrary organic solvent so far as it does not adversely affect the reaction. Although the reaction temperature and reaction time are not particularly limited, the reaction is usually carried out at room temperature overnight. The reaction can be carried out in the presence of a catalyst such as 1-hydroxybenzotriazole (HOBt) and 4-dimethylaminopyridine (DMAP), and/or a condensing agent such as dicyclohexylcarbodiimide (DCC), 1-[3-(dimethyl-amino)propyl-3-ethylcarbodiimide hydrochloride (WSCD.HCl), and carbonyldiimidazole (CDI). Further, the reaction can also be carried out in the presence of N,N-dimethylformamide (DMF) via an acid chloride using thionyl chloride or oxalyl chloride. Alternatively, the reaction can be carried out via an active ester using an acid anhydride such as acetic anhydride, or an acid chloride such as mesyl chloride.
Incidentally, with respect to the compound of the invention, as described above, there may be the case where isomers such as a racemate, an optically active compound, and a diastereomer are present singly or as a mixture. The racemic compound can be introduced into a stereochemically pure isomer by using a proper starting material(s), or by general racemic resolution (such as a method in which the racemic compound is introduced into a diastereomer salt with a general optically active acid (such as tartaric acid), which is then subjected to optical resolution). Further, the mixture of diastereomers can be separated by a customary manner such as fractional crystallization and chromatography.
INDUSTRIAL APPLICABILITY
The invention exhibits a selective anti-bacterial action against Helicobacter pylori and is effective for the therapy of infections of Helicobacter pylori in human being and related bacteria belonging to the genus Helicobacter in animals. Further, the anti-helicobacter pylori agent of the invention is effective for prevention (including prevention of recurrence) or therapy of peptic ulcers (such as gastric or duodenal ulcer), inflammations (such as acute or chronic gastritis or duodenitis), diseases of upper digestive tracts such as gastric cancer, MALT (mucosa-associated lymphoid tissue) lymphoma, or chronic heart disease.
The actions of the compound of the invention were confirmed by the following pharmacological tests.
(1) In Vitro Anti-Bacterial Activity Test:
1) Preparation of Anti-Bacterial Substance-Containing Agar Plate:
A substance to be evaluated was dissolved in 100% DMSO, and the solution was subjected to two-fold series dilution. The diluted solution was charged in a sterilized round Petri dish, to which was then added 10 mL of a brucella agar medium (0.1% β-cyclodextrin or 5% sheep blood) which had been sterilized and kept at 50° C. After intimate mixing, the mixture was solidified. The ultimate concentration of DMSO is 1% or less.
2) Preparation of Inoculation Material and Result Judgment:
Helicobacter pylori, such as Helicobacter pylon ATCC43504, which had been cultured at 37° C. for 3 days in a multigas incubator (N2: 80%, CO2: 15%, O2: 5%) using a brucella agar medium (containing 5% calf serum), was prepared using a brucella broth such that the number of bacteria was about 108 per mL depending on the turbidity. The bacterial solution was similarly diluted with a brucella broth 100-fold, about 1 or 5 μL of which was then inoculated on the surface of a drug-containing agar medium using a micro-planter. The inoculated agar plate was cultured at 37° C. for 3 days (72 hours) in the foregoing multigas incubator. The cultured agar plate was observed, and a minimal drug concentration at which the proliferation was not observed was designated as MIC.
(2) In Vivo Anti-Bacterial Activity Test:
The infection test was carried out by using Mongolian gerbils as reported to be stably infected (J. Gastroenterology 31: supple IX, 24–28, 1996). An overnight cultured inoculum of ATCC43504 was inoculated into a stomach of overnight-fasted Mongolian gerbils (MGS/Sea, male, 4-week-old) About one week after the infection, the therapy was started by orally administering a drug to be evaluated dissolved in a solvent according to the customary manner in a dose of 10 mg/kg, 3 mg/kg or 1 mL/kg twice per day for three days. Next day after completion of the administration, the stomach was taken out and ground. A stomach homogenate solution was subjected to 10-fold series dilution, inoculated on a modified Skirrow medium, and then cultured at 37° C. for six to seven days under microaerophile conditions or 10% CO2 conditions. The number of bacteria within the stomach was calculated from the number of grown bacteria.
Any of the compounds of Examples 1 to 4 of the invention exhibited a bacterial elimination effect from at a dose of 1 mg/kg. On the other hand, the following known compounds exhibited a bacterial elimination effect from at a does of 10 mg/kg.
Accordingly, it was confirmed that the compounds of the invention have a stronger bacterial elimination effect by oral administration to mammals as compared with the known anti-helicobacter pylori agents.
The drug containing the compound (I) of the invention or its salt and a pharmaceutically acceptable carrier can be prepared by a usually employed method using one or two or more of the compound represented by the general formula (I) or its salt and a pharmaceutical carrier, excipient and other additives as used for formulation. The administration may be in any form of oral administration by tablets, pills, capsules, granules, powders, liquids, etc., or parenteral administration by injections such as intravenous or intramuscular injection, suppositories, dermal administration, etc.
As a solid composition for the oral administration according to the invention, tablets, powders, or granules are used. In such a solid composition, one or more active substances are mixed with at least one inert diluent such as lactose, mannitol, glucose, hydroxypropyl cellulose, microcrystalline cellulose, starch, polyvinylpyrrolidone, magnesium metasilicate aluminate. The composition may contain additives other than the inert diluent, such as a lubricant such as magnesium stearate, a disintegrating agent such as cellulose calcium glycolate, a stabilizer such as lactose, and a dissolution aid such as glutamic acid and aspartic acid, according to the customary method. If desired, the tablets or pills may be coated by a sugar coating such as sugar, gelatin, hydroxypropyl cellulose, and hydroxypropylmethyl cellulose phthalate, or a film made of a gastric-soluble or intestinal soluble substance.
The liquid composition for oral administration contains a pharmaceutically acceptable emulsion agent, solution agent, suspending agent, syrup, or elixir and contains a generally employed inert diluent such as purified water and ethanol. In addition to the inert diluent, this composition may contain an auxiliary agent such as a wetting agent and a suspending agent, a sweetener, a flavor, an aromatic, or an antiseptic.
The injection for parenteral administration contains a sterile aqueous or non-aqueous solution agent, suspending agent or emulsion agent. Examples of the aqueous solution agent or suspending agent include distilled water or physiological saline for injection. Examples of the non-aqueous solution agent or suspending agent include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol, and Polysolvate 80 (trade name). Such a composition may also contain an auxiliary agent such as an antiseptic, a wetting agent, an emulsifier, a dispersing agent, a stabilizer (such as lactose), and a dissolution aid (such as glutamic acid and aspartic acid). These compositions are sterilized by, for example, filtration through a bacteria-holding filter, compounding with an anti-bacterial agent, or irradiation. Further, these can be used by producing a sterile solid composition and dissolving it in sterile water or a sterile solvent for injection before the used.
Usually, in the case of the oral administration, it is proper that the dose of the drug per day is from about 0.001 to 30 mg per kg, and preferably from 0.1 to 5 mg per kg of the body weight and that the drug is administered once or dividedly two to four times. In the case of the intravenous administration, it is proper that the dose of the drug per day is from about 0.001 to 30 mg per kg of the body weight and that the drug is administered once or dividedly several times. The dose is properly determined depending on the individuals while taking into consideration the symptom, age and sex.
According to the invention, the compound (I) can be used singly or in combination with other anti-bacterial agents (preferably one to three kinds). Such other anti-bacterial agents can be used in combination simultaneously with the compound of the invention or after elapsing for a while.
Examples of such other anti-bacterial agents include nitroimidazole antibiotics (such as tinidazole and metronidazole), tetracycline series drugs (such as tetracycline, minocycline, and doxycycline), penicillin series drugs (such as amoxicillin, ampicillin, talampicillin, bacampicillin, lenampicillin, mezlocillin, and sultamicillin), cephalosporin series drugs (such as cefaclor, cefadroxil, cefalexin, cefpodoxime proxetil, cefixime, cefdinir, ceftibuten, cefatiam hexetil, cefetamet pivoxil, cefcapene pivoxil, sefiditoren pivoxil, and cefloxime axetil), penenm series drugs (such as faropenem and ritipenem acoxil), macrolide series drugs (such as erythromycin, oleandomycin, josamycin, midecamycin, rokitamycin, clarithromycin, roxithromycin, terithromycin, and azithromycin), lincomycin series drugs (such as lincomycin and clindamycin), aminoglycocide series drygs (such as paromomycin), quinolone series drugs (such as ofloxacin, lebofloxacin, norfloxacin, enoxacin, ciprofloxacin, lomefloxacin, tosufloxacin, fleroxacin, spafloxacin, temafloxacin, nadifloxacin, grepafloxacin, balfloxacin, prulifloxacin, gatifloxacin, sitafloxacin, and pazufloxacin), and nitrofurantoin. Further, combinations of the compound (I) of the invention with PPI (such as omeprazole, rabeprazole, and lansoprazole) or anti-ulcer agents (such as H2 antagonists such as ranitidine, cimetidine, and famotidine, or gastric mucosal protective agents) fall within the scope of the invention.
BEST MODE FOR CARRYING OUT THE INVENTION
The invention will be described below in more detail with reference to the Referential Examples and Examples. As a matter of course, it should not be construed that the invention is limited thereto. In the nuclear magnetic resonance spectra (1H-NMR) described in physical properties, DMSO was used as a measurement solvent, and tetramethyl silane was used as an internal standard (δ: 0.00 ppm). The mass analysis (MS) was made by the fast atom bombardment (FAB).
REFERENTIAL EXAMPLE 1
Ethyl 3-methyl-4-oxo-1,4-dihydroquinoline-2-carboxylate
(1) Aniline (51.1 g) and 21.4 g of Diethyl oxalpropionate were dissolved in 200 mL of benzene, to which was then added 4 mL of acetic acid, and the mixture was refluxed upon heating overnight by a Dean-Stark condenser. The solvent was distilled off to obtain a yellow oily substance.
(2) The compound obtained in (1) was dissolved in 200 mL of diphenyl ether, and the solution was heated at 250° C. for 30 minutes. After allowing to stand for cooling, the reaction mixture was poured into 600 mL of hexane, and precipitates were filtered out. The crystals were rinsed with diethyl ether and dried to obtain 36.7 g (63%) of the titled compound. MS: 232 (M++1).
REFERENTIAL EXAMPLE 2
3-Methyl-4-oxo-1,4-dihydroquinoline-2-carboxylic Acid
To 18.1 g of the compound obtained in Referential Example 1 was added 170 mL of a 1N sodium hydroxide aqueous solution, and the mixture was refluxed upon heating for 30 minutes. After allowing to stand for cooling, hydrochloric acid (concentrated hydrochloric acid:water=1:1) was gradually added, and precipitates were collected by filtration and rinsed with dilute hydrochloric acid, followed by drying. To the resulting crystals was added 100 mL of acetonitrile, and the mixture was refluxed upon heating for 30 minutes. After allowing to stand for cooling, crystals were collected by filtration and dried to obtain 15.6 g (98%) of the titled compound. MS: 202 (M+−1).
EXAMPLE 1
N-Heptyl-3-methyl-4-oxo-1,4-dihydroquinoline-2-carboxamide
The compound (3.252 g) obtained in Referential Example 2, 2.39 g of HOBt, and 3.385 g of WSCD were dissolved in 40 mL of DMF. After dropwise addition of 2.041 g of heptylamine, the reaction mixture was stirred overnight. Saturated sodium chloride aqueous solution and ethyl acetate were added, and the mixture was stirred for one hour. Precipitates were collected by filtration, rinsed with dilute hydrochloric acid, ethyl acetate, and water, and then dried to obtain 4.437 g (92%) of the titled compound.
1H-NMR: 0.87 (t, 2H), 1.28 to 1.34 (m, 8H), 1.51 to 1.58 (m, 2H), 1.98 (s, 3H), 3.27 (q, 2H), 7.27 to 7.31 (m, 1H), 7.58 to 7.65 (m, 2H), 8.08 to 8.10 (m, 1H), 8.81 (t, 1H), 11.84 (s, 1H). MS (m/z): 301 (M++1).
Compounds of the Examples 2 to 5 were obtained in the same manner as in Example 1.
EXAMPLE 2
N-(4-Methylbenzyl)-3-methyl-4-oxo-1,4-dihydroquinoline-2-carboxamide
1H-NMR: 1.96 (s, 3H), 2.30 (s, 3H), 4.46 (d, 2H), 7.18 (d, 2H), 7.27 to 7.31 (m, 3H), 7.60 to 7.65 (m, 2H), 8.09 (d, 1H), 9.32 (s, 1H), 11.89 (s, 1H). MS (m/z): 307 (M++1).
EXAMPLE 3
N-(3-Methoxybenzyl)-3-methyl-4-oxo-1,4-dihydroquinoline-2-carboxamide
1H-NMR: 1.99 (s, 3H), 3.76 (s, 3H), 4.50 (d, 2H), 6.85 to 6.87 (m, 1H), 6.96 to 6.97 (m, 2H), 7.27 to 7.32 (m, 2H), 7.60 to 7.65 (m, 2H), 8.09 (d, 1H), 9.35 (t, 1H), 11.91 (s, 1H). MS (m/z): 323 (M++1).
EXAMPLE 4
N-{2-[Ethyl-(3-methylphenyl)amino]ethyl}-3-methyl-4-oxo-1,4-dihydroquinoline-2-carboxamide
1H-NMR: 1.10 (t, 3H), 1.99 (s, 3H), 2.24 (s, 3H), 3.39 (q, 2H), 3.45 (s, 4H), 6.42 (d, 1H), 6.59 to 6.62 (m, 2H), 7.05 (t, 1H), 7.28 to 7.32 (m, 1H), 7.60 to 7.66 (m, 2H), 8.09 (d, 2H), 8.95 (s, 1H), 11.86 (s, 1H). MS (m/z): 364 (M++1).
EXAMPLE 5
N-(4-Phenylbutyl)-3-methyl-4-oxo-1,4-dihydroquinoline-2-carboxamide
1H-NMR: 1.53 to 1.70 (m, 4H), 1.96 (s, 3H), 2.63 (t, 2H), 3.29 to 3.34 (m, 2H), 7.16 to 7.30 (m, 6H), 7.57 to 7.64 (m, 2H), 8.09 (d, 1H), 8.83 (t, 1H), 11.83 (s, 1H). MS (m/z): 335 (M++1).
EXAMPLE 6
Benzyl 3-methyl-4-oxo-1,4-dihydroquinoline-2-carboxylate
The compound (5.615 g) obtained in Referential Example 2, 4.209 g of HOBt, 5.879 g of WSCD, and 138 mg of DMAP were dissolved in a mixed solvent of 55 mL of methylene chloride and 20 mL of DMF. After dropwise addition of 3.341 g of benzyl alcohol, the reaction mixture was stirred overnight, to which was then added saturated sodium chloride aqueous solution. Precipitates were collected by filtration, rinsed with ethyl acetate and water, and then dried to obtain 5.109 g (63%) of the titled compound.
1H-NMR: 2.19 (s, 3H), 5.49 (s, 3H), 7.31 to 7.35 (m, 1H), 7.37 to 7.46 (m, 3H), 7.53 to 7.55 (m, 2H), 7.65 to 7.69 (m, 1H), 7.81 (d, 1H), 8.08 to 8.10 (m, 1H), 11.78 (s, 1H). MS (m/z): 294 (M++1).
Further, the compounds represented by the chemical structural formulae in the table can be easily produced in substantially same manners as in the Examples or production processes, or by undergoing slight modifications within the range obvious to those skilled in the art. Incidentally, symbols shown in the table have the following meanings. No.: compound number, Me: methyl, Cl: chloro, F: fluoro
(I)
No.
X1
X2
X3
X4
Y
R1
R2
R3
R4
R5
R6
R7
1
C
C
C
C
NH
Me
H
H
H
H
H
2
C
C
C
C
NH
Me
H
H
H
H
H
3
C
C
C
C
NH
Me
H
H
H
H
H
4
C
C
C
C
NH
Me
H
H
H
H
H
5
C
C
C
C
NH
Me
H
H
H
H
H
6
C
C
C
C
NH
Me
H
H
H
H
H
7
C
C
C
C
NH
Me
H
H
H
H
H
8
N
C
C
C
NH
Me
H
—
H
H
H
9
N
C
N
C
NH
Me
H
—
H
—
H
10
C
C
C
C
NH
Me
Me
H
H
H
H
11
C
C
C
C
NH
Me
H
H
H
H
12
C
C
C
C
NH
Me
H
H
H
H
13
C
C
C
C
NH
Me
F
H
14
C
C
C
C
O
Me
Me
H
H
H
H
15
C
C
C
C
O
Me
H
H
H
H
16
C
C
C
C
O
Me
H
H
H
H
17
N
C
C
C
O
Me
H
—
H
H
H
18
C
C
C
N
O
Me
H
H
H
—
H
19
C
N
C
C
O
Me
H
H
—
H
H
20
C
C
C
C
O
Me
F
H
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10992134
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astellas pharma inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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546/156
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Apr 1st, 2022 05:13PM
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Apr 1st, 2022 05:13PM
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Astellas Pharma
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tyo:4503
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Astellas Pharma
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May 2nd, 2006 12:00AM
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Aug 13th, 2004 12:00AM
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https://www.uspto.gov?id=US07037915-20060502
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Fused heteroaryl derivatives
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The present invention provides a pharmaceutical composition which is useful as a phosphatidylinositol 3 kinase (PI3K) inhibitor and an antitumor agent, and it provides a novel bicyclic or tricyclic fused heteroaryl derivative or a salt thereof which possesses an excellent PI3K inhibiting activity and cancer cell growth inhibiting activity.
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7037915
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1. A compound which is a fused heteroaryl derivative of general formula (Ib):
wherein:
B is a thiophene ring;
R1 is a lower alkyl, lower alkenyl, lower alkynyl, cycloalkyl, aryl which may have one or more substituents, heteroaryl which may have one or more substituents, halogen, —NO2,
—CN, a halogenated lower alkyl, —ORb, —SRb, —SO2-Rb, —SO-Rb, —COORb, —CO-Rb,
—CONRaRb, —SO2NRaRb, —NRaRb, —NRa-CORb, —NRa-SO2Rb, —O—CO—NRaRb, —NRaCO—COORb, —NRaCOORb, —NRaCO-lower alkylene-aryl, —NRa-SO2-lower alkylene-aryl,
—NRa-lower alkylene-aryl, -lower alkylene-ORb, -lower alkylene-NRaRb, —CO-a nitrogen-containing saturated heterocyclic group, —CONRa-lower alkylene-ORb,
—CONRa-lower alkylene-NRcRb, —CONRa-lower alkylene-nitrogen-containing saturated heterocyclic group, —O-lower alkylene-ORb, —O-lower alkylene-NRaRb, —O-lower alkylene-nitrogen-containing saturated heterocyclic group, —O-lower alkylene-O-lower alkylene-ORb,
—O-lower alkylene-O-lower alkylene-NRaRb, —O-lower alkylene-NRc-lower alkylene-NRaRb, —NRc-lower alkylene-NRaRb, —N(a lower alkylene-NRaRb)2, —CONRa-ORb, —NRa-CO—NRbRc, or —OCORb;
R2 and R3 are combined together with the N atom adjacent thereto to form a nitrogen-containing saturated heterocyclic group as —NR2R3 which may have one or more substituents;
each of Ra and Rc, which maybe the same or different, represents H or lower alkyl;
Rb is H, lower alkyl, cycloalkyl, aryl which may have one or more substituents or a heteroaryl which may have one or more substituents;
n is 2;
W is N;
R4 is -(aryl which may have one or more substituents), lower alkylene-(aryl which may have one or more substituents), lower alkenylene-(aryl which may have one or more substituents), lower alkynylene-(aryl which may have one or more substituents), -(cycloalkyl which may have one or more substituents), -(cycloalkenyl which may have one or more substituents), lower alkylene-(cycloalkyl which may have one or more substituents), lower alkenylene-(cycloalkyl which may have one or more substituents, lower alkylene-(nitrogen-containing saturated heterocyclic group which may have one or more substituents), lower alkenylene-(nitrogen-containing saturated heterocyclic group which may have one or more substituents), (a heteroaryl which may have one or more substituents and which is a 5 or 6 membered monocyclic heteroaryl containing 1 to 4 heteroatoms selected from N and S or a bicyclic heteroaryl which is a monocyclic heteroaryl as defined above is fused to a benzene ring), lower alkylene-(heteroaryl which may have one or more substituents), or lower alkenylene-(heteroaryl which may have one or more substituents);
or a pharmaceutically acceptable salt thereof.
2. A compound according to claim 1 wherein —NR2R3 is a nitrogen-containing saturated heterocyclic group selected from 1-pyrrolidinyl, 1-piperazinyl, piperidino and morpholino.
3. A compound according to claim 2 wherein —NR2R3 is morpholino.
4. A compound according to claim 1 wherein R4 is aryl which may have one or more substituents or heteroaryl which may have one or wore substituents, wherein the substituents are 1 to 5 groups selected from a) to c) below, which may be the same or different:
a) lower alkyl, lower alkenyl, lower alkynyl, halogen, halogenated lower alkyl, lower alkylene-OR, —NO2, —CN, ═O, —O-halogenated lower alkyl, —SO2-lower alkyl, —SO-lower alkyl, —COOR, —COO-lower alkylene-aryl, —COR, —CO-aryl,
—CONRR′, —SO2NRR′, -Cyc or -Alp-Cyc (wherein Alp represents lower alkylene, lower alkenylene or lower alkynylene and Cyc represents an aryl which may have 1 to 5 substituents selected from Group A as defined below, heteroaryl which may have 1 to 5 substituents selected from Group A as defined below, a nitrogen-containing saturated heterocyclic group which may have 1 to 5 substituents selected from Group A as defined below, a cycloalkyl which may have 1 to 5 substituents selected from Group A as defined below or cycloalkenyl which may have 1 to 5 substituents selected from Group A as defined below;
b) -NR-E-F wherein E represents —CO—, —COO—, —CONR′—, —SO2NR′ or SO2; F represents Cyc or a group selected from lower alkyl, lower alkenyl and lower alkynyl, which group may be substituted by one or more substituents selected from the group consisting of halogen, —NO2,—CN, —OR, —O-lower alkylene-NRR′, —O-lower alkylene-OR, —SR, —SO2-lower alkyl, —SO-lower alkyl, —COOR, —COR, —CO-aryl, —CONRR′, —SO2NRR′, —NRCO-lower alkyl, —NRR′, —NR′-lower alkylene-OR, —NR″-lower alkylene-NRR′ and Cyc; and
c) -Z-R′, -Z-Cyc, -Z-Alp-Cyc, -Z-Alp-Z′-R′, or -Z-Alp-Z′-Cyc wherein each of Z and Z′, which may be the same or different, independently represents O, S or NR; and
wherein Group A is selected from lower alkyl, lower alkenyl, lower alkynyl, halogen, halogenated lower alkyl, lower alkylene-OR, —NO2, —CN, ═O, —OR,
—O-halogenated lower alkyl, —O-lower alkylene-NRR′, —O-lower alkylene-OR, —O-lower alkylene-aryl, —SR, —SO2-lower alkyl, —SO-lower alkyl, —COOR,
—COO-lower alkylene-aryl, —COR, CO-aryl, -aryl, —CONRR′, —SO2NRR′,
—NRR′, —NR″-lower alkylene-NRR′, —NR′-lower alkylene-OR, —NR-lower alkylene-aryl, —NRCO-lower alkyl, —NRSO2-lower alkyl, cycloalkyl and cycloalkenyl; and
each of R, R′ and R″, which are the same or different, represents H or lower alkyl.
5. A pharmaceutical composition comprising a compound as claimed in claim 1 and a pharmaceutically acceptable carrier.
6. A method of treating a disorder in a patient, said disorder having abnormal cell growth associated with phosphatidylinositol 3 kinase, which method comprises administering to the patient a therapeutically effective amount of a compound which is a fused heteroaryl derivative of general formula (Ib):
wherein:
B is a thiophene ring;
R1 is a lower alkyl, lower alkenyl, lower alkynyl, cycloalkyl, aryl which may have one or more substituents, heteroatyl which may have one or more substituents, halogen, —NO2,
—CN, a halogenated lower alkyl, —ORb, —SRb, —SO2-Rb, —SO-Rb, —COORb, —CO-Rb,
—CONRaRb, —SO2NRaRb, —NRaRb, —NRa-CORb, —NRa-SO2Rb, —O—CO—NRaRb, —NRaCO—COORb, —NRaCOORb, —NRaCO-lower alkylene-aryl, —NRa-SO2-lower alkylene-aryl,
—NRa-lower alkylene-aryl, -lower alkylene-ORb, -lower alkylene-NRaRb, —CO-a nitrogen-containing saturated heterocyclic group, —CONRa-lower alkylene-ORb,
—CONRa-lower alkylene-NRcRb, —CONRa-lower alkylene-nitrogen-containing saturated heterocyclic group, —O-lower alkylene-ORb, —O-lower alkylene-NRaRb, —O-lower alkylene-nitrogen-containing saturated heterocyclic group, —O-lower alkylene-O-lower alkylene-ORb,
—O-lower alkylene-O-lower alkylene-NRaRb, —O-lower alkylene-NRc-lower alkylene-NRaRb, —NRc-lower alkylene-NRaRb, —N(a lower alkylene-NRaRb)2, —CONRa-ORb, —NRa-CO—NRbRc, or —OCORb;
R2 and R3 are combined together with the N atom adjacent thereto to form a nitrogen-containing saturated heterocyclic group as —NR2R3 which may have one or more substituents;
each of Ra and Rc, which may be the same or different, represents H or lower alkyl;
Rb is H, lower alkyl, cycloalkyl, aryl which may have one or more substituents or a heteroaryl which may have one or more substituents;
n is 1 or 2;
W is N;
R4 is -(aryl which may have one or more substituents), lower alkylene-(aryl which may have one or more substituents), lower alkenylene-(aryl which may have one or more substituents), lower alkynylene-(aryl which may have one or more substituents), -(cycloalkyl which may have one more substituents), -(cycloalkenyl which may have one or more substituents), lower alkylene-(cycloalkyl which may have one or more substituents), lower alkenylene-(cycloalkyl which may have one or more substituents, lower alkylene-(nitrogen-containing saturated heterocyclic group which may have one or more substituents), lower alkenylene-(nitrogen-containing saturated heterocyclic group which may have one or more substituents), (a heteroaryl which may have one or more substituents and which is a 5 or 6 membered monocyclic heteroaryl containing 1 to 4 heteroatoms selected from N and S or a bicyclic heteroaryl which is a monocyclic heteroaryl as defined above is fused to a benzene ring), lower alkylene-(heteroaryl which may have one or more substituents), or lower alkenylene-(heteroaryl which may have one or more substituents);
or a pharmaceutically acceptable salt thereof.
7. A method according to claim 6, wherein the disorder has abnormal cell growth associated with phosphatidylinositol 3 kinase p110α subtype.
8. A method according to claim 6 wherein the disorder is cancer.
9. A method according to claim 8, wherein the cancer is selected from the group consisting of leukemia, skin cancer, bladder cancer, breast cancer, uterus cancer, lung cancer, colon cancer, prostate cancer, ovary cancer, pancreas cancer, renal cancer, gastric cancer and brain tumor.
10. A method according to claim 6, wherein n is 1.
11. A method according to claim 6, wherein n is 2.
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11
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This application is a continuation application of Ser. No. 10/459,002 filed Jun. 10, 2003 (now U.S. Pat. No. 6,838,457) which is a divisional application of Ser. No. 10/243,416 filed Sep. 13, 2002, (now U.S. Pat. No. 6,608,056) which is a divisional of application Ser. No. 09/843,615 filed Apr. 26, 2001 (now U.S. Pat. No. 6,608,053) which claims priority from application Ser. No. 60/200,537 filed Apr. 27, 2000 and application Ser. No. 60/200,481 filed Apr. 28, 2000.
FIELD OF THE INVENTION
The present invention relates to fused heteroaryl derivatives which are useful as medicaments, more particularly as phosphatidylinositol 3-kinase (PI3K) inhibitors and carcinostatic agents.
BACKGROUND OF THE INVENTION
Phosphatidylinositol (hereinafter abbreviated as “PI”) is one of phospholipids in cell membranes. In recent years it has become clear that PI plays an important role also in intracellular signal transduction. It is well recognized in the art that especially PI (4,5) bisphosphate (PI(4,5)P2) is degraded into diacylglycerol and inositol (1,4,5) triphosphate by phospholipase C to induce activation of protein kinase C and intracellular calcium mobilization, respectively [M. J. Berridge et al., Nature, 312, 315 (1984); Y Nishizuka, Science, 225, 1365 (1984)].
Turning back to the late 1980s, PI3K was found to be an enzyme to phosphorylate the 3-position of the inositol ring of phosphatidylinositol [D. Whitman et al., Nature, 332, 664 (1988)].
PI3K was originally considered to be a single enzyme at the time when PI3K was discovered. Recently it was clarified that a plurality of subtypes are present in the PI3K. Three major classes of PI3Ks have now been identified on the basis of their in vitro substrate specificity [B. Vanhaesebroeck, Trend in Biol. Sci., 22, 267(1997)].
Substrates for class I PI3Ks are PI, PI(4)P and PI(4,5)P2. In these substrates, PI(4,5)P2 is the most advantageous substrate in cells. Class I PI3Ks are further divided into two groups, class Ia and class Ib, in terms of their activation mechanism. Class Ia PI3Ks, which include PI3K p110α, p110β and p110δ subtypes, are activated in the tyrosine kinase system. Class Ib PI3K is a p110γ subtype activated by a G protein-coupled receptor.
PI and PI(4)P are known as substrates for class II PI3Ks but PI(4,5)P2 is not a substrate for the enzymes of this class. Class II PI3Ks include PI3K C2α, C2β and C2γ subtypes, which are characterized by containing C2 domains at the C terminus, implying that their activity will be regulated by calcium ions. The substrate for class III PI3Ks is PI only. A mechanism for activation of the class III PI3Ks is not clarified yet. Since each subtype has its own mechanism for the regulating activity, it is considered that the respective subtypes will be activated depending on their respective stimuli specific to each of them.
In the PI3K subtypes, the class Ia subtype has been most extensively investigated to date. The three subtypes of class Ia are hetero dimers of a catalytic 110 kDa subunit and regulatory subunits of 85 kDa and 55 kDa. The regulatory subunits contain SH2 domains and bind to tyrosine residues phosphorylated by growth factor receptors with a tyrosine kinase activity or oncogene products thereby inducing the PI3K activity of the p110 catalytic subunit. Thus, the class Ia subtypes are considered to be associated with cell proliferation and carcinogenesis. Furthermore, the class Ia PI3K subtypes bind to activated ras oncogene to express their enzyme activity. It has been confirmed that the activated ras oncogene is found to be present in many cancers, suggesting a role of class Ia PI3Ks in carcinogenesis.
As explained above, PI3K inhibitors are expected to be a novel type of medicaments useful against cell proliferation disorders, especially as carcinostatic agents. As for the PI3K inhibitor, wortmannin [H. Yano et al., J. Biol. Chem., 263, 16178 (1993)] and LY294002 [J. Vlahos et al., J. Biol. Chem., 269, 5241(1994)] which is represented by the formula below are known. However, development of PI3K inhibitors having a more potent cancer cell growth inhibiting activity is desired.
Japanese Patent KOKAI (Laid-Open) No. 6-220059 discloses fused heteroaryl derivatives shown by formula (a) below which possess an activity of reducing the blood glucose level. Furthermore, compounds shown by formula (b) and formula (c) below are described in Indian J. Chem., Sect. B (1993), 32B (9), 965–8 and J. Heterocycl. Chem. (1992), 29 (7), 1693–702, respectively. In addition, Al-AzharBull. Sci. (1992), 3(2), 767–75 discloses a compound shown by formula (d) below. However, none of these prior art publications disclose or suggest the PI3K inhibiting activity.
In formula (a) above, Z is O, S or ═N—R0, R1 is an amino which may be substituted, a heterocyclic group which may be substituted, etc.; R2 is cyano, an amino which may be substituted, or a heterocyclic group which may be substituted; and with respect to the remaining substituents, see the specification of the patent. In formula (b) and (c) above, R is a (substituted) amino or a (substituted) nitrogen-containing saturated heterocyclic group.
Publication No. WO98/23613 discloses fused pyrimidine derivatives, such as 7H-pyrrolo[2,3-d]pyrimidine derivatives, which having a tyrosine kinase receptor inhibiting activity and which are useful as carcinostatic agents, wherein the fused pyrimidine derivatives have at its fourth position a particular-heteroaryl-substituted amino, pheny-substituted amino, or indole-1-yl, and have no substituent at its second position.
Following compounds are known among the compounds shown by general formula (I), whereas “A” ring is a ring shown by (b);
(1) Ann. Pharm. Fr. (1974), 32(11), 575–9 discloses 4-(4-morpholinyl)-2-phenylpirido[2,3-d]pyrimidine as a compound having antiinflammatory and spasmolytic activities,
(2) Chem. Pharm. Bull. (1976), 24(9), 2057–77 discloses 4-(4-morpholinyl)-2-phenylpirido[2,3-d]pyrimidine-7(1H)-one as a compound having a diuretic activity,
(3) Khim.-Farm. Zh. (1993), 7(7), 16–19 and Khim. Geterotsiki. Soedin. (1971), 7(3), 418–20 disclose 4-(4-morpholinyl)-2-phenyl-6-quinazolinol and 6-methoxy-4-(4-morpholinyl)-2-phenylquinazoline as compounds having an antibiotic activity,
(4) Publication No. WO2000/41697 discloses 2,4-diamino-6-phenyl-8-piperidinopyrimido[5,4-d]pyrimidine as a compound having celebral ischemia prevention and treatment effects,
(5) Publication No. WO99/32460 discloses, as cardiovascular drugs, compounds of general formula (Ib) described hereinafter wherein B is a benzene ring, W is N, n is 2 or 3, existing R1's are all —OMe, and R4b is an unsubstituted phenyl or a phenyl substituted by 1 to 3 substituents which are selected from -a halogen, NO2, -a lower alkyl, —O-a lower alkyl, -a halogenated lower alkyl and —CONRaRc,
(6) Publication No. BE841669 discloses, as antiparasitics, compounds of general formula (Ib) described hereinafter wherein B is a benzene ring, W is N, n is 1, R1 is -a halogen or -a lower alkyl, and R4b is -(an imidazolyl which may have one or more substituents),
(7) Publication No. WO99/43682 discloses, as antianxiety agents, compounds of general formula (Ib) described hereinafter wherein B is a thiophene ring, and W is CH,
(8) Japanese Patents KOKAI (Laid-Open) Nos. 62-10085 and 61-158983 disclose compounds of general formula (Ib) described hereinafter wherein B is an imidazole ring, and W is N, whereas the compounds have an antiinflammatory activity, a platelet aggregation inhibiting activity, etc.,
(9) U.S. Pat. No. 3,873,545 and Act Pol. Pharm. (1994), 51(4–5), 359–63 disclose compounds of general formula (Ib) described hereinafter wherein B is a pyridine ring, and R4b is an unsubstituted phenyl, an unsubstituted pyridyl, or -a lower alkylene-(a nitrogen-containing saturated heterocyclic group which may have one or more substituents), whereas the compounds have a spasmolytic, diuretic or hypotensive activity,
(10) U.S. Pat. No. 2,940,972 discloses compounds of general formula (Ib) described hereinafter wherein B is a pyrazine ring, and R4b is an unsubstituted phenyl, or a benzyl, whereas the compounds have a coronary dilating or sedative activity,
(11) U.S. Pat. No. 3,753,981 and German Patent Publication No. 2,140,280 disclose compounds of general formula (Ib) described hereinafter wherein B is a benzene ring, and R4b is a styryl or 2-(5-nitro-2-furyl)vinyl, whereas the compounds have an antiinflammatory or antibiotic activity, and
(12) Eur. J. Med. Chem. (1996), 31(5), 417–425, discloses compounds of general formula (Ib) described hereinafter wherein B is a benzene ring, W is CH, and R2 and R3 are bonded together with an adjacent N atom to form -(piperidinyl which may have one or more substituents) or -(piperazinyl which may have one or more substituents), as compounds working as a benzodiazepine receptor ligand, U.S. Pat. No. 4,560,692 discloses them as those having a spasmolytic and ataractic activity, and Japanese Patents KOKAI (Laid-Open) No. 2-129169 discloses them as those having a lipoperoxidation inhibiting activity.
Furthermore, compounds of general formula (Ib) described hereinafter wherein B is a pyridine ring and n is 0 are disclosed in Japanese Patent KOKAI (Laid-Open) No. 51-138689 (antiparasitics), Japanese Patent KOKAI (Laid-Open) No. 56-120768 (a dye component for thermosensitive recording materials), Antimicrob. Agents Chemother., (1975), 8 (2), 216–19 (an antibacterial activity), Cancer Res. (1975), 35 (12), 3611–17 (a mutagenic activity), CA 64: 19608c, Collect. Czech. Chem. Commun., (1994), 59 (6), 1463–6, U.S. Pat. No. 5,304,554 (an anti-HIV activity), Chem. Pharm. Bull., (1982), 30(6), 1974–9, and J. Heterocycl, Chem. (1980), 17(5), 1029–34.However, none of the prior publications teach or even suggest the PI3K inhibiting activity and carcinostatic activity.
SUMMARY OF THE INVENTION
The present inventors have performed extensive investigations on compounds with a PI3K inhibiting activity. As a result, it has been found that novel fused heteroaryl derivatives have an excellent PI3K inhibiting activity as well as a cancer cell growth inhibiting activity. Based on the finding, it has been discovered that the fused heteroaryl derivatives could be excellent PI3K inhibitors and antitumor agents. The present invention has thus been achieved.
Therefore, the present invention relates to pharmaceutical compositions, which are PI3K inhibitors or antitumor agents, comprising a fused heteroaryl derivative represented by general formula (I) below or a salt thereof and a pharmaceutically acceptable carrier.
[wherein:
B represents a benzene ring, or a 5- or 6-membered monocyclic heteroaryl containing 1 to 2 hetero atoms selected from O, S and N;
R1 represents -a lower alkyl, -a lower alkenyl, -a lower alkynyl, -a cycloalkyl, -an aryl which may have one or more substituents, -a heteroaryl which may have one or more substituents, -a halogen, —NO2, —CN, -a halogenated lower alkyl, —ORb, —SRb, —SO2-Rb, —SO—Rb, —COORb, —CO—Rb, —CONRaRb, —SO2NRaRb, —NRaRb, —NRa-CORb, —NRa-SO2Rb, —O—CO—NRaRb or —NRaCO—COORb, —CO-a nitrogen-containing saturated heterocyclic group, —CONRa-a lower alkylene-ORb, —CONRa-a lower alkylene-NRb, —O-a lower alkylene-ORb, —O-a lower alkylene-O-a lower alkylene-ORb, —O-a lower alkylene-NRaRb, —O-a lower alkylene-O-a lower alkylene-NRaRb, —O-a lower alkylene-NRc-a lower alkylene-NRaRb, —NRc-a lower alkylene-NRaRb, —N(a lower alkylene-NRaRb)2, —CONRa-ORb, —NRa-CO—NRbRc, or —OCORb;
each of R2 and R3, which may be the same or different, represents —H, -a lower alkyl, -a lower alkylene-ORa or -a lower alkylene-NRaRc, or R2 and R3 are combined together with the N atom adjacent thereto to form a nitrogen-containing saturated heterocyclic group as —NR2R3 which may have one or more substituents;
each of Ra and Rc, which may be the same or different, represents —H or -a lower alkyl;
Rb represents —H, -a lower alkyl, a cycloalkyl, an aryl which may have one or more substituents or a heteroaryl which may have one or more substituents;
n represents 0, 1, 2 or 3;
each of W and X, which may be same or different, represents N or CH;
Y represents O, S or NH; and,
R4 represents —H, -a lower alkyl, -a lower alkenyl, -a lower alkynyl, -(an aryl which may have one or more substituents), -a lower alkylene-(an aryl which may have one or more substituents), -a lower alkenylene-(an aryl which may have one or more substituents), -a lower alkynylene-(an aryl which may have one or more substituents), -(a cycloalkyl which may have one or more substituents), -(a cycloalkenyl which may have one or more substituents), -a lower alkylene-(a cycloalkyl which may have one or more substituents), -a lower alkenylene-(a cycloalkyl which may have one or more substituents), -a lower alkylene-(a nitrogen-containing saturated heterocyclic group which may have one or more substituents), -a lower alkenylene-(a nitrogen-containing saturated heterocyclic group which may have one or more substituents), -(a heteroaryl which may have one or more substituents), -a lower alkylene-(a heteroaryl which may have one or more substituents), or -a lower alkenylene-(a heteroaryl which may have one or more substituents). The same applies hereinbelow.
The compounds (I) of the present invention encompass the known compounds as well as commercially available compounds later described in Compound Z, which are all included within the definition of formula (I).
The present invention further relates to a novel fused heteroaryl derivative represented by general formula (Ia) or (Ib) or salts thereof, as well as a novel pharmaceutical composition comprising the same and a pharmaceutically acceptable carrier:
[wherein:
R1 represents -a lower alkyl, -a lower alkenyl, -a lower alkynyl, -a cycloalkyl, -an aryl which may have one or more substituents, -a heteroaryl which may have one or more substituents, -a halogen, —NO2, —CN, -a halogenated lower alkyl, —ORb, —SRb, —SO2-Rb, —SO—Rb, —COORb, —CO—Rb, —CONRaRb, —SO2NRaRb, —NRaRb, —NRa-CORb, —NRa-SO2Rb, —O—CO—NRaRb or —NRaCO—COORb, —CO-a nitrogen-containing saturated heterocyclic group, —CONRa-a lower alkyl-ORb, —CONRa-a lower alkylene-ORb, —O-a lower alkylene-NRb, —O-a lower alkylene-O-a lower alkylene-ORb, —O-a lower alkylene-NRaRb, —O-a lower alkylene-O-a lower alkylene-NRaRb, —O-a lower alkylene-NRc-a lower alkylene-NRaRb, —NRc-a lower alkylene-NRaRb, —N(a lower alkylene-NRaRb)2, —CONRa-ORb, —NRa-CO—NRbRc, or —OCORb;
each of R2 and R3, which may be the same or different, represents —H or -a lower alkyl, or R2 and R3 are combined together with the N atom adjacent thereto to form a nitrogen-containing saturated heterocyclic group as —NR2R3 which may have one or more substituents;
Ra and Rc, which may be the same or different, represent —H or -a lower alkyl;
Rb represents —H, -a lower alkyl, a cycloalkyl, an aryl which may have one or more substituents or a heteroaryl which may have one or more substituents;
n represents 0, 1, 2 or 3;
X represents N or CH;
Y represents O, S or NH; and,
R4a represents -(an aryl which may have one or more substituents), -a lower alkylene-(an aryl which may have one or more substituents), -a lower alkenylene-(an aryl which may have one or more substituents), -a lower alkynylene-(an aryl which may have one or more substituents), -(a cycloalkyl which may have one or more substituents), -(a cycloalkenyl which may have one or more substituents), -a lower alkylene-(a cycloalkyl which may have one or more substituents), -a lower alkenylene-(a cycloalkyl which may have one or more substituents), -a lower alkylene-(a nitrogen-containing saturated heterocyclic group which may have one or more substituents), -a lower alkenylene-(a nitrogen-containing saturated heterocyclic group which may have one or more substituents), -(a heteroaryl which may have one or more substituents), -a lower alkylene-(a heteroaryl which may have one or more substituents), or -a lower alkenylene-(a heteroaryl which may have one or more substituents);
with the proviso that the following compounds are excluded:
(1) compounds in which X represents N, Y represents S, n is 3 and R1 represents a combination of —CN, —OEt and phenyl, and R4a represents 2-nitrophenyl;
(2) compounds in which X represents CH, and R4a represents -(a heteroaryl which may have one or more substituents);
(3) compounds in which X represents CH, Y represents O, n is 0 and R4a represents an unsubstituted phenyl; and
(4) compounds in which X represents N, Y represents S, n is 2, R1 represents an unsubstituted phenyl and R4a represents 4-methoxyphenyl or an unsubstituted phenyl. The same applies hereinbelow.
[wherein:
B represents a benzene ring, or a 5- or 6-membered monocyclic heteroaryl containing 1 to 2 hetero atoms selected from O, S and N;
R1 represents -a lower alkyl, -a lower alkenyl, -a lower alkynyl, -a cycloalkyl, -an aryl which may have one or more substituents, -a heteroaryl which may have one or more substituents, -a halogen, —NO2, —CN, -a halogenated lower alkyl, —ORb, —SRb, —SO2-Rb, —SO—Rb, —COORb, —CO—Rb, —CONRaRb, —SO2NRaRb, —NRaRb, —NRa-CORb, —NRa-SO2Rb, —O—CO—NRaRb, —NRaCO—COORb, —NRaCOORb, —NRaCO-a lower alkylene-an aryl, —NRa-SO2-a lower alkylene-an aryl, —NRa-a lower alkylene-an aryl, -a lower alkylene-ORb, -a lower alkylene-NRaRb, —CO-a nitrogen-containing saturated heterocyclic group, —CONRa-a lower alkylene-ORb, —CONRa-a lower alkylene-NRcRb, —CONRa-a lower alkylene-a nitrogen-containing saturated heterocyclic group, —O-a lower alkylene-ORb, —O-a lower alkylene-NRaRb, —O-a lower alkylene-a nitrogen-containing saturated heterocyclic group, —O-a lower alkylene-O-a lower alkylene-ORb, —O-a lower alkylene-O-a lower alkylene-NRaRb, —O-a lower alkylene-NRc-a lower alkylene-NRaRb, —NRc-a lower alkylene-NRaRb, —N(a lower alkylene-NRaRb)2, —CONRa-ORb, —NRa-CO—NRbRc, or —OCORb;
R2 and R3 are combined together with the N atom adjacent thereto to form —NR2R3 which is a nitrogen-containing saturated heterocyclic group which may have one or more substituents;
Ra and Rc, which may be the same or different, represent —H or -a lower alkyl;
Rb represents —H, -a lower alkyl, -a cycloalkyl, -(an aryl which may have one or more substituents) or -(a heteroaryl which may have one or more substituents);
n represents 0, 1, 2 or 3, whereas n represents 1, 2 or 3 when B represents a benzene ring;
W represents N or CH; and,
R4b represents -(an aryl which may have one or more substituents), -a lower alkylene-(an aryl which may have one or more substituents), -a lower alkenylene-(an aryl which may have one or more substituents), -a lower alkynylene-(an aryl which may have one or more substituents), -(a cycloalkyl which may have one or more substituents), -(a cycloalkenyl which may have one or more substituents), -a lower alkylene-(a cycloalkyl which may have one or more substituents), -a lower alkenylene-(a cycloalkyl which may have one or more substituents), -a lower alkylene-(a nitrogen-containing saturated heterocyclic group which may have one or more substituents), -a lower alkenylene-(a nitrogen-containing saturated heterocyclic group which may have one or more substituents), -(a heteroaryl which may have one or more substituents), -a lower alkylene-(a heteroaryl which may have one or more substituents), or -a lower alkenylene-(a heteroaryl which may have one or more substituents);
with the proviso that the following compounds are excluded:
(1) 4-(4-morpholinyl)-2-phenylpyrido[2,3-d]pyrimidine,
(2) 4-(4-morpholinyl)-2-phenylpyrido[2,3-d]pyrimidin-7(1H)-one,
(3) 4-(4-morpholinyl)-2-pheny-6-quinazolinol and 6-methoxy-4-(4-morpholinyl)-2-phenyquinazoline,
(4) 2,4-diamino-6-phenyl-8-piperidinopyrimido[5,4-d]pyrimidine,
(5) compounds in which B represents a benzene ring, W represents N, n is 2 or 3, existing R1's all represent —OMe, and R4b is an unsubstituted phenyl or a phenyl which is substituted by 1 to 3 substituents selected from -halogen, —NO2, -a lower alkyl, —O-a lower alkyl, -a hanogenated lower alkyl and —CONRaRc,
(6) compounds in which B represents a benzene ring, W represents N, n is 1, R1 represents -halogen or -a lower alkyl, and R4b represents -(imidazolyl which may have one or more substituents),
(7) compounds in which B represents a thiophene ring, and W represents CH,
(8) compounds in which B represents an imidazole ring, and W represents N,
(9) compounds in which B represents a pyridine ring, and R4b represents an unsubstituted phenyl, an unsubstituted pyridyl, or -a lower alkylene-(a nitrogen-containing saturated heterocyclic group which may have one or more substituents),
(10) compounds in which B represents a pyrazine ring, and R4b represents an unsubstituted phenyl, or a benzyl,
(11) compounds in which B represents a benzene ring, and R4b represents a styryl or 2-(5-nitro-2-furyl)vinyl, and
(12) compounds in which B represents a benzene ring, W represents CH, and R2 and R3 are combined together with the N atom adjacent thereto to form -(piperidinyl which may have one or more substituents) or -(piperazinyl which may have one or more substituents). The same applies hereinbelow.
Further teaching of the present invention provides a method to treat disorders (especially cancers) which are associated with PI3K, wherein the method comprises of administering to a patient an effective amount of a fused heteroaryl derivative of formula (I), (Ia) or (Ib) above or a salt thereof as well as a use of said fused heteroaryl derivative or a salt thereof for producing a medicament (especially a carcinostatic agent) which inhibit PI3K.
EMBODIMENTS
The compounds of general formula (I), (Ia) or (Ib) are described below in more detail.
The term “lower” throughout the specification is used to mean a straight or branched hydrocarbon chain having 1 to 10, preferably 1 to 6, and more preferably 1 to 3 carbon atoms.
Preferred examples of the “lower alkyl” are an alkyl having 1 to 3 carbon atoms, more preferably methyl and ethyl. Preferred examples of the “lower alkenyl” include vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl and 3-butenyl. Preferred examples of the “lower alkynyl” include ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl and 1-methyl-2-propynyl. The terms “lower alkylene”, “lower alkenylene” and “lower alkynylene” are used to mean bivalent groups of the lower alkyl, lower alkenyl and lower alkynyl described above. Preferred examples of these groups are methylene, ethylene, vinylene, propenylene, ethynylene and propynylene. The terms “cycloalkyl” and “cycloalkenyl” refer to cycloalkyl and cycloalkenyl groups preferably having 3 to 8 carbon atoms. Preferred examples of these groups include cyclopropyl, cyclopentyl, cyclohexyl and cyclopentenyl.
Examples of the “halogen” are F, Cl, Br and I. Examples of the “halogenated lower alkyl” are the aforementioned lower alkyl groups which are further substituted with one or more halogen atoms described above, preferably —CF3.
The term “nitrogen-containing saturated heterocyclic group” throughout the specification refers to a 5- to 7-membered heterocyclic group containing one or two nitrogen atoms on the ring, which may further contain one O or S atom and may form a bridge structure or may be fused with one benzene ring. Preferred examples of such heterocyclic group are pyrrolidinyl, piperazinyl, piperidyl and morpholinyl. Preferred examples of the nitrogen-containing saturated heterocyclic group as —NR2R3 are 1-pyrrolidinyl, 1-piperazinyl, piperidino and morpholino, with particular preference to morpholino.
The term “aryl” is used throughout the specification to mean an aromatic cyclic hydrocarbon group. An aryl having 6 to 14 carbon atoms is preferable. Preferred examples of such aryl are phenyl and naphthyl.
The term “heteroaryl” refers to a 5- or 6-membered monocyclic heteroaryl containing 1 to 4 hetero atoms selected from N, S and O as well as a bicyclic heteroaryl fused to a benzene ring. The heteroaryl may be partially saturated. Preferred examples of the monocyclic heteroaryl are furyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidinyl, pyridazinyl and pyrazinyl. Examples of the bicyclic heteroaryl are preferably benzofuranyl, benzothienyl, benzothiadiazolyl, benzothiazolyl, benzimidazolyl, indolyl, isoindolyl, indazolyl, quinolyl, isoquinolyl, cinnolinyl, quinazolinyl, quinoxalinyl and benzodioxolyl. Specific examples of the partially saturated heteroaryl are 1,2,3,4-tetrahydroquinolyl, etc. Particularly preferred are 5- to 6-membered monocyclic groups, more preferably imidazolyl, thiazolyl, triazolyl, pyridyl and pyrazinyl.
Examples of a “5- or 6-membered monocyclic heteroaryl containing 1 or 2 hetero atoms selected from O, S and N” in B include a furan, thiophene, pyrole, imidazole, pyrazole, thiazole, isothiazole, oxazole, pyridine, pyrimidine, pyridazine and pyrazine ring. Preferably, it is a pyridine, pyrazine or thiophene ring. More preferable, it is a pyridine ring.
The substituents for the “aryl which may have one or more substituents”, “heteroaryl which may have one or more substituents”, “cycloalkyl which may have one or more substituents”, “cycloalkenyl which may have one or more substituents” or “nitrogen-containing saturated heterocyclic group which may have one or more substituents” are 1˜5 substituents, which may be the same or different. Preferably, these substituents are selected from Group A described below. Each of R, R′ and R″, which may be the same or different, represents H or a lower alkyl (the same shall apply hereinafter).
Group A: -a lower alkyl, -a lower alkenyl, -a lower alkynyl, -a halogen, -a halogenated lower alkyl, -a lower alkylene-OR, —NO2, —CN, ═O, —OR, —O— a halogenated lower alkyl, —O-a lower alkylene-NRR′, —O-a lower alkylene-OR, —O-a lower alkylene-an aryl, —SR, —SO2-a lower alkyl, —SO-a lower alkyl, —COOR, —COO-a lower alkylene-an aryl, —COR, —CO-an aryl, -an aryl, —CONRR′, —SO2NRR′, —NRR′, —NR″-a lower alkylene-NRR′, —NR′-a lower alkylene-OR, —NR-a lower alkylene-an aryl, —NRCO-a lower alkyl, —NRSO2-a lower alkyl, -a cycloalkyl and -a cycloalkenyl.
When R4, R4a and R4b represent “an aryl which may have one or more substituents” or “a heteroaryl which may have one or more substituents”, the substituents are 1 to 5 groups selected from a) through c) below, which may be the same or different.
a): -a lower alkyl, -a lower alkenyl, -a lower alkynyl, -a halogen, -a halogenated lower alkyl, -a lower alkylene-OR, —NO2, —CN, ═O, —O-halogenated lower alkyl, —SO2-a lower alkyl, —SO-a lower alkyl, —COOR, —COO-a lower alkylene-an aryl, —COR, —CO-an aryl, —CONRR′, —SO2NRR′, -Cyc or -Alp-Cyc (wherein Alp represents a lower alkylene, a lower alkenylene or a lower alkynylene, and Cyc represents an aryl which may have 1 to 5 substituents selected from Group A, a heteroaryl which may have 1 to 5 substituents selected from Group A, a nitrogen-containing saturated heterocyclic group which may have 1 to 5 substituents selected from Group A, a cycloalkyl which may have 1 to 5 substituents selected from Group A, or a cycloalkenyl which may have 1 to 5 substituents selected from Group A; the same shall apply hereinafter).
b): —NR-E-F (wherein E represents —CO—, —COO—, —CONR′—, —SO2NR′— or —SO2-; F represents -Cyc or -(a lower alkyl, a lower alkenyl or a lower alkynyl which may be substituted by one or more substituents selected from the group comprising of -a halogen, —NO2, —CN, —OR, —O-a lower alkylene-NRR′, —O-a lower alkylene-OR, —SR, —SO2-a lower alkyl, —SO-a lower alkyl, —COOR, —COR, —CO-an aryl, —CONRR′, —SO2NRR′, —NRCO-a lower alkyl, —NRR′, —NR′-a lower alkylene-OR, —NR″-a lower alkylene-NRR′ and -Cyc) and the same shall apply hereinafter).
c): -Z-R′, -Z-Cyc, -Z-Alp-Cyc, -Z-Alp-Z′-R′ or -Z-Alp-Z′-Cyc (wherein each of Z and Z′, which may be the same or different, independently represents O, S or NR; and the same shall apply hereinafter).
The particularly preferred ones are -a lower alkylene-OR, —CONRR′, —NR—CO-Cyc1 (wherein Cyc1 is -an aryl which may have 1˜5 substituents selected from Group A, -a heteroaryl which may have 1˜5 substituents selected from Group A, or -a nitrogen-containing saturated heterocyclic group which may have 1˜5 substituents selected from Group A, and the same applies hereinbelow), —NR—SO2-Cyc1, —OR, —NRR′, —O-a lower alkylene-NRR′ and —O-a lower alkylene-(a nitrogen-containing saturated ring which may have 1˜5 substituents selected from Group A).
When n is 2 to 4, each R1 group may be the same or different, independently.
In the compounds which are shown by formulas (I), (Ia) and (Ib) of the present invention, the following compounds are preferred:
(1) Compounds in which R2 and R3 forms —NR2R3 which is a nitrogen-containing saturated heterocyclic group which may have 1˜2 substituents selected from the group comprising of —OH, ═O and -a lower alkyl;
(2) Compounds in which R2 and R3 forms —NR2R3 which is -morpholino;
(3) Compounds in which W is N;
(4) Compounds in which R4, R4a or R4b represents -(an aryl which may have one or more substituents) or -(a heteroaryl which may have one or more substituents);
(5) Compounds in which B represents a benzene ring; R1 represents -a lower alkyl, -a lower alkenyl, -a lower alkynyl, -a cycloalkyl, -an aryl which may have one or more substituents, -a heteroaryl which may have one or more substituents, -a halogen, —NO2, —CN, -a halogenated lower alkyl, —ORb, —SRb, —SO2-Rb, —SO—Rb, —COORb, —CO—Rb, —CONRaRb, —SO2NRaRb, —NRaRb, —NRa-CORb, —NRa-SO2Rb, —O—CO—NRaRb or —NRaCO—COORb;
(6) Compounds in which B is a pyridine, pyrazine or thiophene ring and n is 0;
(7) Compounds in which X represents N, Y represents O and n is 0;and
(8) Compounds in which R4, R4a or R4b represents an aryl which has one or more substituents selected from the group comprising of -a lower alkylene-OR, —CONRR′, —NR—CO—Cyc1, —NR—SO2-Cyc1, —OR, —NRR′, —O-a lower alkylene-NRR′ and —O-a lower alkylene-(a nitrogen-containing saturated heterocyclic group which may have 1˜5 substituents selected from Group A).
The particularly preferred compounds shown by general formula (Ia) are those having R4a which is a phenyl having at least one substituent which is selected from of the group comprising of —OH, —NH2, —NH-a lower alkyl, —N(a lower alkyl)2, —O-a lower alkylene-NH2 and —O-a lower alkylene-(a nitrogen-containing saturated heterocyclic group which may be substituted by a lower alkyl).
Moreover, the following compounds shown by general formula (Ib) are particularly preferred:
(1) Compounds in which W represents N, R4b represents -(an aryl which may have one or more substituents), and R2 and R3 form —NR2R3 which is -morpholino;
(2) Compounds in which B represents a benzene ring, n is 1 or 2, and R1 represents -a halogen, —NO2, —CN, -a halogenated lower alkyl, —ORb, —SRb, —NRaRb, —NRa-CORb or —NRa-SO2Rb; and
(3) Compounds in which B represents a pyridine, pyrazine or thiophene ring, n is 0, and R4b represents a phenyl which has at least one substituent which is selected from —OH, —CH2OH and —CONH2.
Among the compounds of the present invention, the preferred ones which are shown by general formula (Ia) are (Co 17) 6-amino-3′-(4-morpholinopyrido[3′,2′:4,5]furo[3,2-d]pyrimidin-2-yl)nicotinanilide, (Co 33) 4-(4-morpholinopyrido[3′,2′:4,5]furo[3,2-d]pyrimidin-2-yl)aniline, (Co 50) 3-(4-morpholinopyrido[3′,2′:4,5]furo[3,2-d]pyrimidin-2-yl)phenol, (Co 69) 4-morpholino-2-[3-(2-piperazin-1-ylethoxy)phenyl]pyrido[3′,2′:4,5]furo[3,2-d]pyrimidine, (Co 73) 3′-(4-morpholinopyrido[3′,2′:4,5]furo[3,2-d]pyrimidin-2-yl)acrylanilide, and salts thereof. The preferred ones which are shown by general formula (Ib) are (Co 144) N-[2-(3-benzenesulfonylaminophenyl)-4-morphoniloquinazolin-6-yl]acetamide, (Co 164) 3-(4-morpholinopyrido[4,3-d]pyrimidin-2-yl)phenol, (Co 172) 3-(4-morpholinopyrido[3,2-d]pyrimidin-2-yl)phenol, (Co 174) 3-(4-morpholinopyrido[3,4-d]pyrimidin-2-yl)phenol, (Co 186) 3-(6-methoxy-4-morpholinoquinazolin-2-yl)phenol, (Co 190) 3-(4-morpholinothieno[3,2-d]pyrimidin-2-yl)phenol, (Co 191) 3-(4-morpholinopteridin-2-yl)phenol, and salts thereof.
The compound of this invention may exist in the form of geometrical isomers or tautomers depending on the kinds of substituent groups, and these isomers in separated forms or mixtures thereof are included in the present invention. Also, the compound of the present invention may have asymmetric carbon atoms, so that optical isomer forms may exist based on such carbon atoms. All of the mixtures and the isolated forms of these optical isomers are included in the present invention.
Some of the compounds of the invention may form salts. There is no particular limitation so long as the formed salts are pharmacologically acceptable. Specific examples of acid salts are salts with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid, etc., organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, aspartic acid, glutamic acid, etc. Specific examples of basic salts include salts with inorganic bases containing metals such as sodium, potassium, magnesium, calcium, aluminum, etc., or salts with organic bases such as methylamine, ethylamine, ethanolamine, lysine, ornithine, etc. In addition, various hydrates and solvates and polymorphism of the compound (I), (Ia) or (Ib) and salts thereof are also included in this invention.
(Processes for Producing Compounds)
Hereinafter representative processes for producing the compounds of the present invention are described below. In these processes, functional groups present in the starting materials or intermediates may be suitably protected with protective groups, depending upon the kinds of functional groups. In view of the preparation technique, it may be advantageous to protect the functional groups with groups that can be readily reverted to the original functional groups. When required, the protective groups are removed to give the desired products. Examples of such functional groups are amino, hydroxy, carboxyl, etc. Examples of the protective groups which may be used to protect these functional groups are shown in, e.g., Greene and Wuts, “Protective Groups in Organic Synthesis”, second edition. These protective groups maybe appropriately employed depending upon reaction conditions.
Production Method 1
(Here and hereinafter, L represents a leaving group.)
This process for producing the compounds (I) of the present invention comprises converting the compounds shown by general formula (II) to reactive derivatives thereof (III) in a conventional manner and then reacting al amine (IV) with the reactive derivatives. When another reactive site containing the leaving group L also exists on the ring A or the substituent R4 in the reactive derivatives (III), the same or different amine (IV) may be reacted again, if necessary. In a similar manner, when the A ring or R4 of the compounds of the present invention has a leaving group L such as a chloro or fluoro, transformations of functional groups may be conducted such as a hydrolysis reaction according to a method described in J. Am. Chem. Soc., 68, 1288 (1946) or an ipso-substitution reaction using alkoxide as a reacting agent according to a method described in Tetrahedron Lett., 40, 675 (1999).
The leaving group shown by L is preferably a halogen, or an organic sulfonyloxy group, e.g., methanesulfonyloxy, p-toluenesulfonyloxy, etc.
The reaction for preparing the reactive derivatives (III) can be carried out by the usual procedures. Where the leaving group is chlorine, phosphorus oxychloride, oxalyl chloride or thionyl chloride can be reacted under cooling or heating or at room temperature in an inert organic solvent or without. As such an inert organic solvent, there is an aromatic hydrocarbon solvent such as benzene or toluene; an ethereal solvent such as tetrahydrofuran (THF), 1,4-dioxane, etc.; a halogenated hydrocarbon solvent such as dichloromethane, chloroform, etc.; and a basic solvent such as pyridine or collidine. These solvents may be used alone or as a mixture of two or more. The solvent is optionally selected depending on the kinds of starting compounds. The addition of a base (preferably a dialkylaniline, triethylamine, ammonia, lutidine, collidine, etc.), phosphorus chloride (e.g., phosphorus pentachloride), a quaternary ammonium salt (e.g., tetraethylammonium chloride), or an N,N-dialkylamide compound (e.g., dimethylformamide (DMF)) may be advantageous in some cases from the viewpoint of accelerating the reaction. Where the leaving group is sulfonyloxy, the active intermediates (III) can be synthesized from the corresponding sulfonyl chloride by the usual procedures, e.g., using a method described in Tetrahedron Lett. 23 (22), 2253 (1982) or Tetrahedron Lett. 27 (34), 4047 (1986).
The reaction for producing the compounds (I) from the reactive derivatives (III) and the amine (IV) can be carried out by reacting the amine (IV) in an inert organic solvent or in the absence of any solvents under cooling or heating or at room temperature. The solvent described above is available and it may be used singly or as a mixture of two or more. The addition of an inorganic base such as sodium hydride, or an organic base such as triethylamine (TEA), pyridine or 2,6-lutidine, may be advantageous in some cases from the viewpoint of accelerating the reaction
Production Method 2
(Wherein Rd is a lower alkyl which may have one or more substituents and Rb has the same definition as defined above; and the same shall apply hereinafter.)
This process comprises O-alkylation of the hydroxy-substituted compounds shown by general formula (Ia) or (Ic) in a conventional manner to obtain the compounds (Ib) or (Id). The reaction may be carried out, e.g., by reacting the compounds (Ia) or (Ic) with an alkylating agent such as an alkyl halide or a sulfonic acid ester in the presence of a base such as triethylamine, potassium carbonate, sodium carbonate, cesium carbonate, sodium hydroxide, sodium hydride or potassium t-butoxide. The reaction temperature can be under cooling or heating or at room temperature. and can be appropriately chosen depending on the kinds of starting compounds. When water is used or contained as a solvent in an O-alkylation reaction, the reaction may be accelerated by the addition of a phase transfer catalyst such as tetra n-butylammonium hydrogensulfate.
Another method for the O-alkylation reaction is Mitsunobu reaction. For example, methods described in Synthesis, 1 (1981) or modified methods may be used. For the hydroxyethylation of a hydroxyl group, methods using carbonate ester such as [1,3]dioxolane-2-one are also effective. As an example, methods described in J. Am. Chem. Soc., 68, 781 (1946) can be used.
Moreover, when functional groups exist on Rb and Rd of the compounds (Ib) and (Id) of the present invention, known reactions may be employed to convert the functional group. For example, when a hydroxyl group is present on Rb and Rd, the aforementioned O-alkylation reaction can be conducted, and when a leaving group is present such as a halogen, an appropriate alcohol or amine can be reacted with utilizing the conditions of said O-alkylation or N-alkylation described hereinafter in Production Method 4.When an ester group is present, the functional group can be converted to a carboxylic acid, hydroxymethyl group, and amido, using a method described hereinafter in Production Method 3.
The starting compounds (Ia) and (Ic) used in this process can be prepared by the method described for Production Method 1, using starting compounds whose OH group has been protected by an acyl type protective group (e.g., acetyl or tosyl). Further, when phosphorus oxychloride is used as a reacting agent for synthesizing reactive derivatives (III) and then a desired amino is reacted to synthesize (I), protective groups for OH group may be removed and O-phosphoramides may be produced, depending on the kind of starting compounds, a protective group, reaction conditions and conditions for work-up. In that case, for example, using a method described in Chem. Pharm. Bull., 37, 2564 (1989), phosphoramides groups can be removed. Other general protective groups can be introduced and removed by the methods described in “Protective Groups in Organic Synthesis” supra.
Production Method 3
(Wherein Rf is a lower alkyl and Rg is a lower alkyl which may have one or more substituents; and the same shall apply hereinafter.)
Production Method 3 comprises transformations of the functional groups of the ester compounds of the present invention shown by general formula (Ie) to produce the hydroxymethyl compounds (If), carboxylic acid derivatives (Ig) and amide derivatives (Ih) of the present invention, respectively. Each of the reactions can be carried out in a conventional maimer, e.g., as described in Jikken Kagaku Kouza (Encyclopedia for Experimental Chemistry) edited by Nihon Kagaku Kai (Japanese Association of Chemistry) and published by Maruzen Co., Ltd., and “Protective Groups in Organic Synthesis” supra.
Preferably, the reduction to give the hydroxymethyl compounds (If) can be conducted in an inert organic solvent to the reactions, e.g., an ethereal solvent or an aromatic hydrocarbon solvent, using a reducing agent such as lithium aluminum hydride, lithium borohydride, zinc borohydride, boran, Vitride, etc. The hydrolysis to give the carboxylic acid derivatives (Ik) can be conducted by reacting with lithium hydroxide, sodium hydroxide or potassium hydroxide in a single solvent selected from methanol, ethanol, THF and water, or a mixture of two or more. The amidation to give the amide compounds (Ih) may be performed by converting carboxylic acids to reactive derivatives such as acyl halides (acyl chlorides, etc.) or acid anhydrides, and then reacting the reactive derivatives with amines. In the reaction with amines, it is preferred to conduct the reaction in an inert organic solvent in the presence of a base (an inorganic base such as sodium hydroxide, or an organic base such as TEA, diisopropylethylamine or pyridine). Furthermore, the amidation using the carboxylic acid as a starting compound can also be carried out in an inert organic solvent in the presence of a condensation agent such as (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI), 1,1′-carbonylbis-1H-imidazole (CDI), etc.). In this case, an additive such as 1-hydroxybenzotriazol (HOBt) or the like may also be added to the reaction. The reaction temperature and solvent can be appropriately chosen depending on the kinds or the like of starting compounds.
Production Method 4
(Wherein R′ has the same definition as defined above, Rh is -a lower alkyl which may have one or more substituents, Ri is -Cyc or -Alp which may have one or more substituents, a C ring is a nitrogen-containing saturated heterocyclic group which may have one or more substituents, and Rj is —H, -a lower alkyl, -an aryl, etc.; and the same shall apply hereinafter.)
Production Method 4 comprises the reduction of the nitro compounds shown by general formula (Ii) to the corresponding amino compounds (Ij) and then subjecting the amino compounds (Ij) to various modification reactions including N-alkylation, amidation, sulfonamidation, conversion to the corresponding urea, conversion to the corresponding carbamic acid, imidation or conversion to the corresponding thiazoles, to give the compounds (Ik), (Im), (In), (Io), (Ip), (Iq) and (Ir), respectively. These products can be appropriately subjected to further known modification reactions such as N-alkylation, if necessary.
These reactions can all be carried out in a conventional manner, e.g., using the methods described in “Jikken Kagaku Kouza” supra, or “Protective Groups in Organic Synthesis” supra. Preferred procedures in these methods are described below.
The reduction of the nitro compounds can be carried out in an alcoholic solvent such as methanol in a gaseous hydrogen atmosphere using palladium on carbon (Pd—C).
When various aldehydes are employed as the starting compounds, the N-alkylation can be conducted by reductive amination using aldehydes and reducing agents such as sodium borohydride, sodium triacetoxyborohydride or sodium cyanoborohydride. Reducting amination using Dean-Stark apparatus could be useful, too. When an alkyl halide such as methyl iodide or benzyl bromide, or dimethyl sulfate is employed as an alkylating agent, the reaction can be carried out in an inert organic solvent, e.g., DMF, acetonitrile or toluene, in the presence of base such as potassium carbonate, sodium hydroxide or sodium hydride, under cooling or heating or at room temperature. For monoalkylation, an example of useful procedure is as follows: protection of amino group by acyl group such as trifluoroacetyl, alkylation of acylamide by conventional methods using halogenated alkyl, and removal of protection. The dialkylation can be conducted by reacting 2 equivalents or more of a halogenated alkyl. For dimethylation, the reaction with formalin in formic acid at room temperature or under heating is also useful.
The amidation reaction may be performed in a similar manner to that described above for Production Method 3.The sulfonamidation can be carried out in an inert organic solvent using a reactive derivative such as an acid halide (acid chloride, etc.) or an acid anhydride. The conversion to the corresponding urea can be conducted by reacting with isocyanates in an inert organic solvent, e.g., an aromatic hydrocarbon solvent, under cooling or heating or at room temperature. The conversion to the corresponding carbamic acids can be conducted by reacting chloroformate derivatives in an inert organic solvent under cooling or heating or at room temperature. The imidation can be carried out using agents such as succinic anhydride or maleic anhydride.
The conversion to the corresponding aminothiazole compounds can be conducted by converting the amino compounds to the corresponding thiourea derivatives and then reacting the derivatives with an α-halogenated ketone. Compounds (Ij) can be converted into the thiourea derivatives by methods described in, e.g., Synth. Commun. 1998, 28 (8), 1451;J. Org. Chem., 1984, 49 (6), 997, Org. Synth., 1963, IV, 180;J. Am. Chem. Soc., 1934, 56, 1408, etc. The conversion of the thiourea derivatives into the thiazole derivatives can be conducted by reacting the thiourea derivatives with the (X-halogenated ketone in an alcoholic solvent such as ethanol or a carbonyl solvent such as methyl ethyl ketone, under cooling or heating or at room temperature. The addition of a base (potassium carbonate, sodium carbonate, etc.) may be effective in some cases from the viewpoint of accelerating the reaction.
Production Method 5
(Wherein Rk and Rm each represents -a lower alkyl which may have one or more substituents.)
Production Method 5 comprises converting the nitro compounds of the invention shown by general formula (Is) to the corresponding amino compounds (It) and then subjecting them to various modification reactions to obtain the other compounds of the present invention. Each reaction can be carried out as described for Production Method 4.
Other Production Method
Other compounds included in the present invention can be obtained in the same manner as described above or by using methods well known to those skilled in the art. For instance, the reactions are carried out appropriately using methods described in “Jikken Kagaku Kouza” supra, or “Protective Groups in Organic Synthesis” supra.
For example, the demethylation reaction of compounds with an aryl group into the corresponding phenol derivatives can be carried out by the methods described in “Protective Groups in Organic Synthesis” supra, i.e., the method of reacting with a demethylating agent such as sodium cyanide or potassium cyanide in a solvent such as dimethylsulfoxide (DMSO), etc., at room temperature or under heating.
Processes for Preparing Starting Compounds
The starting compounds (II) for the synthesis of the present invention can be performed in conventional manners, e.g., by the reactions shown in the following synthetic routes.
Process 1
(Wherein Rn is a lower alkyl; and the same shall apply hereinafter.)
The starting compounds (IIc) can be synthesized by a cyclization reaction of amide intermediates (5) or cyclization conducted by reacting anthranylic acid derivatives (1) as the starting compounds with imidates (6). Conventional cyclization reactions for preparing pyrimidine ring are available for the cyclization reaction for this purpose. For instance, the method described in Chem. Pharm. Bull., 39 (2), 352 (1991) can be used for the cyclization of the intermediates (5) and the intermediates (1) and (6) as the starting materials can be cyclized by the method described in J. Med. Chem., 9, 408 (1966). The amide intermediates (5) can be synthesized by amidation of the aniline derivatives (4) in a conventional manner, or by sequential conversions of esterification of a carboxylic acid in (1), acylation of an amino, and amidation of the ester group according to conventional methods. For example, the amide intermediates (5) can be obtained in accordance with the methods described in J. Med. Chem., 33, 1722 (1990), Eur. J. Med. Chem.-Chim. Ther., 9(3), 305 (1974), etc. When (3) is obtained by acylation using (2) as the starting materials, diacylation may take place depending on the starting compounds and reaction conditions. In such a case, treatment with basic conditions will give desired monoacyl compounds (3).
Process 2
The starting compounds (IId) can be synthesized by cyclization of the amide intermediates (12) or by cyclization of the ester intermediates (7) and the amide compounds (10). The intermediates (12) can be cyclized in the same maimer as described above; where the intermediates (7) and (10) are used as the starting compounds, the cyclization can be carried out by the method described in, e.g., J. Med. Chem., 37, 2106 (1994). The amide intermediates (12) can be prepared by conversion of the functional group in ester compounds (7) in a conventional manner. The bicyclic ester intermediates (9) can be synthesized by formation of a 5-membered ring by reacting nitrile compounds (7) with ester compounds (8) in the presence of a base, for example, in accordance with the method described in J. Org. Chem., 37, 3224 (1972) or J. Heterocycl. Chem., 11 (6), 975 (1974), etc.
Process 3
The starting compounds, (IIe) can be synthesized, e.g., by cyclization of the starting compounds (14). Preferably, the compounds (14) are heated in a solvent with a high boiling point such as diphenyl ether or in the absence of any solvents. The starting compounds (14) can be synthesized in a conventional manner, e.g., by condensation of the corresponding anilines (13) with the compounds (15).
Each of the reaction products obtained by the aforementioned production methods is isolated and purified as a free base or a salt thereof. The salt can be produced by a usual salt forming method. The isolation and purification are carried out by employing usually used chemical techniques such as extraction, concentration, evaporation, crystallization, filtration, recrystallization, various types of chromatography and the like.
Various forms of isomers can be isolated by the usual procedures making use of physicochemical differences among isomers. For instance, racemic compounds can be separated by means of a conventional optical resolution method (e.g., by forming diastereomer salts with a conventional optically active acid such as tartaric acid, etc. and then optically resolving the salts) to give optically pure isomers. A mixture of diastereomers can be separated by conventional means, e.g., fractional crystallization or chromatography. In addition, an optical isomer can also be synthesized from an appropriate optically active starting compound.
INDUSTRIAL APPLICABILITY
The compounds of the present invention exhibit a PI3K inhibitory activity and therefore, can be utilized in order to inhibit abnormal cell growths in which PI3K plays a role. Thus, the compounds are effective in the treatment of disorders with which abnormal cell growth actions of PI3K are associated, such as restenosis, atherosclerosis, bone disorders, arthritis, diabetic retinopathy, psoriasis, benign prostatic hypertrophy, atherosclerosis, inflamation, angiogenesis immunological disorders, pancreatitis, kidney disease, cancer, etc. In particular, the compounds of the present invention possess excellent cancer cell growth inhibiting effects and are effective in treating cancers, preferably all types of solid cancers and malignant lymphomas, and especially, leukemia, skin cancer, bladder cancer, breast cancer, uterus cancer, ovary cancer, prostate cancer, lung cancer, colon cancer, pancreas cancer, renal cancer, gastric cancer, brain tumor, etc.
The pharmacological effect of the compounds according to the invention have been verified by the following pharmacological tests.
Test Example 1 Inhibition of PI3K (p110α Subtype)
Inhibition was determined using enzyme (bovine p110α) prepared in the baculovirus expression system. Bovine p110 was prepared according to a modification from the method by I. Hiles et al., Cell, 70, 419 (1992). Each compound to be assayed was dissolved in DMSO and the obtained 10 mM DMSO solution was serially diluted with DMSO.
The compound (0.5 μl) to be assayed and enzyme were mixed in 25 μl of buffer solution (40 mM Tris-HCl (pH 7.4), 200 mM NaCl, 2 mM dithiothreitol, 5 mM MgCl2). Then, 25 μl of 5 mM Tris-HCl (pH 7.4) buffered solution supplemented with 10 μg PI (Sigma), 2 μCi [γ-32P] ATP (Amersham Pharmacia) and 80 μM non-radiolabeled ATP (Sigma) was added to the mixture to initiate the reaction. After reacting at 37° C. for 15 minutes, 200 μl of 1M HCl and 400 μl of CHCl3/MeOH (1:1) were added to the reaction mixture. The resulting mixture was stirred and then centrifuged. After the organic layer was again extracted twice with 150 μl of MeOH/1M HCl (1:1). The radioactivity was measured using Cerenkov light.
The IC50 inhibition activity was defined by a 50% inhibition concentration of each compound assayed, which was converted from the radioactivity determined as 100% when DMSO alone was added and as 0% when no enzyme was added.
The compounds of the prevent invention exhibited an excellent p110α subtype inhibition activity. For example, IC50 of Compound (hereinafter, abbreviated as Co) 10, Co 17, and Co 24 were less than 1 μM.
Moreover, compounds of the prevent invention were confirmed to have inhibiting activities against other subtypes (such as a C2 β subtype).
Test Example 2 Colon Cancer Cell Growth Inhibition
HCT116 cells from a colon cancer cell line were cultured in McCoy's 5A medium (GIBCO) supplemented with 10% fetal bovine serum. HCT116 cells were inoculated on a 96 well plate (5000 cells/well) followed by overnight incubation. The test compound diluted with the medium was added to the medium in a final concentration of 0.1 to 30M (final DMSO concentration, 1%). After incubation over 72 hours, Alamar Blue reagent was added to the medium. Two hours after the addition, a ratio of fluorescent intensity at an excitation wavelength of 530 nm to that at an emission wavelength of 590 nm was measured to determine the IC50.Co 14, Co 24, Co 25, Co 31, Co 46 and Co 47 of the present invention exerted an excellent cancer cell growth inhibition activity.
Test Example 3 Melanoma Cell Growth Inhibition
A375 cells from a melanoma cell line were cultured in DMEM medium (GIBCO) supplemented with 10% fetal bovine serum. A375 cells at 10,000 cells/100 μl were added to a 96 well plate which contained 1 μl/well of the test compounds (final concentration of 0.001˜30 μM). After incubation for over 46 hours, Alamar Blue reagent was added to the medium (10 μl/well). Two hours after the addition, a ratio of fluorescent intensity at an excitation wavelength of 530 nm to that at an emission wavelength of 590 nm was measured to determine the IC50 of the test compounds in the same manner as in the above examples.
The compounds of the prevent invention exhibited an excellent cancer cell growth inhibition activity. For example, Co 17, Co 33, Co 50, Co 69, Co 164, Co 172, Co 174, Co 186, Co 190 and Co 191 exerted a good melanoma cell growth inhibition activity. Their IC50 values were 0.33˜4.26 μM. Contrarily, the known PI3K inhibitor LY294002 showed a value of 8.39 μM.
In addition to the above cancer cell lines, the compounds of the present invention exhibited excellent cancer cell growth inhibiting activities against Hela cells from a cervix cancer cell line, A549, H460 cells from a lung cancer cell line, COLO205, WiDr, Lovo cells from a colon cancer cell line, PC3, LNCap cells from a prostate cancer cell line, SKOV-3, OVCAR-3, CH1 cells from an ovary cancer cell line, U87 MG cells from a glioma cell line and B×PC-3 cells from a pancreas cancer cell line.
Test Example 4 in vivo Cancer Cell Growth Inhibition
A single-cell suspension of HelaS3 (5×106 cells), a human cervix cancer cell line, was inoculated into the flank of female Balb/c nude mice by subcutaneously injection. When the tumor reached 100˜200 mm3 in volume, test compounds were intraperitoneally administered once a day for two weeks. 20% Hydroxypropyl-β-cyclodextrin/saline was intraperitoneally administered with the same schedule as a control group. The diameter of the tumors was measured with a vernier caliper at certain time intervals until one day after the final doze administration. The tumor volume was calculated by the following formula: ½×(a shorter diameter)2×(a longer diameter).
In the present test, test compounds exhibited superior anti-tumor activities as compared with the control group.
The pharmaceutical composition of the present invention can be prepared in a conventional manner by mixing one or more compounds of the invention shown by general formula (I) with a carrier for medical use, a filler and other additives usually used in pharmaceutical preparation. The pharmaceutical composition of the invention may be administered either orally in the form of tablets, pills, capsules, granules, powders, liquid, etc., or parenterally such as by intravenous or intramuscular injection, in the form of suppositories, or through pernasal, permucosal or subcutaneous route.
For oral administration of the composition in the present invention, a solid composition in the form of, e.g., tablets, powders or granules is available. In such a solid composition, one or more active or effective ingredients are blended with at least one inert diluent such as lactose, mannitol, glucose, hydroxypropyl cellulose, microcrystalline cellulose, starch, polyvinyl pyrrolidone or magnesium aluminate metasilicate. The composition may further contain additives other than the inert diluent by the usual procedures. Examples of such additives include a lubricant such as magnesium stearate, a disintegrating agent such as calcium cellulose glycolate, a solubilization assisting agent such as glutamic acid or aspartic acid. Tablets or pills may be coated, if necessary, with films of sugar or a gastric or enteric substance such as sucrose, gelatin, hydroxypropyl cellulose, hydroxypropylmethyl cellulose phthalate, etc.
A liquid composition for oral administration includes pharmaceutically acceptable emulsions, solutions, suspensions, syrups, elixirs, etc. and contains an inert diluent conventionally employed, e.g., purified water or ethanol. In addition to the inert diluent above, the liquid composition may further contain an auxiliary agent such as a moistening agent or a suspending agent, a sweetener, a flavor and/or a preservative.
A composition for parenteral administration contains a sterile aqueous or non-aqueous solution, a suspension and an emulsion. Examples of the aqueous solution and suspension include distilled water for injection use and physiological saline. Typical examples of the non-aqueous solution and suspension are propylene glycol, polyethylene glycol, vegetable oil such as olive oil, an alcohol such as ethanol, polysorbate 80, and the like. These compositions may further contain a preservative, a moistening agent, an emulsifier, a dispersing agent, a stabilizer and a solubilization assisting agent. These compositions are sterilized, e.g., by filtering them through a bacteria retention filter, incorporating a bactericide or through irradiation. Alternatively, they may be prepared into a sterile solid composition, which is dissolved in sterile water or a sterile solvent for injection prior to use.
In the case of oral administration, suitable daily does is usually about 0.0001 to 50 mg/kg body weight, preferably about 0.001 to 10 mg/kg, more preferably about 0.01 to 1 mg/kg, and the daily does is administered once a day or divided into 2 to 4 doses per day. In the case of intravenous injection, suitable daily dose is usually about 0.0001 to 1 mg/kg body weight, preferably about 0.0001 to 0.1 mg/kg. And the daily does is administered once a day or divided into a plurality of doses per day. The dose may be appropriately determined for each case, depending on conditions, age, sex, etc.
The compounds of the present invention can be utilized alone, or in conjunction with other treatments (e.g., radiotherapy and surgery). Moreover, they can be utilized in conjunction with other antitumor agents, such as alkylation agents (cisplatin, carboplatin, etc.), antimetabolites (methotrexate, 5-FU, etc.), antitumor antibiotics (adriamymycin, bleomycin, etc.), antitumor vegetable alkaloids (taxol, etoposide, etc.), antitumor hormones (dexamethasone, tamoxifen, etc.), antitumor immunological agents (interferon α, β, γ, etc.), and so forth.
EXAMPLES
The present invention will be described in more detail by referring to the following EXAMPLES but is not deemed to be limited thereto.
The following Tables 1˜3 and 13˜16 show starting compounds which were used in EXAMPLES, and Tables 4˜11 and 17˜24 show structural formulas as well as physicochemical properties of the compounds of the present invention. Moreover, the compounds of the present invention with structural formulas shown in Tables 12 and 25˜26 can be easily produced in the same manner as in the EXAMPLES mentioned hereinafter or in accordance with the Production Methods mentioned hereinabove, or by applying thereto some modifications which are obvious to those skilled in the art.
In the tables, abbreviations are used to mean the following.
Rco:starting compounds number
Rex:Production method of Reference Example compounds (a following number represents a Reference Example number described hereinafter, indicating that the compound was prepared using the method described in the Reference Example or the one similar thereto.)
Co:compounds number of the present invention
Str:structural formula
Sal:salt
Syn:production method (a following number represents a number of an EXAMPLE described hereinbelow, indicating that the associated compound is produced using the method described in the EXAMPLE or a similar method.)
Dat:physicochemical properties wherein:
F: FAB-MS (M+H)+
FN:FAB-MS (M−H)−
E:EI-MS
M:melting point [° C.]
(dec.):Decomposition
N1:characteristic peaks δ ppm of NMR (DMSO-d6, TMS internal standard)
Ac:acetyl
Bn:benzyl
Ph:phenyl
Ts:4-toluenesulfonyl
Ms:methanesulfonyl
Me:methyl
Et:ethyl
Where two or more positions to permit substitution are present, the position substituted is indicated as a prefix (e.g., 6-MeO-7-HO represents 6-methoxy-7-hydroxy.).
TABLE 1
Rco
Rex
Str
DAT
1
1
F: 249
2
1
F: 277
3
2
F: 206
4
3
F: 327
5
3
F: 353
6
3
F: 341
7
3
F: 398
8
3
F: 381
9
3
F: 310
10
3
F: 341
11
4
F: 356
12
4
F: 375
13
5
F: 299
14
5
FN: 311
15
5
FN: 281
16
5
FN: 311
TABLE 2
Rco
Rex
Str
DAT
17
5
FN: 326
18
5
F: 282
19
5
F: 311
20
5
FN: 326
21
6
F: 298
22
6
FN: 279
23
6
FN: 325
24
6
F: 282
25
6
F: 310
26
6
F: 312
27
6
F: 312
28
6
FN: 325
29
7
F: 188
30
8
E: 279
31
8
E: 308
32
8
F: 264
TABLE 3
Rco
Rex
Str
DAT
33
8
F: 309
34
8
F: 292
35
8
F: 263
36
8
F: 294
37
8
E: 293
38
9
F: 322
39
9
E: 321
40
3
F: 341
41
4
FN: 344
42
5
FN: 311
43
5
FN: 316
44
6
F: 312
45
6
F: 317
46
8
F: 293
47
8
FN: 297
48
9
F: 322
49
19
F: 207
TABLE 4
Co
Syn
Str
DAT
1
1
F: 378
2
1
E: 377
3
2
F: 593
4
2
N1: 4.15(4H, t;J=4.4 Hz),8.52(1H, s),10.41(1H, s).
5
2
F: 593
6
2
F: 497
7
12
M: 206–208
Z
—
: SPECSand BioSPECSB.V. ( :AE-848/3855062)
TABLE 5
(Ia)
Co
Syn
X
Y
NR2R3
R4
Sal
DAT
8
3
N
O
—
M: 229–230; N1: 3.85 (4H, t, J=4.8Hz), 7.22–7.25(1H, m), 10.47(1H, s)
9
3
N
O
—
M: 291–293; N1: 2.74(3H, s), 7.39(1H, t, J=7.8 Hz), 8.23(1H, s)
10
4
N
O
—
M: 266–268; N1: 3.87(4H, t, J=4.8Hz), 7.51(1H, t, J=7.8 Hz), 10.78(1H, s)
11
4
N
O
—
F: 487
12
5
N
O
HCl
N1: 3.87(4H, t, J=4.8 Hz), 8.87(1H,s), 11.06(1H, s)
13
5
N
O
HCl
N1: 3.88(4H, t, J=4.8 Hz), 9.34(1H,s), 10.88(1H, s)
14
5
N
O
2HCl
N1: 3.86(4H, t, J=4.8 Hz), 8.14(1H,s), 10.85(1H, s)
15
5
N
O
2HCl
N1: 3.87(4H, t, J=4.4 Hz), 7.56(1H,t, J=7.8 Hz), 11.26(1H, s)
16
5
N
O
HCl
N1: 3.87(4H, t, J=4.4 Hz), 8.84(1H,s), 10.72(1H, s)
17
5
N
O
2HCl
M: 203–207; N1: 3.86(4H, t, J=4.9Hz), 7.52(1H, t, J=7.8 Hz), 10.71(1H, s)
18
5
N
O
—
N1: 1.35–1.48(1H, m), 3.85(4H, t,J=4.4 Hz), 10.19(1H, s)
TABLE 6
Co
Syn
X
Y
NR2R3
R4
Sal
DAT
19
5
N
O
2HCl
M: 203–206
20
5
N
O
—
F: 487
21
5
N
O
2HCl
M: 173–175; N1: 7.53(1H, t, J=7.8Hz), 8.24–8.29(2H, m), 11.01(1H, s)
22
5
N
O
HCl
N1: 3.87(4H, m), 8.30(2H, s), 10.06(1H, s)
23
5
N
O
2HCl
N1: 4.39(2H, s), 7.47(1H, t, J=7.7Hz), 10.87(1H, s)
24
6
N
O
—
N1: 2.09(3H, s), 3.87(4H, t, J=4.9Hz), 10.11(1H, s)
25
7
N
O
2HCl
M: 213–217
26
7
N
O
3HCl
M: 203–205
27
7
N
O
2HCl
N1: 1.50–1.90(5H, m), 3.86(4H, t,J=4.9 Hz), 11.00(1H, s)
28
7
N
O
2HCl
N1: 1.83–2.06(4H, m), 3.86(4H, t,J=4.4 Hz), 10.37(1H, s)
29
8
N
O
—
N1: 2.84(4H, s), 3.85(4H, t, J=4.9Hz), 7.38–7.40(1H, m)
30
9
N
O
HCl
M: 293–295
31
10
N
O
2HCl
M: 237–240
TABLE 7
Co
Syn
X
Y
NR2R3
R4
Sal
DAT
32
10
N
O
2HCl
N1: 3.87(4H, t, J=4.4 Hz), 7.51–7.55(2H, m), 10.68(1H, s)
33
10
N
O
HCl
M: 262–266; N1: 3.86(4H, t, J=4.4Hz), 8.37(2H, d, J=8.8 Hz), 8.70(1H, dd,J=1.5, 4.9 Hz)
34
11
N
O
NH2
H
—
N1: 7.59(1H, dd, J=4.8, 7.8Hz),
7.72(2H, br s), 8.45(1H, s)
35
12
N
O
—
M: 237–239
36
12
N
NH
—
M: 248–250
37
12
N
S
—
M: 201–202
38
12
N
O
H
—
M: 182–183
39
12
CH
S
H
HCl
M: 202–205
40
13
N
O
2HCl
M: 237–240; N1: 3.09–3.14(2H, m),7.50(1H, t, J=7.8 Hz), 10.59(1H, s)
41
13
N
O
3HCl
M: 178(dec.); N1: 3.13–3.16(2H,m), 7.54(1H, t, J=7.8 Hz), 11.04(1H, s)
42
13
N
O
2HCl
M: 282–285; N1: 3.09(3H, s), 7.51(1H, t, J=7.8 Hz), 10.79(1H, s)
43
13
N
O
2HCl
M: 257–261; N1: 4.81(2H, s), 7.33–7.53(6H, m), 10.76(1H, s)
44
14
N
O
2HCl
M: 234–237; N1: 3.32(6H, s), 7.53(1H, t, J=7.8 Hz), 10.75(1H, s)
45
15
N
O
HCl
M: 244–245; N1: 3.87(4H, t, J=4.9Hz), 7.49–7.67(5H, m), 10.47(1H, s)
TABLE 8
Co
Syn
X
Y
NR2R3
R4
Sal
DAT
46
15
N
O
2HCl
N1: 3.87(4H, t, J=4.9 Hz), 7.53(1H, t,J=7.8 Hz), 10.73(1H, s)
47
15
N
O
2HCl
N1: 3.87(4H, t, J=4.9 Hz), 7.55(1H, t,J=7.8 Hz), 10.86(1H, s)
48
15
N
O
HCl
M: 195–197; N1: 3.62(3H, s),(1H, t, J=7.8 Hz), 10.25(1H, s)
49
16
N
O
HCl
M: 164–167; N1: 2.55–2.64(4H, m),7.43(1H, t, J=7.8 Hz), 10.17(1H, s)
50
17
N
O
HCl
M: 270–272; N1: 4.15(4H, t, J=4.8Hz), 7.32(1H, t, J=7.8 Hz), 8.70(1H,dd, J=1.9, 4.9 Hz)
51
17
N
O
HCl
M: 182–184; N1: 3.87(3H, s), 7.45(1H, t, J=7.8 Hz), 8.69(1H, dd, J=1.5,4.9 Hz)
52
17
N
O
HCl
M: 306(dec.); N1: 3.85(4H, t, J=4.9Hz), 6.91(2H, d, J=8.8 Hz), 8.32(2H,d, J=8.8 Hz)
53
17
N
O
HCl
N1: 2.18(3H, s), 8.11(1H, s), 12.50(1H, s)
54
18
N
O
2HCl
M: 186–190; N1: 4.15(4H, t, J=4.4Hz), 4.59(2H, t, J=4.8 Hz), 7.49(1H, t,J=7.8 Hz)
55
18
N
O
2HCl
M: 283–286; N1: 1.35–1.47(1H, m),4.56(2H, t, 1=4.9 Hz), 7.49(1H, t,J=7.8 Hz)
56
18
N
O
2HCl
M: 233–235; N1: 1.30(6H, t, J=7.3Hz), 4.53(2H, t, J=4.9 Hz), 7.49(1H, t,J=7.8 Hz)
57
18
N
O
2HCl
M: 275–277; N1: 3.19–3.28(2H, m),7.15(2H, d, J=8.8 Hz), 8.46(2H, d,J=8.8 Hz)
TABLE 9
(Ia)
Co
Syn
R
Sal
DAT
58
47
HCl
N1: 3.29–3.34(2H, m), 7.46(1H, t, J=7.8Hz), 8.68–8.71(2H, m)
59
48
2HCl
M: 206–210; N1: 4.17(4H, t, J=4.97.73(1H, d, J=7.8 Hz), 7.81(1H, s)
60
49
—NHCOCF3
—
N1: 3.86(4H, t, J=4.9 Hz), 7.64(1H, dd,J=5.0, 7.7Hz), 11.43(1H, s)
61
18
—
N1: 7.56–7.67(3H, m), 8.48–8.53(2H, m),8.62–8.65(1H, m)
62
50
—
N1: 3.83–3.88(6H, m), 7.45(1H, t, J=7.9Hz), 8.67–8.72(2H, m)
63
50
—
N1: 1.85–2.07(4H, m), 4.10–4.14(6H, m),8.65–8.70(2H, m)
64
51
3HCl
N1: 2.85(3H, brs), 4.16(4H, t, J=4.4 Hz),7.66(1H, dd, J=4.9, 7.8 Hz)
65
18
—
N1: 4.19(2H, t, J=4.9 Hz), 7.62(1H, t, J=7.8Hz), 7.96(1H, s)
66
51
2HCl
M: 196–198; N1: 3.33(6H, s), 7.50(1H, t,J=7.8 Hz), 8.07–8.11(2H, m)
67
51
HCl
N1: 4.12(4H, t, J=4.3 Hz), 8.09(1H, d,J=8.0 Hz), 8.65–8.69(2H, m)
68
51
2HCl
M: 243–248; N1: 3.01–3.10(2H, m), 3.15–3.20(2H, m), 7.65(1H, dd, J=4.9, 7.8 Hz)
69
52
3HCl
M: 250–253; N1: 4.56(2H, t, J=4.9 Hz),7.49(1H, t, J=7.8 Hz), 8.10(1H, d, J=7.8Hz)
70
53
2HCl
N1: 3.05(3H, s), 4.19(4H, t, J=4.4 Hz),7.91(1H, br s)
TABLE 10
Co
Syn
R
Sal
DAT
71
15
2HCl
M: 244–245; N1: 1.84(3H, s), 4.11–4.18(6H, m), 7.64–7.72(3H, m)
72
51
2HCl
M: 196–201; N1: 4.15(4H, t, J=4.4 Hz),7.74(1H, s), 9.34(1H, s)
73
15
HCl
N1: 5.79(1H, d, J=10.8 Hz), 6.30(1H, d,J=17.1 Hz), 10.40(1H, s)
74
51
2HCl
N1: 4.15(4H, t, J=4.9 Hz), 4.53–4.56(2H,m), 8.05(1H, s)
75
18
HCl
N1: 1.24(3H, t, J=6.8 Hz), 4.22(2H, q,J=6.8 Hz), 4.89(2H, s)
76
16
HCl
M: 264–267; N1: 4.79(2H, s), 7.45(1H, t,J=7.8 Hz), 7.64(1H, dd, J=4.9, 7.8 Hz)
77
54
HCl
M: 243–244; N1: 3.78(2H, t, J=4.9 Hz),4.14(4H, t, J=4.9 Hz), 7.98–7.99(1H, m)
TABLE 11
(Ia)
Co
Syn
R4
Sal
DAT
78
47
2HCl
Na: 3.05–3.14 (2H, m), 3.26–3.31 (2H, m), 7.10(2H, d, J=8.8 Hz)
79
55
—
N1: 4.15(4H, t, J=4.9 Hz), 7.66 (1H, dd, J=4.9,7.8 Hz), 8.15 (1H, dd, J=1.5, 7.8 Hz)
80
12
—
N1: 4.16 (4H, t, J=4.9 Hz), 7.67 (1H, dd, J=4.9,7.3 Hz), 8.34–8.35 (2H, m)
81
56
—
M: 343–347; N1: 7.64 (1H, dd, J=4.9, 7.3 Hz),8.66–8.69 (2H, m), 11.72 (1H, br)
TABLE 12
(Ia)
Co
X
Y
R4
A1
N
S
A2
N
S
A3
N
S
A4
N
S
A5
N
S
A6
N
S
A7
N
S
A8
N
O
A9
N
O
A10
N
S
A11
N
S
A12
N
S
A13
N
O
A14
N
S
A15
N
O
A16
N
S
A17
N
O
A18
N
S
A19
N
O
A20
N
S
A21
N
O
A22
N
S
A23
N
O
A24
N
S
A25
N
O
A26
N
S
TABLE 13
Rco
Rex
Str
DAT
50
10
F: 208
51
10
F: 240
52
11
E: 324
53
11
F: 321
54
11
F: 283
55
11
F: 253
56
11
F: 241
57
11
F: 321
58
11
F: 280
59
11
FN: 282
60
11
FN: 282
61
11
F: 297
62
11
FN: 227
63
11
F: 297
64
11
F: 329
65
11
F: 290
66
11
F: 329
67
12
F: 259
68
12
F: 313
69
12
F: 279
TABLE 14
Rco
Rex
Str
DAT
70
12
F: 271
71
12
F: 254
72
13
F: 255
73
13
F: 293
74
14
F: 407
75
14
F: 317
76
14
F: 393
77
15
F: 299
78
15
F: 281
79
15
F: 397
80
15
F: 287
81
15
F: 321
82
15
F: 282
83
16
FN: 324
84
16
F: 271
85
16
F: 371
86
16
F: 339
87
16
FN: 369
88
16
F: 332
89
16
F: 339
TABLE 15
Rco
Rex
Str
DAT
90
16
F: 326
91
16
F: 400
92
17
F: 339
93
17
F: 341
94
17
F: 369
95
18
FN: 359
96
3
F: 286
97
3
F: 287
98
3
F: 287
99
3
F: 292
100
3
F: 302
101
3
F: 422
102
8
F: 254
103
8
F: 254
104
8
F: 382
105
8
F: 269
106
9
F: 281
107
9
F: 283
108
9
F: 282
109
9
F: 282
TABLE 16
Rco
Rex
Str
DAT
110
9
F: 299
111
9
F: 410
112
9
F: 286
113
9
F: 282
114
9
F: 282
115
11
F: 286
116
20
F: 254
117
21
F: 272
118
21
F: 272
119
22
F: 329
120
22
F: 400
121
23
F: 311
122
24
F: 259
123
24
F: 255
124
24
F: 269
TABLE 17
(Ib)
Co
Syn
X
R1
NR2R3
R4
Sal
DAT
82
19
N
H
—
M: 111–112; N2: 3.86 (4H, m), 3.95(4H, m), 8.56 (2H, m)
83
19
N
6-F
—
M: 157–159; N2: 3.80 (4H, t, J=4.7 Hz), 3.94 (4H, t, J=4.7 Hz), 8.50–8.54 (2H, m)
84
19
N
6-MeO-7-MeO
—
M: 182–186
85
19
N
6-NO2
—
M: 238–240; N1: 3.83 (4H, t, J=4.9 Hz), 7.99 (1H, d, J=8.8 Hz), 8.82 (1H, d, J=2.4 Hz)
86
19
N
6-AcHN
—
M: 121–124
87
19
N
6-MeO
—
M: 145–146; N1: 3.79 (4H, m), 3.87 (4H, m), 3.94 (3H, s)
88
19
N
6-AcHN
—
N1: 8.52 (1H, s), 8.86 (1H, m), 10.36 (1H, s)
89
19
N
6-MeO
—
M: 161–163
89
19
N
6-MeO
—
M: 218–220
90
19
N
6-MsHN
—
N1: 3.10 (3H, s), 3.80–3.90 (8H, m), 10.18 (1H, br)
91
19
CH
6-MeO
—
N1: 3.92 (8H, m), 7.96 (1H, d, J=8.8 Hz), 8.22 (2H, m)
92
20
N
6-MeO-7-OH
—
M: 202–204; N1: 3.70 (4H, t, J=4.4 Hz), 3.98(3H, s), 7.07(1H, s)
93
20
N
6-HO
—
M: 203–205; N1: 3.79 (4H, m), 3.87 (4H, m), 10.22 (1H, s)
94
20
N
6-HO
—
M: 222–225 (dec.); N1: 3.72 (4H,m), 4.82 (1H, brs), 8.01 (1H, brs)
TABLE 18
Co
Syn
X
R1
NR2R3
R4
Sal
DAT
96
20
N
6-HO
—
M: 296–305 (dec.)
97
21
N
6-H2N
—
M: 184–186
98
22
N
6-OHCNH—
—
M: 218–222; N1: 3.79 (4H, t, J=4.2 Hz), 8.41 (1H, d, J=1.5 Hz),10.59 (1H, s)
99
23
N
6-HO
—
M: 243–249; N1: 4.07 (2H, s), 7.67(1H, d, J=8.8 Hz), 10.00 (1H, s)
100
23
N
6-HO
—
M: 258–262 (dec.)
101
23
N
6-HO
H
—
M: 259–260; N1: 3.57 (4H, t, J=4.7 Hz), 8.55 (1H, s) 10.12 (1H, s)
102
23
N
6-HO
—
M: 249–250; N1: 3.82 (1H, m),7.77 (1H, d, J=9.6 Hz), 10.08 (1H,s)
103
23
N
6-HO
—
M: 221–225; N1: 1.20 (H, d, J=6.4 Hz), 7.80 (1H, d, J=8.8 Hz),10.12 (1H, s)
104
23
N
6-HO
—
M: 139–141
105
24
N
6-AcMeN—
—
M: 204–206
106
25
N
6-TsHN—
—
M: 199–200; N1: 2.32 (3H, s), 3.62(4H, t, J=4.4 Hz), 10.65 (1H, s)
107
26
N
6-Me2N—
—
M: 124–125
108
27
N
6-HO
0.5HCl
M: 268–271
109
28
N
6-HO
Me
—
M: 281–284
TABLE 19
Co
Ex.
X
R1
NR2R3
R4
Sal
DAT
110
29
N
7-HO
—
M: 245–246
111
29
N
6-HO
—
M: 266–269; N1: 3.74 (4H, t, J=4.4 Hz), 8.66 (2H, d, J=9.1 Hz), 10.29 (1H, s)
112
29
N
6-HO
—
M: 226–227
113
29
N
6-HO
—
N1: 1.94 (2H, m), 2.69 (1H, m), 7.65 (1H, d, J=8.8 Hz)
114
29
N
6-HO
—
M: 275–277; N1: 7.83 (1H, d, J=8.8 Hz), 9.58( (1H, d, J=1.5 Hz),10.09 (1H, brs)
115
29
N
6-HO
—
M: 280 (dec.)
116
29
N
6-HO
—
M: 239–241
117
29
N
6-HO
—
M: 184–186; N1: 3.92 (3H, s), 9.03(1H, br), 10.19 (1H, s)
118
29
N
6-HO
—
NM: 280–284; N1: 3.68 (4H, t, J=4.5 Hz), 9.49 (1H, s), 10.12 (1H, s)
119
29
N
6-HO
—
M: 306–311 (dec.); N1: 7.75 (1H,d, J=8.8 Hz), 9.29 (2H, s), 10.10(1H, s)
120
29
N
6-HO
—
M: 254–255
121
29
N
6-HO
—
M: 288–290
122
29
N
6-HO
—
M: 188–190; N1: 3.80 (3H, s), 13.83 (3H, s), 10.06 (1H, s)
123
29
N
6-HO
—
M: 224–227
TABLE 20
Co
Syn
X
R1
NR2R3
R4
Sal
DAT
124
29
N
6-HO
—
M: 285–288; N1: 3.88 (3H, s), 9.37(1H, s), 10.03 (1H, s)
125
29
N
6-HO
—
M: 310–313; N1: 3.71 (4H, m), 3.87(4H, m), 10.23 (1H, s)
126
29
N
6-HO
—
M: 178–180
127
30
N
6-HO
—
M: 260–263; N1: 3.64 (4H, m), 3.86(4H, m), 1H, 10.12 (1H, s)
128
30
N
6-HO
—
M: 280–282
129
31
N
6-HO
—
M: 285 (dec.); N1: 3.62 (4H, t, J=4.7 Hz), 5.51 (2H, br), 9.95 (1H, s)
130
32
N
6-HO
—
M: 305 (dec.)
131
32
N
6-HO
—
M: 306–309; N1: 3.71 (4H, t, J=4.9Hz), 10.18 (1H, s), 13.08 (1H, s)
132
33
N
6-HO
—
M: 204–206; N1: 4.78 (2H, d, J=5.9Hz), 5.28 (1H, t, J = 5.9 Hz), 10.13(1H, s)
133
34
N
6-HO
—
M: 274–277; N1: 5.17 (2H, brs), 6.66(1H, m), 10.08 (1H, s)
134
34
N
6-MsHN
—
N1: 3.07 (3H, s), 3.72–3.77 (4H, m),10.07 (1H, br s)
135
35
N
6-HO
—
M: 266–267
136
36
N
6-HO
—
M: 261–264; N1: 8.10 (1H, br), 8.91(1H, t, J=1.4), 10.17 (1H, s)
137
36
N
6-HO
—
M: 306–309
138
37
N
6-HO
—
M: 245–248
TABLE 21
Co
Syn
X
R1
NR2R3
R4
Sal
DAT
139
38
N
6-HO
—
M: 296–299; N1: 2.08 (3H, s),10.08 (1H, s), 10.11 (1H, s)
140
39
N
6-HO
—
M: 152–157
141
40
N
6-HO
—
M: 225–228; N1: 8.21 (1H, m),10.16 (1H, brs), 10.44 (1H, brs)
142
40
N
6-HO
—
M: 206–207; N1: 8.33 (1H, s),10.12 (1H, s), 10.18 (1H, s)
143
40
N
6-HO
—
M: 172–174
144
40
N
6-AcHN
—
M: 145–150; N1: 8.48 (1H, d, J =2.0 Hz), 10.33 (1H, brs), 10.44(1H, brs)
145
40
N
6-MsHN
—
M: 234–236; N1: 3.08 (3H, s),3.74–3.79 (4H, m), 10.30 (2H, br)
146
40
N
6-AcHN
—
M: 145–148; N1: 2.12 (3H, s),10.34 (1H, s), 10.56 (1H, s)
147
40
N
6-AcHN
—
M: 290 (d); N1: 2.12 (3H, s),10.32 (1H, s), 10.83 (1H, s)
148
41
N
6-HO
—
M: 167–169
149
42
N
6-HO
—
M: 144–147
150
43
N
6-HO
—
M: 175–178; N1: 3.71–3.73 (4H,m), 10.17 (1H, s), 10.68 (1H, s)
151
43
N
6-HO
—
M: 239–243; N1: 2.33–2.42 (1H,m), 3.66–3.69 (4H, m), 9.96 (1H, s)
TABLE 22
Co
Ex.
X
R1
NR2R3
R4
Sal
DAT
152
43
N
6-HO
—
M: 214–216; N1: 3.68–3.70(6H, m), 10.14 (1H, s), 10.34(1H, s)
153
43
N
6-HO
—
M: 246–247
154
43
N
6-HO
—
M: 251–252
155
43
N
6-HO
—
N1: 3.86 (3H, s), 10.14 (1H,s), 10.26 (1H, s)
156
43
N
6-HO
—
M: 182–183
157
43
N
6-HO
—
N1: 3.72–3.74 (4H, m), 9.70–9.99 (1H, br), 10.45 (1H, brs)
158
43
N
6-HO
—
M: 232–233
159
44
N
—
M: 182–183
160
45
N
—
M: 224–227
161
45
N
—
M: 199–202; N1: 8.76 (1H,d, J = 2.4 Hz), 8.49 (2H, m),10.74 (1H, brs)
162
46
CH
6-HO
—
M: 250–253
TABLE 23
(Ib)
Co
Syn
B
R
Sal
DAT
163
17
OMe
2HCl
N1: 3.84 (4H, t, J=4.9 Hz), 3.89(3H, s), 9.55 (1H, s)
164
17
OH
HCl
M: 261–266; N1: 3.84 (4H, t, J=4.9Hz), 7.91 (1H, s), 9.53 (1H, s)
165
18
3HCl
M: 167–170; N1: 3.61 (2H, br s),4.61 (2H, t, J=4.9 Hz); 9.54 (1H, s)
166
47
3HCl
N1: 3.26–3.31 (2H, m), 7.54 (1H, t,J=7.8 Hz), 9.53 (1H, s)
167
17
NO2
HCl
M: 272–273; N1: 4.20 (4H, t, J=4.9Hz), 7.97 (1H, d, J=6.4 Hz), 9.52(1H, s)
168
34
NH2
2HCl
M: 195–200; N1: 4.25 (4H, t, J=4.9Hz), 7.64 (1H, t, J=7.8 Hz), 9.55(1H, s)
169
57
NHAc
HCl
N1: 2.10 (3H, s), 9.52 (1H, s), 10.32(1H, s)
170
3
NHSO2Ph
HCl
N1: 8.75 (1H, d, J=6.4 Hz), 9.49(1H, s), 10.62 (1H, s)
171
2
2HCl
M: 200–203; N1: 8.89–8.90 (1H,m), 9.53 (1H, s), 10.84 (1H, s)
172
17
OH
HCl
M: 233–238; N1: 4.73 (4H, br),7.43 (1H, t, J=7.8 Hz), 10.02 (1H,br)
173
18
2HCl
M: 201–206; N1: 3.19–3.29 (2H,m), 7.55 (1H, t, J=7.8 Hz), 8.50(1H, br)
174
17
OH
HCl
M: 269–274; N1: 7.39 (1H, t, J=7.8Hz), 8.06 (1H, d, J=5.9 Hz), 9.44(1H, s)
175
18
2HCl
N1: 3.20–3.29 (2H, m), 4.60 (2H, t,J=4.9 Hz), 9.50 (1H, s)
TABLE 24
Co
Syn
B
R
Sal
DAT
176
17
OMe
HCl
M: 159–162; N1: 3.89 (3H, s), 7.55(1H, t, J=7.8 Hz), 8.02 (1H, d,J=7.8 Hz)
177
17
OH
HCl
M: 274–279; N1: 4.22 (4H, t, J=4.9Hz), 7.41 (1H, t, J=7.8 Hz), 10.05(1H, br)
178
18
2HCl
N1: 4.59 (2H, t, J=4.9 Hz), 7.57(1H, t, J=7.8 Hz), 7.68 (1H, dd,J=4.9, 8.3 Hz)
179
17
OH
HCl
M: 235–237; N1: 4.19 (4H, br s),7.43 (1H, t, J=7.8 Hz), 8.27–8.341H, m)
180
18
2HCl
N1: 4.19 (4H, br s), 8.28 (1H, br s),8.57 (1H, br)
181
17
NO2
HCl
N1: 3.78–3.79 (4H, m), 7.83–7.89(3H, m), 8.88 (1H, d, J=7.8 Hz)
182
34
NH2
2HCl
N1: 4.11 (4H, br s), 7.47 (1H, d,J=7.8 Hz), 7.62 (1H, t, J=7.8 Hz)
183
57
NHAc
HCl
N1: 2.10 (3H, s), 7.52 (1H, t, J=7.8Hz), 10.25 (1H, s)
184
3
NHSO2Ph
HCl
N1: 4.10 (4H, br s), 8.17–8.28 (3H,m), 10.83 (1H, s)
185
2
2HCl
M: 196–201 N1: 4.15 (4H, br s),8.85–8.87 (2H, m), 10.97 (1H, s)
186
17
OH
HCl
M: 252–258 N1: 3.95 (3H, s), 4.23(4H, br s), 10.04 (1H, br)
187
18
2HCl
N1: 4.67 (2H, t, J=4.9 Hz), 8.12(1H, d, J=7.8 Hz), 8.49 (1H, br)
188
17
2HCl
M: 266–267; N1: 4.60–4.63 (2H,m), 8.16 (1H, s), 10.68 (1H, br)
189
17
OH
HCl
M: 214–220; N1: 4.26 (4H, br),7.45 (1H, t, J=7.8 Hz), 7.82 (1H, s)
190
17
OH
HCl
M: 207–210; N1: 7.40 (1H, t, J=7.8Hz), 7.86 (1H, d, J=7.8 Hz), 8.51(1H, d, J=5.3 Hz)
191
17
OH
HCl
M: 262–268 (d); N1: 4.58 (4H, br),8.87 (1H, d, J=2.0 Hz), 9.09 (1H, d,J=2.0 Hz)
192
58
OH
2HCl
M: 270–273; N1: 4.66–4.68 (2H,m), 7.55 (1H, br s), 10.05 (1H, br)
TABLE 25
(Ib)
Co
R4
B1
B2
B3
B4
B5
B6
TABLE 26
(Ib)
Co
B
R
B7
CO2NHMe
B8
OH
B9
OH
B10
CH2OH
B11
CH2OH
B12
CH2OH
B13
CONH2
B14
CONH2
B15
CONH2
B16
OH
B17
OH
B18
OH
B19
CH2OH
B20
CH2OH
B21
CH2OH
B22
CONH2
B23
CONH2
B24
CONH2
B25
OH
B26
OH
B27
OH
B28
CH2OH
B29
CH2OH
B30
CH2OH
B31
CONH2
B32
CONH2
B33
CONH2
Production methods of the starting compounds shown in the foregoing tables are explained in the following Reference Examples.
Reference Example 1
A suspension of 2-chloro-3-cyanopyridine, ethyl glycolate and sodium carbonate in 3-methyl-1-butanol was refluxed for 3 days. The solvent was evaporated and water was added to the residue to crystallize to give Reference Example Compound (hereinbelow, abbreviated as Rco) 1.
Reference Example 2
A suspension of 2-chloro-3-cyanopyridine, glycine ethyl ester hydrochloride and sodium carbonate in 3-methyl-1-butanol was refluxed for 6 days. The solvent was evaporated. After the obtained residue was diluted with ethyl acetate and water, insoluble solids were filtered off. The separated organic layer was concentrated under reduced pressure. The residue was dissolved in ethanol, sodium ethoxide was added, and the mixture was stirred at room temperature for 15 minutes. The reaction solution was concentrated, and ethyl acetate saturated aqueous sodium hydrogencarbonate were added. The separated organic layer was concentrated under reduced pressure and the residue was purified with silica gel column chromatography to give Rco 3.
Reference Example 3
Dimethylaminopyridine and benzoyl chloride were added to a solution of 3-aminothieno[2,3-b]pyridine-2-carboxylic acid ethyl ester in pyridine. The reaction mixture was stirred at room temperature for 18 hours, and concentrated. 1M Hydrochloric acid was added, and the mixture was extracted with chloroform. The organic layer was concentrated under reduced pressure. The obtained residue was purified with silica gel column chromatography to give Rco 4.
Reference Example 4
Phosphorous oxychloride was added to a solution of Rco 49 and 4-nitrobenzoic acid and the reaction mixture was stirred at −15 C for 15 minutes. Ice and water was added to this reaction mixture. The precipitated crystals were collected to give Rco 11.
Reference Example 5
1M Sodium hydroxide was added to a solution of Rco 4 in methanol. The reaction mixture was stirred at room temperature for 2 hours, and 1M hydrochloric acid was added. The precipitated crystals were collected to give Rco 13.
Reference Example 6
Thionyl chloride was added to Rco 13.The mixture was refluxed for 2 hours, cooled to room temperature and then concentrated. DMF and aqueous ammonia were added to the obtained residue and the reaction mixture was stirred at room temperature for 2 hours. Water was added to the resulting mixture and extracted with chloroform. The organic layer was concentrated under reduced pressure to give Rco 21.
Reference Example 7
Formamide was added to Rco 1 and the mixture was stirred at 200 C for 2 hours. After the mixture was cooled to room temperature, the precipitated crystals were collected to give Rco 29.
Reference Example 8
2M potassium hydroxide was added to a solution of Rco 21 in methanol and the mixture was stirred at 100 C for 1 hour. After being cooled to room temperature, hydrochloric acid was added. The precipitated crystals were collected to give Rco 30.
Reference Example 9
Acetic acid and 48% hydrobromic acid were added to Rco 36 and the mixture was refluxed for 17 hours. After the reaction solution was concentrated under reduced pressure, diethyl ether was added and the reaction mixture was concentrated under reduced pressure. Sodium acetate and acetic anhydride were added to the obtained residue, and the mixture was stirred at 110 C for 2 hours. Ice and then water were added to this reaction mixture under ice cooling. The precipitated crystals were collected to give Rco 38.
Reference Example 10
Ethanol was added to a solution of 3-cyanobenzoic acid methyl ester in chloroform and gaseous hydrogen chloride was passed into the mixture at 0 C for 15 minutes. Further, the solution was sealed and the solution was stirred at 0 C for 17 hours. The reaction mixture was concentrated, ether was added, and the precipitated crystals were collected to give Rco 50.
Reference Example 11
2-Propanol was added to a mixture of 5-acetoamidoanthranilic acid, 3-nitrobenzimidic acid ethyl ester hydrochloride and sodium methoxide, and the mixture was refluxed for 3 days. The reaction solution was allowed to cool to room temperature. The obtained solid was collected to give Rco 52.
Reference Example 12
A solution of cyclohexanecarbonyl chloride in benzene was added in dropwise to a solution of 2-amino-5-methoxybenzamide and dimethylaminopyridine in pyridine at room temperature, and the mixture was stirred for 2 hours. The reaction mixture was concentrated, and the residue was dissolved with ethyl acetate. After the organic layer was washed with 1M hydrochloric acid and saturated aqueous sodium hydrogencarbonate, it was concentrated and the obtained residue was dissolved with methanol. 2M Sodium hydroxide was added. After the reaction solution was refluxed for 2 hours, it was neutralized with 12M hydrochloric acid. The solvent was evaporated and the crystals were filtered to give Rco 67.
Reference Example 13
THF and DMF were added to a mixture of 2-amino-5-methoxybenzamide, EDCI hydrochloride, HOBt and pyrazinecarboxylic acid, and the mixture was stirred at room temperature for 3 days. The solvents were evaporated, and the crystals were collected and dissolved in methanol and 2M sodium hydroxide. The reaction solution was refluxed for 3 hours and neutralized with 12M hydrochloric acid. The obtained crystals were collected to give Rco 72.
Reference Example 14
Dimethylaminopyridine, TEA, ethanol and tosyl chloride were added to a suspension of Rco 55 in chloroform, and the reaction mixture was stirred at room temperature for 12 hours. DMSO was added to it to give a solution. Then, the reaction solution was stirred for 12 hours. Again, dimethylaminopyridine, TEA and tosyl chloride were added and the reaction solution was stirred for 18 hours. The reaction solution was concentrated, and the residue was diluted with ethyl acetate and purified according to a conventional method to give Rco 74.
Reference Example 15
48% Hydrobromic acid was added to a solution of Rco 70 in acetic acid, and the mixture was refluxed for 2 days. After the reaction solution was allowed to cool, it was concentrated, and sodium acetate and acetic anhydride were added to the obtained residue. The reaction solution was refluxed for 3 hours. After the reaction solution was allowed to cool, it was concentrated and ether was added to the solution, followed by collection of crystals to give Rco 77.
Reference Example 16
Sodium acetate and acetic anhydride were added to Rco 60, and the mixture was refluxed for 40 minutes. After the reaction mixture was allowed to cool, the precipitated crystals were collected to give Rco 83.
Reference Example 17
Sodium methoxide was added to a solution of 3-hydroxybenzimidate ethyl ester hydrochloride and 5-hydroxyanthranilic acid in methanol, and the mixture was refluxed for 30 minutes. After the reaction solution was cooled to room temperature, the precipitate was collected. Sodium acetate and acetate anhydride were added to the obtained precipitate and the mixture was refluxed for 30 minutes. After the reaction solution was allowed to cool, the precipitated crystals were collected to give Rco 92.
Reference Example 18
Concentrated hydrochloric acid was added to Rco 52, and the mixture was stirred at 80 C. The reaction mixture was allowed to cool, filtered, and concentrated under reduced pressure to give 6-amino-2-(3-nitrophenyl)-3H-quinazoline-4-one hydrochloride. After the obtained compound was neutralized, pyridine, dimethylaminopyridine and methanesulfonyl chloride were added. The reaction solution was stirred at room temperature for 20 hours, the solvent was evaporated and the crystals were collected to give Rco 95.
Reference Example 19
1,8-diazabicyclo[5,4,0]-7-undecene (DBU) was added to a solution of 2-chloro-3-cyanopyridine and ethyl glycolate in ethanol and the reaction mixture was refluxed for 21 hours. The resulting mixture was evaporated under reduced pressure, diluted with ethyl acetate and then, washed with water and brine. The organic layer was concentrated under reduced pressure and crystallized to give Rco 49.
Reference Example 20
Aqueous ammonia was added to a solution of Rco 96 in methanol, and the reaction solution was stirred at room temperature for 3 hours. Methanol in the reaction mixture was evaporated under reduced pressure and the crystals were collected to give Rco 116.
Reference Example 21
Aqueous ammonia was added to a solution of Rco 98 in methanol, and the reaction solution was stirred at room temperature overnight. Methanol in the reaction mixture was evaporated under reduced pressure and the crystals were collected to give Rco 117.
Reference Example 22
EDCI hydrochloride and HOBt were added to a solution of 3-acetoxybenzoic acid in DMF, and the reaction mixture was stirred at room temperature for 10 minutes. Then, 2-amino-5-methoxybenzamide was added, and the reaction mixture was stirred at room temperature for 1 hour. The solvent was evaporated under reduced pressure, and waster and THF were added. After the extraction with ethyl acetate, the organic layer was washed with brine, evaporated under reduced pressure, and crystallized to give Rco 119.
Reference Example 23
Acetic anhydride was added to Rco 105 and sodium acetate, and the reaction mixture was stirred at 110 C for 1 hour and 15 minutes. Under cooling, the precipitated crystals were collected to give Rco 121.
Reference Example 24
Aqueous ammonia was added to a solution of Rco 99 in dioxane, and the reaction mixture was stirred at room temperature for 13 days. The solvent was evaporated under reduced pressure and the crystals were collected to give a mixture of amido 3-(3-methoxybenzoylamino)thiophene-2-carboxylate and Rco 122. 2M aqueous sodium hydroxide was added to a solution of this mixture in 2-propanol and the reaction solution was refluxed for 21 hours. After being cooled, it was neutralized and the precipitated crystals were collected to give Rco 122.
Example 1
Phosphorous oxychloride was added to Rco 33 and the mixture was refluxed for 20 minutes. The reaction mixture was concentrated under reduced pressure, and was azeotropically concentrated with toluene. Morpholine was added to the obtained residue and the mixture was refluxed for 10 minutes. The reaction solution was concentrated under reduced pressure and the obtained crystals were washed with chloroform and water to give Compound (hereinafter, abbreviated as Co) 1.
Example 2
1-Benzyl piperidine-1,2-dicarboxylate, HOBt and EDCI hydrochloride were added to a solution of a free form of Co 31 in DMF, and the mixture was stirred at room temperature for 7 hour. The solvent was evaporated under reduced pressure. After being diluted with ethyl acetate, the organic layer was washed with saturated aqueous sodium hydrogencarbonate and brine. After the solution was dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure. The obtained residue was purified with silica gel column chromatography and crystallized to give Co 3.
Example 3
Benzenesulfonyl chloride (6.02 ml) was added to a solution of a free form of C 31 (13.6 g) in pyridine (480 ml) at 0 C and the mixture was stirred at room temperature for 1.5 hours. The solvent was evaporated under reduced pressure. The obtained residue was dissolved in ethyl acetate and the solution was washed with saturated aqueous sodium hydrogencarbonate and brine. After the solution was dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure. The obtained residue was purified with silica gel column chromatography (eluent; chloroform methanol=96:4) and recrystallized (ethanol) to give Co 8 (15.6 g).
Example 4
Picolinoyl chloride hydrochloride (9.40 g) and TEA (14.7 ml) were added to a solution of a free form of Co 31 (15.3 g) in THF (1L) at 0 C and the mixture was stirred at room temperature for 1.5 hours. Then, additional picolinoyl chloride hydrochloride (4.00 g) was added and the mixture was stirred at room temperature for 30 minutes. The reaction mixture was concentrated under reduced pressure. After dissolving in ethyl acetate, the solution was washed with saturated aqueous sodium hydrogencarbonate and brine. After being dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure and the obtained residue was purified with silica gel column chromatography (chloroform:methanol=96:4) and recrystallized (ethanol) to give Co 10 (15.4 g).
Example 5
3-Hydroxypicolinic acid (140 mg), EDCI hydrochloride (190 mg) and HOBt (135 mg) were added to a solution of a free form of Co 31 (300 mg) in DMF (20 ml) and the mixture was stirred at room temperature for 2 hours. The solvent was evaporated under reduced pressure. The obtained residue was dissolved with ethyl acetate and THF. The solution was washed with water and saturated aqueous sodium hydrogencarbonate. After the organic layer was dried over anhydrous magnesium sulfate, the solvent was evaporated under reduced pressure. The obtained residue was purified with silica gel column chromatography (chloroform:methanol=100:1). 4M Hydrogen chloride/ethyl acetate was added to a solution of the obtained residue in chloroform and methanol. The solvent was evaporated under reduced pressure, and the obtained solid was crystallized from methanol to give Co 12 (207 mg).
Example 6
A suspension of a free form of Co 31 (300 mg), succinic anhydride (519 mg) and acetic acid (1 ml) was stirred at 100 C for 30 minutes. The solvent was evaporated under reduced pressure. The obtained residue was purified with silica gel column chromatography (chloroform: methanol=98:2) and recrystallized (methanol) to give Co 24 (106 mg).
Example 7
To a solution of Co 18 (300 mg) in THF (12 ml) and ethanol (12 ml), 10% Pd—C (35 mg) was added, and the mixture was stirred at room-temperature under hydrogen atmosphere (1 atm) for 4 hours. The reaction solution was filtered by Celite. After the filtrate was concentrated under reduced pressure, the obtained residue was purified with silica gel column chromatography (chloroform:methanol 98:2-80:20) to give a free form of Co 25 (178 mg). 1M Hydrochloric acid (1.00 ml) was added to a solution of the obtained free form (153 mg) in THF (35 ml) and methanol (20 ml). The reaction solution was stirred at room temperature, then concentrated under reduced pressure, and recrystallized (methanol) to give dihydrochloride of Co 25 (119 mg).
Example 8
A suspension of a free form of Co 31 (300 mg), succinic anhydride (519 mg) and acetic acid (1 ml) was stirred at 100 C for 30 minutes. The solvent was evaporated under reduced pressure. The obtained residue was purified with silica gel column chromatography (chloroform:methanol=98:2) and recrystallized (methanol) to give Co 29 (57 mg).
Example 9
N-Carboethoxyphthalimide (262 mg) and TEA (0.166 ml) were added to a solution of a free form of Co 31 (346 mg) in THF (60 ml) and the mixture was stirred at 80 C for 1 day. After the reaction mixture was allowed to cool, water was added, and collected to give a free form of Co 30 (374 mg). 1M Hydrochloric acid (1.55 ml) was added to a solution of the obtained free form (371 mg) in THF (200 ml) and the solution was stirred at room temperature. The precipitated crystals were collected to give hydrochloride of Co 30 (287 mg).
Example 10
Co 1 (22.4 g) and ammonium chloride (1.59 g) were suspended in a mixture of ethanol (717 ml) and water (269 ml). Then, iron (33.2 g) was added and the solution was refluxed for 9 hours. While the reaction solution was still hot, hot THF was added, and the mixture was filtered with Celite. After most of the solvent was evaporated under reduced pressure, the precipitate was collected, and washed with diethyl ether to give a free form of Co 31 (18.9 g). 1M Hydrochloric acid (0.870 ml) was added to a solution of the obtained free form (101 mg) in THF (25 ml) and the solution was stirred at room temperature. The precipitated crystals were collected, and washed with methanol to give dihydrochloride of Co 31 (75 mg).
Example 11
A solution of Rco 1 (1.03 g) in formamide (12 ml) was refluxed for 2 hours. After the reaction mixture was cooled to room temperature, the obtained solids were collected to give Rco 29 (648 mg). Phosphorous oxychloride (7 ml) was added to a solution of obtained Rco 29 (630 mg) in pyridne (3.5 ml). The reaction mixture was refluxed for 2.5 hours. After being cooled to room temperature, the solvent was evaporated. Toluene (7 ml) was added to the obtained residue. After morpholine (7 ml) was slowly added in dropwise under ice cooling, the reaction mixture was refluxed for 3.5 hours. Further, THF (3 ml) and morpholine (20 ml) were added, and the reaction mixture was refluxed for 5 days, and then concentrated under reduced pressure. After the residue was diluted with ethyl acetate, the crystals were collected, washed with ethyl acetate, saturated aqueous sodium hydrogencarbonate and water, and recrystallized (ethanol) to give Co 34 (372 mg).
Example 12
Phosphorous oxychloride (5 ml) was added to Rco 32 (396 mg) and the mixture was refluxed for 50 minutes. The solvent was evaporated under reduced pressure. After morpholine (10 ml) was slowly added in dropwise into the obtained residue under ice cooling, the reaction mixture was refluxed for 30 minutes. The reaction mixture was concentrated under reduced pressure. The obtained crystals were washed with ethyl acetate and water, and recrystallized (ethanol) to give Co 35 (411 mg).
Example 13
Ethylenediamine (1.85 ml) was added to Co 11 (303 mg) and the mixture was stirred at 90 C for 2 hours. The solvent was evaporated under reduced pressure. The obtained residue was purified with silica gel column chromatography (chloroform:methanol=80:20) and crystallized (methanol) to give a free form of Co 40 (269 mg). The obtained free form (266 mg) was subjected to salt formation as described in EXAMPLE 7, and the obtained residue was recrystallized (methanol) to give dihydrochloride of Co 40 (153 mg).
Example 14
DMF (10 ml) was added to Co 11 (285 mg) and the solution was stirred at 110 C for 2 hours and at 80 C for 27 hours. The solvent was evaporated under reduced pressure. The obtained residue was dissolved in ethyl acetate and THF. The reaction solution was washed with aqueous sodium hydrogencarbonate and brine, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The obtained residue was purified with silica gel column chromatography (chloroform:methanol=98:2) to give a free form of Co 44 (167 mg). 1M Hydrochloric acid (0.283 ml) was added to a solution of the obtained free form (70 mg) in THF (5 ml) and methanol (10 ml), and the solution was stirred at room temperature. The precipitated crystals were collected to give dihydrochloride of Co 44 (72 mg).
Example 15
Benzoyl chloride (0.118 ml) was added to a solution of a free form of Co 31 (297 mg) in pyridine (20 ml) under ice cooling and the mixture was stirred at room temperature for 20 minutes. The solvent was evaporated under reduced pressure. The obtained residue was dissolved in ethyl acetate and THF. The reaction solution was washed with aqueous sodium hydrogencarbonate and brine. After being dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure. The obtained residue was recrystallized (methanol) to give a free form of Co 45 (301 mg). The obtained free form (287 mg) was subjected to salt formation as described in EXAMPLE 7 to give hydrochloride crystals of Co 45 (249 mg).
Example 16
1M Sodium hydroxide (11 ml) was added in two portions to a solution of a free form of Co 48 (171 mg) in methanol (60 ml) and THF (30 ml), and the mixture was stirred at room temperature for 2.5 hours. The reaction mixture was acidified with 1M hydrochloric acid, and the organic solvent was evaporated under reduced pressure. The precipitate was collected, and washed with water and diethyl ether. The obtained crystals were recrystallized (methanol/diethyl ether) to give Co 49 (116 mg).
Example 17
Phosphorous oxychloride (5 ml) was added to Rco 38 (452 mg), and the mixture was refluxed for 30 minutes. After the reaction solution was cooled to room temperature, it was concentrated under reduced pressure. After adding THF (5 ml) and then slowly adding morpholine (4 ml) in dropwise to the obtained residue under ice cooling, the ice bath was removed and the solution was refluxed for 1 hour. After the reaction mixture was cooled to room temperature the solvent was evaporated under reduced pressure. The obtained solid was washed with water and diethyl ether and purified with silica gel column chromatography (chloroform:methanol=98:2) to give a free form of Co 50 (411 mg). The obtained free form (183 mg) was subjected to salt formation as described in EXAMPLE 7 and recrystallized (methanol) to give hydrochloride of Co 50 (129 mg).
Example 18
4-(2-Chloroethyl)morpholine hydrochloride (1.53 g) and potassium carbonate (1.90 g) were added to a solution of a free form of Co 50 (956 mg) in DMF (35 ml), and the mixture was stirred at 70 C for 2.5 days. After the reaction solution was cooled to room temperature, the solvent was evaporated under reduced pressure. The obtained solid was washed with water and diethyl ether and purified with silica gel column chromatography (chloroform:methanol=98:2) and crystallized (methanol) to give a free form of Co 54 (1.16 g). 4M Hydrochloric acid/ethyl acetate (1.14 ml) was added to a solution of the obtained free form (1.05 g) in THF (140 ml) and methanol (70 ml), and the solution was stirred at room temperature. The solvents were evaporated under reduced pressure, and the material was recrystallized (methanol) to give dihydrochloride crystals of Co 54 (1.18 g).
Example 19
Phosphorous oxychloride (5 ml) was added to 2-phenyl-3H-quinazoline-4-one (450 mg), and the mixture was refluxed for 3 hours. The reaction mixture was concentrated. Saturated aqueous sodium hydrogencarbonate was added and extracted with ethyl acetate. After the organic layer was dried over anhydrous magnesium sulfate, the solvent was evaporated under reduced pressure. The obtained colorless crystals were dissolved in benzene (10 ml) and morpholine (325 mg) was added. The reaction mixture was refluxed overnight. Insoluble materials were filtered off and the filtrate was diluted with ethyl acetate. The organic layer was washed with saturated aqueous sodium hydrogencarbonate, dried over anhydrous magnesium sulfate, and then concentrated under reduced pressure. The obtained residue was purified with column chromatography (hexane:ethyl acetate=5:1) and recrystallized (hexane-benzene) to give Co 82 (136 mg).
Example 20
Sodium cyanide (132 mg) was added to a solution of Co 84 (190 mg) in DMSO (5 ml), and the mixture was stirred at 180 C for 2 hours. After the reaction mixture was allowed to cool, water was added to it and the mixture was extracted with ethyl acetate. After the organic layer was washed with brine and dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure. The obtained residue was recrystallized (hexane/ethyl acetate) to give Co 92 (40 mg).
Example 21
Iron (415 mg) was added to a solution of Co 85 (500 mg) in acetic acid (12 ml), and the mixture was stirred at 105 C for 1 hour. After the reaction solution was allowed to cool, chloroform and 1M sodium hydroxide were added. The solution was filtered with Celite and the filtrate was extracted with chloroform. The organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. 1M Hydrochloric acid (10 ml) was added to the obtained residue and the mixture was stirred at 85 C for 90 minutes. After the mixture was allowed to cool, 1M sodium hydroxide was added and extracted with chloroform, and the organic layer was washed with brine. After the solution was dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure. The obtained residue was purified with silica gel column chromatography (eluent; chloroform:methanol=50:1), and recrystallized (chloroform/hexane) to give Co 97 (374 mg).
Example 22
Acetic anhydride (3 ml) was added to a solution of Co 97 (149 mg) in formic acid (3 ml), and the mixture was stirred at room temperature for 2 hours. Water was added and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated aqueous sodium hydrogencarbonate and brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified with silica gel column chromatography (eluent; chloroform:methanol=50:1), and recrystallized (chloroform/hexane) to give Co 98 (46 mg).
Example 23
Phosphorous oxychloride (3 ml) was added to Rco 74 (270 mg), and the mixture was refluxed for 0.5 hours. The reaction mixture was concentrated under reduced pressure, and morpholine (10 ml) was added. After the reaction mixture was refluxed for 1 hour, the reaction mixture was concentrated under reduced pressure and diluted with ethyl acetate. The organic layer was washed with saturated aqueous sodium hydrogencarbonate, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. After purifying by silica gel column chromatography (eluent; hexane:ethyl acetate=5:1), ethanol (2 ml) and 20% potassium hydroxide (100 mg) were added, and the mixture was stirred at room temperature for 40 minutes. The reaction mixture was extracted with ethyl acetate, and the organic layer was washed with saturated aqueous sodium hydrogencarbonate, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The obtained crystals were washed with a mixture of ethyl acetate and hexane, and recrystallized (ethyl acetate/hexane) to give Co 99 (30 mg).
Example 24
Sodium hydroxide (43 mg), potassium carbonate (37 mg) and tetra-n-butylammonium hydrogensulfate (2 mg) were added to a solution of Co 86 (95 mg) in toluene (15 ml), and the mixture was stirred at 35 C for 0.5 hour, followed by addition of dimethylsulfate (34 mg) and stirring at 35 C for 2 hours. The reaction solution was filtered and the filtrated was concentrated under reduced pressure. The residue was purified with silica gel column chromatography (eluent; chloroform:methanol=20:1), and recrystallized (chloroform/hexane) to give Co 105 (62 mg).
Example 25
p-Toluenesulfonyl chloride (124 mg) and pyridine (1 ml) were added to a solution of Co 97 (200 mg) in chloroform (6 ml), and the mixture was stirred at room temperature for 45 minutes. 1M Hydrochloric acid was added and extracted with ethyl acetate. The organic layer was washed with saturated aqueous sodium hydrogencarbonate and brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was recrystallized (chloroform/hexane) to give Co 106 (165 mg).
Example 26
35% Formalin (5 ml) and formic acid (5 ml) were added to Co 97 (250 mg), and the mixture was stirred at 100 C for 90 minutes. After the mixture was allowed to cool, 1M sodium hydroxide was added and the reaction mixture was extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified with silica gel column chromatography (eluent; hexane:ethyl acetate=4:1), and recrystallized (chloroform/hexane) to give Co 107 (133 mg).
Example 27
Phosphorous oxychloride (50 ml) was added to Rco 76 (6.2 g), and the mixture was refluxed for 1 hour. The reaction solution was concentrated under reduced pressure and the residue was dissolved in chloroform. The chloroform layer was washed twice with saturated aqueous sodium hydrogencarbonate, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to give 6.0 g of crystals. To 802 mg of the crystals, thiomorpholine (500 mg) and benzene (10 ml) were added. The reaction mixture was heated with stirring at 70 C for 1 hour, and then diluted with ethyl acetate. The organic layer was washed with saturated aqueous sodium hydrogencarbonate and brine, dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and purified with silica gel column chromatography (hexane:ethyl acetate=5:1) to give p-toluenesulfonic acid 4-thiomorpholino 2-phenylquinazoline-6-yl (828 mg). 802 mg of this compound was dissolved in methanol (10 ml) and THF (10 ml). 20% Potassium hydroxide (1.0 g) was added to the solution and the mixture was stirred at 70 C for 1 hour. Water and 1M hydrochloric acid was added to neutralize the solution. Then, the solution was extracted with ethyl acetate, and the organic layer was washed with brine. After the solution was dried over anhydrous magnesium sulfate, the solution was concentrated under reduced pressure and recrystallized (ethyl acetate-hexane) to give Co 108 (151 mg).
Example 28
Ammonium acetate (1.2 g) was added to acetic acid 2-methyl-4-oxo-4H-benzo[d][1,3]oxazine-6-yl ester (3 g), and the mixture was stirred at 150 C for 30 minutes. After cooling to 80 C, methanol was added to the mixture and the mixture was stirred at 80 C for 1 hour. After the mixture was allowed to cool, the precipitated crystals were collected and washed with methanol to give crystals (930 mg). To a solution of the obtained crystals (918 mg) in DMSO (10 ml)/chloroform (5 ml), toluenesulfonyl chloride (1 ml), TEA (1 ml) and a catalytic amount of dimethylaminopyridine were added. The mixture was stirred at room temperature for 8 hours, and then extracted with ethyl acetate. The organic layer was washed with saturated aqueous sodium hydrogencarbonate and brine, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. Phosphorous oxychloride (15 ml) was added to the obtained residue and the mixture was refluxed for 15 hours. After the reaction solution was allowed to cool, phosphorous oxychloride was evaporated under reduced pressure. The residue was extracted with chloroform and washed with saturated aqueous sodium hydrogencarbonate. The organic layer was dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified with silica gel column chromatography (eluent; chloroform:methanol=50:1) to give a liquid material (577 mg). To a solution of the obtained material (577 mg) in toluene (20 ml), morpholine (2 g) was added, and the mixture was refluxed for 16 hours. The reaction solution was allowed to cool, and concentrated under reduced pressure. To a solution of the obtained residue in ethanol (15 ml), 20% potassium hydroxide (1 ml) was added, and the mixture was stirred at room temperature for 1 hour. The solvent was evaporated under reduced pressure. The residue was purified with silica gel column chromatography (eluent; chloroform:methanol=20:1), and recrystallized (chloroform/methanol/hexane) to give Co 109 (207 mg).
Example 29
Phosphorous oxychloride (10 mg) was added to Rco 78 (590 mg), and the mixture was refluxed for 0.5 hours. The reaction mixture was concentrated, diluted with chloroform, and washed with saturated aqueous sodium hydrogencarbonate. The organic layer was dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. To the obtained colorless crystals, morpholine (10 ml) was added, and the mixture was refluxed for 12 hours. The reaction mixture was diluted with chloroform, washed with water and saturated aqueous sodium hydrogencarbonate, and dried over anhydrous magnesium sulfate. The obtained organic layer was concentrated under reduce pressure. The residue was crystallized from chloroform-methanol, and further recrystallized to give Co 110 (126 mg).
Example 30
Acetic acid (20 ml) and 48% hydrogen bromide (20 ml) were added to Rco 67 (957 mg), and the mixture was stirred at an oil bath temperature of 135 C for 13 hours. After the mixture was allowed to cool to room temperature, precipitate was collected as a mixture of a starting material and a desired compound. To the obtained solid, acetic anhydride (30 ml) and sodium acetate (112 mg) were added and the reaction mixture was refluxed for 30 minutes. The reaction solution was allowed to cool, precipitate was collected. To the obtained solids, phosphorous oxychloride (10 ml) was added, and the mixture was refluxed for 30 minutes and concentrated under reduced pressure. The obtained residue was dissolved in chloroform and the solution was washed with saturated aqueous sodium hydrogencarbonate. The organic layer was dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. To the obtained residue, morpholine (20 ml) was added and the mixture was refluxed for 15 hours. The reaction mixture was concentrated under reduced pressure and purified with silica gel column chromatography (eluent; chloroform:methanol=50:1). The obtained crystals were washed with a mixture of chloroform and ether to give Co 127 (193 mg).
Example 31
Iron (396 mg) was added to a solution of Co 111 (500 mg) in acetic acid (12 ml), and the mixture was stirred at 105 C for 45 minutes. The reaction solution was allowed to cool. Chloroform and 1M sodium hydroxide were added to it. The mixture was filtered and extracted with chloroform. The organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The obtained residue was purified with silica gel column chromatography (eluent; chloroform:methanol=10:1) and recrystallized (chloroform/methanol/hexane) to give Co 129 (120 mg).
Example 32
1M Sodium hydroxide (8 ml) was added to a solution of Co 116 (564 mg) in ethanol (8 ml) and THF (8 ml), and the reaction mixture was stirred at room temperature for 15 hours. 1M Hydrochloric acid (8 ml) was added and the solution was extracted with ether. The organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was recrystallized (methanol/ether/hexane) to give Co 130 (163 mg).
Example 33
Lithium aluminum hydride (67 mg) was added to a solution of Co 117 (325 mg) in THF (40 ml), and the reaction mixture was stirred at 0 C for 2 hours. Water (0.1 ml), 1M sodium hydroxide (0.1 ml), and then water (0.3 ml) were added and the mixture was stirred at room temperature for 30 minutes. The mixture was dried over anhydrous sodium sulfate, filtered through silica gel, and concentrated under reduced pressure. The residue was recrystallized (THF/hexane) to give Co 131 (158 mg).
Example 34
Co 112 (860 mg) was dissolved in a mixture of THF (30 ml), methanol (30 ml) and ethanol (30 ml). 10% Pd—C (130 mg) was added and the reaction mixture was stirred under hydrogen atmosphere (1 atm) at room temperature for 2 hours. Insoluble materials were removed by filtration and the filtrate was concentrated to give 780 mg of solid. Of the solid, 202 mg was recrystallized (ethanol/methanol) to give Co 133 (148 mg).
Example 35
Co 133 (202 mg) was dissolved in pyridine (10 ml). Methanesulfonyl chloride (96 mg) was added to the reaction solution. The reaction mixture was stirred for 15 hours, and concentrated under reduced pressure. The obtained residue was dissolved in ethyl acetate. The organic layer was washed with water, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The obtained crystals were purified with column chromatography (chloroform-methanol=100:1) and recrystallized (ethanol-ethyl acetate-hexane) to give Co 135.
Example 36
HOBt (75 mg) and EDCI hydrochloride (106 mg) were added to a solution of Co 131 (177 mg) in DMF (12 ml), and the reaction solution was stirred at 0 C for 30 minutes and then at room temperature for 30 minutes. Aqueous ammonia (2 ml) was added and the mixture was stirred at room temperature for 3 hours. The reaction mixture was extracted with chloroform, and the organic layer was washed with water, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified with silica gel column chromatography (eluent; chloroform:methanol=10:1) and recrystallized (chloroform/methanol/hexane) to give Co 136 (39 mg).
Example 37
Phosphorous oxychloride (15 ml) was added to Rco 87 (1.1 g), and the mixture was refluxed for 1 hour. The reaction mixture was allowed to cool, and concentrated under reduced pressure. The mixture was diluted with chloroform and the organic layer was washed with saturated aqueous sodium hydrogencarbonate, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. To a solution of the obtained residue in toluene (40 ml), morpholine (5 g) was added and the reaction solution was refluxed for 17 hours, allowed to cool, and concentrated under reduced pressure. The residue was purified with silica gel column chromatography (eluent; chloroform:methanol=30:1), and recrystallized (chloroform/ether/hexane) to give Co 138 (869 mg).
Example 38
Acetic anhydride (0.75 ml) and pyridine (1 ml) were added to a solution of Co 129 (457 mg) in DMF (10 ml), and the reaction mixture was stirred at room temperature for 1 hour. 1M Sodium hydroxide (15 ml) and water were added and the reaction mixture was extracted with chloroform. The organic layer was dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified with silica gel column chromatography (eluent; chloroform:methanol=10:1), recrystallized (chloroform/methanol/hexane), and washed with ether to give Co 139 (358 mg).
Example 39
Benzene (10 ml), benzaldehyde (249 mg) and THF (10 ml) were added to Co 133 (476 mg). The reaction mixture was azeotropically refluxed for 2 hours and concentrated under reduced pressure, and the obtained residue was dissolved in methanol (20 ml). Under ice cooling, sodium borohydride (50 mg) was added. The reaction mixture was stirred at room temperature for 1 hour, and concentrated under reduced pressure. The residue was dissolved in ethyl acetate, and the organic layer was washed with water and brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The residue was purified with silica gel column chromatography (chloroform:methanol=100:1), and recrystallized (ethyl acetate/hexane) to give Co 140 (259 mg).
Example 40
Co 133 (3.0 g) was dissolved in pyridine (50 ml). Benzenesulfonyl chloride (1.9 g) was added to the mixture at room temperature. After stirring the mixture for 2 hours, the solvent was evaporated under reduced pressure. Ethyl acetate and water were added to the obtained residue and then the organic layer was washed with brine three times. The obtained organic layer was dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The obtained solid was purified with silica gel column chromatography (chloroform:methanol=100:1) and recrystallized (ethanol) to give Co 141 (3.0 g).
Example 41
THF (15 ml) and phenyl isocyanate (207 mg) were added to Co 133 (540 mg). After the mixture was refluxed for 3 hours, phenyl isocyanate (500 mg) was again added. The mixture was then refluxed for 4 hours. 1M Sodium hydroxide (5 ml) was added. After the mixture was stirred for 15 minutes, 1M hydrochloric acid (5 ml) was added to neutralize it. The reaction mixture was extracted with chloroform, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The obtained residue was purified with silica gel column chromatography (chloroform: methanol=50:1) and recrystallized (2-propanol/diethyl ether/hexane) to give Co 148 (180 mg).
Example 42
THF (15 ml) and TEA (520 mg) were added to Co 133 (440 mg). Phenyl chloroformate (498 mg) was added in dropwise to the reaction solution at room temperature, and stirred at room temperature for 2 hours. After adding methanol, 1M sodium hydroxide (5 ml) was added under ice cooling and the reaction mixture was stirred for 20 minutes. 1M Hydrochloric acid (5 ml) was added to neutralize it. The reaction mixture was extracted with chloroform, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The obtained residue was purified with silica gel column chromatography (chloroform:methanol=50:1) and recrystallized (ethyl acetate) to give Co 149 (189 mg).
Example 43
Isonicotinoyl chloride hydrochloride (280 mg) and pyridine (0.127 ml) were added to a solution of Co 133 (253 mg) in THF (10 ml), and the reaction mixture was stirred at room temperature for 5.5 hours. Further, TEA (0.1 ml) was added and the mixture was stirred at room temperature for 2.5 hours. Then, 1M sodium hydroxide (0.786 ml) and methanol (4 ml) were added, and the reaction mixture was stirred at room temperature for 30 minutes to cleave the ester. After neutralization, most of the solvent was evaporated, the resulting precipitate was collected, washed with ethyl acetate and water, and recrystallized (ethanol) to give Co 150 (71 mg).
Example 44
Chlorooxoacetic acid ethyl ester (190 mg) and TEA (1 ml) were added to a solution of Co 97 (285 mg) in chloroform (12 ml), and the mixture was stirred at room temperature for 16 hours. The reaction mixture was extracted with chloroform, washed with water, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified with silica gel column chromatography (eluent; chloroform:methanol=50:1), and recrystallized (chloroform/methanol/hexane) to give Co 159 (103 mg).
Example 45
Benzoyl isothiocyanate (1.2 ml) was added to a solution of Co 97 (1.2 g), in chloroform (30 ml) under ice cooling, and the mixture was stirred at room temperature for 3 hours. The precipitated crystals were collected. To the obtained crystals, 40% methylamine/methanol was added and the reaction mixture was stirred at room temperature for 1 hour, concentrated under reduced pressure, and purified with silica gel column chromatography to give 1-(4-morpholino-2-phenylquinazoline-6-yl)thiourea (1.1 g). To 266 mg of this compound, ethanol (5 ml), methanol (3 ml) and 40% chloroacetaldehyde (300 mg) were added. The reaction mixture was stirred for 4 days, diluted with ethyl acetate, washed with saturated aqueous sodium hydrogencarbonate, and dried over anhydrous magnesium sulfate. The residue was purified with silica gel column chromatography (hexane:ethyl acetate=2:1), and recrystallized (ethanol/hexane) to give Co 160 (49 mg).
Example 46
Acetic acid (5 ml) and 48% hydrobromic acid (5 ml) were added to Co 91 (500 mg), and the reaction solution was refluxed for 13 hours, and then concentrated. The reaction mixture was neutralized with 1M sodium hydroxide and saturated aqueous sodium hydrogencarbonate, and extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The obtained solid was purified with silica gel column chromatography (hexane:ethyl acetate=1:1), and recrystallized (ethanol) to give Co 162 (112 mg).
Example 47
3-Morpholinopropanol (93 mg) and a free form of Co 50 (203 mg) were added to a solution of diethyl azodicarboxylate (0.101 ml) and triphenylphosphine (168 mg) in THF (20 ml) and the mixture solution was stirred at 60 C for 13 hours. Additional diethyl azodicarboxylate (0.1 ml), triphenylphosphine (170 mg) and 3-morpholinopropanol (93 mg) were added and the mixture was stirred at 60 C. This addition of the reagents was repeated again. After the reaction mixture was allowed to cool, water and ethyl acetate were added, and the reaction mixture was basified with saturated aqueous sodium hydrogencarbonate. After extraction with ethyl acetate, the solution was washed with brine. After the solution was dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure. The obtained residue was purified with silica gel column chromatography (chloroform:methanol=98:2), and recrystallized (methanol) to give a free form of Co 58 (174 mg). The obtained free form (71 mg) was subjected to salt formation as described in EXAMPLE 18, and recrystallized (methanol) to give hydrochloride of Co 58 (68 mg).
Example 48
Potassium carbonate (1.10 g) was added to a solution of Co 61 (1.48 g) in methanol (15 ml) and water (15 ml). After the mixture was stirred at 80 C, additional methanol (8 ml) and water (8 ml) were added and the mixture was stirred at 80 C for 12 hours. After the reaction mixture was allowed to cool, crystals were collected, purified with silica gel column chromatography (chloroform:methanol=98:2), and crystallized (methanol) to give a free form of Co 59 (1.13 g). The obtained free form (160 mg) was subjected to salt formation as described in EXAMPLE 18, and recrystallized (methanol) to give dihydrochloride of Co 59 (146 mg).
Example 49
Anhydrous trifluoroacetic acid (1.07 ml) and dimethylaminopyridine (78 mg) were added to a solution of a free form of Co 31 (2.21 g) in pyridine (70 ml) under ice cooling. After the mixture solution was stirred for 1 hour, more anhydrous trifluoroacetic acid (0.5 ml) and dimethylaminopyridine (30 mg) were added, and the mixture was stirred under ice cooling for 1 hour. The solvent was evaporated under reduced pressure, water and ethyl acetate were added, and the precipitated crystals were filtered and washed with ethyl acetate to give Co 60 (2.66 g).
Example 50
Water (0.645 ml), dibromoethane (1.11 ml), tetrabutylammonium hydrogensulfate (22 mg) and 2M aqueous sodium hydroxide (2.58 ml) were added to a free form of Co 50 (1.29 g), and the mixture was stirred 60 C for 6 hours. Chloroform was added to the mixture, unsoluble materials were filtered, and the filtrate was extracted with chloroform and then washed with brine. After the solution was dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure, and the obtained residue was purified with silica gel column chromatography (chloroform:methanol=98:2) to give Co 62 (376 mg).
Example 51
1-Methylpiperazine (139 mg) and potassium carbonate (256 mg) were added to a solution of Co 62 (211 mg) in DMF (5 ml) and the mixture solution was stirred at 60 C for 4 hours. The solvent was evaporated under reduced pressure, water and THE were added to the obtained residue, the mixture was extracted with ethyl acetate, and then the solvent was evaporated under reduced pressure. The obtained residue was purified with silica gel column chromatography (chloroform:methanol=97:3˜95:5) to give a fi-ee form of Co 64 (198 mg). The obtained free form (198 mg) was subjected to salt formation as described in EXAMPLE 18, and recrystallized (methanol) to give trihydrochloride of Co 64 (160 mg).
Example 52
tert-Butyl piperazine-1-carboxylate (285 mg) and potassium carbonate (282 mg) were added to a solution of Co 62 (232 mg) in DMF (5 ml) and the mixture solution was stirred at 60 C for 17 hours. The solvent was evaporated under reduced pressure, water was added to the obtained residue, and the resulting mixture was extracted with ethyl acetate and then washed with brine. After the solution was dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure and the obtained residue was purified with silica gel column chromatography (chloroform:methanol=99:1) to give solid (227 mg). 4M Hydrogen chloride/ethyl acetate (1 ml) were added to a solution of the obtained solid (212 mg) in dioxane (3 ml) and methanol (3 ml), and the mixture was stirred at room temperature for 4 hours. The resulting mixture was concentrated and the residue was recrystallized (methanol) to give Co 69 (144 mg).
Example 53
Paraformaldehyde (15 mg) and acetic acid (81 ml) were added to a solution of a free form of Co 59 (216 mg) in THF (3 ml). After the mixture was stirred at room temperature for 10 minutes, sodium triacetoxyborohydride (199 mg) was added and the mixture was stirred at room temperature for 21 hours. Then, liquid formaldehyde (0.44 ml), acetic acid (5.5 ml) and sodium triacetoxyborohydride (704 mg) were added in 3 divided portions, and the reaction solution was stirred at room temperature for 4 days. The reaction mixture was neutralized with 2M aqueous sodium hydroxide, and THF was added. After the reaction solution was extracted with ethyl acetate, it was washed with brine. After it was dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure, and the obtained residue was purified with silica gel column chromatography (chloroform:methanol=98:2) to give a free form of Co 70 (162 mg). The obtained free form (48 mg) was subjected to salt formation as described in EXAMPLE 18, and recrystallized (methanol) to give dihydrochloride of Co 70 (53 mg).
Example 54
After a free form of Co 50 (322 mg), 1,3-dioxolane-2-one (814 mg) and potassium carbonate (192 mg) were stirred at 100 C for 2 hours, more 1,3-dioxolane-2-one (680 mg) was added and the mixture was stirred at 100 C for 17 hours. Then, DMF (3 ml) was added and the mixture was stirred at 100 C for 2 hours, and further 1,3-dioxolane-2-one (670 mg) was added and the mixture was stirred at 100 C for 20 hours. After the reaction mixture was allowed to cool, the solvent was evaporated under reduced pressure, and water was added. Then, 1M aqueous hydrochloric acid was added until bubbles no longer appeared. The precipitated crystals were collected and recrystallized (methanol) to give Co 77 (164 mg).
Example 55
Phosphorus oxychloride (10 ml) was added to Rco 48 (1.07 g), and the mixture was refluxed for 2.5 hours. The solvent was evaporated, and the reaction mixture was azeotropically concentrated with toluene. THF (15 ml) was added to the obtained residue. After morpholine (10 ml) was slowly added in dropwise under ice cooling, the ice bath was removed and the reaction mixture was refluxed for 30 minutes. Ethyl acetate and THF were added to the reaction mixture and the mixture was washed with water and brine. After it was dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure and the obtained residue was purified with silica gel column chromatography (chloroform:methanol=98:2) to give bis(morpholinoamido) 2-(4-morpholinopyrido[3′,2′:4,5]furo[3,2-d]pyrimidine-2-yl)phenylphosphonate (594 mg). Formic acid (4 ml) was added to this compound (360 mg) and the mixture was stirred at 100 C for 3 days. The solvent was evaporated under reduced pressure, ethyl acetate and water were added, and the mixture was neutralized with saturated aqueous sodium hydrogencarbonate under ice cooling. The precipitated crystals were filtered to give crystals (162 mg). The obtained crystals (123mg) were recrystallized (methanol-THF) to give Co 79 (122 mg).
Example 56
Dioxane (3.9 ml) and 6M hydrochloric acid (5.5 ml) were added to Co 80 (220 mg), and the mixture was refluxed for 3 days. After the reaction mixture was allowed to cool, it was neutralized, extracted with a mixture solution of ethyl acetate and THF, and washed with brine. After it was dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure, and the obtained residue was purified with silica gel column chromatography (chloroform:methanol=96:4) to give crystals (83 mg). The obtained crystals (81 mg) were recrystallized (THF-methanol) to give Co 81 (53 mg).
Example 57
After a solution of a free form of Co 168 (151 mg) in pyridine (9 ml) was cooled in an ice bath, acetic anhydride (4.5 ml) was added, and the mixture was stirred under ice cooling. After the reaction completed, the reaction mixture was poured into water with ice, extracted with ethyl acetate, and washed with brine. After it was dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure to give a free form of Co 183 (158 mg). The obtained free form (156 mg) was subjected to salt formation as described in EXAMPLE 18, and the obtained crystals were recrystallized (methanol) to give hydrochloride of Co 169 (93 mg).
Example 58
2-Morpholinoethanol (806 mg) was added in dropwise to a solution of 60% sodium hydroxide (63 mg) in DMF (5 ml), and the mixture solution was stirred at room temperature for 15 minutes. Then, a free form of Co 179 (285 mg) was added and the mixture was stirred at 60 C for 23 hours. Then, a mixture, which was prepared by adding 2-morpholinoethanol (806 mg) in dropwise to 60% sodium hydroxide (63 mg) in DMF (1 ml) and stirring at room temperature for 15 minutes, was added in dropwise to the reaction mixture and the resulting mixture was stirred at 60 C. This addition of sodium 2-morpholinoethoxide was conducted 3 times. The solvent was evaporated under reduced pressure, water and THF were added to the obtained residue, and the mixture was extracted with ethyl acetate and then washed with brine. After it was dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure, and the obtained residue was purified with silica gel column chromatography (chloroform:methanol=95:5) to give a free form of Co 192 (529 mg). The obtained free form (404 mg) was subjected to salt formation as described in EXAMPLE 18, and the obtained crystals were recrystallized (methanol) to give dihydrochloride of Co 192 (320 mg).
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10918094
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astellas pharma inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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514/260.1
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Apr 1st, 2022 05:13PM
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Apr 1st, 2022 05:13PM
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Astellas Pharma
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tyo:4503
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Astellas Pharma
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Jun 20th, 2017 12:00AM
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Apr 21st, 2016 12:00AM
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https://www.uspto.gov?id=US09683051-20170620
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Anti-human tie-2 antibody
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Provided is an anti-human Tie2 antibody for preventing or treating diabetic macular edema, diabetic retinopathy, and critical limb ischemia by binding to a human Tie2 to activate the human Tie2. The present inventors have conducted investigations on an anti-human Tie2 antibody, and have thus provided an anti-human Tie2 antibody comprising four heavy chain variable regions and four light chain variable regions, in which the heavy chain variable region consists of the amino acid sequence of the amino acid numbers 1 to 122 of SEQ ID NO: 2, the light chain variable region consists of the amino acid sequence of the amino acid numbers 1 to 113 of SEQ ID NO: 4, the one heavy chain variable region and the one light chain variable region constitute one antigen-binding site, and the antibody comprises four antigen-binding sites.
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9683051
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1. An anti-human Tie2 antibody or an antigen-binding fragment thereof, comprising four heavy chain variable regions and four light chain variable regions, wherein
the heavy chain variable region comprises CDR1 consisting of the amino acid sequence of the amino acid numbers 31 to 35 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequence of the amino acid numbers 50 to 66 of SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence of the amino acid numbers 99 to 111 of SEQ ID NO: 2;
the light chain variable region comprises CDR1 consisting of the amino acid sequence of the amino acid numbers 24 to 39 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence of the amino acid numbers 55 to 61 of SEQ ID NO: 4, and CDR3 consisting of the amino acid sequence of the amino acid numbers 94 to 102 of SEQ ID NO: 4; and
the one heavy chain variable region and the one light chain variable region constitute one antigen-binding site, and the antibody or the antigen-binding fragment comprises four antigen-binding sites.
2. The anti-human Tie2 antibody or the antigen-binding fragment thereof according to claim 1, selected from the group consisting of:
(i) an anti-human Tie2 antibody or an antigen-binding fragment thereof, comprising four heavy chain variable regions and four light chain variable regions, in which
the heavy chain variable region consists of the amino acid sequence of the amino acid numbers 1 to 122 of SEQ ID NO: 2,
the light chain variable region consists of the amino acid sequence of the amino acid numbers 1 to 113 of SEQ ID NO: 4, and
the one heavy chain variable region and the one light chain variable region constitute one antigen-binding site, and the antibody or the antigen-binding fragment comprises four antigen-binding sites; and
(ii) an anti-human Tie2 antibody or an antigen-binding fragment thereof wherein the antibody or the antigen-binding fragment thereof has undergone pyroglutamylation at the N-terminal of the heavy chain variable region of the anti-human Tie2 antibody or the antigen-binding fragment thereof of (i).
3. A pharmaceutical composition comprising the anti-human Tie2 antibody or the antigen-binding fragment thereof according to claim 2 and a pharmaceutically acceptable excipient.
4. A method for treating diabetic macular edema, diabetic retinopathy, or critical limb ischemia, comprising administering a therapeutically effective amount of the anti-human Tie2 antibody or the antigen-binding fragment thereof according to claim 2.
5. The anti-human Tie2 antibody or the antigen-binding fragment thereof according to claim 2, comprising an anti-human Tie2 antibody or an antigen-binding fragment thereof, comprising four heavy chain variable regions and four light chain variable regions, in which
the heavy chain variable region consists of the amino acid sequence of the amino acid numbers 1 to 122 of SEQ ID NO: 2,
the light chain variable region consists of the amino acid sequence of the amino acid numbers 1 to 113 of SEQ ID NO: 4, and
the one heavy chain variable region and the one light chain variable region constitute one antigen-binding site, and the antibody or the antigen-binding fragment comprises four antigen-binding sites.
6. An anti-human Tie2 antibody or an antigen-binding fragment thereof, produced by a method comprising culturing host cell(s) selected from the group consisting of (a) to (c) below to express a tetravalent anti-human Tie2 antibody or an antigen-binding fragment thereof:
(a) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human Tie2 antibody or the antigen-binding fragment thereof according to claim 5 and a polynucleotide comprising a base sequence encoding the light chain variable region of the antibody or the antigen-binding fragment thereof according to claim 5;
(b) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human Tie2 antibody or the antigen-binding fragment thereof according to claim 5 and an expression vector comprising a polynucleotide comprising a base sequence encoding the light chain variable region of the antibody or the antigen-binding fragment thereof according to claim 5; and
(c) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human Tie2 antibody or the antigen-binding fragment thereof according to claim 5 and a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the light chain variable region of the antibody or the antigen-binding fragment thereof according to claim 5.
7. The anti-human Tie2 antibody according to claim 1, wherein the antibody comprises two heavy chains and four light chains;
each heavy chain comprises two structures consisting of a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of the amino acid numbers 31 to 35 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequence of the amino acid numbers 50 to 66 of SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence of the amino acid numbers 99 to 111 of SEQ ID NO: 2 and a CH1 region, a CH2 region, and a CH3 region, and the carboxy terminus (C terminus) of one of the structures is linked to the amino terminus (N terminus) of the other structure through a linker; and
each light chain comprises a light chain variable region comprising CDR1 consisting of the amino acid sequence of the amino acid numbers 24 to 39 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence of the amino acid numbers 55 to 61 of SEQ ID NO: 4, and CDR3 consisting of the amino acid sequence of the amino acid numbers 94 to 102 of SEQ ID NO: 4, and a light chain constant region.
8. The anti-human Tie2 antibody according to claim 7, selected from the group consisting of:
(i) an anti-human Tie2 antibody comprising two heavy chains and four light chains, in which
each heavy chain comprises two structures consisting of a heavy chain variable region consisting of the amino acid sequence of the amino acid numbers 1 to 122 of SEQ ID NO: 2 and a CH1 region, a CH2 region, and a CH3 region, and the C terminus of one of the structures is linked to the N terminus of the other structure through a linker; and
each light chain comprises a light chain variable region consisting of the amino acid sequence of the amino acid numbers 1 to 113 of SEQ ID NO: 4, and a light chain constant region; and
(ii) an anti-human Tie2 antibody wherein of the antibody has undergone pyroglutamylation at the N-terminal of the heavy chain variable region of the anti-human Tie2 antibody or the antigen-binding fragment thereof of (i).
9. The anti-human Tie2 antibody according to claim 8, selected from (i) or (ii) below:
(i) an anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence shown by SEQ ID NO: 2 and four light chains consisting of the amino acid sequence shown by SEQ ID NO: 4; or
(ii) an anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence in which glutamic acid of the amino acid number 1 of SEQ ID NO: 2 is modified to pyroglutamic acid and/or lysine of the amino acid number 679 of SEQ ID NO: 2 is deleted and four light chains consisting of the amino acid sequence shown by SEQ ID NO: 4.
10. The anti-human Tie2 antibody according to claim 9, comprising two heavy chains consisting of the amino acid sequence shown by SEQ ID NO: 2 and four light chains consisting of the amino acid sequence shown by SEQ ID NO: 4.
11. An anti-human Tie2 antibody produced by a method comprising culturing host cell(s) selected from the group consisting of (a) to (c) below to express an anti-human Tie2 antibody:
(a) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human Tie2 antibody according to claim 10 and a polynucleotide comprising a base sequence encoding the light chain of the antibody according to claim 10;
(b) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human Tie2 antibody according to claim 10 and an expression vector comprising a polynucleotide comprising a base sequence encoding the light chain of the antibody according to claim 10; and
(c) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human Tie2 antibody according to claim 10 and a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the light chain of the anti-human Tie2 antibody according to claim 10.
12. The anti-human Tie2 antibody according to claim 9, comprising an anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence in which glutamic acid of the amino acid number 1 of SEQ ID NO: 2 is modified to pyroglutamic acid and/or lysine of the amino acid number 679 of SEQ ID NO: 2 is deleted and four light chains consisting of the amino acid sequence shown by SEQ ID NO: 4.
13. The anti-human Tie2 antibody according to claim 9, comprising two heavy chains consisting of the amino acid sequence of the amino acid numbers 1 to 678 of SEQ ID NO: 2 and four light chains consisting of the amino acid sequence shown by SEQ ID NO: 4.
14. A pharmaceutical composition comprising,
an anti-human Tie2 antibody comprising two heavy chains and four light chains, each heavy chain comprising two structures consisting of a heavy chain variable region consisting of the amino acid sequence of the amino acid numbers 1 to 122 of SEQ ID NO: 2 and a CH1 region, a CH2 region, and a CH3 region, and the C terminus of one of the structures is linked to the N terminus of the other structure through a linker, and each light chain comprising a light chain variable region consisting of the amino acid sequence of the amino acid numbers 1 to 113 of SEQ ID NO: 4, and a light chain constant region; and/or
an anti-human Tie2 antibody wherein the antibody has undergone pyroglutamylation at the N-terminal of the heavy chain variable region of the anti-human Tie2 antibody which comprises two heavy chains and four light chains, each heavy chain comprising two structures consisting of a heavy chain variable region consisting of the amino acid sequence of the amino acid numbers 1 to 122 of SEQ ID NO: 2 and a CH1 region, a CH2 region, and a CH3 region, and the C terminus of one of the structures is linked to the N terminus of the other structure through a linker, and each light chain comprising a light chain variable region consisting of the amino acid sequence of the amino acid numbers 1 to 113 of SEQ ID NO: 4, and a light chain constant region; and
a pharmaceutically acceptable excipient.
15. A pharmaceutical composition comprising an anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence shown by SEQ ID NO: 2 and four light chains consisting of the amino acid sequence shown by SEQ ID NO: 4, and/or an anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence of the amino acid numbers 1 to 678 of SEQ ID NO: 2 and four light chains consisting of the amino acid sequence shown by SEQ ID NO: 4, and a pharmaceutically acceptable excipient.
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15
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CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a Continuation Application of PCT/JP2015/070089, filed Jul. 14, 2015, which claims priority from Japanese application JP 2014-145135, filed Jul. 15, 2014, the entire contents of which are incorporated herein by reference.
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-WEB and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 18, 2016, is named sequence.txt and is 89 KB.
TECHNICAL FIELD
The present invention relates to a novel anti-human Tie-2 antibody.
BACKGROUND ART
A tyrosine kinase with Ig and EGF homology domains 2 (Tie2) is a receptor type tyrosine kinase. Tie2 is mainly known to be expressed in vascular endothelial cells. As the ligand, Angiopoietin-1 (Ang-1) and Angiopoietin-2 (Ang-2), which are multimer type secreted glycoproteins, are known.
Ang-1 functions as an agonist for Tie2. It has been found that when Tie2 binds to Ang-1, it is autophosphorylated by forming a multimer and transmits a signal into a cell, thereby promoting an anti-apoptotic action of vascular endothelial cells, vascular stabilization via a permeation inhibitory action of blood vessels, maturation and remodeling (Cell, 1996, Vol. 87, pp. 1171-1180; Genes Dev., 1994, Vol. 8, pp. 1897-1909; Science, 1999, Vol. 286, pp. 2511-2514; and Nat. Struct. Biol., 2003, Vol. 10, pp. 38-44). Further, it has also been known that Ang-1 exerts vasodilating and blood flow-enhancing actions by the production of nitric oxide through Tie2 activation (Pharmacol. Res., 2014, Vol. 80, pp. 43-51). In addition, it is believed that Ang-1 contributes to the stabilization of blood vessels by inhibiting the internalization of vascular endothelial cadherin through Tie2 activation (Dev. Cell, 2008, Vol. 14, pp. 25-36). On the other hand, it is believed that Ang-2 is capable of activating Tie2 on vascular endothelial cells, but its activation is believed to be partial, as compared to Ang-1 (Mol. Cell Biol., 2009, Vol. 29, pp. 2011-2022). Ang-2 binds to the same site of Tie2 with substantially the same affinity as Ang-1, and as a result, it has been suggested that Ang-2 functions as an endogenous Tie2 antagonist from the viewpoint that the activation of Tie2 by Ang-1 is replaced by partial activation of Ang-2 (Science, 1997, Vol. 277, pp. 55-60).
An increase in the concentration of Ang-2 in the blood has been reported in a disease induced by vascular vulnerability which is considered to be one of the causes of the disease, such as diabetes, diabetic retinopathy, sepsis, and acute renal failure (Atherosclerosis, 2005, Vol. 180, pp. 113-118; Br. J. Ophthalmol., 2004, Vol. 88, pp. 1543-1546; Critical Care, 2009, Vol. 13, p. 207; and Intensive Care Med., 2010, Vol. 36, pp. 462-470).
Regarding relevance to diabetic retinopathy and diabetic macular edema, it has been reported that the concentration of Ang-2 in the blood plasma or the vitreous humor of patients has risen (Br. J. Ophthalmol., 2004, Vol. 88, pp. 1543-1546; and Br. J. Ophthalmol., 2005, Vol. 89, pp. 480-483). Further, in the retinal blood vessel of patients with diabetic retinopathy, the loss of pericytes which are the main Ang-1 producing cells (Cell, 1996, Vol. 87, pp. 1161-1169) has also been known to be one of the characteristic lesions (Retina, 2013, Fifth edition, pp. 925-939). Diabetic macular edema is known for involving the thickening of the macular area as one of the conditions thereof, but it has also been reported that in patients with an increase in the intraocular Ang-1 concentration due to vitreous removal surgery, the thickening of the macular area is decreased (Br. J. Ophthalmol., 2005, Vol. 89, pp. 480-483). Further, from the viewpoints that in retinal edema mouse models with the loss of pericytes in the retinal blood vessels, retinal edema and retinal bleeding are observed, and the pathology onset is inhibited by the intravitreal administration of Ang-1 (J. Clin. Invest., 2002, Vol. 110, pp. 1619-1628), and that in a test using a mouse model with diabetic retinopathy, vascular endothelial cell disorders in the retina are inhibited by the administration of an adenovirus containing a gene encoding Ang-1 (Am. J. Pathol., 2002, Vol. 160, pp. 1683-1693), it has been suggested that Ang-1 has an action of improving the conditions. Meanwhile, it has been reported that in genetically modified mice having Ang-2 specifically over-expressed in the retina, retinal cell damage is increased (Acta. Diabetol. 2010, Vol. 47, pp. 59-64).
It has been reported that with regard to critical limb ischemia, the amount of Ang-2 in the blood plasma increases in patients with peripheral arterial diseases, and the amount of Ang-2 expressed in the ischemic limb muscles or the skin tissues in patients with critical limb ischemia is high (J. Am. Coll. Cardial., 2008, Vol. 52, pp. 387-393; and Int. Angiol., 2011, Vol. 30, pp. 25-34). Moreover, in a test using a rat model with hindlimb ischemia, blood flow recovery and anti-apoptotic effect in the ischemic limb is promoted by the administration of a viral vector containing a gene encoding Ang-1 (Angiogenesis, 2009, Vol. 12, pp. 243-249). From the viewpoint that it has been reported that mature blood vessels covered by the smooth muscle cells are increased in the border zone of infarcted area by the administration of a virus containing a gene encoding Ang-1 in a coronary artery ligation model of a db/db mouse as an animal model with type 2 diabetes (Diabetes, 2008, Vol. 57, pp. 3335-3343), an effect of promoting the maturation of unstable neovascular vessels can be expected by the activation of Tie2 signals.
As an antibody showing an agonistic action on a human Tie2, a murine monoclonal antibody 15B8 (Patent Document 1) has been reported. It has been reported that 15B8 binds to the human Tie2 to induce an anti-apoptotic action in a human vascular endothelial cell HUVEC (Patent Document 1)
RELATED ART
Patent Document
[Patent Document 1] WO 2000/018804
DISCLOSURE OF INVENTION
Problems to be Solved by the Invention
An object of the present invention is to provide an anti-human Tie2 antibody for preventing or treating diabetic macular edema, diabetic retinopathy, or critical limb ischemia by binding to a human Tie2 to activate the human Tie2.
Means for Solving the Problems
The present inventors have repeatedly conducted substantial and inventive studies in preparation of an anti-human Tie2 antibody, and as a result, they have found that a tetravalent anti-human Tie2 antibody comprising a heavy chain variable region consisting of the amino acid sequence of the amino acid numbers 1 to 122 of SEQ ID NO: 2 and a light chain variable region consisting of the amino acid sequence of the amino acid numbers 1 to 113 of SEQ ID NO: 4 is prepared (Examples 1 to 8), and thus, the anti-human Tie2 antibody binds to the human Tie2 (Example 12), induces the anti-apoptotic action in a human Tie2-expressing BaF3 cell (Examples 9 and 11), and inhibits the vascular hyperpermeability in a rat model with vascular hyperpermeability (Examples 10 and 13). As a result, they have provided such an anti-human Tie2 antibody, thereby completing the present invention.
That is, the present invention may include the following invention as a material or a method which is medically or industrially applicable.
[1] An anti-human Tie2 antibody or an antigen-binding fragment thereof, comprising four heavy chain variable regions and four light chain variable regions, wherein
the heavy chain variable region comprises CDR1 consisting of the amino acid sequence of the amino acid numbers 31 to 35 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequence of the amino acid numbers 50 to 66 of SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence of the amino acid numbers 99 to 111 of SEQ ID NO: 2;
the light chain variable region comprises CDR1 consisting of the amino acid sequence of the amino acid numbers 24 to 39 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence of the amino acid numbers 55 to 61 of SEQ ID NO: 4, and CDR3 consisting of the amino acid sequence of the amino acid numbers 94 to 102 of SEQ ID NO: 4; and
the one heavy chain variable region and the one light chain variable region constitute one antigen-binding site, and the antibody or the antigen-binding fragment thereof comprises four antigen-binding sites.
[2] The anti-human Tie2 antibody or the antigen-binding fragment thereof of [1], selected from (1) or (2) below:
(1) an anti-human Tie2 antibody or an antigen-binding fragment thereof, comprising four heavy chain variable regions and four light chain variable regions, in which
the heavy chain variable region consists of the amino acid sequence of the amino acid numbers 1 to 122 of SEQ ID NO: 2,
the light chain variable region consists of the amino acid sequence of the amino acid numbers 1 to 113 of SEQ ID NO: 4, and
the one heavy chain variable region and the one light chain variable region constitute one antigen-binding site, and the antibody or the antigen-binding fragment thereof comprises four antigen-binding sites; and
(2) an anti-human Tie2 antibody or an antigen-binding fragment thereof which is an antibody or an antigen-binding fragment thereof derived from posttranslational modification of the anti-human Tie2 antibody or the antigen-binding fragment thereof of (1).
[3] The anti-human Tie2 antibody of [1], wherein
the antibody comprises two heavy chains and four light chains;
each heavy chain comprises two structures consisting of a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of the amino acid numbers 31 to 35 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequence of the amino acid numbers 50 to 66 of SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence of the amino acid numbers 99 to 111 of SEQ ID NO: 2 and a CH1 region, a CH2 region, and a CH3 region, and the carboxy terminus (C terminus) of one of the structures is linked to the amino terminus (N terminus) of the other structure through a linker; and
each light chain comprises a light chain variable region comprising CDR1 consisting of the amino acid sequence of the amino acid numbers 24 to 39 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence of the amino acid numbers 55 to 61 of SEQ ID NO: 4, and CDR3 consisting of the amino acid sequence of the amino acid numbers 94 to 102 of SEQ ID NO: 4, and a light chain constant region.
[4] The anti-human Tie2 antibody of [3], selected from (1) or (2) below:
(1) an anti-human Tie2 antibody comprising two heavy chains and four light chains, in which
each heavy chain comprises two structures consisting of a heavy chain variable region consisting of the amino acid sequence of the amino acid numbers 1 to 122 of SEQ ID NO: 2 and a CH1 region, a CH2 region, and a CH3 region, and the C terminus of one of the structures is linked to the N terminus of the other structure through a linker; and
each light chain comprises a light chain variable region consisting of the amino acid sequence of the amino acid numbers 1 to 113 of SEQ ID NO: 4, and a light chain constant region; and
(2) an anti-human Tie2 antibody, which is an antibody derived from posttranslational modification of the anti-human Tie2 antibody of (1).
[5] The anti-human Tie2 antibody of [4], wherein
the anti-human Tie2 antibody comprises two heavy chains and four light chains;
each heavy chain comprises two structures consisting of a heavy chain variable region consisting of the amino acid sequence of the amino acid numbers 1 to 122 of SEQ ID NO: 2 and a CH1 region, a CH2 region, and a CH3 region, and the C terminus of one of the structures is linked to the N terminus of the other structure through a linker; and
each light chain comprises a light chain variable region consisting of the amino acid sequence of the amino acid numbers 1 to 113 of SEQ ID NO: 4, and a light chain constant region.
[6] An anti-human Tie2 antibody which is an antibody derived from posttranslational modification of the anti-human Tie2 antibody of [5].
[7] The anti-human Tie2 antibody of [6], wherein the posttranslational modification is pyroglutamylation at the N terminus of the heavy chain variable region and/or deletion of lysine at the C terminus of the heavy chain.
[8] The anti-human Tie2 antibody of any one of [3] to [7], comprising a heavy chain constant region which is a human Igγ1 constant region or a human Igγ4 constant region.
[9] The anti-human Tie2 antibody of [8], in which the human Igγ1 constant region is a human Igγ1 constant region having amino acid variations of L234A, L235A, and P331S, or a human Igγ1 constant region having amino acid variations of L234A, L235A, P331S, and I253A.
[10] The anti-human Tie2 antibody of [8], in which the human Igγ4 constant region is a human Igγ4 constant region having amino acid variations of S228P and L235E.
[11] The anti-human Tie2 antibody of any one of [3] to [7], comprising a light chain constant region which is a human ID(constant region.
[12] The anti-human Tie2 antibody of any one of [3] to [7], comprising a heavy chain constant region which is a human Igγ1 constant region or a human Igγ4 constant region and a light chain constant region which is a human Igκ constant region.
[13] The anti-human Tie2 antibody of [12], in which the human Igγ1 constant region is a human Igγ1 constant region having amino acid variations of L234A, L235A, and P331S, or a human Igγ1 constant region having amino acid variations of L234A, L235A, P331S, and I253A.
[14] The anti-human Tie2 antibody of [12], in which the human Igγ4 constant region is a human Igγ4 constant region having amino acid variations of S228P and L235E.
[15] The anti-human Tie2 antibody of any one of [3] to [7], in which the linker is a peptide linker comprising 5 to 70 amino acids.
[16] The anti-human Tie2 antibody of [15], in which the linker comprises the amino acid sequence of a hinge region or a portion thereof
[17] The anti-human Tie2 antibody of [16], in which the linker comprises the amino acid sequence shown by SEQ ID NO: 13.
[18] The anti-human Tie2 antibody of [4], comprising two heavy chains consisting of the amino acid sequence shown by SEQ ID NO: 2 and four light chains consisting of the amino acid sequence shown by SEQ ID NO: 4.
[19] The anti-human Tie2 antibody of [4], comprising two heavy chains consisting of the amino acid sequence shown by SEQ ID NO: 6 and four light chains consisting of the amino acid sequence shown by SEQ ID NO: 4.
[20] The anti-human Tie2 antibody of [4], comprising two heavy chains consisting of the amino acid sequence shown by SEQ ID NO: 10 and four light chains consisting of the amino acid sequence shown by SEQ ID NO: 4.
[21] An anti-human Tie2 antibody which is an antibody derived from posttranslational modification of the anti-human Tie2 antibody of any one of [18] to [20]
[22] The anti-human Tie2 antibody of [21], wherein the posttranslational modification is pyroglutamylation at the N terminus of the heavy chain variable region and/or deletion of lysine at the C terminus of the heavy chain.
[23] The anti-human Tie2 antibody of [21], comprising two heavy chains consisting of the amino acid sequence of the amino acid numbers 1 to 678 of SEQ ID NO: 2 and four light chains consisting of the amino acid sequence shown by SEQ ID NO: 4.
[24] A tetravalent anti-human Tie2 antibody or an antigen-binding fragment thereof, binding to the same human Tie2 epitope as the anti-human Tie2 antibody of [18] or [23].
[25] The tetravalent anti-human Tie2 antibody or the antigen-binding fragment thereof of [24], wherein the human Tie2 epitope is the human Tie2 epitope containing the amino acid of the amino acid numbers 192, 195 and 197 of Accession No. NP_000450.2.
[26] A polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human Tie2 antibody or the antigen-binding fragment thereof of [2].
[27] A polynucleotide comprising a base sequence encoding the light chain variable region of the anti-human Tie2 antibody or the antigen-binding fragment thereof of [2].
[28] A polynucleotide comprising a base sequence encoding the heavy chain of the anti-human Tie2 antibody of any one of [18] to [20].
[29] A polynucleotide comprising a base sequence encoding the light chain of the anti-human Tie2 antibody of any one of [18] to [20].
[30] An expression vector comprising the polynucleotide of [26] and/or [27].
[31] An expression vector comprising the polynucleotide of [28] and/or [29].
[32] A host cell transformed with the expression vector of [30], which is selected from the group consisting of (a) to (d) below:
(a) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human Tie2 antibody or the antigen-binding fragment thereof of [2], and a polynucleotide comprising a base sequence encoding the light chain variable region of the antibody or an antigen-binding fragment thereof;
(b) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human Tie2 antibody or the antigen-biding fragment thereof of [2] and an expression vector comprising a polynucleotide comprising a base sequence encoding the light chain variable region of the antibody or an antigen-binding fragment thereof;
(c) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human Tie2 antibody or the antigen-binding fragment thereof of [2]; and
(d) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the light chain variable region of the anti-human Tie2 antibody or the antigen-binding fragment thereof of [2].
[33] A host cell transformed with the expression vector of [31], selected from the group consisting of (a) to (d) below:
(a) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human Tie2 antibody of any one of [18] to [20] and a polynucleotide comprising a base sequence encoding the light chain of the antibody;
(b) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human Tie2 antibody of any one of [18] to [20] and an expression vector comprising a polynucleotide comprising a base sequence encoding the light chain of the antibody;
(c) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human Tie2 antibody of any one of [18] to [20]; and
(d) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the light chain of the anti-human Tie2 antibody of any one of [18] to [20].
[34] A method for producing an anti-human Tie2 antibody or an antigen-binding fragment thereof, comprising culturing host cell(s) selected from the group consisting of (a) to (c) below to express a tetravalent anti-human Tie2 antibody or an antigen-binding fragment thereof:
(a) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human Tie2 antibody or the antigen-binding fragment thereof of [2] and a polynucleotide comprising a base sequence encoding the light chain variable region of the antibody or the antigen-binding fragment thereof;
(b) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human Tie2 antibody or the antigen-binding fragment thereof of [2] and an expression vector comprising a polynucleotide comprising a base sequence encoding the light chain variable region of the antibody or the antigen-binding fragment thereof, and
(c) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human Tie2 antibody or the antigen-binding fragment thereof of [2] and a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the light chain variable region of the antibody or the antigen-binding fragment thereof
[35] A method for producing an anti-human Tie2 antibody, comprising culturing host cell(s) selected from the group consisting of (a) to (c) below to express an anti-human Tie2 antibody:
(a) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human Tie2 antibody of any one of [18] to [20] and a polynucleotide comprising a base sequence encoding the light chain of the antibody;
(b) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human Tie2 antibody of any one of [18] to [20] and an expression vector comprising a polynucleotide comprising a base sequence encoding the light chain of the antibody; and
(c) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human Tie2 antibody of any one of [18] to [20] and a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the light chain of the anti-human Tie2 antibody.
[36] An anti-human Tie2 antibody or an antigen-binding fragment thereof, produced by the method of [34].
[37] An anti-human Tie2 antibody produced by the method of [35].
[38] A pharmaceutical composition comprising the anti-human Tie2 antibody or the antigen-binding fragment thereof of any one of [1] to [23], [36], and [37], and a pharmaceutically acceptable excipient.
[39] A pharmaceutical composition comprising the anti-human Tie2 antibody of [5], the anti-human Tie2 antibody of [6], and a pharmaceutically acceptable excipient.
[40] A pharmaceutical composition comprising the anti-human Tie2 antibody of [18], the anti-human Tie2 antibody of [23], and a pharmaceutically acceptable excipient.
[41] The pharmaceutical composition of any one of [38] to [40], which is a pharmaceutical composition for preventing or treating diabetic macular edema, diabetic retinopathy, or critical limb ischemia.
[42] A method for preventing or treating diabetic macular edema, diabetic retinopathy, or critical limb ischemia, comprising administering a therapeutically effective amount of the anti-human Tie2 antibody or the antigen-binding fragment thereof of any one of [1] to [23], [36], and [37].
[43] The anti-human Tie2 antibody or the antigen-binding fragment thereof of any one of [1] to [23], [36], and [37], for preventing or treating diabetic macular edema, diabetic retinopathy, or critical limb ischemia.
[44] Use of the anti-human Tie2 antibody or the antigen-binding fragment thereof of any one of [1] to [23], [36], and [37] for manufacture of a pharmaceutical composition for preventing or treating diabetic macular edema, diabetic retinopathy, or critical limb ischemia.
The anti-human Tie-2 antibody or the antigen-binding fragment thereof includes a fusion of the antibody with another peptide or protein, and a modification having a modifying agent bound thereto.
Effects of the Invention
The anti-human Tie2 antibody of the present invention can be used as an agent for preventing or treating diabetic macular edema, diabetic retinopathy, or critical limb ischemia by binding to a human Tie2 to activate the human Tie2.
BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 shows an example of the format of a tetravalent anti-human Tie2 antibody of the present invention.
FIG. 2 shows the vascular permeability inhibitory action of the fully human 2-16A2 and TIE-1-Igγ1-WT in a rat model with vascular permeability. The vertical axis indicates the amount of leakage of an Evans Blue dye (****: p<0.0001 vs a vehicle group).
FIG. 3 shows the vascular permeability inhibitory action of TIE-1-Igγ1-LALA in a rat model with vascular permeability. The vertical axis indicates the amount of leakage of an Evans Blue dye (****: p<0.0001 vs a vehicle group).
FIG. 4 shows the retinal edema inhibitory action of TIE-1-Igγ1-LALA in a mouse model with the loss of pericytes in the retinal blood vessel. The vertical axis indicates a sum of a retinal nerve fiber layer and a retinal ganglion cell layer (##: p<0.005 vs Cont. group, *: p<0.05 vs Veh. group).
FIG. 5 shows the blood flow improving action of TIE-1-Igγ1-LALA in a mouse model with hindlimb ischemia. The vertical axis indicates the amount of blood flow. (*: p<0.05 vs control group, **: p<0.01 vs control group).
FIGS. 6A and 6B show a representative example of the results of a surface plasmon resonance phenomenon as epitope analysis of TIE-1-Igγ1-LALA. The vertical axis indicates a binding responsiveness (Resonance Unit: RU) and the horizontal axis indicates time (seconds).
FIG. 7 shows the results of ELISA as epitope analysis of TIE-1-Igγ1-LALA. The vertical axis indicates a luminescent intensity and the horizontal axis indicates a concentration of TIE-1-Igγ1-LALA (ng/mL).
EMBODIMENTS FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in detail.
There are five classes of IgG, IgM, IgA, IgD, and IgE in an antibody. The basic structure of an antibody molecule is configured of heavy chains having a molecular weight of 50000 to 70000 and light chains having a molecular weight of 20000 to 30000 in each of the classes in common. Heavy chain usually consists of a polypeptide chain comprising approximately 440 amino acids, has a distinctive structure for each of the classes, and is referred to as Igγ, Igμ, Igα, and Igδ corresponding to IgG, IgM, IgA, IgD, and IgE, respectively. Further, four subclasses of IgG1, IgG2, IgG3, and IgG4 are present in IgG; and the heavy chains respectively corresponding thereto are referred to as Igγ1, Igγ2, Igγ3, and Igγ4. Light chain usually consists of a polypeptide chain comprising approximately 220 amino acids, two types of which, type L and type K are known, and are referred to as Igλ and Igκ. In a peptide configuration of the basic structure of antibody molecules, two homologous heavy chains and two homologous light chains are bound by disulfide bonds (S—S bond) and non-covalent bonds, and the molecular weight thereof is 150000 to 190000. Two kinds of light chains can be paired with any heavy chain.
With regard to intrachain S—S bonds, four of the S—S bonds are present in the heavy chain (five in Igμ and Igε) and two of them are present in the light chain; one loop is formed per 100 to 110 amino acid residues, and this steric structure is similar among the loops and are referred to as a structural unit or a domain. The domain located at the amino-terminal side (N terminal side) in both of the heavy chain and the light chain, whose amino acid sequence is not constant even in a case of a sample from the same class (sub class) of the same kind of animal is referred to as a variable region, and respective domains are referred to as a heavy chain variable region and a light chain variable region. The amino acid sequence of the carboxy-terminal side (C terminal side) from the variable region is nearly constant in each class or subclass and is referred to as a constant region.
An antigen binding site of an antibody is configured of heavy chain variable region (VH) and the light chain variable region (VL), and the binding specificity depends on the amino acid sequence of this site. On the other hand, biological activities such as binding to complements and various cells reflect differences in the constant region structures among each class Ig. It is understood that the variability of variable regions of the light chains and the heavy chains is mostly limited to three small hypervariable regions present in both chains and these regions are referred to as complementarity determining regions (CDR: CDR1, CDR2, and CDR3 from the N terminal side). The remaining portion of the variable region is referred to as a framework region (FR) and is relatively constant.
With regard to the constant region, the heavy chain constant region consists of three regions, which are each called a CH1 region, a CH2 region, and a CH3 region in order from the variable region side. The light chain constant region consists of one region. A peptide sequence called a hinge region is present between the CH1 region and the CH2 region. The hinge region contributes to the mobility of a structure consisting of the heavy chain variable region and the CH1 region.
Further, various kinds of antigen-binding fragments comprising VH and VL of an antibody have antigen binding activity. For example, a single-chain variable region fragment (scFv), Fab, Fab′, and F(ab′)2 are exemplified as typical antigen-binding fragments. A Fab is a monovalent antigen-binding fragment which is constituted with a light-chain and a heavy-chain fragment comprising a VH, a CH1 region, and a portion of the hinge region. A Fab′ is a monovalent antigen-binding fragment which is constituted with a light-chain and a heavy-chain fragment comprising a VH, a CH1 region, and a portion of the hinge region, and cysteine residues constituting the inter-heavy-chain S—S bond are comprised in the portion of the hinge region. A F(ab′)2 is a bivalent antigen-binding fragment having a dimeric structure in which two Fab′ fragments bind to each other via the inter-heavy-chain S—S bond in the hinge region. An scFv is a monovalent antigen-binding fragment which is constituted with a VH and VL connected with a linker peptide.
An antibody having two or more antigen-binding sites is referred to as a multivalent antibody. Among these, an antibody having four antigen-binding sites is referred to as a tetravalent antibody. For the tetravalent antibody, various formats (structures) have been reported (Nat. Rev. Immunol. 2010, Vol. 10, pp. 301-316; J. Immunol., 2003, Vol. 170, pp. 4854-4861; Mol. Immunol., 2000, Vol. 37, pp. 1067-1077; Biochem. J., 2007, Vol. 406, pp. 237-246; and J. Immunol. Methods, 2003, Vol. 279, pp. 219-232). For example, a tetravalent antibody in which the N terminals of a heavy chain variable region and a light chain variable region of a bivalent antibody are each linked to the C terminals of the heavy chain variable region and the light chain variable region through a linker; a tetravalent antibody comprising two heavy chains and four light chains, in which each heavy chain comprises two structures consisting of a heavy chain variable region and a CH1 region; a tetravalent antibody in which the C terminals of scFv are bonded to each streptavidin of a tetrameric streptavidin one by one; a tetravalent antibody in which the C terminals of scFv are bonded to each p53 of a tetrameric p53 one by one; and a tetravalent antibody in which the N terminals of a CH3 region are linked to the C terminals of a dimeric scFv through a linker have been reported.
<Anti-Human Tie2 Antibody of the Present Invention>
The anti-human Tie2 antibody or the antigen-binding fragment thereof of the present invention includes an anti-human Tie2 antibody or an antigen-binding fragment thereof, having the following characteristics.
An anti-human Tie2 antibody or an antigen-binding fragment thereof, comprising four heavy chain variable regions and four light chain variable regions, in which
the heavy chain variable region consists of the amino acid sequence of the amino acid numbers 1 to 122 of SEQ ID NO: 2,
the light chain variable region consists of the amino acid sequence of the amino acid numbers 1 to 113 of SEQ ID NO: 4, and
the one heavy chain variable region and the one light chain variable region constitute one antigen-binding site, and the antibody or the antigen-binding fragment thereof comprises four antigen-binding sites.
The anti-human Tie2 antibody or the antigen-binding fragment thereof of the present invention is not particularly limited as long as it is a tetravalent antibody, and various formats of tetravalent antibodies described in, for example, Nat. Rev. Immunol. 2010, Vol. 10, pp. 301-316, J. Immunol., 2003, Vol. 170, pp. 4854-4861; Mol. Immunol., 2000, Vol. 37, pp. 1067-1077; Biochem. J., 2007, Vol. 406, pp. 237-246; J. Immunol. Methods, 2003, Vol. 279, pp. 219-232; and the like can be used for the anti-human Tie2 antibody or the antigen-binding fragment thereof of the present invention.
Preferably, the anti-human Tie2 antibody of the present invention comprises two heavy chains and four light chains,
each heavy chain comprises two structures consisting of a heavy chain variable region consisting of the amino acid sequence of the amino acid numbers 1 to 122 of SEQ ID NO: 2 and a CH1 region, a CH2 region, and a CH3 region, and the C terminus of one of the structures is linked to the N terminus of the other structure through a linker, and
each light chain comprises a light chain variable region consisting of the amino acid sequence of the amino acid numbers 1 to 113 of SEQ ID NO: 4, and a light chain constant region.
Hereinafter, a tetravalent antibody in the format is referred to as a tandem antibody, and an example thereof is shown in FIG. 1.
In the case where the anti-human Tie2 antibody of the present invention is a tandem antibody, a constant region (for example, a constant region of Igγ1, Igγ2, Igγ3 or Igγ4 as a heavy chain constant region, and a constant region of Igλ or Igκ as a light chain constant region) in any subclass can be selected as the constant region. The heavy chain constant region (including a CH1 region, a CH2 region, and a CH3 region) is preferably a human Igγ1 constant region or a human Igγ4 constant region. The light chain constant region is preferably a human Igκ constant region.
In the case where a human Igγ1 constant region is used as the heavy chain constant region of the anti-human Tie2 antibody of the present invention, examples of the CH1 region, the CH2 region, and the CH3 region of the human Igγ1 constant region comprise a CH1 region consisting of the amino acid sequence of the amino acid numbers 350 to 447 of SEQ ID NO: 8, a CH2 region consisting of the amino acid sequence of the amino acid numbers 463 to 572 of SEQ ID NO: 8, and a CH3 region consisting of the amino acid sequence of the amino acid numbers 573 to 679 of SEQ ID NO: 8.
In the case where a human Igγ1 constant region is used as the heavy chain constant region of the anti-human Tie2 antibody of the present invention, a human Igγ1 constant region having introduction of amino acid variation, such as L234A (having substitution of leucine at the amino acid 234th position with alanine according to an EU index such as Kabat), L235A (having substitution of leucine at the amino acid 235th position with alanine according to an EU index such as Kabat), and P331S (having substitution of proline at the amino acid 331st position with serine according to an EU index such as Kabat) can also be used in order to reduce the antibody-dependent cellular cytotoxicity or the complement-dependent cytotoxicity activity of an antibody (Mol. Immunol., 1992, Vol. 29, No. 5, pp. 633-639). Further, from the viewpoint of pharmacokinetics, a human Igγ1 constant region to which amino acid variations has been introduced, such as I253A (having substitution of isoleucine at the amino acid 253th position with alanine according to an EU index such as Kabat) can also be used in order to attain a rapid loss in the blood (J. Immunol., 1997, Vol. 158, pp. 2211-2217). The residue numbers with respect to the introduction of amino acid variation in the constant region of the antibody used in the present specification are in accordance with an EU index (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institute of Health, Bethesda).
In the case where a human Igγ1 constant region is used as the heavy chain constant region of the anti-human Tie2 antibody of the present invention, the human Igγ1 constant region is preferably a human Igγ1 constant region having amino acid variations of L234A, L235A, and P331S, or L234A, L235A, P331S and I253A. Examples of the CH1 region, the CH2 region, and CH3 region of the human Igγ1 constant region having amino acid variations of L234A, L235A, and P331S comprise a CH1 region consisting of the amino acid sequence of the amino acid numbers 350 to 447 of SEQ ID NO: 2, a CH2 region consisting of the amino acid sequence of the amino acid numbers 463 to 572 of SEQ ID NO: 2, and a CH3 region consisting of the amino acid sequence of the amino acid numbers 573 to 679 of SEQ ID NO: 2. Examples of the CH1 region, the CH2 region, and the CH3 region of the human Igγ1 constant region having amino acid variations of L234A, L235A, P331S, and I253A comprise a CH1 region consisting of the amino acid sequence of the amino acid numbers 350 to 447 of SEQ ID NO: 6, a CH2 region consisting of the amino acid sequence of the amino acid numbers 463 to 572 of SEQ ID NO: 6, and a CH3 region consisting of the amino acid sequence of the amino acid numbers 573 to 679 of SEQ ID NO: 6.
In the case where a human Igγ4 constant region is used as the heavy chain constant region of the anti-human Tie2 antibody of the present invention, a human Igγ4 constant region having introduction of amino acid variations such as S228P (having substitution of serine at the amino acid 228th position with proline according to an EU index such as Kabat) and L235E (having substitution of leucine at the amino acid 235st position with glutamic acid according to an EU index such as Kabat) can also be used in order to inhibit Fab arm exchange (Drug Metab. Dispos., 2010, Vol. 38, No. 1, pp. 84-91).
In the case where a human Igγ4 constant region is used as the heavy chain constant region of the anti-human Tie2 antibody of the present invention, the human Igγ4 constant region is preferably a human Igγ4 constant region having amino acid variations of S228P and L235E. Examples of the CH1 region, the CH2 region, and the CH3 region of the human Igγ4 constant region having amino acid variations of S228P and L235E comprise a CH1 region consisting of the amino acid sequence of the amino acid numbers 350 to 447 of SEQ ID NO: 10, a CH2 region consisting of the amino acid sequence of the amino acid numbers 460 to 569 of SEQ ID NO: 10, and a CH3 region consisting of the amino acid sequence of the amino acid numbers 570 to 676 of SEQ ID NO: 10.
Examples of the human Igκ constant region include a human Igκ (constant region consisting of the amino acid sequence of the amino acid numbers 114 to 219 of SEQ ID NO: 4.
Preferably, in the case where the anti-human Tie2 antibody of the present invention is a tandem antibody, the heavy chain constant region is a human Igγ1 constant region or a human Igγ4 constant region, and the light chain constant region is a human Igκ constant region. In the case where the heavy chain constant region is a human Igγ1 constant region, the human Igγ1 constant region is preferably a human Igγ1 constant region having amino acid variations of L234A, L235A, and P331 S, or a human Igγ1 constant region having amino acid variations of L234A, L235A, P331 S, and I253A. In the case where the heavy chain constant region is a human Igγ4 constant region, the human Igγ4 constant region is preferably a human Igγ4 constant region having amino acid variations of S228P and L235E.
In the case where the anti-human Tie2 antibody of the present invention is a tandem antibody, as a linker that links the structures consisting of a heavy chain variable region and a CH1 region, any peptide (peptide linker) can be used as long as the antibody has such a function. The length of the peptide linker and the amino acid sequence can be appropriately selected by a person skilled in the art. The peptide linker preferably has 5 to 70 amino acids in length. The peptide linker preferably comprises the amino acid sequence of a hinge region or a portion thereof. The hinge region means a region that exists between the CH1 region and the CH2 region of an antibody, and examples of the hinge region to be used comprise a hinge region of IgG1 or IgG3. A portion of the hinge region means a region having at least 5 successive amino acids in the hinge region, and preferably means a region having at least 5 successive amino acids from the N terminus of the hinge region. Examples of a part of the hinge region include a region having 5 successive amino acids from the N terminal (consisting of the amino acid sequence of the amino acid numbers 1 to 5 of SEQ ID NO: 13) in the case of the hinge region of IgG1 and a region having 12 successive amino acids from the N terminal (consisting of the amino acid sequence of the amino acid numbers 1 to 12 of SEQ ID NO: 14) in the case of the hinge region of IgG3. In one embodiment, the linker comprises the amino acid sequence of a region having at least 5 successive amino acids from the N terminus of the hinge region and comprises amino acid sequence GlySer at the C terminus of the linker. Examples of such a linker comprise a peptide linker consisting of the amino acid sequence shown by any one of SEQ ID NOS: 13 to 20, and the linker preferably consists of the amino acid sequence shown by SEQ ID NO: 13.
In one embodiment, the anti-human Tie2 antibody of the present invention is an anti-human Tie2 antibody having any one of the following characteristics i) to iv).
i) An anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence shown by SEQ ID NO: 2 and four light chains consisting of the amino acid sequence shown by SEQ ID NO: 4.
ii) An anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence shown by SEQ ID NO: 6 and four light chains consisting of the amino acid sequence shown by SEQ ID NO: 4.
iii) An anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence shown by SEQ ID NO: 8 and four light chains consisting of the amino acid sequence shown by SEQ ID NO: 4.
iv) An anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence shown by SEQ ID NO: 10 and four light chains consisting of the amino acid sequence shown by SEQ ID NO: 4.
It is known that when an antibody is expressed in cells, the antibody is modified after translation. Examples of the posttranslational modification include cleavage of lysine at the C terminal of the heavy chain by a carboxypeptidase; modification of glutamine or glutamic acid at the N terminal of the heavy chain and the light chain to pyroglutamic acid by pyroglutamylation; glycosylation; oxidation; deamidation; and glycation, and it is known that such posttranslational modifications occur in various antibodies (Journal of Pharmaceutical Sciences, 2008, Vol. 97, p. 2426-2447).
The anti-human Tie2 antibody or the antigen-binding fragment thereof of the present invention includes an anti-human Tie2 antibody or an antigen-binding fragment thereof, which has undergone posttranslational modification. Examples of the anti-human Tie2 antibody or the antigen-binding fragment thereof of the present invention, which undergoes posttranslational modification, include anti-human Tie2 antibodies or antigen-binding fragments thereof, which have undergone pyroglutamylation at the N terminal of the heavy chain variable region and/or deletion of lysine at the C terminal of the heavy chain. It is known in the field that such posttranslational modification due to pyroglutamylation at the N terminal and deletion of lysine at the C terminal does not have any influence on the activity of the antibody (Analytical Biochemistry, 2006, Vol. 348, p. 24-39).
In one embodiment, the anti-human Tie2 antibody of the present invention is an anti-human Tie2 antibody having any one of the following characteristics (1) to (4).
(1) An anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence in which glutamic acid of the amino acid number 1 of SEQ ID NO: 2 is modified to pyroglutamic acid and/or lysine of the amino acid number 679 of SEQ ID NO: 2 is deleted and four light chains consisting of the amino acid sequence shown by SEQ ID NO: 4.
(2) An anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence in which glutamic acid of the amino acid number 1 of SEQ ID NO: 6 is modified to pyroglutamic acid and/or lysine of the amino acid number 679 of SEQ ID NO: 6 is deleted and four light chains consisting of the amino acid sequence shown by SEQ ID NO: 4.
(3) An anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence in which glutamic acid of the amino acid number 1 of SEQ ID NO: 8 is modified to pyroglutamic acid and/or lysine of the amino acid number 679 of SEQ ID NO: 8 is deleted and four light chains consisting of the amino acid sequence shown by SEQ ID NO: 4.
(4) An anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence in which glutamic acid of the amino acid number 1 of SEQ ID NO: 10 is modified to pyroglutamic acid and/or lysine of the amino acid number 676 of SEQ ID NO: 10 is deleted and four light chains consisting of the amino acid sequence shown by SEQ ID NO: 4.
In one embodiment, the anti-human Tie2 antibody of the present invention is an anti-human Tie2 antibody having the following characteristics.
An anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence of the amino acid numbers 1 to 678 of SEQ ID NO: 2 and four light chains consisting of the amino acid sequence shown by SEQ ID NO: 4.
The present invention further includes an anti-human Tie2 antibody or an antigen-binding fragment thereof, having the following characteristics.
An anti-human Tie2 antibody or an antigen-binding fragment thereof, comprising four heavy chain variable regions and four light chain variable regions, in which the heavy chain variable region comprises CDR1 consisting of the amino acid sequence of the amino acid numbers 31 to 35 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequence of the amino acid numbers 50 to 66 of SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence of the amino acid numbers 99 to 111 of SEQ ID NO: 2.
the light chain variable region comprises CDR1 consisting of the amino acid sequence of the amino acid numbers 24 to 39 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence of the amino acid numbers 55 to 61 of SEQ ID NO: 4, and CDR3 consisting of the amino acid sequence of the amino acid numbers 94 to 102 of SEQ ID NO: 4, and
the one heavy chain variable region and the one light chain variable region constitute one antigen-binding site, and the antibody or the antigen-binding fragment thereof comprises four antigen-binding sites.
In addition, the present invention further includes an anti-human Tie2 antibody having the following characteristics.
An anti-human Tie2 antibody comprising two heavy chains and four light chains, in which
each heavy chain comprises two structures consisting of a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of the amino acid numbers 31 to 35 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequence of the amino acid numbers 50 to 66 of SEQ ID NO: 2, and CDR3 consisting of the amino acid sequence of the amino acid numbers 99 to 111 of SEQ ID NO: 2 and a CH1 region, a CH2 region, and a CH3 region, and the carboxy terminus of one of the structures is linked to the amino terminus of the other structure through a linker, and
each light chain comprises a light chain variable region comprising CDR1 consisting of the amino acid sequence of the amino acid numbers 24 to 39 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence of the amino acid numbers 55 to 61 of SEQ ID NO: 4, and CDR3 consisting of the amino acid sequence of the amino acid numbers 94 to 102 of SEQ ID NO: 4, and a light chain constant region.
The anti-human Tie2 antibody of the present invention is an antibody that binds to a human Tie2. Whether the antibody binds to the human Tie2 (Accession No. NP_000450.2) can be confirmed by using a known binding activity measurement method. Examples of the binding activity measurement method include a method of Enzyme-Linked ImmunoSorbent Assay (ELISA) or the like. In a case of using the ELISA, in an exemplary method, a protein formed by fusion of the human Tie2 with a human Fc is immobilized on an ELISA plate, and a test antibody is added thereto to be reacted. A secondary antibody such as a biotin-labeled anti-IgG antibody is reacted with the resultant, washed, and then reacted with streptavidin to which an enzyme such as an alkaline phosphatase is bound. After washing, it is possible to confirm whether the test antibody binds to the human Tie2 by carrying out activity measurement using an activity-detecting reagent (for example, in the case of the alkaline phosphatase, Chemiluminescent Ultra Sensitive AP Microwell and/or Membrane Substrate (450 nm) (BioFX, APU4-0100-01) or the like)). As a specific method for evaluating the activity, the same method as the one described in Example 12 as described later, for example, can be used.
The anti-human Tie2 antibody of the present invention further includes an antibody binding to Tie2 derived from other animals (for example, monkey Tie2) in addition to binding to a human Tie2 as long as it is an antibody binding to a human Tie2.
Preferably, the anti-human Tie2 antibody of the present invention binds to a human Tie2, and further, has anti-apoptotic activity with respect to a human Tie2-expressing cell. As a specific method for evaluating whether the antibody has anti-apoptotic activity with respect to a human Tie2-expressing cell, for example, the same method as the one described in Example 4 as described later can be used.
The anti-human Tie2 antibody or the antigen-binding fragment thereof of the present invention includes a tetravalent anti-human Tie2 antibody or an antigen-binding fragment thereof which binds to the same human Tie2 epitope as the anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence shown by SEQ ID NO: 2 and four light chains consisting of the amino acid sequence shown by SEQ ID NO: 4, or as the anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence of the amino acid numbers 1 to 678 of SEQ ID NO: 2 and four light chains consisting of the amino acid sequence shown by SEQ ID NO: 4. Here, the epitope refers to an antigen site recognized by an antibody.
The anti-human Tie2 antibody or the antigen-binding fragment thereof of the present invention includes a tetravalent anti-human Tie2 antibody or an antigen-binding fragment thereof, which binds to an epitope comprising at least one amino acid of the amino acids of the amino acid numbers 192, 195 and 197 of a human Tie2 (Accession No. NP_000450.2).
Moreover, the anti-human Tie2 antibody or the antigen-binding fragment thereof of the present invention includes a tetravalent anti-human Tie2 antibody or an antigen-binding fragment thereof, which binds to an epitope comprising the amino acids of the amino acid numbers 192, 195 and 197 of a human Tie2 (Accession No. NP_000450.2).
The tetravalent anti-human Tie2 antibody or the antigen-binding fragment thereof, which binds to the same human Tie2 epitope as the anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence shown by SEQ ID NO: 2 and four light chains consisting of the amino acid sequence shown by SEQ ID NO: 4, or as the anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence of the amino acid numbers 1 to 678 of SEQ ID NO: 2 and four light chains consisting of the amino acid sequence shown by SEQ ID NO: 4 can be acquired by using a known method for determining an epitope. Examples of the method for determining an epitope include hydrogen/deuterium exchange mass spectrometry, X-ray crystal structure analysis, ELISA and a surface plasmon resonance phenomenon using an amino acid substitution mutant of a human Tie2, a partial peptide of human Tie2, or the like, and the like.
It is possible to check whether the test antibody binds to the same human Tie2 epitope as the anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence shown by SEQ ID NO: 2 and four light chains consisting of the amino acid sequence shown by SEQ ID NO: 4, or as the anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence of the amino acid numbers 1 to 678 of SEQ ID NO: 2 and four light chains consisting of the amino acid sequence shown by SEQ ID NO: 4 by using the well-known method for determining an epitope as described above. In the case of using hydrogen/deuterium exchange mass spectrometry, a human Tie2 with deuterium substitution in the absence of a test antibody and a human Tie2 with deuterium substitution in the presence of a test antibody are each decomposed by peptides, and the amount of molecules of each peptide is measured to calculate the ratio of deuterium substitution. The human Tie2 epitope of the test antibody can be determined from the difference in the ratios of deuterium substitution of the human Tie2 according to the presence or absence of the test antibody. In the case of using ELISA, a point mutant of a human Tie2 is prepared. The mutant human Tie2 is immobilized and a test antibody is added thereto to undergo a reaction. After the reaction, a secondary antibody such as a biotin-labeled anti-human kappa light chain antibody is reacted and washed. Thereafter, an alkaline phosphatase-labeled streptavidin (Thermo Fisher Scientific, 21324) is reacted therewith and washed. Further, it is possible to identify whether or not the test antibody binds to the mutant human Tie2 by carrying out activity measurement using Chemiluminescent Ultra Sensitive AP Microwell and/or Membrane Substrate (450 nm), or the like. It is possible to determine an epitope of the test antibody by evaluating the binding activity to various types of mutant human Tie2. In the case where the epitope of the test antibody comprises at least one amino acid in the epitope of the anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence shown by SEQ ID NO: 2 and four light chains consisting of the amino acid sequence shown by SEQ ID NO: 4, or the anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence of the amino acid numbers 1 to 678 of SEQ ID NO: 2 and four light chains consisting of the amino acid sequence shown by SEQ ID NO: 4, it can be determined that the test antibody binds to the same human Tie2 epitope as the anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence shown by SEQ ID NO: 2 and four light chains consisting of the amino acid sequence shown by SEQ ID NO: 4, or as the anti-human Tie2 antibody comprising a heavy chain consisting of the amino acid sequence of the amino acid numbers 1 to 678 of SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown by SEQ ID NO: 4.
The anti-human Tie2 antibody or the antigen-binding fragment thereof of the present invention can be easily prepared by a person skilled in the art, using a method known in the art, based on the sequence information of the heavy chain variable region and the light chain variable region of the antibody of the present invention, as disclosed in the present specification. The anti-human Tie2 antibody or the antigen-binding fragment thereof of the present invention is not particularly limited, but can be produced in accordance with the method described in <Method for Producing Anti-Human Tie2 Antibody of the Present Invention and Anti-Human Tie2 Antibody Produced by the Method> as described later, for example.
The anti-human Tie2 antibody or the antigen-binding fragment thereof of the present invention is further purified as needed, and formulated according to a conventional method. It may be used for the prevention or the treatment of blood vessel-related diseases such as diabetic retinopathy, diabetic macular edema, sepsis, acute hepatic disorders, acute renal disorders, acute pulmonary disorders, systemic inflammatory reaction syndrome, peripheral arterial occlusive disease, or critical limb ischemia.
<Polynucleotide of the Present Invention>
The polynucleotide of the present invention includes a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human Tie2 antibody or the antigen-binding fragment thereof of the present invention and a polynucleotide comprising a base sequence encoding the light chain variable region of the anti-human Tie2 antibody or the antigen-binding fragment thereof of the present invention.
In one embodiment, the polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human Tie2 antibody or the antigen-binding fragment thereof of the present invention is a polynucleotide comprising a base sequence encoding the heavy chain variable region consisting of the amino acid sequence of the amino acid numbers 1 to 122 of SEQ ID NO: 2.
Examples of the polynucleotide comprising a base sequence encoding the heavy chain variable region shown by the amino acid sequence of the amino acid numbers 1 to 122 of SEQ ID NO: 2 include a polynucleotide comprising the base sequence of the base numbers 1 to 366 of SEQ ID NO: 1.
In a preferred embodiment, the polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human Tie2 antibody or the antigen-binding fragment thereof of the present invention is a polynucleotide comprising a base sequence encoding the heavy chain consisting of the amino acid sequence shown by SEQ ID NO: 2, a polynucleotide comprising a base sequence encoding the heavy chain consisting of the amino acid sequence shown by SEQ ID NO: 6, a polynucleotide comprising a base sequence encoding the heavy chain consisting of the amino acid sequence shown by SEQ ID NO: 8, or a polynucleotide comprising a base sequence encoding the heavy chain consisting of the amino acid sequence shown by SEQ ID NO: 10.
Examples of the polynucleotide comprising a base sequence encoding the heavy chain consisting of the amino acid sequence shown by SEQ ID NO: 2 include a polynucleotide comprising the base sequence shown by SEQ ID NO: 1. Examples of the polynucleotide comprising a base sequence encoding the heavy chain consisting of the amino acid sequence shown by SEQ ID NO: 6 include a polynucleotide comprising the base sequence shown by SEQ ID NO: 5. Examples of the polynucleotide comprising a base sequence encoding the heavy chain consisting of the amino acid sequence shown by SEQ ID NO: 8 include a polynucleotide comprising the base sequence shown by SEQ ID NO: 7. Examples of the polynucleotide comprising a base sequence encoding the heavy chain consisting of the amino acid sequence shown by SEQ ID NO: 10 include a polynucleotide comprising the base sequence shown by SEQ ID NO: 9.
In one embodiment, the polynucleotide comprising a base sequence encoding the light chain variable region of the anti-human Tie2 antibody or the antigen-binding fragment thereof of the present invention is a polynucleotide comprising a base sequence encoding the light chain variable region consisting of the amino acid sequence of the amino acid numbers 1 to 113 of SEQ ID NO: 4.
Examples of the polynucleotide comprising a base sequence encoding the light chain variable region shown by the amino acid sequence of the amino acid numbers 1 to 113 of SEQ ID NO: 4 include a polynucleotide comprising the base sequence of the base numbers 1 to 339 of SEQ ID NO: 3.
In a preferred embodiment, the polynucleotide comprising a base sequence encoding the light chain variable region of the anti-human Tie2 antibody or the antigen-binding fragment thereof of the present invention is a polynucleotide comprising a base sequence encoding the light chain consisting of the amino acid sequence shown by SEQ ID NO: 4.
Examples of the polynucleotide comprising a base sequence encoding the light chain consisting of the amino acid sequence shown by SEQ ID NO: 4 include a polynucleotide comprising a base sequence shown by SEQ ID NO: 3.
The polynucleotide of the present invention can be easily prepared by a person skilled in the art using a known method in the field based on the base sequence. For example, the polynucleotide of the present invention can be synthesized using a known gene synthesis method in the field. As the gene synthesis method, various methods such as a synthesis method of antibody genes described in WO90/07861 known by a person skilled in the art can be used. Further, once the polynucleotide of the present invention is acquired, it is possible to acquire other polynucleotides of the present invention by introducing a variation into a predetermined site of the polynucleotide. As such a method for introducing the variation, various methods known to a person skilled in the art, such as a site-specific mutagenesis method (Current Protocols in Molecular Biology edit, 1987, John Wiley & Sons Section 8.1-8.5), can be used.
<Expression Vector of the Present Invention>
The expression vector of the present invention includes the polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human Tie2 antibody or the antigen-binding fragment thereof of the present invention and/or the polynucleotide comprising a base sequence encoding the light chain variable region of the anti-human Tie2 antibody or the antigen-binding fragment thereof of the present invention. Tetravalent antibodies in various formats and methods for producing the same are well-known in the art, and the expression vector of the present invention can be easily established by a person skilled in the art according to such production methods or the formats of the tetravalent antibodies to be expressed.
Preferred examples of the expression vector of the present invention include an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human Tie2 antibody of the present invention, an expression vector comprising a polynucleotide comprising a base sequence encoding the light chain of the anti-human Tie2 antibody of the present invention, and an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human Tie2 antibody of the present invention and a polynucleotide comprising a base sequence encoding the light chain of the antibody.
The expression vector used to express the polynucleotide of the present invention are not particularly limited as long as a polynucleotide comprising the base sequence encoding the heavy chain variable region of the anti-human Tie2 antibody or the antigen-biding fragment thereof of the present invention and/or a polynucleotide comprising the base sequence encoding the light chain variable region of the anti-human Tie2 antibody or the antigen-biding fragment thereof of the present invention can be expressed in various host cells of eukaryotic cells (for example, animal cells, insect cells, plant cells, and yeast) and/or prokaryotic cells (for example, Escherichia coli), and the polypeptides encoded by these can be produced. Examples of the expression vector include plasmid vectors, viral vectors (for example, adenovirus, adeno-associated virus, Sendai virus or retrovirus), and the like. Preferably pEE6.4 or pEE12.4 (Lonza, Inc.) can be used. Further, antibody genes can be expressed by using expression vectors comprising human Ig constant region genes in advance such as AG-γ1 or AG-κ (for example, see WO94/20632).
The expression vector of the present invention may comprise a promoter that is operably linked to the polynucleotide of the present invention. Examples of the promoter for expressing the polynucleotide of the invention with animal cells include a virus-derived promoter such as CMV, RSV, or SV40, an actin promoter, an EF (elongation factor) la promoter, and a heat shock promoter. Examples of promoters for expression by bacteria (for example, Escherichia) include a trp promoter, a lac promoter, λPL promoter, and tac promoter. Further, examples of promoters for expression by yeast include a GAL1 promoter, a GAL10 promoter, a PH05 promoter, a PGK promoter, a GAP promoter, and an ADH promoter.
In the case of using an animal cell, an insect cell, or yeast as the host cell, the expression vector of the present invention may comprise initiation codon and termination codon. In this case, the expression vector of the present invention may comprise an enhancer sequence, an untranslated region on the 5′ side and the 3′ side of genes encoding the antibody of the present invention or the heavy chain variable region or the light chain variable region, a secretory signal sequence, a splicing junction, a polyadenylation site, or a replicable unit. When Escherichia coli is used as the host cell, the expression vector of the present invention may comprise an initiation codon, a termination codon, a terminator region, and a replicable unit. In this case, the expression vector of the present invention may comprise a selection marker (for example, tetracycline resistant genes, ampicillin resistant genes, kanamycin resistant genes, neomycin resistant genes, or dihydrofolate reductase genes) which is generally used according to the necessity.
<Transformed Host Cell of the Present Invention>
The transformed host cell of the present invention includes a host cell transformed with the expression vector of the present invention which is selected from the group consisting of (a) to (d) below:
(a) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human Tie2 antibody or the antigen-binding fragment thereof of the present invention, and a polynucleotide comprising a base sequence encoding the light chain variable region of the antibody or the antigen-binding fragment thereof;
(b) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human Tie2 antibody or the antigen-binding fragment thereof of the present invention and an expression vector comprising a polynucleotide comprising a base sequence encoding the light chain variable region of the antibody or the antigen-binding fragment thereof;
(c) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human Tie2 antibody or the antigen-binding fragment thereof of the present invention; and
(d) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the light chain variable region of the anti-human Tie2 antibody or the antigen-binding fragment thereof of the present invention.
In one embodiment, the transformed host cell of the present invention is a host cell transformed with the expression vector of the present invention, which is selected from the group consisting of (a) to (d) below:
(a) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human Tie2 antibody of the present invention and a polynucleotide comprising a base sequence encoding the light chain of the antibody;
(b) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human Tie2 antibody of the present invention and an expression vector comprising a polynucleotide comprising a base sequence encoding the light chain of the antibody;
(c) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human Tie2 antibody of the present invention; and
(d) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the light chain of the anti-human Tie2 antibody of the present invention.
Preferred examples of the transformed host cell of the present invention include a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human Tie2 antibody of the present invention and a polynucleotide comprising a base sequence encoding the light chain of the antibody, and a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human Tie2 antibody of the present invention and an expression vector comprising a polynucleotide comprising a base sequence encoding the light chain of the antibody.
The transformed host cell is not particularly limited as long as the host cell is appropriate for the expression vector being used, transformed with the expression vector, and can express the antibody. Examples of the transformed host cell include various cells such as natural cells or artificially established cells which are generally used in the field of the present invention (for example, animal cells (for example, CHO—K1SV cells), insect cells (for example, Sf9), bacteria (for example, Escherichia), yeast (for example, Saccharomyces or Pichia) or the like). Preferably cultured cells such as CHO cells (CHO—K1SV cells, CHO-DG 44 cells, or the like) 293 cells, or NSO cells can be used.
A method of transforming the host cell is not particularly limited, but, for example, a calcium phosphate method or an electroporation method can be used.
<Method for Producing Anti-Human Tie2 Antibody of the Present Invention and Anti-Human Tie2 Antibody Produced by the Method>
Examples of the method for producing the anti-human Tie2 antibody or the antigen-binding fragment thereof of the present invention include a method for producing an anti-human Tie2 antibody or a antigen-binding fragment thereof, comprising culturing host cell(s) selected from the group consisting of (a) to (c) below to express a tetravalent anti-human Tie2 antibody or an antigen-binding fragment thereof:
(a) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human Tie2 antibody or the antigen-binding fragment thereof of the present invention and a polynucleotide comprising a base sequence encoding the light chain variable region of the antibody or the antigen-binding fragment thereof;
(b) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human Tie2 antibody or the antigen-binding fragment thereof of the present invention and an expression vector comprising a polynucleotide comprising a base sequence encoding the light chain variable region of the antibody or the antigen-binding fragment thereof; and
(c) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human Tie2 antibody or the antigen-binding fragment thereof of the present invention and a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the light chain variable region of the antibody or the antigen-binding fragment thereof.
In one embodiment, examples of the method for producing the anti-human Tie2 antibody of the present invention include a method for producing an anti-human Tie2 antibody, comprising culturing host cell(s) selected from the group consisting of (a) to (c) below to express an anti-human Tie2 antibody:
(a) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human Tie2 antibody of the present invention and a polynucleotide comprising a base sequence encoding the light chain of the antibody;
(b) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human Tie2 antibody of the present invention and an expression vector comprising a polynucleotide comprising a base sequence encoding the light chain of the antibody; and
(c) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human Tie2 antibody of the present invention and a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the light chain of the antibody.
The method for producing the anti-human Tie2 antibody of the present invention is not particularly limited as long as it comprises a step of culturing the transformed host cells of the present invention to express the anti-human Tie2 antibody. Examples of the preferred host cells for use in the method include the preferred transformed host cells of the present invention as described above.
The transformed host cell can be cultured by known methods. Culture conditions, for example, the temperature, pH of culture medium, and the culture time are appropriately selected. In a case where the host cell is an animal cell, examples of the culture medium include MEM culture medium supplemented with approximately 5% to 20% of fetal bovine serum (Science, 1959, Vol. 130, No. 3373, p. 432 to 7), DMEM culture medium (Virology, 1959, Vol. 8, p. 396), and RPMI1640 culture medium (J. Am. Mde. Assoc., 1967, Vol. 199, p. 519), a 199 culture medium (Exp. Biol. Med., 1950, Vol. 73, p. 1 to 8). The pH of the culture medium is preferably approximately 6 to 8, and the culture is generally carried out at approximately 30° C. to 40° C. for approximately 15 hours to 72 hours while air ventilating and stirring if necessary. In a case where the host cell is an insect cell, as the culture medium, for example, Grace's culture medium (Proc. Natl. Acad. Sci. USA, 1985, Vol. 82, p. 8404) supplemented with fetal bovine serum can be used. The pH of the culture medium is preferably approximately 5 to 8, and the culture is generally carried out at approximately 20° C. to 40° C. for approximately 15 hours to 100 hours while air ventilating and stirring if necessary. In a case where the host cell is Escherichia coli or yeast, as the culture medium, for example, liquid culture medium supplemented with a source of nutrients is appropriate. It is preferable that the nutrient culture medium contain a carbon source, an inorganic nitrogen source, or an organic nitrogen source necessary for the growth of the transformed host cell. Examples of the carbon source include glucose, dextran, soluble starch, and sucrose and examples of the inorganic nitrogen source or the organic nitrogen source include ammonium salts, nitrate salts, amino acids, corn steep liquor, peptone, casein, meat extract, soybean meal, and potato extract. Other nutrients (for example, inorganic salts (for example, calcium chloride, sodium dihydrogen phosphate, and magnesium chloride), vitamins), and antibiotics (for example, tetracycline, neomycin, ampicillin, and kanamycin) may be contained as desired. The pH of the culture medium is preferably approximately 5 to 8. In a case where the host cell is Escherichia coli, preferred examples of the culture medium include LB culture medium and M9 culture medium (Mol. Clo., Cold Spring Harbor Laboratory, Vol. 3, A2.2). The culture is generally carried out at approximately 14° C. to 39° C. for approximately 3 hours to 24 hours while air ventilating and stirring if necessary. In a case where the host cell is yeast, as the culture medium, for example, Burkholder minimal medium (Proc. Natl. Acad, Sci, USA, 1980, Vol. 77, p. 4505) can be used. The culture is generally carried out at approximately 20° C. to 35° C. for approximately 14 hours to 144 hours while air ventilating and stirring if necessary. By carrying out the culture in the above-described manner, it is possible to express the anti-human Tie2 antibody or the antigen-binding fragment thereof of the present invention.
The method of producing the anti-human Tie2 antibody or the antigen-binding fragment thereof of the present invention may comprise recovering, preferably isolating or purifying the anti-human Tie2 antibody or the antigen-binding fragment thereof from the transformed host cell in addition to culturing the transformed host cell of the present invention to express the anti-human Tie2 antibody or the antigen-binding fragment thereof. Examples of the isolation or purification method include methods using solubility such as salting-out and the solvent precipitation method, methods using the difference in molecular weight such as dialysis, ultrafiltration, and gel filtration, methods using an electric charge such as ion exchange chromatography and hydroxylapatite chromatography, methods using specific affinity such as affinity chromatography, methods using the difference in hydrophobicity such as reverse phase high performance liquid chromatography, and methods using the difference in the isoelectric point such as isoelectric focusing phoresis. Preferably, the antibody accumulated in a culture supernatant can be purified by various chromatographies, for example, column chromatography using Protein A column or Protein G column.
The anti-human Tie2 antibody or the antigen-binding fragment thereof of the present invention also includes an anti-human Tie2 antibody or an antigen-binding fragment thereof produced by the method for producing the anti-human Tie2 antibody or the antigen-binding fragment thereof of the present invention.
<Pharmaceutical Composition of the Present Invention>
The pharmaceutical compositions of the present invention include a pharmaceutical composition comprising the anti-human Tie2 antibody or the antigen-binding fragment thereof of the present invention and pharmaceutically acceptable excipients. The pharmaceutical composition of the present invention can be prepared by a method being generally used with excipients being generally used in the field, that is, excipients for medicine or carriers for medicine. Examples of dosage forms of the pharmaceutical compositions include parenteral drug such as an injection drug and a drip infusion drug, and these can be administered by intravenous administration, subcutaneous administration, intraocular administration, or the like. In drug preparation, excipients, carriers, and additives in accordance with the dosage forms can be used within the pharmaceutically acceptable range.
The pharmaceutical compositions of the present invention may comprise plural kinds of anti-human Tie2 antibodies or antigen-binding fragments thereof of the present invention. For example, the present invention includes a pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof, which does not undergo posttranslational modification and an antibody or an antigen-binding fragment thereof derived from posttranslational modification of the antibody or the antigen-binding fragment thereof.
In one embodiment, the pharmaceutical composition of the present invention comprising an anti-human Tie2 antibody or an antigen-binding fragment thereof, includes a pharmaceutical composition as described below.
A pharmaceutical composition comprising an anti-human Tie2 antibody or an antigen-binding fragment thereof, in which the anti-human Tie2 antibody or the antigen-binding fragment thereof comprises four heavy chain variable regions and four light chain variable regions, the heavy chain variable region consists of the amino acid sequence of the amino acid numbers 1 to 122 of SEQ ID NO: 2, the light chain variable region consists of the amino acid sequence of the amino acid numbers 1 to 113 of SEQ ID NO: 4, the one heavy chain variable region and the one light chain variable region constitute one antigen-binding site, and the antibody or the antigen-binding fragment thereof comprises four antigen-binding sites, and an antibody or an antigen-binding fragment thereof derived from posttranslational modification of the antibody or the antigen-binding fragment thereof.
In one embodiment, the pharmaceutical composition comprising the anti-human Tie2 antibody of the present invention includes the pharmaceutical composition as described below.
A pharmaceutical composition comprising an anti-human Tie2 antibody which is an anti-human Tie2 antibody and an antibody formed by posttranslational modification of the antibody, comprising two heavy chains and four light chains, in which each heavy chain comprises two structures consisting of a heavy chain variable region consisting of the amino acid sequence of the amino acid numbers 1 to 122 of SEQ ID NO: 2 and a CH1 region, a CH2 region, and a CH3 region, and the C terminus of one of the structures is linked to the N terminus of the other structure through a linker, and each light chain comprises a light chain variable region consisting of the amino acid sequence of the amino acid numbers 1 to 113 of SEQ ID NO: 4, and a light chain constant region, and the antibody comprises four antigen-binding sites, and an antibody derived from posttranslational modification of the antibody.
The pharmaceutical compositions of the present invention also include a pharmaceutical composition comprising an antibody in which lysine of the C terminus of the heavy chain is deleted, an antibody or an antigen-binding fragment thereof with posttranslational modification to N terminal, an antibody in which lysine of the C terminus of the heavy chain is deleted and posttranslation modification to N terminal is made, and/or an antibody which has lysine in the C terminus of the heavy chain and does not have posttranslational modification to N terminal.
In one embodiment, the pharmaceutical composition of the present invention comprising an anti-human Tie2 antibody includes a pharmaceutical composition comprising at least two kinds of anti-human Tie2 antibodies selected from (1) to (4) below.
(1) An anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence of the amino acid numbers 1 to 678 of SEQ ID NO: 2 and four light chains consisting of the amino acid sequence shown by SEQ ID NO: 4.
(2) An anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence of SEQ ID NO: 2 in which glutamic acid of amino acid number 1 is modified to pyroglutamic acid and four light chains consisting of the amino acid sequence shown by SEQ ID NO: 4.
(3) An anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence of the amino acid numbers 1 to 678 of SEQ ID NO: 2 in which glutamic acid of amino acid number 1 is modified to pyroglutamic acid and four light chains consisting of the amino acid sequence shown by SEQ ID NO: 4.
(4) An anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence shown by SEQ ID NO: 2 and four light chains consisting of the amino acid sequence shown by SEQ ID NO: 4.
In one embodiment, the pharmaceutical composition of the present invention comprising an anti-human Tie2 antibody includes a pharmaceutical composition comprising at least two kinds of anti-human Tie2 antibodies selected from (1) to (4) below.
(1) An anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence of the amino acid numbers 1 to 678 of SEQ ID NO: 6 and four light chains consisting of the amino acid sequence shown by SEQ ID NO: 4.
(2) An anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence of SEQ ID NO: 6 in which glutamic acid of amino acid number 1 is modified to pyroglutamic acid and four light chains consisting of the amino acid sequence shown by SEQ ID NO: 4.
(3) An anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence of the amino acid numbers 1 to 678 of SEQ ID NO: 6 in which glutamic acid of amino acid number 1 is modified to pyroglutamic acid and four light chains consisting of the amino acid sequence shown by SEQ ID NO: 4.
(4) An anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence shown by SEQ ID NO: 6 and four light chains consisting of the amino acid sequence shown by SEQ ID NO: 4.
In one embodiment, the pharmaceutical composition of the present invention comprising an anti-human Tie2 antibody includes a pharmaceutical composition comprising at least two kinds of anti-human Tie2 antibodies selected from (1) to (4) below.
(1) An anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence of the amino acid numbers 1 to 678 of SEQ ID NO: 8 and four light chains consisting of the amino acid sequence shown by SEQ ID NO: 4.
(2) An anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence of SEQ ID NO: 8 in which glutamic acid of amino acid number 1 is modified to pyroglutamic acid and four light chains consisting of the amino acid sequence shown by SEQ ID NO: 4.
(3) An anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence of the amino acid numbers 1 to 678 of SEQ ID NO: 8 in which glutamic acid of amino acid number 1 is modified to pyroglutamic acid and four light chains consisting of the amino acid sequence shown by SEQ ID NO: 4.
(4) An anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence shown by SEQ ID NO: 8 and four light chains consisting of the amino acid sequence shown by SEQ ID NO: 4.
In one embodiment, the pharmaceutical composition of the present invention comprising an anti-human Tie2 antibody includes a pharmaceutical composition comprising at least two kinds of anti-human Tie2 antibodies selected from (1) to (4) below.
(1) An anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence of the amino acid numbers 1 to 675 of SEQ ID NO: 10 and four light chains consisting of the amino acid sequence shown by SEQ ID NO: 4.
(2) An anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence of SEQ ID NO: 10 in which glutamic acid of amino acid number 1 is modified to pyroglutamic acid and four light chains consisting of the amino acid sequence shown by SEQ ID NO: 4.
(3) An anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence of the amino acid numbers 1 to 675 of SEQ ID NO: 10 in which glutamic acid of amino acid number 1 is modified to pyroglutamic acid and four light chains consisting of the amino acid sequence shown by SEQ ID NO: 4.
(4) An anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence shown by SEQ ID NO: 10 and four light chains consisting of the amino acid sequence shown by SEQ ID NO: 4.
In one embodiment, the pharmaceutical composition of the present invention comprising an anti-human Tie2 antibody or an antigen-binding fragment thereof also includes the pharmaceutical composition as described below.
A pharmaceutical composition comprising an anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence shown by SEQ ID NO: 2 and four light chains consisting of the amino acid sequence shown by SEQ ID NO: 4, an anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence of the amino acid numbers 1 to 678 of SEQ ID NO: 2 and four light chains consisting of the amino acid sequence shown by SEQ ID NO: 4, and a pharmaceutically acceptable excipient.
The amount of the anti-human Tie2 antibody or the antigen-binding fragment thereof of the present invention added in formulation varies depending on the degree of symptoms and the age of a patient, a dosage form of a preparation to be used, the binding titer of an antibody, or the like, and for example, an amount added of approximately 0.001 mg/kg to 100 mg/kg can be used.
The pharmaceutical composition of the present invention can be used as an agent for preventing or treating blood vessel-related diseases, for example, diabetic retinopathy, diabetic macular edema, sepsis, acute hepatic disorders, acute renal disorders, acute pulmonary disorders, systemic inflammatory reaction syndrome, peripheral arterial occlusive disease, or critical limb ischemia.
The present invention includes a pharmaceutical composition for preventing or treating diabetic macular edema, diabetic retinopathy, or critical limb ischemia, comprising the anti-human Tie2 antibody of the present invention. Further, the present invention includes a method for preventing or treating diabetic macular edema, diabetic retinopathy, or critical limb ischemia, comprising administering a therapeutically effective amount of the anti-human Tie2 antibody of the present invention. Further, the present invention includes the anti-human Tie2 antibody of the present invention for use in preventing or treating diabetic macular edema, diabetic retinopathy, or critical limb ischemia. In addition, the present invention includes use of the anti-human Tie2 antibody of the present invention for preparation of a pharmaceutical composition for preventing or treating diabetic macular edema, diabetic retinopathy, or critical limb ischemia.
<Fusion Antibody and Modification Antibody>
Any person skilled in the art can prepare a fusion antibody in which an antibody or an antigen-binding fragment thereof is fused with another peptide or protein, and can also prepare a modification antibody to which a modifying agent is bound, using a known method in the field. The anti-human Tie2 antibody or the antigen-binding fragment thereof of the present invention includes the antibody and the antigen-binding fragment thereof in the form of such a fusion or a modification. For example, the anti-human Tie2 antibody or an antigen-binding fragment thereof, comprising four heavy chain variable regions and four light chain variable regions, in which the heavy chain variable region consists of the amino acid sequence of the amino acid numbers 1 to 122 of SEQ ID NO: 2, the light chain variable region consists of the amino acid sequence of the amino acid numbers 1 to 113 of SEQ ID NO: 4, the one heavy chain variable region and the one light chain variable region constitute one antigen-binding site, and the antibody or the antigen-binding fragment thereof comprises four antigen-binding sites, includes an anti-human Tie2 antibody or an antigen-binding fragment thereof fused with another peptide or protein, and an anti-human Tie2 antibody or an antigen-binding fragment thereof having a modifying agent bound thereto. The other peptide or protein for use in the fusion is not particularly limited as long as the antibody or the antigen-binding fragment thereof of the present invention as a fusion has binding activity to a human Tie2, and examples thereof include human serum albumin, various tag peptides, artificial helix motif peptides, maltose-binding protein, a glutathione S transferase, various toxins, and other peptides or proteins capable of promoting multimerization. The modifying agent for use in the modification is not particularly limited as long as the antibody or an antigen-binding fragment thereof of the present invention as a modification antibody has binding activity to a human Tie2, and examples thereof include polyethylene glycol, sugar chains, phospholipids, liposomes, and low-molecular compounds.
The present invention has been described and specific examples referred to for better understanding will be provided, but these are merely examples and the present invention is not limited thereto.
EXAMPLES
With regard to parts using commercially available kits or reagents, the experiments were carried out according to the described protocol unless specifically otherwise noted. For the sake of convenience, a concentration in mol/L is represented by M. For example, a 1 M aqueous sodium oxide solution means a 1 mol/L aqueous sodium oxide solution.
Example 1: Preparation of Hybridoma Producing Anti-Human Tie2 Antibody
Antibody was prepared by using the “VelocImmune” (VelocImmune antibody technology: Regeneron, Inc. (U.S. Pat. No. 6,596,541))—human monoclonal antibody developing technology-mouse. A recombinant human Tie2-Fc chimeric protein (R&D, 313-TI-100) was injected into the VelocImmune mouse, together with an adjuvant for causing an immune reaction, so as to perform immunization. According to an ordinary method, the lymph node of the immunized mouse was extracted, and the lymphocytes were collected and cell-fused with mouse-derived myeloma cell SP2/0 (ATCC: CRL-1581), thereby preparing a hybridoma. The hybridoma was monocloned and each clone was cultured in a CD Hybridoma Medium (Invitrogen) which is a serum-free culture medium. The antibody was purified from the obtained culture supernatant using a Protein G Column (GE Healthcare). The antibody obtained by using the VelocImmune technology is an antibody having a variable region of the human antibody and a constant region of the mouse antibody (also referred to a chimeric antibody).
Example 2: Cell ELISA Assay
In order to measure the antigen-binding activity of the antibody, the bindings of the antibody to a human Tie2, a monkey Tie2, a rat Tie2, and a mouse Tie2 were each evaluated by cell ELISA assay using a human Tie2-expressing CHO cell, a monkey Tie2-expressing CHO cell, a rat Tie2-expressing CHO cell, and a mouse Tie2-expressing CHO cell.
Example 3: Evaluation of Competitive Activity Using Modified Ang-1
In order to evaluate the Ang-2 competitive activity of the antibody, the inhibition of the binding of a modified Ang-1 (Proc. Natl. Acad. Sci., 2004, Vol. 101, pp. 5547-5552, also referred to as COMP-Ang1.) to Tie2 was evaluated. The COMP-Ang1 is a modified Ang-1 in which a site not involved in the binding to Tie2 is modified, and its competitive action against Ang-2 can be evaluated by evaluating the competitive action against COMP-Ang1 from the viewpoints that the binding capacity of COMP-Ang1 to Tie2 is maintained (Proc. Natl. Acad. Sci. 2004, Vol. 101, pp. 5547-5552), and Ang-1 and Ang-2 bind to the same site of Tie2 with the same level of affinity (Science, 1997, Vol. 277, pp. 55-60).
An expression vector of COMP-Ang1 was introduced into an HEK293 cell. The COMP-Ang1 was purified from a culture supernatant of the HEK293 cell, and biotin-labeled. The biotin-labeled COMP-Ang1 and the purified antibody obtained in Example 1 were mixed, and the mixture was added to a plate immobilized with a recombinant human Tie2-Fc chimeric protein. For the detection of the biotin-labeled COMP-Ang1 thus bound, a streptavidin-labeled HRP was used. A TMB color developing reagent (Dako, 51599) was added thereto and left to stand. Further, a 2 M sulfuric acid was then added thereto to stop the reaction and an absorbance at 450 nm was measured. In this manner, the competitive action of the antibody against the COMP-Ang1 was evaluated.
Example 4: Evaluation of Anti-Apoptotic Activity Using Human Tie2-Expressing BaF3 Cell
A mouse pro-B cell strain BaF3 cell which stably expresses a human Tie2 (hereinafter also referred to as a human Tie2-expressing BaF3 cell) was prepared by introducing a plasmid containing a human Tie2 gene shown by SEQ ID NO: 21 to the cell by electroporation according to the method described in Immunity, 1998, Vol. 9, pp. 677-686. Thereafter, the anti-apoptotic activity of the antibody was evaluated using the same cell.
The human Tie2-expressing BaF3 cell was suspended in an RPMI1640 medium (Life Technologies) supplemented with 0.05% fetal bovine serum albumin at 2×105 cells/mL, and distributed in the amount of 80 μL per well in a 96-well plate for floating cells (Sumitomo Bakelite Co., Ltd., MS-8096R). Thereafter, 20 μL of the purified antibody obtained in Example 1 or Ang-1 was added thereto. After culturing for 72 hours in a CO2 incubator set to 37° C., 50 μL of the cell suspension was transferred to a white 96-well plate (Nunc, 236108). According to an intracellular ATP quantification reagent CellTiter Glo Luminescent Cell Viability Kit (Promega), by adding 50 μL of a substrate solution diluted with an attached buffer to the cell suspension, the viability of the cell was measured, thereby evaluating anti-apoptotic activity.
From the results of Examples 2 to 4, antibodies having a binding activity to a human Tie2, a monkey Tie2, a rat Tie2, and a mouse Tie2, a COMP-Ang1 competitive activity, and anti-apoptotic activity to a human Tie2 were found. The purified antibody solution comprising an anti-human Tie2 antibody nominated as 2-16 which will be described later exhibited substantially the same anti-apoptotic activity as Ang-1 in Example 4, but the purified antibody solution comprising the mouse anti-human Tie2 antibody 15B8 (Patent Document 1) exhibited only approximately 60% of the maximum activity of the Ang-1.
Example 5: Analysis of Purified Antibody Solution Using Size Exclusion Chromatography and Electrophoresis
The purified antibody solutions identified in Examples 2 to 4 above were analyzed by size exclusion chromatography. As a result, three fractions were detected from the respective purified antibody solutions. As a result of the analysis of the respective fraction solutions by electrophoresis, it was found that the respective fractions include monomers, dimers, trimers or higher-valent multimers of the antibodies, respectively.
Next, the respective fraction solutions were evaluated regarding the anti-apoptotic activity by the method shown in Example 4. As a result, in the fractions comprising the dimers and the fractions comprising the trimers or higher-valent multimers, potent anti-apoptotic activity was recognized. On the other hand, in the fraction comprising monomers from the respective antibodies, anti-apoptotic activity was substantially unrecognized. 15B8 was also analyzed by size exclusion chromatography as described above, and as a result, fractions showing dimers or higher-valent multimers were detected, but fractions comprising monomers were substantially undetected.
From the above, it was found that in any antibody identified in Examples 2 to 4, the fractions comprising the antibodies formed into dimers or higher-order multimers pertained potent anti-apoptotic activities. It is suggested that antibodies having four or higher valences have the strong anti-apoptotic activity through Tie2 activation as a dimer is a tetravalent antibody.
Example 6: Evaluation of Anti-Apoptotic Activity by Cross-Linking Antibody
From the investigations in Example 5, it is considered that the valence of the anti-human Tie2 antibody adjusted to be 4 or higher is important to induce the anti-apoptotic activity through Tie2. Thus, the anti-apoptotic activity of the anti-human Tie2 antibody which was multimerized by performing cross-linking with an anti-mouse IgG antibody was evaluated. As a cell, a human Tie2-expressing BaF3 cell and a human vascular endothelial cell HUVEC that endogenously expresses a human Tie2 were used.
The human Tie2-expressing BaF3 cell and HUVEC were cultured in an RPMI1640 medium and an EBM-2 serum-free medium (Lonza), respectively, to which an antibody solution comprising the anti-human Tie2 antibodies identified in Examples 2 to 4 had been added. An anti-mouse IgG antibody was added thereto to cross-link the antibodies. By employing CellTiter Glo Luminescent Cell Viability Assay, the viability of the cells was measured. By measuring the viability, the anti-apoptotic activity was evaluated.
As a result, it was found that the cross-linking antibody of the anti-human Tie2 antibody (chimeric antibody) nominated as 2-16 has a potent anti-apoptotic activity on the human Tie2.
Example 7: Sequencing of Bivalent Anti-Human Tie2 Antibody
A gene encoding the heavy and light chains of the antibody was cloned from a hybridoma producing the anti-human Tie2 antibody 2-16, and sequenced.
After sequencing the antibody, the framework region (FR) of the light and heavy chains of 2-16 was replaced with the FR of another human antibody in order to improve the physical properties and the stability of the antibody, thereby preparing a modified variable region of anti-human Tie2 antibody 2-16A2.
A gene encoding a signal sequence (Protein Engineering, 1987, Vol. 1, No. 6, pp. 499-505) and a human Igγ1 constant region gene (consisting of the base sequence of base numbers 367 to 1356 of SEQ ID NO: 11) were linked to the 5′ side and the 3′ side, respectively, of the heavy chain variable region gene of 2-16A2, and the heavy chain gene was inserted into a GS vector pEE6.4. Further, a gene encoding a signal sequence (Protein Engineering, 1987, Vol. 1, No. 6, pp. 499-505) and a constant region gene (consisting of the base sequence of base numbers 340 to 657 of SEQ ID NO: 3) of a human chain were connected to the 5′ side and the 3′ side, respectively, of the light chain variable region gene. This light chain gene was inserted into GS vector pEE12.4. The heavy chain gene sequence and the light chain gene sequence of the prepared antibody were analyzed using a sequencer.
The base sequence of the heavy chain of the fully human antibody of 2-16A2 (fully human 2-16A2) and the amino acid sequence encoded by the base sequence are shown by SEQ ID NOS: 11 and 12, respectively. Further, the base sequence of the light chain of the antibody and the amino acid sequence encoded by the base sequence are shown by SEQ ID NOS: 3 and 4, respectively. The variable region of the heavy chain shown by SEQ ID NO: 12 consists of the amino acid sequence of the amino acid numbers 1 to 122 of SEQ ID NO: 12, and the variable region of the light chain shown by SEQ ID NO: 4 consists of the amino acid sequence of the amino acid numbers 1 to 113 of SEQ ID NO: 4.
By using the GS vector as described above, into which the genes of the heavy chain and the light chain of the fully human 2-16A2 had each been inserted, the antibody expression was performed by using two types of methods, that is, transient expression and stably expression. With regard to transient expression, FreeStyle 293 cells (Invitrogen) cultured in a FreeStyle 293 Expression medium (Invitrogen) at about 1,000,000 cells/mL were transfected with both expression vectors of the heavy chain and the light chain as described above using a transfection kit, 293 Fectin (Invitrogen), and cultured for 5 days. Alternatively, Expi 293 cells (Invitrogen) cultured in an Expi 293 Expression medium (Invitrogen) at about 3,000,000 cells/mL were transfected by both expression vectors of the heavy chain and the light chain as described above using a transfection kit, ExpiFectamine 293 Transfection kit (Invitrogen), and cultured for 7 days. Alternatively, CHO—K1SV cells (Lonza) cultured in a CD-CHO medium (Invitrogen) at about 10,000,000 cells/mL were transfected both expression vectors of the heavy chain and the light chain as described above using an electroporation method, and cultured for 7 days. The fully human antibody was purified from each of the culture supernatants using a Protein A column or a Protein G column (GE HealthCare). With regard to stable expression, the GS vector as described above, into which the genes of the heavy chain and the light chain of the antibody had been each inserted, was digested with restriction enzymes of NotI and PvuI, and ligated using a Ligation-Convenience Kit (NIPPONGENE) as a kit for ligation or a ligation reagent, Ligation high Ver. 2 (TOYOBO), thereby constructing a GS vector, into which both genes of the heavy chain and the light chain had been inserted. The antibody was expressed by transfection of the expression vector into the CHO—K1SV cells. The fully human antibody was purified from culture supernatant by a Protein A column, a Protein G column, or a MabSelect SuRe (GE Healthcare, 17-5438-02).
Example 8: Preparation of Tetravalent Anti-Human Tie2 Antibody
A tetravalent anti-human Tie2 antibody was prepared. The tetravalent antibody prepared in the present Example includes two heavy chains and four light chains. Each heavy chain comprises two structures consisting of a heavy chain variable region and a CH1 region, and further comprises a CH2 region, and a CH3 region, in which the C terminus of one structure consisting of the heavy chain variable region and the CH1 region is linked to the N terminus of the other structure through a linker. Each light chain comprises a light chain variable region and a light chain constant region. The format of the present tetravalent antibody is shown in FIG. 1.
A gene encoding a tetravalent anti-human Tie2 antibody heavy chain, in which the C terminus of a structure (consisting of the amino acid sequence of the amino acid numbers 1 to 220 of SEQ ID NO: 12) consisting of the heavy chain variable region and the CH1 region of the fully human 2-16A2 was linked to the N terminus of the fully human 2-16A2 heavy chain through a linker consisting of the amino acid sequence shown by SEQ ID NO: 13, was prepared. A gene encoding a signal sequence (Protein Engineering, 1987, Vol. 1, No. 6, pp. 499-505) was linked to the 5′ side of the prepared heavy chain gene, and inserted into a GS vector pEE6.4. The above heavy chain vector and the GS vector pEE12.4, into which the light chain gene of the fully human antibody 2-16A2 prepared in Example 7 had been inserted, were combined to prepare a tetravalent anti-human Tie2 antibody using the same antibody expression and purification method as described in Example 7. The tetravalent anti-human Tie2 antibody is referred to as TIE-1-Igγ1-WT.
A gene encoding a tetravalent anti-human Tie2 antibody heavy chain having a constant region of the heavy chain of TIE-1-Igγ1-WT substituted with a human Igγ4 constant region (consisting of the amino acid sequence of the amino acid numbers 123 to 220 of SEQ ID NO: 10, and consisting of the amino acid sequence of the amino acid numbers 350 to 676 of SEQ ID NO: 10) with amino acid mutations of S228P and L235E, was prepared. A gene encoding a signal sequence (Protein Engineering, 1987, Vol. 1, No. 6, pp. 499-505) was linked to the 5′ side of the prepared heavy chain gene and inserted into a GS vector pEE6.4. The above heavy chain vector and the GS vector pEE12.4, into which the light chain gene of the fully human 2-16A2 prepared in Example 7 had been inserted, were combined to prepare a tetravalent anti-human Tie2 antibody using the same antibody expression and purification method as described in Example 7. The tetravalent anti-human Tie2 antibody with IgG4 is referred to as TIE-1-Igγ4-PE.
The base sequence of the heavy chain of TIE-1-Igγ1-WT and the amino acid sequence encoded by the base sequence are shown by SEQ ID NOS: 7 and 8, respectively. The base sequence of the heavy chain of TIE-1-Igγ4-PE and the amino acid sequence encoded by the base sequence are shown by SEQ ID NOS: 9 and 10, respectively. The light chain of both the antibodies are the same as the light chain of the fully human antibody 2-16A2, and the base sequence of the light chain and the amino acid sequence encoded by the base sequence of the antibody are shown by SEQ ID NOS: 3 and 4, respectively.
By using the same method, a tetravalent anti-human Tie2 antibody, in which amino acid variations of L234A, L235A, and P331S had been introduced to the constant region of the heavy chain of TIE-1-Igγ1-WT (referred to as TIE-1-Igγ1-LALA), and a tetravalent anti-human Tie2 antibody, in which amino acid variations of L234A, L235A, P331S, and I253A had been introduced to the constant region of the heavy chain of TIE-1-Igγ1-WT (referred to as TIE-1-Igγ1-I253A), were prepared.
The base sequence of the heavy chain and the amino acid sequence encoded by the base sequence of TIE-1-Igγ1-LALA are shown by SEQ ID NOS: 1 and 2, respectively. The base sequence of the heavy chain and the amino acid sequence encoded by the base sequence of TIE-1-Igγ1-I253A are shown by SEQ ID NOS: 5 and 6, respectively. The light chains of both the antibodies were the same as the light chain of the fully human 2-16A2, and the base sequence of the light chain and the amino acid sequence encoded by the base sequence of the antibody were shown by SEQ ID NOS: 3 and 4, respectively.
The variable regions of the heavy chains of four kinds of the tetravalent anti-human Tie2 antibodies shown by SEQ ID NOS: 2, 6, 8, and 10 are common and consist of the amino acid sequence of the amino acid numbers 1 to 122 of SEQ ID NO: 2. The CDR1, CDR2, and CDR3 of the heavy chain variable regions each consist of the amino acid sequence of the amino acid numbers 31 to 35, 50 to 66, and 99 to 111 of SEQ ID NO: 2.
The variable regions of the light chains of four kinds of the tetravalent anti-human Tie2 antibodies shown by SEQ ID NO: 4 each consist of the amino acid sequence of the amino acid numbers 1 to 113 of SEQ ID NO: 4. The CDR1, CDR2, and CDR3 of the light chain variable regions consist of the amino acid sequence of the amino acid numbers 24 to 39, 55 to 61, and 94 to 102 of SEQ ID NO: 4, respectively.
As a result of the analysis of the amino acid modifications of the purified TIE-1-Igγ1-LALA, it was found that in most of the purified antibodies, deletion of lysine at the C terminus of the heavy chain occurred.
In addition, by using the same method, tetravalent anti-human Tie2 antibodies, in which with respect to TIE-1-Igγ1-WT and TIE-1-Igγ4-PE, the linker (consisting of the amino acid sequence shown by SEQ ID NO: 13) was substituted with other linkers (four kinds of linkers consisting of the amino acid sequences shown by SEQ ID NOS: 17 to 20 with respect to TIE-1-Igγ1-WT, and seven kinds of linkers consisting of the amino acid sequences shown by SEQ ID NOS: 14 to 20 with respect to TIE-1-Igγ4-PE), were prepared (total 11 kinds). A linker having a length of 7 amino acids (a linker consisting of the amino acid sequence shown by SEQ ID NO: 13) to a linker having a length of 64 amino acids (a linker consisting of the amino acid sequence shown by SEQ ID NO: 20) were investigated.
As a result of the investigations on TIE-1-Igγ1-WT, TIE-1-Igγ4-PE and 11 kinds of antibodies in which the linker were substituted, it was found that all of the anti-human Tie2 antibodies had substantially the same anti-apoptotic activities in accordance with the method of Example 4.
Example 9: Evaluation of Anti-Apoptotic Action of Bivalent Anti-Human Tie2 Antibody and Tetravalent Anti-Human Tie2 Antibody
From the results of Example 5, it was suggested that a tetravalent or higher-valent antibody has a potent anti-apoptotic activity through a human Tie2 activation. Thus, the efficacy of the bivalent anti-human Tie2 antibody was compared with that of the tetravalent anti-human Tie2 antibody by measuring the anti-apoptotic action on the human Tie2-expressing BaF3 cell as an index.
According to the method of Example 4, the anti-apoptotic action of the fully human 2-16A2 which is a bivalent antibody and TIE-1-Igγ1-WT which is a tetravalent antibody was evaluated by using the human Tie2-expressing BaF3 cell. The fully human 2-16A2 and TIE-1-Igγ1-WT which were tested antibodies were purified by MabSelect SuRe and fractionized into monomer fractions by size exclusion chromatography, thereby acquiring monomer purities of 99.98% and 99.74%, respectively. The respective antibodies were diluted with phosphate buffer saline (PBS) to from 5 ng/mL to 5000 ng/mL at an about 3-fold common ratio through seven steps, and added in the amount of 20 μL per well. As a control, PBS, or Ang-1 diluted with PBS (R&D, 923-AN-025/CF, a final concentration of 1 ng/mL to 1000 ng/mL, diluted at an about 3-fold common ratio through 7 steps) had been added instead of the test antibodies, were prepared, respectively. For calculation of the anti-apoptotic activity at each of the concentrations of the test antibodies, the measured value of the well to which PBS had been added instead of the test antibody was set to 0%, and the average value of the measured values of the wells to which Ang-1 had been added at the concentration of 300 ng/mL and 1000 ng/mL, respectively, instead of the test antibodies, was set to 100%. The EC50 value of the test antibody was calculated by analyzing the calculated anti-apoptotic activity using Sigmoid-Emax model non-linear regression analysis.
TABLE 1
Anti-Apoptotic Activities of Bivalent Anti-Human Tie2
Antibody and Tetravalent Anti-Human Tie2 Antibody
Maximum activity of anti-
EC50 value
apoptotic activities
TIE-1-Igγ1-WT
7.10 ng/mL
104%
Fully human 2-16A2
37.6 ng/mL
22%
As a result, it was found that TIE-1-Igγ1-WT which is a tetravalent antibody has potent anti-apoptotic action. From the above, it was found that the tetravalent antibody has a superior anti-apoptotic activity, as compared with the bivalent antibody.
Example 10: Evaluation of Vascular Permeability Inhibitory Action of Bivalent Anti-Human Tie2 Antibody and Tetravalent Anti-Human Tie2 Antibody in Rat
A mustard oil-induced vascular permeability model is a model with a modification applied to a Miles assay (J. Physiol., 1952, Vol. 118, pp. 228-257) which has been widely used as a plasma leakage evaluation system, and it has been reported that Ang-1 inhibits the vascular hyperpermeability in the present model (Nature Medicine, 2000, Vol. 6, pp. 460-463). Accordingly, in order to compare the vascular permeability inhibitory action of the bivalent anti-human Tie2 antibody with that of the tetravalent anti-human Tie2 antibody, the fully human 2-16A2 and TIE-1-Igγ1-WT were evaluated using the present model.
The fully human 2-16A2 or TIE-1-Igγ1-WT diluted with PBS was subcutaneously administered to an SD rat (Male, 4-5-week-old, Charles River Laboratories Japan, Inc.). The treated groups were set as follows.
[Treated group (6 rats per group)]
Vehicle Group:
Group to which PBS instead of the antibody was administered
Fully human 2-16A2 administration group:
Group to which the fully human 2-16A2 was administered (0.3 mg/kg)
TIE-1-Igγ1-WT administration group:
Group to which TIE-1-Igγ1-WT was administered (0.3 mg/kg)
At 48 hours after the administration of the antibody, an Evans Blue dye dissolved in physiological saline (45 mg/kg, Sigma-Aldrich Corporation, E2129) was intravenously administered, immediately allyl isothiocyanate (also referred to as a mustard oil, Nacalai Tesque, Inc., 01415-92) diluted with a mineral oil (Sigma-Aldrich Corporation, M8410) of 5% was applied onto one ear, while the mineral oil was applied onto the contralateral ear, in the amount of 20 μl. After 30 minutes, both of the ears were sampled, weighed, then immersed in 1 mL of formamide, and incubated at 70° C. overnight to extract the Evans Blue dye in the ear tissue. The Evans Blue dye concentration was determined from the absorbance (a measurement wavelength of 620 nm and a control wavelength of 740 nm) of the extract to calculate the amount of the Evans Blue dye in the extract. Thereafter, by dividing the amount of the Evans Blue dye by the weight of the ear, the dye leakage amount per weight of the ear was calculated. A value obtained by subtracting the leakage amount of the Evans Blue dye of the ear having the mineral oil applied thereon from the leakage amount of the Evans Blue dye of the ear having the mustard oil applied thereon in the same individual was calculated as a final leakage amount of the Evans Blue dye of each individual. The leakage amount of the Evans Blue dye was used as an index of vascular permeability. The results are shown in FIG. 2.
The mean value and the standard error of each group were determined. A Student t-test was used to determine a significant difference between the vehicle group and each group to which an antibody had been administrated. A case with p<0.05 was intended to indicate that there was a significant difference.
As shown in FIG. 2, compared with a vehicle group, the fully human 2-16A2 which is a bivalent antibody did not inhibit the dye leakage, whereas TIE-1-Igγ1-WT which is a tetravalent antibody significantly inhibited the dye leakage. It was found that TIE-1-Igγ1-WT which is a tetravalent antibody inhibited vascular hyperpermeability. From above, it was found that the tetravalent antibody has a superior vascular hyperpermeability inhibitory action, as compared with the bivalent antibody.
From the results of Examples 9 and 10, it was found that the tetravalent anti-Tie2 antibody strongly induced an action through Tie2.
Example 11: Evaluation of Anti-Apoptotic Action of Tetravalent Anti-Human Tie2 Antibody (2)
For TIE-1-Igγ1-LALA and TIE-1-Igγ1-I253A, according to the method of Example 9, the anti-apoptotic activity of the antibody on the human Tie2-expressing BaF3 cell was evaluated. In the same concentration range as in Example 9, evaluation of each tetravalent anti-human Tie2 antibody was carried out. In this regard, when the average value of the measured values of the wells, to which each of 100 ng/mL, 300 ng/mL, and 1000 ng/mL of Ang-1 had been added, was taken as 100%, the EC50 value and the maximum activity of the anti-apoptotic activity of each antibody were evaluated.
TABLE 2
Anti-Apoptotic Activity of Each Tetravalent
Anti-Human Tie2 Antibody
Maximum activity of anti-
EC50 value
apoptotic activities
TIE-1-Igγ1-LALA
3.65 ng/mL
88%
TIE-1-Igγ1-I253A
5.06 ng/mL
94%
As a result, it was found that both the TIE-1-Igγ1-LALA and the TIE-1-Igγ1-I253A exhibited substantially equivalent anti-apoptotic activity as Ang-1.
Reference Example 1: Evaluation of Anti-Apoptotic Action of 15B8
For 15B8, according to the method of Example 9, the anti-apoptotic activity on the human Tie2-expressing BaF3 cell was evaluated. Evaluation of 15B8 (Patent Document 1) was carried out in the same antibody concentration range as in Example 9. Evaluation of Ang-2 (R&D, 623-AN-025) was carried out in the same manner as that for Ang-1. In this regard, when the average value of the measured values of the wells, to which 1000 ng/mL of Ang-1 had been added, was taken as 100%, the EC50 value and the maximum activity of the anti-apoptotic activity were evaluated.
TABLE 3
Anti-Apoptotic Activity of 15B8
Maximum activity of anti-
EC50 value
apoptotic activities
15B8
26.6 ng/mL
64%
Ang-2
39.3 ng/mL
67%
As a result, it was found that the anti-apoptotic activity of 15B8 was about 64% of Ang-1 and had substantially equivalent anti-apoptotic activity as Ang-2.
As combined with the results of Example 11, it was found that TIE-1-Igγ1-LALA exhibited substantially equivalent anti-apoptotic activity as Ang-1, whereas 15B8 exhibited substantially equivalent partial anti-apoptotic activity as Ang-2.
Example 12: Evaluation of Binding Activity of TIE-1-Igγ1-LALA to Tie2
For TIE-1-Igγ1-LALA, the binding activities to each species Tie2 proteins were evaluated. A recombinant human Tie2-Fc chimeric protein (R&D, 313-TI-100), a recombinant monkey Tie2-Fc chimeric protein (Sino Biological Inc., 90292-C02H), a recombinant rat Tie2-Fc chimeric protein (R&D, 3874-T2-100), or a recombinant mouse Tie2-Fc chimeric protein (R&D, 762-T2-100) was prepared in PBS at 1 μg/mL, added to a white Maxisorp 384-well plate (Nunc, 460372) in the amount of 20 μL per well, and incubated at 4° C. overnight to perform immobilization. The next day, the immobilized solution was removed, and 20% Blocking One (Nacalai Tesque Inc., 03953-95)—containing Tris Buffer Saline (TBS)—0.05% Tween (Wako, 310-7375) (hereinafter referred to as a TBS-T solution) was added thereto in the amount of 50 μL per well, and left to stand at room temperature for 1 hour. TIE-1-Igγ1-LALA as a test antibody was diluted with a TBS-T solution containing 5% Blocking One from 0.03 ng/mL to 100 ng/mL at an about 3-fold common ratio through 8 steps, and added in the amount of 20 μL per well. As a control, a well to which a TBS-T solution had been added instead of the test antibody was prepared. The resultant was incubated at room temperature for 1.5 hours, and then washed with a TBS-T solution. As a secondary antibody, a biotin-labeled anti-human kappa light chain antibody (Immuno-Biological Laboratories Co., Ltd., 17249), which had been diluted to 0.1 μg/mL with a TBS-T solution containing 5% Blocking One, was added thereto in the amount of 20 μL per well. The resultant was incubated at room temperature for 1 hour and then washed with a TBS-T solution, and alkaline phosphatase-labeled streptavidin (Thermo Fisher Scientific Inc., 21324), which had been diluted to 0.1 μg/mL with 5% Blocking One-containing TBS-T solution, was added thereto in the amount of 20 μL per well. The resultant was incubated at room temperature for 1 hour and then washed with a TBS-T solution, and Chemiluminescent Ultra Sensitive AP Microwell and/or Membrane Substrate (450 nm) (BioFX, APU4-0100-01), which had been 5-fold diluted with 1 mM MgCl2-containing 20 mM TBS (pH 9.8) as a substrate, was added thereto in the amount of 20 μL. The resultant was incubated at room temperature for 30 minutes, and then the chemiluminescence thereof was measured by an EnVision™ multi-label counter (PerkinElmer, Inc.). The EC50 value of the test antibody was calculated by analyzing the calculated binding activity using Sigmoid-Emax model non-linear regression.
TABLE 4
Binding Activity of TIE-1-Igγ1-LALA
EC50 value (ng/mL)
Human
Monkey
Rat
Mouse
TIE-1-Igγ1-LALA
0.565
0.545
0.633
0.696
As a result, it was found that TIE-1-Igγ1-LALA has substantially the same high binding activity as a human Tie2, a monkey Tie2, a rat Tie2, and a mouse Tie2.
Reference Example 2: Evaluation of Binding Activity of 15B8 to Tie2
According to the method of Examples 12, the binding activities of 15B8 to each species Tie2 proteins were evaluated. In this regard, the absorbance at 450 nm was measured using an HRP-labeled anti-mouse kappa light chain antibody (SouthernBiotech, 1050-05) as a second antibody, a TMB color development reagent as a substrate, and an ARVO multi-label reader (PerkinElmer Inc.) as a measuring apparatus. In addition, 15B8 antibody concentration was adjusted to be from 1000 ng/mL to 0.3 ng/mL at an about 3-fold common ratio (diluted through eight steps), as a test antibody. The EC50 value of the test antibody was calculated by analyzing the calculated binding activity using Sigmoid-Emax model non-linear regression (Table 5).
TABLE 5
Binding Activity of 15B8
EC50 value (ng/mL)
Human
Monkey
Rat
Mouse
15B8
218.7
224.3
>1000
>1000
As a result, it was observed that 15B8 had binding activity to a human Tie2 and a monkey Tie2, but it was found that 15B8 has low binding activity to a rat Tie2 and a mouse Tie2.
From the results of Example 12, it was observed that TIE-1-Igγ1-LALA had high binding activity to a human Tie2, a monkey Tie2, a rat Tie2, and a mouse Tie2 without a species difference therein. On the other hand, it was observed that 15B8 had a species difference in the binding activity. From the above, it was suggested that the human Tie2 epitope of TIE-1-Igγ1-LALA was different from the epitope of 15B8.
Example 13: Evaluation of Vascular Permeability Inhibitory Action of TIE-1-Igγ1-LALA in Rat
According to the method of Example 10, the vascular permeability inhibitory action of TIE-1-Igγ1-LALA in rats was evaluated. In this regard, TIE-1-Igγ1-LALA was used as a test antibody, and the antibody dose was adjusted to be 0.1 mg/kg and 0.3 mg/kg. The results are shown in FIG. 3.
The mean value and the standard error of each group were determined. A Dunnett multiple comparison test was employed to determine a significant difference between the vehicle group and each group to which the antibody had been administrated. A case in which p<0.05 was intended to indicate that there was a significant difference.
As shown in FIG. 3, compared to the vehicle group, TIE-1-Igγ1-LALA significantly inhibited the dye leakage. From the above, it was found that TIE-1-Igγ1-LALA inhibited the vascular hyperpermeability.
Example 14: Retinal Edema Inhibitory Action in Mouse with Loss of Pericytes
In the retinal blood vessels of a patient with diabetic retinopathy, the loss of pericytes is one of characteristic lesions (Retina, 2013, Fifth edition, pp. 925-939). Although rat models with Streptozotocin-induced diabetes are widely used on diabetic retinopathy studies, there is a limitation in the usefulness of the models in the following aspects: a period of several months is taken until the loss of pericytes is observed, retinal microaneurysm which is thought to be caused by the loss of pericytes is not observed, the ratio of the pericytes to the endothelial cells is different from that of a human (Retina, 2013, Fifth edition, pp. 925-939), and apparent retinal edema is not observed (Diabetes Metab. J., 2013, Vol. 37, pp. 217-224). On the other hand, in a mouse having the retinal blood vessels with the loss of pericytes by administration of an anti-PDGF receptor β (PDGFR β) antibody, the lesions similar to those seen in diabetic retinopathy and diabetic macular edema, such as expansion of retinal blood vessel, retinal edema, and bleeding are observed, suggesting that the blood vessels are weakened like diabetic retinopathy and diabetic macular edema due to the loss of pericytes, although hyperglycemia is not observed (J. Clin. Invest., 2002, Vol. 110, pp. 1619-1628). Therefore, evaluation of the inhibitory action on retinal edema using a model with a condition showing the loss of pericytes, which is a characteristic lesion in a patient with diabetic retinopathy, is useful to evaluate the effectiveness on diabetic retinopathy and diabetic macular edema.
The retinal edema induced by loss of pericytes was prepared with a slight modification to the method reported in J. Clin. Invest., 2002, Vol. 110, pp. 1619-1628. That is, anti-PDGFR β monoclonal antibody 1B3 (WO 2008/130704) diluted with PBS was subcutaneously administered at 25 mg/kg to C57BL/6J mouse (Charles River Laboratories Japan, Inc.) on the 2nd day after birth to induce the loss of pericytes in the retinal blood vessels.
[Treated Group]
Control Group (also referred to as Cont. group): 17 mice Group to which an anti-PDGFR β antibody was not administered and PBS was administered
Vehicle group (also referred to as Veh. group): 24 mice
Group to which an anti-PDGFR β antibody was administered and PBS was administered, instead of TIE-1-Igγ1-LALA
TIE-1-Igγ1-LALA Group (0.1 mg/kg, 0.3 mg/kg, and 1 mg/kg): each 23 mice, 21 mice, and 21 mice
Group to which an anti-PDGFR β antibody was administered and each dose of TIE-1-Igγ1-LALA was administered
At 90 minutes before administration of the anti-PDGFR β antibody, TIE-1-Igγ1-LALA diluted with PBS was subcutaneously administered at 0.1 mg/kg, 0.3 mg/kg and 1 mg/kg. At 1 week after administration of the antibody, retinal edema was evaluated. Specifically, the eyeball was extracted and fixed with 1% glutaraldehyde and 2.5% formalin containing solutions, and then a paraffin-embedded slice graft was prepared. Hematoxylin-eosin stained specimens were scanned to convert image data using a virtual slide scanner (NanoZoomer XR, Hamamatsu Photonics K. K.). In this model, retinal edema in the retinal nerve fiber layer (NFL) is reported (J. Clin. Invest., 2002, Vol. 110, pp. 1619-1628), thereby quantification of retinal edema was carried out by measuring the areas of NFL and adjacent retinal ganglion cell layer with an NPD view 2 (Hamamatsu Photonics K. K.). The results are shown in FIG. 4.
The mean value and the standard error of each group were determined. A Dunnett multiple comparison test was employed as an assay for determining a significant difference between the vehicle group and each group to which TIE-1-Igγ1-LALA had been administrated. A Student t-test was used as an assay for determining a significant difference between the Cont. group and the Veh. group. A case in which p<0.05 was intended to indicate that there was a significant difference in each case.
As shown in FIG. 4, it was found that the TIE-1-Igγ1-LALA group (1 mg/kg) significantly inhibited the retinal edema having retinal blood vessels with the loss of pericytes as compared with the vehicle group. From the viewpoint that TIE-1-Igγ1-LALA inhibited the retinal edema caused by the retinal blood vessels with the loss of pericytes, it was suggested that TIE-1-Igγ1-LALA is effective on diabetic macular edema and diabetic retinopathy.
Example 15: Ischemia Limb Blood Flow Improving Action in Mouse with Hindlimb Ischemia
The model with hindlimb ischemia is a model having ischemia in the hindlimb tissue induced by ligation and excision of the blood vessel in the hindlimb on one side, and is also a representative model for evaluating the improving the ischemia symptoms (J. Vasc. Surg., 2012, Vol. 56, pp. 1669-1679).
The inguinal region of the femoral artery and vein and the saphenous artery and vein on the left hindlimb were ligated in a 10-week C57BL/6J mouse (CLEA Japan, Inc.). Further, after the branch vessel therebetween was ligated, and the blood vessel between the ligated points was excised. Surgery was carried out under anesthesia with pentobarbital sodium (60 mg/kg, Tokyo Chemical Industry Co., Ltd.). At one week after excision of the vessel, the blood flow in the hindlimb was measured by using a laser Doppler perfusion imager MoorLDI2 (Moor Instruments Inc.) under anesthesia with pentobarbital. After confirming a decrease in the blood flow in the limb to be treated, the treated group was set as follows.
[Treated Group (10 mice per group)]
Control Group:
Group to which PBS was administered instead of an antibody
TIE-1-Igγ1-LALA Group (1 mg/kg):
Group to which TIE-1-Igγ1-LALA was administered
TIE-1-Igγ1-LALA diluted with PBS was subcutaneously administered at 1 mg/kg, and the amount of skin blood flow of the normal limb and the ischemic limb at one week after administration of the antibody were measured. Specifically, pentobarbital sodium (60 mg/kg) was intraperitoneally administered, followed by placing on a heating plate, so as to measure the skin blood flow of the foot at 15 minutes after administration of anesthesia. The results of the blood flow measured by taking the bottom part of the foot as a region of interest (ROI), are shown in FIG. 5.
The mean value and the standard error of each group were determined. A Student t-test was used to determine a significant difference between the control group and the TIE-1-Igγ1-LALA group. A case in which p<0.05 is intended to indicate that there was a significant difference.
As shown in FIG. 5, it was found that compared with the control group, the TIE-1-Igγ1-LALA group significantly improved the amount of blood flow of the normal limb and the ischemic limb. Accordingly, the effectiveness of TIE-1-Igγ1-LALA on peripheral arterial diseases such as critical limb ischemia was suggested.
Example 16: Evaluation of Epitope of TIE-1-Igγ1-LALA: Hydrogen Deuterium Exchange Mass Spectrometry
In order to identify the recognition epitope of TIE-1-Igγ1-LALA, Fab of the fully human 2-16A2 in Example 7 (hereinafter referred to as fully human 2-16A2-Fab) was prepared. Since the fully human 2-16A2-Fab has the same variable region as TIE-1-Igγ1-LALA, these antibodies recognize the same epitope. As an antigen, a human Tie2 protein consisting of the amino acid numbers 1 to 452 of Accession No. NP_000450.2 (hereinafter referred to as a human Tie2 (1-452)) was prepared. The amino acid sequence is the same site used when the Tie2 binding site of Ang-2 was identified (Nat. Struct. Mol. Biol., Vol. 13, pp. 524-532).
Specifically, the fully human 2-16A2-Fab was prepared by combining a GS vector pEE6.4 in which a heavy chain gene encoding a structure (consisting of the amino acid sequence of the amino acid numbers 1 to 221 of SEQ ID NO: 12) consisting of the heavy chain variable region and the CH1 region of the fully human 2-16A2 was inserted, and the GS vector pEE12.4 in which a light chain gene of the fully human 2-16A2 was inserted, and using the same method as the expression method and the purification method for the antibody described in Example 7.
In order to obtain human Tie2 (1-452), first, human Tie2 (1-452) obtained by fusing human Fc (consisting of the amino acid sequence shown by SEQ ID NO: 23) with a thrombin recognition sequence (consisting of the amino acid sequence shown by SEQ ID NO: 22) as a linker (hereinafter referred to as a human Tie2 (1-452)-Fc chimeric protein) was prepared. Specifically, by inserting a gene encoding the human Tie2 (1-452)-Fc chimeric protein into a GS vector pEE12.4, and using the same expression method and the purification method described in Example 7, the human Tie2 (1-452)-Fc chimeric protein was prepared. Next, the prepared human Tie2 (1-452)-Fc chimeric protein was incubated with thrombin (GE Healthcare, 27-0846-01) at 22° C. for 16 hours to cut the Fc portion, and thrombin and human Fc were removed by Benzamidine Sepharose 4 Fast Flow (high sub) (GE Healthcare) and MabSelect SuRe, thereby preparing a human Tie2 (1-452).
For the purpose of identifying the epitope site, hydrogen/deuterium exchange mass spectrometry (hereinafter referred to as H/D exchange mass spectrometry, Anal. Bioanal. Chem., 2010, Vol. 397, pp. 967-979) was carried out by using NanoAQUITY UPLC HDX Systems (Waters).
Specifically, the fully human 2-16A2-Fab and human Tie2 (1-452) mixed liquid (final concentration of 50 μM and 25 μM, respectively) was prepared using a 20 mM citric acid buffer (pH 6) containing 120 mM sodium chloride, and incubated at 37° C. overnight. As a control, a solution with only human Tie2 (1-452) was prepared using 20 mM citric acid buffer (pH 6) containing 120 mM sodium chloride. Thereafter, the solution was added to a PBS buffer solution prepared using deuterium water (Kanto Chemical Co., Inc.), and incubated for 20 seconds, 1 minute, 10 minutes, 60 minutes, and 120 minutes, respectively, and deuteration was carried out. Then, an aqueous solution (pH 2.5) containing 100 mM dithiothreitol (Nacalai Tesque) and 4 M guanidine hydrochloride (Wako Pure Chemical Industries, Ltd.) was added thereto at 0° C., and then digestion was carried out using a Pepsin Column (Proszyme (registered trademark) Immobilized Pepsin Cartridge, Applied Biosystems), and the peptide digested with a trap column (ACQUITY UPLC BEH C18 1.7 μm VanGuard Pre-Column, Waters) was captured. Then, separation was carried out by reverse phase chromatography using C18 column (AQUITY UPLC BEH C18 1.7 μm, Waters) and the molecular weight was measured with a mass spectrometer (SynaptG2-Si, Waters). The centroid value of the isotopic distribution of all the detected peptides was calculated, and compared with centroid value of the isotopic distribution of only human Tie2 (1-452) which had undergone deuterium exchange, and the change amount with occurrence of deuterium substitution was calculated in terms of each deuteration period.
As a result of the H/D exchange mass spectrometry, it was demonstrated the peptides of the amino acid numbers 27 to 37, 29 to 37, 29 to 38, 43 to 60, 82 to 100, 98 to 107, 111 to 124, 116 to 125, 116 to 129, 119 to 129, 189 to 198 and 190 to 198 of Accession No. NP_000450.2 have inhibited deuteration in the coexistence of the antibody. The redundant domains of these peptides are arranged, further, the information of the peptides having not inhibited deuteration was added thereof and taking into consideration that two amino acids on the N-terminal side easily undergo reverse change (Proteins, 1993, Vol. 17, 75-86), five regions having inhibited deuterium substitution, that is, amino acid numbers 29 to 38, 84 to 102, 113 to 120, 126 to 129, and 191 to 198 of Accession No. NP_000450.2 as the epitope candidate sites were found. Further, as a result of the H/D exchange mass spectrometry, it was found that in the case where TIE-1-Igγ1-LALA interacts with a region consisting of these five amino acid segments or where a change in the steric structure or an allosteric effect by the antibody binding occurs, these residues are protected from hydrogen/deuterium exchange.
Example 17: Evaluation of Epitope of TIE-1-Igγ1-LALA: Surface Plasmon Resonance Analysis and ELISA
An epitope candidate for human Tie2 of TIE-1-Igγ1-LALA was identified from H/D exchange mass spectrometry of Example 16. In order to predict the epitope portion in detail, amino acid mutants of the human Tie2 (1-452)-Fc chimeric protein were prepared, and the binding activity was evaluated using surface plasmon resonance analysis (SPR analysis) and ELISA.
Based on the result of the H/D exchange mass spectrometry and the report of a region in which Ang-1 and Ang-2 bind to Tie2 (Nat. Struct. Mol. Biol., 2006, Vol. 13, pp. 524-532. Proc. Natl. Acad. Sci. USA, 2013, Vol. 110, 7205-7210), 23 amino acid mutant proteins in which one to four amino acids were substituted with alanine (in one case, glutamic acid) of the human Tie2 (1-452) in the human Tie2 (1-452)-Fc chimeric protein as amino acid mutant proteins of the human Tie2 (1-452) were prepared (Table 6). Various mutants were prepared by the same preparation method for the human Tie2 (1-452)-Fc chimeric protein prepared in Example 16.
TABLE 6
Mutant human Tie2 (1-452)-Fc chimeric proteins
Name of Mutant
Amino acid variation site
Human Tie2 (1-452)r1-Fc
R167A, H168A, E169A
Human Tie2 (1-452)r2-Fc
D172A, I173A
Human Tie2 (1-452)r3-Fc
R167A
Human Tie2 (1-452)r4-Fc
H168A
Human Tie2 (1-452)r5-Fc
E169A
Human Tie2 (1-452)r6-Fc
D172A
Human Tie2 (1-452)r7-Fc
I173A
Human Tie2 (1-452)g1-Fc
I194A, N197A, L198A
Human Tie2 (1-452)g2-Fc
R192A
Human Tie2 (1-452)g3-Fc
I194A
Human Tie2 (1-452)g4-Fc
G195E
Human Tie2 (1-452)g5-Fc
N197A
Human Tie2 (1-452)g6-Fc
L198A
Human Tie2 (1-452)m1-Fc
W82A, K84A
Human Tie2 (1-452)m3-Fc
S94A, K95A
Human Tie2 (1-452)y1-Fc
D37A
Human Tie2 (1-452)c1-Fc
R50A, H52A, E53A, P54A
Human Tie2 (1-452)A1-Fc
E151A
Human Tie2 (1-452)A2-Fc
V154A
Human Tie2 (1-452)A3-Fc
Y156A
Human Tie2 (1-452)A4-Fc
F161A
Human Tie2 (1-452)A5-Fc
S164A
Human Tie2 (1-452)A6-Fc
P166A
SPR analysis was carried out in order to evaluate the binding activity of the human Tie2 (1-452)-Fc chimera protein and 23 mutant proteins thereof to the fully human 2-16A2-Fab.
For SPR analysis, Biacore T200 (GE Healthcare) was used. An anti-human IgG (Fc) antibody (Human Antibody Capture Kit, GE Healthcare) was fixed onto a CM5 sensor chip. The human Tie2 (1-452)-Fc chimeric protein and 23 mutant proteins thereof, diluted with HBS-EP (GE Healthcare) at 5 μg/mL, were each allowed for immobilization, and the capture-amount was measured. Thereafter, the fully human 2-16A2-Fab diluted with HBS-EP to 50 nM, the binding amount thereof to the human Tie2 (1-452)-Fc chimeric protein and 23 mutant proteins thereof were measured. Further, by dividing the binding amount with the capture-amount, the binding amount of the antibody in the unit immobilized antigen (hereinafter referred to as a binding ratio) was calculated. The arithmetic mean of three experiments and the relative value of the binding ratio of each mutant proteins when the binding ratio of the human Tie2 (1-452)-Fc chimeric protein was taken as 100% are shown in Table 7. Further, the representative measurement data is shown in FIGS. 6A and 6B. The method for relative comparison of the binding amounts in Biacore is described in, for example, Analytical Biochemistry, 2003, Vol. 312, pp. 113-124.
As a result, it was found that the binding of the fully human 2-16A2-Fab was decreased in the human Tie2 (1-452)g1-Fc, the human Tie2 (1-452)g2-Fc, the human Tie2 (1-452)g3-Fc, the human Tie2 (1-452)g4-Fc, the human Tie2 (1-452)g5-Fc, the human Tie2 (1-452)m3-Fc, the human Tie2 (1-452)A1-Fc, the human Tie2 (1-452)A2-Fc, the human Tie2 (1-452)A3-Fc and the human Tie2 (1-452)A4-Fc, compared with the human Tie2 (1-452)-Fc chimeric protein.
TABLE 7
Results of SPR Analysis
Binding ratio
Relative value (%)
Human Tie2 (1-452)-Fc
0.29
100
chimeric protein
Human Tie2 (1-452)r1-Fc
0.29
102
Human Tie2 (1-452)r2-Fc
0.28
98
Human Tie2 (1-452)r3-Fc
0.34
119
Human Tie2 (1-452)r4-Fc
0.33
113
Human Tie2 (1-452)r5-Fc
0.29
99
Human Tie2 (1-452)r6-Fc
0.29
100
Human Tie2 (1-452)r7-Fc
0.30
106
Human Tie2 (1-452)g1-Fc
0.00
0
Human Tie2 (1-452)g2-Fc
0.03
9
Human Tie2 (1-452)g3-Fc
0.06
23
Human Tie2 (1-452)g4-Fc
0.02
7
Human Tie2 (1-452)g5-Fc
0.03
9
Human Tie2 (1-452)g6-Fc
0.38
131
Human Tie2 (1-452)m1-Fc
0.31
107
Human Tie2 (1-452)m3-Fc
0.04
12
Human Tie2 (1-452)y1-Fc
0.25
87
Human Tie2 (1-452)c1-Fc
0.54
189
Human Tie2 (1-452)A1-Fc
0.10
34
Human Tie2 (1-452)A2-Fc
0.08
29
Human Tie2 (1-452)A3-Fc
0.17
59
Human Tie2 (1-452)A4-Fc
0.13
44
Human Tie2 (1-452)A5-Fc
0.39
135
Human Tie2 (1-452)A6-Fc
0.31
109
ELSA was carried out by the method as in Example 12 in order to evaluate the binding activity of TIE-1-Igγ1-LALA to the human Tie2 (1-452)-Fc chimeric protein and 23 mutant proteins thereof.
The human Tie2 (1-452)-Fc chimeric protein and 23 mutant proteins thereof were diluted with PBS to 1 μg/mL, added to a white Maxisorp 384-well plate in the amount of 20 μL per well, and incubated at 4° C. overnight to perform immobilization. The next day, the immobilized solution was removed, and the plate was washed with a TBS-T solution, and incubated for 60 minutes by the addition of 50 μL of a Blocker™ Casein in TBS (Thermo Fisher Scientific Inc., 37532) to perform blocking. The resultant was washed with a TBS-T solution, and TIE-1-Igγ1-LALA, diluted with 0.05% Tween 20 (Nacalai Tesque Inc., 28353-85)—containing Blocker™ Casein in TBS from 0.03 ng/mL to 100 ng/mL through eight steps, was added thereto in the amount of 20 μL per well. The resultant was incubated at room temperature for 90 minutes and then washed with a TBS-T solution three times, and 20 μL of a biotin-labeled anti-human kappa light chain antibody, which had been diluted to 0.1 μg/mL with 0.05% Tween 20-containing Blocker™ Casein in TBS, was added thereto. The resultant was incubated at room temperature for 60 minutes and then washed with a TBS-T solution three times, and 20 μL of alkaline phosphatase-labeled streptavidin, which had been diluted to 0.1 μg/mL with 0.05% Tween 20-containing Blocker™ Casein in TBS, was added thereto. The resultant was incubated at room temperature for 60 minutes and then washed with a TBS-T solution three times, and 50 μL of Chemiluminescent Ultra Sensitive AP Microwell and/or Membrane Substrate (450 nm), which had been 5-fold diluted with 1 mM MgCl2-containing 20 mM TBS (pH 9.8) as a substrate, was added thereto. The resultant was incubated at room temperature under light-shielding for 40 minutes, and then luminescent intensity thereof was measured with an EnVision™ multi-label counter. The EC50 value of TIE-1-Igγ1-LALA with respect to the human Tie2 (1-452)-Fc chimeric protein and 23 mutant proteins thereof were calculated. The relative value of luminescent intensity of 100 ng/mL TIE-1-Igγ1-LALA as maximum concentration point with respect to human Tie2 (1-452)-Fc chimeric protein and 23 mutant proteins thereof when the convergence value of the sigmoid curve of TIE-1-Igγ1-LALA binding to the human Tie2 (1-452)-Fc chimeric protein which was taken as 100% was calculated (Table 8 and Table 9). Further, the EC50 value and the convergence value were calculated by Sigmoid-Emax model non-linear regression analysis. The results of ELISA are shown in FIG. 7.
As a result, it has been found that compared with the human Tie2 (1-452)-Fc chimeric protein, TIE-1-Igγ1-LALA had a remarkably decreased relative value with respect to Tie2 (1-452)g1-Fc, Tie2 (1-452)g2-Fc and Tie2 (1-452)g4-Fc, which are mutant proteins. Further, it has been found that compared with the human Tie2 (1-452)-Fc chimeric protein, TIE-1-Igγ1-LALA had a decreased relative value and an increased EC50 value with respect to Tie2 (1-452)g5-Fc, which is a mutant protein. From the result, it was found that TIE-1-Igγ1-LALA had a decreased binding activity to Tie2 (1-452)g1-Fc, Tie2 (1-452)g2-Fc, Tie2 (1-452)g4-Fc, and Tie2 (1-452)g5-Fc, unlike the human Tie2 (1-452)-Fc chimeric protein. Since TIE-1-Igγ1-LALA had a decreased relative value and similar EC50 value with respect to Tie2 (1-452)A1-Fc, it was determined that TIE-1-Igγ1-LALA had no change in the binding activity to Tie2 (1-452)A1-Fc.
TABLE 8
Results of ELISA
Relative value (%) of
EC50 value
luminescent intensity
(ng/mL)
Human Tie2 (1-452)-Fc
97
1.1
chimeric protein
Human Tie2 (1-452)r1-Fc
97
1.1
Human Tie2 (1-452)r2-Fc
96
0.9
Human Tie2 (1-452)r3-Fc
102
1.0
Human Tie2 (1-452)r4-Fc
101
1.0
Human Tie2 (1-452)r5-Fc
102
1.0
Human Tie2 (1-452)r6-Fc
100
1.0
Human Tie2 (1-452)r7-Fc
100
1.1
Human Tie2 (1-452)g1-Fc
3
12.3
Human Tie2 (1-452)g2-Fc
72
5.5
Human Tie2 (1-452)g3-Fc
96
1.1
Human Tie2 (1-452)g4-Fc
53
18
Human Tie2 (1-452)g5-Fc
91
2.2
Human Tie2 (1-452)g6-Fc
105
1.3
Human Tie2 (1-452)m1-Fc
104
1.0
Human Tie2 (1-452)m3-Fc
103
1.0
Human Tie2 (1-452)y1-Fc
109
1.0
Human Tie2 (1-452)c1-Fc
104
1.0
TABLE 9
Results of ELISA
Relative value (%) of
EC50 value
luminescent intensity
(ng/mL)
Human Tie2 (1-452)-Fc
98
1.0
chimeric protein
Human Tie2 (1-452)A1-Fc
90
1.3
Human Tie2 (1-452)A2-Fc
95
1.3
Human Tie2 (1-452)A3-Fc
98
1.0
Human Tie2 (1-452)A4-Fc
100
1.0
Human Tie2 (1-452)A5-Fc
98
0.9
Human Tie2 (1-452)A6-Fc
99
1.1
From the results of the two independent experiments, the ELISA and the SPR analysis, Tie2 (1-452)g1-Fc, Tie2 (1-452)g2-Fc, Tie2 (1-452)g4-Fc, and Tie2 (1-452)g5-Fc were identified as the mutant proteins to which the binding activity of TIE-1-Igγ1-LALA or the fully human 2-16A2-Fab was decreased in both experiments. It was found that the amino acids numbers 192, 194, 195, 197 and 198 in four mutant proteins are very important epitope candidates for TIE-1-Igγ1-LALA to bind to human Tie2. Herein, the binding activity of Tie2 (1-452)g1-Fc, which has the amino acid variations of I194A, N197A and L198A, decreased in ELISA assay, while the binding activity of Tie2 (1-452)g3-Fc which has the amino acid variation of I194A to TIE-1-Igγ1-LALA did not altered in ELISA assay. The binding activity of Tie2 (1-452)g6-Fc which has the amino acid variation of L198A altered neither in ELISA assay nor in SPR analysis. These results indicated that the mutation of amino acid number 197 in Tie2 (1-452)g1-Fc was the most critical amino acid as epitope. Finally, it was found that TIE-1-Igγ1-LALA binds to amino acid numbers 192, 195 and 197 of Accession No. NP_000450.2 as the epitopes.
INDUSTRIAL APPLICABILITY
The anti-human Tie2 antibody of the present invention is useful for preventing or treating various blood vessel-related diseases. Further, the polynucleotide, the expression vectors, the transformed host cell, and the methods for producing the antibody of the present invention are useful for producing the anti-human Tie2 antibody.
Sequence List Free Text
In the number heading <223> of the sequence list below, description of “Artificial Sequence” is made. Specifically, the base sequence shown by SEQ ID NO: 1 in the sequence list is the base sequence of the heavy chain of TIE-1-Igγ1-LALA and the amino acid sequence shown by SEQ ID NO: 2 is the amino acid sequence of the heavy chain encoded by SEQ ID NO: 1. The base sequence shown by SEQ ID NO: 3 in the sequence list is the base sequence of the light chain of TIE-1-Igγ1-LALA, TIE-1-Igγ1-I253A, TIE-1-Igγ1-WT, TIE-1-Igγ4-PE, and fully human 2-16A2, and the amino acid sequence shown by SEQ ID NO: 4 is the amino acid sequence of the light chain encoded by SEQ ID NO: 3. The base sequence shown by SEQ ID NO: 5 in the sequence list is the base sequence of the heavy chain of TIE-1-Igγ1-I253A and the amino acid sequence shown by SEQ ID NO: 6 is the amino acid sequence of the heavy chain encoded by SEQ ID NO: 5. The base sequence shown by SEQ ID NO: 7 in the sequence list is the base sequence of the heavy chain of TIE-1-Igγ1-WT, and the amino acid sequence shown by SEQ ID NO: 8 is the amino acid sequence of the heavy chain encoded by SEQ ID NO: 7. The base sequence shown by SEQ ID NO: 9 in the sequence list is the base sequence of the heavy chain of TIE-1-Igγ4-PE, and the amino acid sequence shown by SEQ ID NO: 10 is the amino acid sequence of the heavy chain encoded by SEQ ID NO: 9. The base sequence shown by SEQ ID NO: 11 in the sequence list is the base sequence of the heavy chain of the fully human 2-16A2, and the amino acid sequence shown by SEQ ID NO: 12 is the amino acid sequence of the heavy chain encoded by SEQ ID NO: 11. The amino acid sequences shown by SEQ ID NOS: 13 to 20 in the sequence list are the amino acid sequences of the linker. The amino acid sequence shown by SEQ ID NO: 22 in the sequence list is a thrombin recognition site.
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15134803
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astellas pharma inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Apr 1st, 2022 05:13PM
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Apr 1st, 2022 05:13PM
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Astellas Pharma
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tyo:4503
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Astellas Pharma
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Nov 29th, 2011 12:00AM
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Feb 22nd, 2007 12:00AM
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https://www.uspto.gov?id=US08067446-20111129
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Methods for treating an ulcer of the small intestine and stomach
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A method for treating a digestive ulcer of the small intestine or stomach is disclosed with a non-purine xanthine oxidase inhibitor that is a carboxylic acid compound, wherein the non-purine xanthine oxidase inhibitor is a carboxylic acid compound of formula (I) or its salt and wherein the terms of formula (I) are herein defined:
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8067446
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1. A method for treating a stomach or small intestine ulcer, comprising administering an effective amount of at least one non-purine xanthine oxidase inhibitor or a salt thereof to a patient in need thereof, wherein said non-purine xanthine oxidase inhibitor is selected from the group consisting of
2-(2-cyanobiphenyl-4-yl)isonicotinic acid;
5-(2-cyanobiphenyl-4-yl)thiophene-2-carboxylic acid;
2-(2-cyanobiphenyl-4-yl)-4-methyl-1,3-thiazole-5-carboxylic acid;
2-(2-cyano-4′-methoxybiphenyl-4-yl)-4-methyl-1,3-thiazole-5-carboxylic acid;
1-(2-cyanobiphenyl-4-yl)-1H-pyrazole-4-carboxylic acid;
1-(2-cyano-4′-methoxybiphenyl-4-yl)-1H-pyrazole-4-carboxylic acid;
2-(2-cyanobiphenyl-4-yl)-1,3-thiazole-5-carboxylic acid;
2-[2-cyano-4′-(trifluoromethoxy)biphenyl-4-yl]-4-methyl-1,3-thiazole-5-carboxylic acid;
2-(2-cyano-3′-methoxybiphenyl-4-yl)-4-methyl-1,3-thiazole-5-carboxylic acid; and
2-[2-cyano-4′-(dimethylamino)biphenyl-4-yl]-4-methyl-1,3-thiazole-5-carboxylic acid.
2. A method according to claim 1, which comprises administering 2-(2-cyanobiphenyl-4-yl)isonicotinic acid or a salt thereof.
3. A method according to claim 1, which comprises administering 5-(2-cyanobiphenyl-4-yl)thiophene-2-carboxylic acid or a salt thereof.
4. A method according to claim 1, which comprises administering 2-(2-cyanobiphenyl-4-yl)-4-methyl-1,3-thiazole-5-carboxylic acid or a salt thereof.
5. A method according to claim 1, which comprises administering 2-(2-cyano-4′-methoxybiphenyl-4-yl)-4-methyl-1,3-thiazole-5-carboxylic acid or a salt thereof.
6. A method according to claim 1, which comprises administering 1-(2-cyanobiphenyl-4-yl)-1H-pyrazole-4-carboxylic acid or a salt thereof.
7. A method according to claim 1, which comprises administering 1-(2-cyano-4′-methoxybiphenyl-4-yl)-1H-pyrazole-4-carboxylic acid or a salt thereof.
8. A method according to claim 1, which comprises administering 2-(2-cyanobiphenyl-4-yl)-1,3-thiazole-5-carboxylic acid or a salt thereof.
9. A method according to claim 1, wherein said ulcer is that formed in small intestine.
10. A method according to claim 9, wherein said ulcer is caused by a non-steroidal anti-inflammatory drug.
11. A method according to 1, which comprises administering 2-[2-cyano-4′-(trifluoromethoxy)biphenyl-4-yl]-4-methyl-1,3-thiazole-5-carboxylic acid or a salt thereof.
12. A method according to claim 1, which comprises administering 2-(2-cyano-3′-methoxybiphenyl-4-yl)-4-methyl-1,3-thiazole-5-carboxylic acid or a salt thereof.
13. A method according to claim 1, which comprises administering 2-[2-cyano-4′-(dimethylamino)biphenyl-4-yl]-4-methyl-1,3-thiazole-5-carboxylic acid or a salt thereof.
14. A method according to claim 1, wherein said ulcer is that formed in stomach.
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14
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CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a 371 of PCT/JP2007/053310 filed Feb. 22, 2007 and claims the benefit of JP 2006-048914 filed Feb. 24, 2006.
TECHNICAL FIELD
The present invention relates to a pharmaceutical composition for treatment or prevention for digestive ulcer, comprising a non-purine-structure xanthine oxidase inhibitor as an active ingredient, in particular to a pharmaceutical composition for treatment or prevention for ulcer that are formed in digestive tracts by the attack thereto such as gastric acid, pepsin, stress, Helicobacter pylori, NSAID (non-steroidal antiinflammatory drug), etc.
BACKGROUND ART
The digestive ulcer indicates an ulcer in which the partial defect of the epithelial tissue in the mucosal layer of a digestive tract has reached the depth. The basic consideration for the reasons for the pathogenesis is a balance theory that the balance between the aggressive factor such as gastric acid, pepsin, stress, Helicobacter pylori, NSAID, etc. and the mucosal membrane protecting factors for digestive tracts, i.e., the equilibrium of the function of mucus/mucosa barrier and blood flow/microcirculation, growth factor and prostaglandin, may be lost to form an ulcer (Chiryogaku, 2005; 39(5): 8-10; Can. J. Gastroenterol., 1999, November; 13(9): 753-9). It has been considered that the main pathogenic sites of this disorder are upper digestive tracts such as stomach and duodenum; but with the recent technical development of capsule endoscopes, double balloon endoscopes, etc., it has been known that some ulcer may be formed even in small intestine which has heretofore been difficult to inspect (World J. Gastroenterol., 2005 Aug. 21; 11(31):4861-4). However, it has been reported that administration of the proton pump inhibitor, which is a standard medicine for a gastric/duodenal ulcer, is not effective for a small intestine ulcer (Gastroenterology, 2005 May; 128(5): 1172-8). From these, a pharmaceutical composition having a mechanism of widely effective for digestive ulcers not limited only to stomach/duodenum is desired.
Allopurinol is known as an agent for treating gout and hyperuricemia, and this compound inhibits xanthine oxidase to thereby exhibit the action of lowering the serum uric acid level (Am. J. Manag. Care., 2005 November; 11(15 Suppl.): S451-8). On the other hand, the compound has a nucleic acid-derived structure (purine-like structure), and many side effects have been reported, which are considered to be based on the inhibition of the nucleic acid metabolism (Am. J. Med. 1984 January; 76(1):47-56; Isr. Med. Assoc. J., 2005 October; 7(10): 656-60; Biol. Pharm. Bull., 1999 August; 22(8): 810-5). Therefore, recently, development of a non-nucleic acid structure xanthine oxidase inhibitor has been much studied; and for example, there are mentioned phenylazole-carboxylic acid derivatives such as 2-phenylthiazole derivatives (Patent References 1 to 3), 3-phenylisothiazole derivatives (Patent Reference 4 and Patent Reference 5), phenylpyrazole derivatives (Patent Reference 6, Patent Reference 7 and Patent Reference 8), 2-phenyloxazole derivatives (Patent Reference 9), 2-phenylimidazole derivatives (Patent Reference 9). There is no report showing the indication of these xanthine oxidase inhibitors for digestive ulcer.
Some reports say that allopurinol is effective for models with upper and lower digestive ulcers at an extremely high-level dose (Non-Patent References 1 to 3). Non-Patent Reference 1 has a description discussing that allopurinol has a xanthine oxidase-inhibiting effect and expresses the effect by inhibiting the production of free radicals. There is a clinical test report saying that a combination of allopurinol and cimetidine enhances the effect of curing duodenal ulcer (Non-Patent Reference 4). On the other hand, there is a report saying that allopurinol does not inhibit NSAID ulcer at a clinical dose to human (Non-Patent Reference 5). In addition, it is also reported that allopurinol has a purine-like structure, and its structure itself has a strong action of scavenging free radicals (Non-Patent Reference 6). As in the above, the effectiveness and the functional mechanism of allopurinol for animal models with digestive ulcer have been unclear.
Patent Reference 1: International Publication WO 92/09279
Patent Reference 2: JP-A 2002-105067
[Patent Reference 3: International Publication WO 96/31211
Patent Reference 4: JP-A 57-85379
Patent Reference 5: JP-A 6-211815
Patent Reference 6: JP-A 59-95272
Patent Reference 7: International Publication WO 98/18765
Patent Reference 8: JP-A 10-310578
Patent Reference 9: JP-A 6-65210
Non-Patent Reference 1: Biochemical Pharmacology, 2003, Vol. 65, pp. 683-695
Non-Patent Reference 2: Digestive Diseases and Sciences, 1998, Vol. 43, No. 9 (extra issue, 1998 September), pp. 30S-34S
Non-Patent Reference 3: Journal of Pediatric Gastroenterology and Nutrition, 1995, Vol. 21, pp. 154-157
Non-Patent Reference 4: Journal of Surgical Research, 1994, Vol. 56, No. 1, pp. 45-52
Non-Patent Reference 5: Gut, 1996, Vol. 38, pp. 518-524
Non-Patent Reference 6: FEBS LETTERS, 1987, Vol. 213, No. 1, pp. 23-28
DISCLOSURE OF THE INVENTION
Problems that the Invention is to Solve
An object of the present invention is to provide an agent for treating or preventing digestive ulcer, which is effective also for ulcer in small intestine on which gastric secretion inhibitors such as proton pump inhibitors exert no effect, and which is superior to allopurinol in efficaciousness and safety.
Means for Solving the Problems
The present inventors have made extensive studies and, as a result, found that a non-purine xanthine oxidase inhibitor shows a stronger antiulcer effect over allopurinol having a radical-scavenging effect based on a purine-like structure, and exhibits an excellent treating effect for gastric ulcer such as NSAID ulcer, and have completed the present invention. The above-mentioned Non-Patent References 1 to 3 describes that allopurinol is effective for a model with digestive ulcer but does not show the effectiveness for human (Non-Patent Reference 1), and animal models require a large amount of administration of 100 mg/kg. However, since allopurinol has a purine-like structure, its side effects have been reported (Am. J. Med., 1984 January; 76(1): 47-56; Isr. Med. Assoc. J., 2005 October; 7(10): 656-60; Biol. Pharm. Bull., 1999 August; 22(8): 810-5), and a high dose of 100 mg/kg effective for models with digestive ulcer cannot be employed in clinical practice. The above-mentioned Non-Patent Reference 4 describes that a combination of allopurinol and cimetidine enhanced the effect of treating human duodenal ulcer. On the other hand, the above-mentioned Patent Reference 5 describes that allopurinol did not inhibit NSAID-induced ulcer in human. Further, it has heretofore been known that allopurinol has a strong action of scavenging radicals based on the purine-like structure (Non-Patent Reference 6), but it is unclear on what the effect for suppressing digestive ulcer in animal is based. Accordingly, use of a compound having only a xanthine oxidase inhibiting effect for treatment of digestive ulcer has not been specifically noted. The present inventors have found that a group of compounds not having a purine-like structure but having a xanthine oxidase-inhibiting activity that differs from the type of allopurinol exhibit an excellent effect for treating gastric ulcer even though not having a radical scavenging effect based on the structure, and have completed the present invention.
The compounds described in Preparation Examples hereinunder are described in patent applications filed by the present patent applicant prior to the present patent application (PCT publications Nos. WO 2006/022374, WO 2006/022375 and PCT application PCT/JP2006/320061). These patent applications describe the applicability to autoimmune diseases such as inflammatory bowel disease, but has no disclosure relating to the applicability to digestive ulcer found in the present application.
Thus, the present invention relates to an agent for treating or preventing digestive ulcer, comprising a non-purine xanthine oxidase inhibitor as an active ingredient.
The present invention also relates to an agent for treating or preventing digestive ulcer, comprising a non-purine xanthine oxidase inhibitor as an active ingredient, wherein the inhibitor is a carboxylic acid derivative of the following general formula (I) or a salt thereof:
(wherein the symbols in the formula have the following meanings:
R1: H or halogen,
R2: —CN, —NO2, halogeno-lower alkyl or —CO-lower alkyl,
A: linear or branched alkyl having from 1 to 8 carbon atoms, linear or branched alkenyl having from 2 to 8 carbon atoms, —Y-cycloalkyl, —Y-nitrogen-containing saturated heterocyclic group, —Y-oxygen-containing saturated heterocyclic group, —Y-optionally-condensed aryl, or —Y-heteroaryl;
wherein the linear or branched alkyl having from 1 to 8 carbon atoms and the linear or branched alkenyl having from 2 to 8 carbon atoms may be substituted with from 1 to 3, the same or different substituents selected from the following group G1; and the cycloalkyl, the nitrogen-containing saturated heterocyclic group and the oxygen-containing saturated heterocyclic group may be substituted with from 1 to 4, the same or different groups selected from lower alkyl and the following group G1; and the optionally-condensed aryl and the heteroaryl may be substituted with from 1 to 3, the same or different groups selected from the following group G2;
G1 group: hydroxy, —CN, —O-lower alkyl, —S-lower alkyl, —NR3R4, —(CO)NR3(R4), —CO2—R5 and halogen,
G2 group: halogen, —CN, —NO2, lower alkyl, halogeno-lower alkyl, —O—R5, —O-halogeno-lower alkyl, —O—CO—R5, —O-benzyl, —O-phenyl, —NR3R4, —NH—CO—R5, —CO2—R5, —CO—R5, —CO—NR3R4, —CO-phenyl, —S—R5, —SO2-lower alkyl, —SO2-phenyl, —NH—SO2-naphthalene-NR3R4, phenyl, cycloalkyl and -lower alkylene-O—R5;
R5: H or lower alkyl;
R3 and R4: the same or different, each representing H or lower alkyl, and R3 and R4, taken together with the nitrogen atom to which they bond, may form a monocyclic nitrogen-containing saturated heterocyclic group;
Y: bond, lower alkylene, lower alkenylene, -(lower alkylene)-O— or -(lower alkylene)-O-(lower alkylene)-;
X: bond, —O—, —N(R6)— or —S—;
R6: H or lower alkyl,
ring B: monocyclic heteroaryl, wherein the monocyclic heteroaryl may be substituted with a group selected from lower alkyl, —OH and halogen; and
R7: H or lower alkyl, and the same shall apply hereinunder).
The present invention further includes the following embodiments:
[1] Use of a non-purine xanthine oxidase inhibitor for the manufacture of an agent for treating or preventing digestive ulcer.
[2] A method of treating or preventing digestive ulcer, comprising administering an effective amount of a non-purine xanthine oxidase inhibitor to a patient.
[3] An agent for treating or preventing digestive ulcer, comprising a non-purine xanthine oxidase inhibitor as an active ingredient, which is administered to a patient under administration with a non-steroidal antiinflammatory drug.
[4] A combined preparation containing a non-purine xanthine oxidase inhibitor and a non-steroidal antiinflammatory drug.
[5] A combination for treating or preventing digestive ulcer, which is a combination of a preparation comprising a non-purine xanthine oxidase inhibitor as an active ingredient and a preparation comprising a non-steroidal antiinflammatory drug as an active ingredient, wherein the preparations are administered simultaneously or separately.
Effects of the Invention
The pharmaceutical composition of the present invention is useful as an agent for treating or preventing ulcer that formed in digestive tracts by the attack by gastric acid, pepsin, stress, Helicobacter pylori, NSAID, etc. The pharmaceutical composition of the present invention is more advantageous to conventional ulcer-treating agents such as proton pump inhibitors, in that it is effective even for ulcer in small intestine, for which gastric/duodenal ulcer-treating agents that inhibit gastric acid secretion such as proton pump inhibitors are ineffective.
BEST MODE FOR CARRYING OUT THE INVENTION
The present invention is described in detail hereinunder.
“Digestive ulcer” indicates an ulcer in which the partial defect of the epithelial tissue in the mucosal layer of a digestive tract has reached the depth. In particular, it means an ulcer formed in a digestive tract by the attack of gastric acid, pepsin, stress, Helicobacter pylori, NSAID, etc. “Digestive ulcer” quite differs from an ulcer formed in a digestive tract in autoimmune diseases such as inflammatory bowel disease. “Upper digestive ulcer” means the above-mentioned digestive ulcer that formed in esophagus, stomach and duodenum; and “lower digestive ulcer” means the above-mentioned digestive ulcer that formed in small intestine and large intestine. The agent for treating or preventing digestive ulcer of the present invention is an agent for treating or preventing the above-mentioned “digestive ulcer”, and especially preferably, it is an agent for treating digestive ulcer in small intestine for which the treating effect of conventional gastric secretion inhibitors cannot be expected.
“Non-purine” means that the compound has no purine skeleton in the molecule. In this description, the purine skeleton means a ring skeleton of a bicyclic unsaturated hydrocarbon ring formed through condensation of a 5-membered ring and a 6-membered ring, which contains four nitrogen atoms. The purine skeleton includes “purine”, which is the basic structure of purine nucleoside, or that is, “a ring skeleton formed through condensation of a pyridine ring and an imidazole ring”, and in addition, analogues that differ in the position of the nitrogen atom, for example, pyrazolopyrimidine, etc. Accordingly, “non-purine xanthine oxidase inhibitor” is a xanthine oxidase inhibitor not having the above-mentioned purine skeleton in the molecule, and means other xanthine oxidase inhibitors than purine skeleton-having xanthine oxidase inhibitors (purine-type xanthine oxidase inhibitors) such as allopurinol or oxypurinol. The non-purine xanthine oxidase inhibitor does not have a purine-like structure, and therefore differs from the purine-type xanthine oxidase inhibitor in the manner of the enzyme inhibition and, in addition, has a common characteristic in that the structure-derived side effect is weak. The non-purine xanthine oxidase inhibitor includes, for example, the compounds of the above formula (I), the compounds described in the above-mentioned Patent References 1 to 9, and benzoxazole derivatives (WO 03/042185), triazole derivatives (WO 03/064410) and tetrazole derivatives (WO 2004/009563), but not limited thereto, and the inhibitor may be any others not having a purine skeleton and substantially not having an action of inhibiting purine biosynthesis.
In case where the agent for treating or preventing digestive ulcer of the present invention is used as combined preparations or drug combinations with NSAID for reducing the side effect of NSAID (non-steroidal antiinflammatory drug), the NSAID includes salicylic acid compounds (aspirin, salicylic acid), anthranilic acid compounds (mefenamic acid), phenylacetic acid compounds (dichlofenac, fenbufen), indole-acetic acid compounds (indomethacin, sulindac), isoxazole-acetic acid compounds (mofezolac), pyrano-acetic acid compounds (etodolac), naphthalene compounds (nabumetone), propionylacetic acid compounds (ibuprofen, ketopurofen, loxoprofen, naproxen, zaltoprofen), oxicam compounds (piroxicam, meloxicam, lornoxicam), and basic antiinflammatory drugs (tiaramide hydrochloride, emorfazone), etc. In addition, also mentioned are NSAIDs described in Konnichinochiryoyaku, 2005, pp. 86-115 (Nanko-do); Drugs 49(1): 51-70, 1995; and Drugs 52 (Suppl. 5): 13-23, 1996. However, NSAID is not limited to these, and can include any others that may induce ulceration by their administration.
In the definition of the general formulae in this description, the term “lower” means a linear or branched carbon chain having from 1 to 6 carbon atoms (hereinafter this may be abbreviated as “C1-6”), unless otherwise specifically indicated. Accordingly, “lower alkyl” is C1-6 alkyl, preferably linear alkyl such as methyl, ethyl, n-propyl, n-butyl, and branched alkyl such as isopropyl, isobutyl, tert-butyl, neopentyl. More preferred is C1-4 alkyl; and even more preferred are methyl, ethyl, n-propyl, isopropyl and tert-butyl. “Lower alkylene” is C1-6 alkylene, preferably linear alkylene such as methylene, ethylene, trimethylene, tetramethylene, and branched alkylene such as propylene, ethylethylene, 1,2-dimethylethylene, 1,1,2,2-tetramethylethylene. More preferred is C1-4 alkylene.
The linear or branched alkyl having from 1 to 8 carbon atoms as A is preferably ethyl, n-propyl, isopropyl, n-butyl, isobutyl, isopentyl, neopentyl.
“Alkenyl” is a group having at least one double bond at any position of “alkyl”, preferably C2-8 alkenyl, more preferably C2-8 alkenyl having at most 3 branches, even more preferably C3-6 alkenyl having one double bond.
“Lower alkenylene” is a group having at least one double bond at any position of C2-6 alkylene, preferably propenylene, butenylene, pentenylene, hexenylene, 1,3-butadienylene, more preferably C3-4 alkenylene.
The linear or branched alkenyl having from 2 to 8 carbon atoms for A is preferably propenyl, butenyl, pentenyl, hexenyl, 1,3-butadienyl, isoprenyl, 3,3-dimethylpropen-2-yl.
“Halogen” indicates F, Cl, Br and I. Preferably, it is F and Cl. “Halogeno-lower alkyl” means C1-6 alkyl substituted with at least one halogen, and is preferably C1-6 alkyl substituted with at least one F, more preferably trifluoromethyl.
“Cycloalkyl” is a C3-10 saturated hydrocarbon ring group, and may be bridged. Preferred is C3-8 cycloalkyl; more preferred are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and adamantyl; and even more preferred are cyclopentyl, cyclohexyl and cycloheptyl.
“Aryl” is a C6-14 monocyclic to tricyclic aromatic hydrocarbon ring group, and is preferably phenyl and naphthyl, more preferably phenyl. “Optionally condensed aryl” is a generic term for the above-mentioned “aryl” and, in addition, for phenyl condensed with a 5- to 7-membered saturated monocyclic hetero ring containing 1 or 2 O atoms, and phenyl condensed with a C3-8 saturated hydrocarbon ring. “Optionally-condensed aryl” is preferably phenyl condensed with at least one ring selected from tetrahydrofuran, 1,3-dioxolane, 1,4-dioxepine, cyclohexane and cyclopentane, or non-condensed phenyl, and is more preferably non-condensed phenyl.
“Heteroaryl” is a generic term for a 5- or 6-membered monocyclic aromatic ring group having from 1 to 3 hetero atoms selected from O, S and N (monocyclic heteroaryl), and bicyclic or tricyclic heteroaryl constructed through condensation of the monocyclic heteroaryl rings or condensation of benzene ring and monocyclic heteroaryl ring. The monocyclic heteroaryl is preferably pyridyl, pyrrolyl, pyrazinyl, pyrimidinyl, pyridazinyl, imidazolyl, triazolyl, thienyl, furyl, thiazolyl, pyrazolyl, isothiazolyl, oxazolyl and isoxazolyl, more preferably thienyl, furyl, pyridyl, pyrrol-3-yl and pyrazol-4-yl. The bicyclic hetero aryl is preferably benzothienyl, benzofuryl, indazolyl, benzothiazolyl, benzoxazolyl, indolyl, benzimidazolyl, quinazolyl, quinoxalinyl, quinolyl, isoquinolyl, cinnolinyl and phthalazinyl, more preferably benzothienyl, benzofuryl, indazolyl and indolyl. The tricyclic heteroaryl is preferably carbazolyl, dibenzo[b,d]furanyl and dibenzo[b,d]thienyl.
In the above-mentioned “heteroaryl”, the ring atom S may be oxidized to form an oxide or dioxide, or N may be oxidized to form an oxide.
The monocyclic heteroaryl of the ring B is preferably pyridyl, pyrrolyl, pyrazinyl, pyrimidinyl, pyridazinyl, imidazolyl, triazolyl, thienyl, furyl, thiazolyl, pyrazolyl, isothiazolyl, oxazolyl and isoxazolyl, more preferably pyridyl, thienyl, thiazolyl, pyrazolyl, isothiazolyl and isoxazolyl. The above-mentioned “monocyclic heteroaryl of the ring B” is described as a nomenclature of a monovalent group for the convenience′ sake, but the cyclic group is a divalent group that bonds to benzene ring and carboxyl group.
“Oxygen-containing saturated heterocyclic group” is a 5- to 7-membered saturated monocyclic heterocyclic group containing one or two O atoms, and is preferably oxiranyl, oxetanyl, tetrahydrofuranyl and tetrahydropyranyl.
“Nitrogen-containing saturated heterocyclic group” is a 5- to 8-membered saturated or partially-unsaturated monocyclic heterocyclic group containing one N atom and optionally further containing one hetero atom of N, S and O (monocyclic nitrogen-containing saturated heterocyclic group), or a cyclic group constructed through condensation of the monocyclic nitrogen-containing saturated heterocyclic group with benzene ring. Preferred are pyrrolidinyl, piperidinyl, piperazinyl, azepanyl, diazepanyl, azocanyl, morpholinyl, thiomorpholinyl, tetrahydropyridyl, indolinyl, isoindolinyl, tetrahydroquinolyl, tetrahydroisoquinolyl and dihydrobenzoxazinyl. More preferred are pyrrolidinyl, piperidinyl, azepanyl, azocanyl and morpholinyl.
In the above-mentioned “nitrogen-containing saturated heterocyclic group”, the ring atom S may be oxidized to form an oxide or dioxide, or N may be oxidized to form an oxide. In this, in addition, a carbon atom may be substituted with an oxo group.
Preferred embodiments of the compounds of formula (I) as an active ingredient of the pharmaceutical composition of the present invention are described below.
1) The compound where the benzene ring and —COOR7 bonding to the ring B bonds to the ring B at any other positions than the adjacent positions.
2) The compound where the relative arrangement of the benzene ring and —COOR7 bonding to the ring B is 1,3-positions.
3) More preferably, the compound of above 2) where the ring B is a divalent group of the following formula:
(wherein the symbols have the following meanings:
W: CH or N,
Z: O, S or NRd,
Ra to Rc: H, lower alkyl, or halogen, and
Rd: H or lower alkyl, the same shall apply hereinunder).
4) More preferably, the compound of above 3) wherein B is a cyclic group selected from pyridine, thiophene, thiazole and pyrazole rings.
5) More preferably, the compound of above 4) wherein X is —O— and A is linear or branched alkyl having from 1 to 8 carbon atoms and optionally substituted with a group of G1; the compound of above 4) where X is a bond and A is a monocyclic nitrogen-containing saturated heterocyclic group optionally substituted with a group of G1; or the compound of above 4) where X is a bond and A is aryl or heteroaryl optionally substituted with a group of G2.
6) More preferably, the compound of above 4) where X is —O— and A is lower alkyl; the compound of above 4) where X is a bond and A is a monocyclic nitrogen-containing saturated heterocyclic group; or the compound of above 4) where X is a bond and A is phenyl optionally substituted with a group of G2.
7) As another preferred embodiment, the compound of above 4) where R1 is H, R2 is CN, R7 is H, Ra to Rc are H or methyl, X is —O— or a bond, A is “—O-lower alkyl” substituted phenyl, phenyl, or lower alkyl.
8) More preferably, a compound selected from 2-(2-cyanobiphenyl-4-yl)isonicotinic acid, 5-(2-cyanobiphenyl-4-yl)thiophene-2-carboxylic acid, 2-(2-cyanobiphenyl-4-yl)-4-methyl-1,3-thiazole-5-carboxylic acid, 2-(2-cyano-4′-methoxybiphenyl-4-yl)-4-methyl-1,3-thiazole-5-carboxylic acid, 1-(2-cyanobiphenyl-4-yl)-1H-pyrazole-4-carboxylic acid, 1-(2-cyano-4′-methoxybiphenyl-4-yl)-1H-pyrazole-4-carboxylic acid, 2-(2-cyanobiphenyl-4-yl)-1,3-thiazole-5-carboxylic acid, 2-(3-cyano-4-isobutyloxyphenyl)-4-methyl-1,3-thiazole-5-carboxylic acid, and 1-(3-cyano-4-neopentyloxyphenyl)-1H-pyrazole-4-carboxylic acid.
The monocyclic nitrogen-containing saturated heterocyclic group is preferably pyrrolidinyl, piperidinyl, azepanyl, azocanyl or morpholinyl. Preferred groups of G2 are halogen, —CN, lower alkyl, halogeno-lower alkyl, —O—R5, —O-halogeno-lower alkyl, —S—R5, —NR3R4, —CO2—R5 and -lower alkylene-O—R5. The substituents for Ra to Rc are preferably H and methyl.
X is preferably a bond or —O—. A is preferably lower alkyl, monocyclic nitrogen-containing saturated heterocyclic group and phenyl. R2 is preferably —CN and —NO2, more preferably —CN. R7 is preferably H.
The compounds as the active ingredient of the pharmaceutical composition of the present invention may include tautomers and optical isomers depending on the type of the substituent therein; and the present invention includes mixtures of those isomers or isolated isomers.
The compounds as the active ingredient of the pharmaceutical composition of the present invention include “pharmaceutically-acceptable prodrugs”. “Pharmaceutically-acceptable prodrugs” are compounds that are metabolized in living bodies to give compounds having a group of CO2H, NH2, OH or the like for the active ingredient of the pharmaceutical composition of the present invention. The group of forming prodrugs includes those described in Prog. Med. 5:2157-2161 (1985); and those described in “Development of Medicines”, Vol. 7, Molecular Design, pp. 163-198, Hirokawa Publishing, 1990.
Salts of the compounds as the active ingredient of the pharmaceutical composition of the present invention are pharmaceutically-acceptable salts, concretely including acid-addition salts with a mineral acid such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid, or an organic acid such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, aspartic acid, glutamic acid. Depending on the type of the substituent therein, the compounds may form salts with a base, including, for example, salts with an inorganic base containing a metal such as sodium, potassium, magnesium, calcium, aluminium, lithium, or an organic base such as methylamine, ethylamine, ethanolamine, lysine, ornithine, and ammonium salts.
Further, the compounds or their salts as the active ingredient of the pharmaceutical composition of the present invention include various hydrates, solvates and polymorphic crystal substances.
(Production Method)
Typical production methods for the compounds as the active ingredient of the pharmaceutical composition of the present invention are described below. Of known non-purine xanthine oxidase inhibitors, those shown in the above-mentioned Patent References 1 to 9 may be produced with reference to the production methods described in those patent publications. Benzotriazole derivatives (WO 03/042185), triazole derivatives (WO 03/064410) and tetrazole derivatives (WO 2004/009563) may be produced with reference to the production methods described in the corresponding patent publications.
On the other hand, the compounds of formula (I) that are used as the active ingredient of the pharmaceutical composition of the present invention may be produced according to various known production methods, taking advantage of the characteristics based on the basic skeleton or the type of the substituent therein. In this case, depending on the type of the functional group therein, it may be often effective for the production technique to protect the functional group with a suitable protective group in a stage of starting compound or intermediate, or to substitute it with a group readily convertible into the functional group. The functional group includes, for example, an amino group, a hydroxyl group and a carboxyl group, and their protective groups are, for example, protective groups described in Protective Groups in Organic Synthesis (by T. W. Greene and P. G. M. Wuts), 3rd Ed., 1999. These may be used, as suitably selected in accordance with the reaction condition. The method comprises introducing the protective group, then reacting the compound, and if desired, removing the protective group or converting it into a desired group, thereby obtaining a desired compound.
The prodrugs of the compounds of formula (I) or their salts may be produced by introducing a specific group into the starting compound or intermediate, like the above-mentioned protective group, or by directly processing the compounds of formula (I). The reaction may be any ordinary esterification, amidation, acylation or the like known to those skilled in the art.
First Production Method:
(In the formula, Q1 represents —B(OH)2 or —B(OR11)OR12; L1 represents a leaving group. In this, R11 and R12 are the same or different, each representing lower alkyl, or R11 and R12, taken together, form lower alkylene. The same shall apply hereinunder.)
This production method is for producing the compounds (I) of the present invention through coupling reaction of the compound (1) and the compound (2).
The leaving group for L1 includes halogen, methanesulfonyloxy, p-toluenesulfonyloxy, trifluoromethanesulfonyloxy. In this production method, the compounds (1) and (2) are used in the same amount or any one of them is used excessively, and they are reacted in a solvent inert to the reaction in the presence of a base and a palladium catalyst at room temperature or under heat with reflux generally for 0.1 hours to 5 days. Preferably, the reaction is attained in an inert gas atmosphere. As the case may be, microwave radiation may be favorable for the heating in the reaction. Not specifically defined, the solvent includes, for example, aromatic hydrocarbons such as benzene, toluene, xylene; ethers such as diethyl ether, tetrahydrofuran (THF), 1,4-dioxane, 1,2-dimethoxyethane, 1,2-diethoxyethane; halogenohydrocarbons such as dichloromethane, 1,2-dichloroethane, chloroform; alcohols such as methanol, ethanol, 2-propanol, butanol; N,N-dimethylformamide (DMF), N-methylpyrrolidone (NMP), dimethylsulfoxide (DMSO), water; and their mixed solvents. The base is preferably an inorganic base such as sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, sodium ethoxide, sodium methoxide. Also usable are other bases such as potassium fluoride, cesium fluoride. In this case, the reaction is preferably attained in an aprotic solvent. The palladium catalyst is preferably tetrakis(triphenylphosphine)palladium, dichlorobis(triphenylphosphine)palladium, palladium chloride-1,1′-bis(diphenylphosphino)ferrocene.
Second Production Method:
(In the formula, A1 represents aryl or heteroaryl of the groups of A in formula (I); Q2 is the same as Q1; and L2 is the same as L1. The same shall apply hereinunder.)
This production method is a method for producing the compound (Ia) of the present invention where A is aryl or heteroaryl in formula (I), through coupling reaction of the compound (3) and the compound (4). The reaction reagents and the reaction conditions for the first production method are applicable also to this production method.
Third Production Method:
(In the formula, X1 represents —O—, —N(R6)— or —S—; L3 represents a leaving group or OH. The same shall apply hereinunder.)
This production method is a method for producing the compound (Ib) of the present invention where X is —O—, —N(R6)— or —S— in formula (I), through alkylation of the compound of formula (5).
The leaving group for L3 includes halogen, methanesulfonyloxy, p-toluenesulfonyloxy, trifluoromethanesulfonyloxy.
In case where L3 is a leaving group, this production method is as follows: The compound (5) and the alkylating agent (6) are used in the same amount of the alkylating agent (6) is used excessively, and they are reacted in a solvent inert to the reaction at room temperature or under heat with reflux generally for 0.1 hours to 5 days. Not specifically defined, the solvent includes aromatic hydrocarbons, ethers, halogenohydrocarbons, DMF, NMP, DMSO, and their mixed solvents, such as those mentioned hereinabove. As the case may be, the reaction is preferably attained in the presence of a base or a phase transfer catalyst. In this case, the base includes organic bases such as triethylamine, diisopropylethylamine (DIPEA), 1,8-diazabicyclo[5.4.0]-7-undecene (DBU); and inorganic bases such as sodium carbonate, potassium carbonate, cesium carbonate, sodium hydride. The phase transfer catalyst includes tetra-n-butylammonium chloride, tetra-n-butylammonium bromide, 18-crown-6.
In case where L3 is OH and X1 is O, the alkylation may be attained by using the compound (5) and the alkylating agent (6) in the same amount or by using the alkylating agent (6) excessively, and by processing them with an azodicarboxylic acid derivative such as ethyl azodicarboxylate or 1,1′-(azodicarbonyl)dipiperidine, and a phosphorus compound such as triphenyl phosphine or tributyl phosphine. Concrete reaction conditions and reaction reagents are described in detail in “Organic Reactions 42, 335-656, (1992); and “Journal of the Synthetic Organic Chemistry, Japan”, 53, 631-641 (1997); and the alkylation may be attained according to the described methods or according to methods similar thereto.
Fourth Production Method:
(In the formula, A2 represents a nitrogen-containing heterocyclic group or heteroaryl containing at least one nitrogen atoms of the groups of A in formula (I), and this is a cyclic group bonding to the benzene ring via the nitrogen atom. The same shall apply hereinunder.)
This production method is a method for producing the compound (Ic) of the present invention through ipso-substitution between the compound (7) and the compound (8). The same condition for the alkylation of the above-mentioned third production method where L1 is a leaving group, is applicable to this production method.
When A-N(R6)H (A and R6 have the same meanings as above) is used in place of the compound of formula (7) in the same reaction as above, then a compound of formula (I) where X is —N(R6)— may be produced.
In the reaction of the above-mentioned first to fourth production methods, where a compound having a group of CO2H is used, it is desirable that the group is previously protected with a protective group and, after the intended reaction, the protective group is removed. Regarding the condition for the selection, protection and removal of the protective group, referred to are the method described in the above-mentioned Protective Groups in Organic Synthesis, 3rd Ed., 1999.
Other Production Methods:
The compounds of the present invention having various functional groups may also be produced according to methods obvious to those skilled in the art, or according to known production methods, or according to modifications of such methods. For example, the compounds of the present invention produced in the above-mentioned production methods may be further subjected to substituent modification reaction, thereby producing desired compounds of the present invention. Typical reactions are mentioned below.
(1) Amidation and Esterification:
Of the compounds (I) of the present invention, those having an amide group or those having an ester group may be produced, starting from a compound having a hydroxyl group or an amino group and reacting it with a carboxylic acid or its reactive derivative. For the reaction, for example, referred to are the methods described in “Courses in Experimental Chemistry”, 4th Ed., Vol. 22 (1992), edited by the Chemical Society of Japan, Maruzen.
(2) Oxidation:
Of the compounds (I) of the present invention, those having a sulfonyl group or a sulfenyl group may be produced through oxidation of a compound having a sulfide group. For example, it may be attained according to the methods described in “Courses in Experimental Chemistry”, 4th Ed., Vol. 23 (1991), edited by the Chemical Society of Japan, Maruzen.
(3) Alkylation:
Of the compounds (I) of the present invention, those having a lower alkoxy group or a lower alkylamino group may be produced through alkylation of a compound having a hydroxyl group or an amino group. The reaction may be attained under the same condition as in the third production method.
Production Methods for Starting Compounds:
(In the formulae, Hal represents Br or Cl. The same shall apply hereinunder.)
Of the starting compounds of formula (I) in the above-mentioned first production method, the compound (1a) where X is X1, the compound (1a) where X is a bond and A is A2, and the compound (1c) where X is a bond and A is A1 may be produced according to the above-mentioned reaction routes.
In the reaction routes, the same condition as in the above-mentioned third production method is applicable to the alkylation. The ipso-substitution may be attained under the same condition as that for the alkylation in the above-mentioned third production method where L3 is a leaving group. To the coupling reaction, the same condition as that for the above-mentioned first production method is applicable. The boration may be attained according to the methods described in “Chem. Rev., 95, 2547-2483, (1995)”, “J. Org. Chem., 67, 5394-5397 (2002)”, “J. Org. Chem., 65, 164-168 (2000)” or “J. Org. Chem., 60, 7508-7510 (1995)”.
Hydrolyzing the compound (1a), (1a) or (1c) gives a compound where R11 and R12 are both hydrogen atoms. The reaction may be attained according to the methods described in “Chem. Rev., 95, 2547-2483 (1995)” or “J. Org. Chem., 67, 5394-5397 (2002)”.
(In the formula, L4 represents sulfonyloxy such as methanesulfonyloxy, p-toluenesulfonyloxy or trifluoromethanesulfonyloxy.)
The starting compound (4a) can be produced according to the above-mentioned reaction route.
In the reaction route, the boration and the hydrolysis are the same as those in the above-mentioned production methods for starting compounds; and the coupling reaction may be attained under the same condition as in the above-mentioned first production method. The sulfonylesterification may be attained in an ordinary manner. In the reaction route, the phenolic hydroxyl group and the carboxyl group of the compounds are preferably protected with a protective group. Regarding the protective group and the condition for protection and deprotection, referred to are the methods described in the above-mentioned “Protective Groups in Organic Synthesis”, 3rd Ed., 1999.
Using a compound derived from the compound (14) by substituting the hydroxyl group therein with halogen followed by reacting it similarly gives a compound corresponding to the compound (17) in which the hydroxyl group is substituted with halogen.
The compounds produced in the manner as above may be isolated and purified directly as they are free compounds or after salt formation in an ordinary manner as salts. The isolation and purification may be attained in ordinary chemical operation of extraction, concentration, distillation, crystallization, filtration, recrystallization or various chromatography.
Various isomers may be isolated in an ordinary manner, utilizing the difference in physicochemical properties between the isomers. For example, optical isomers may be separated and purified according to a method comprising introducing a racemic compound into a diastereomer salt with an optically-active organic acid (tartaric acid, etc.) followed by fractionation and crystallization, or a method of column chromatography using a chiral filler. Optically-active compounds may be produced, using suitable optically-active compounds as starting compounds. A diastereomer mixture may also be isolated through fractionating crystallization or chromatography.
The pharmaceutical composition of the present invention containing a non-purine xanthine oxidase inhibitor as the active ingredient may be formulated, using a carrier, a vehicle and other additives that are generally usable in ordinary pharmaceutical preparations.
The administration may be attained in any route of oral administration with tablets, pills, capsules, granules, powders or liquids, or parenteral administration with intravenous or injections for intramuscular injections, or suppositories, percutaneous preparations, nasal preparations or inhalants. The dose may be suitably determined for individuals, depending on the conditions, the age and the sex of the patients to which they are administered, but is, in general, from 0.001 to 100 mg/kg adult/day, more preferably from 0.01 to 30 mg/kg adult/day for oral administration, and this is administered all at a time or, as divided in portions, administered 2 to 4 times a day. In case of intravenous administration depending on the condition, in general, the dose may be from 0.0001 to 10 mg/kg adult/day, more preferably from 0.001 to 1 mg/kg adult/day, and this is administered all at a time or, as divided in portions, administered plural times a day. For inhalation, the dose may be from 0.0001 to 1 mg/kg adult/day, and this is administered all at a time or, as divided in portions, administered plural times a day. The content of the active ingredient in the preparation may be from 0.0001 to 80%, more preferably from 0.001 to 50%.
As the solid composition for oral administration of the present invention, employed are tablets, powders, granules, etc. The solid composition of those types comprises one or more active substances along with at least one inert diluent, such as lactose, mannitol, glucose, hydroxypropyl cellulose, microcrystalline cellulose, starch, polyvinyl pyrrolidone, magnesium meta-silicate aluminate. In an ordinary manner, the composition may contain any other additives, for example, a lubricant such as magnesium stearate, a disintegrator such as sodium carboxymethyl starch, and a dissolution promoter. If desired, the tablets and pills may be coated with sugar or with a gastric or enteric coating agent.
The liquid composition for oral administration includes pharmaceutically-acceptable emulsions, solutions, suspensions, syrups, elixirs and the like, which contain ordinary inert solvents such as purified water or ethanol. In addition to the inert solvents, those compositions may further contain pharmaceutical aids such as solubilizers, wetting promoters, suspension promoters, and also sweeteners, flavorings, aromas, and preservatives.
The injection for parenteral administration includes germ-free, water-base or water-free solutions, suspensions and emulsions. The water-base solvents include, for example, distilled water for injection and physiological saline water. The water-free solvents include, for example, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethyl alcohol, Polysolvate 80 (Japan Pharmacopeia name), etc. Those compositions may further contain additives such as isotonicating promoters, preservatives, wetting promoters, emulsifiers, dispersants, stabilizers, dissolution promoters. These are sterilized by filtering them through bacteria-trapping filters, or by adding microbicides thereto, or by exposing them to radiations. The germ-free, solid compositions thus produced may be dissolved or suspended in germ-free water or in germ-free solvents for injection, before using them.
Transmucosal preparations such as inhalants or nasal preparations may be solid, liquid or semisolid, and may be produced in any conventional known methods. For example, a vehicle such as lactose or starch, and further a pH-controlling agent, a preservative, a surfactant, a lubricant, a stabilizer and a tackifier may be suitably added thereto. For administration, usable are suitable device for inhalation of insufflation. For example, using a known device or spray such as device for metering administration inhalation, the compound may be administered, as a powder of itself alone or a formulated mixture, or as a solution or suspension combined with a pharmaceutically-acceptable carrier. The dry powder inhalator may be for single use or repeated multiple use, for which usable are dry powder or powder-containing capsules. Further, it may also be a pressure aerosol spray or the like that uses a suitable propellant, for example, a suitable vapor such as chlorofluoroalkane, hydrofluoroalkane or carbon dioxide.
In producing suppositories, a low-melting point wax, for example, a fatty acid glyceride mixture or cocoa butter is melted, an active ingredient is added thereto, and uniformly dispersed by stirring. Next, this is injected into a suitable mold, cooled and solidified therein. The liquid preparation includes solution, suspension, retention enemas and emulsion, as well as water or aqueous propylene glycol solution.
In case where the pharmaceutical composition of the present invention is a combination or a mixture of an agent for treating or preventing digestive ulcer and a non-steroidal antiinflammatory drug, the amount of each ingredient may be suitably determined as the clinical effective amount thereof in its administration as a single preparation depending on the condition of each patient.
The agent for treating or preventing digestive ulcer of the present invention may be suitably combined with any other therapeutically-effective gastric secretion inhibitor, for example, a proton pump inhibitor or an H2 blocker. In case of such combination, the two may form a combined preparation for simultaneous administration, or may form different preparations for separate administration.
PREPARATION EXAMPLES
The following Preparation Examples are to concretely demonstrate the production methods for the compounds of formula (I) as the active ingredient of the pharmaceutical composition of the present invention.
The abbreviations in Reference Preparation Examples, Preparation Examples and Tables Shown Below are as Follows:
Ex: Preparation Example Number, REx: Reference Preparation Example Number,
Dat: Physicochemical data (F:FAB-MS (M+H)+,
FN: FAB-MS (M−H)−, ES:ESI-MS (M+H)+, ESN:ESI-MS (M−H)−,
EI: EI-MS (M)+, AP:API-ES-MS (M+H)+, APN:API-ES-MS (M−H)−, [the compound with (Na) after its mass-spectrometric data means that its Na salt or Na adduct gave the data; and the compound with (G-2W) after its mass-spectrometric data means that its glycerin adduct didehydrate gave the data],
NMR: δ (ppm) of characteristic peaks in 1H NMR in DMSO-d6,
NMRC: δ (ppm) of characteristic peaks in 1H NMR in CDCl3),
Anal: Elementary analysis, Calc. calculated data, Found: found data, H: Retention time (min) in HPLC under the following condition, [HPLC condition: column, Wakosil-II 5C18AR 5 μm, 2.0×30 mm; detection wavelength, 254 nm; measuring temperature, 35.0° C.; solvent, started in aqueous 5 mM trifluoroacetic acid solution/MeOH=9/1, and the ratio was changed to 0/10 within 4 minutes, and afterwards, the sample was eluted at 0/10 for 0.5 minutes, the flow rate was 1.2 ml/min]),
Str: Structural formula,
Syn: Production method (Numeral means the Preparation Example Number as referred to in producing the compound in the same manner),
Sal: Salt (the compound with no mark is a free base, the compound designated by 0.3HCl means that it is a mixture of monohydrochloride and free base in a molar ratio of 0.3/0.7),
Me: methyl; Et ethyl; nPr: n-propyl; iPr: isopropyl; cPr: cyclopropyl; nBu: n-butyl; iBu: isobutyl; tBu: tert-butyl; cBu: cyclobutyl; nPen: n-pentyl, iPen: isopentyl, cPen: cyclopentyl, nHex: n-hexyl, cHex: cyclohexyl, cHep: cycloheptyl, cOct: cyclooctyl, Bn: Benzyl, Ph: Phenyl, 2Py: 2-Pyridyl, 3Py: 3-Pyridyl, 4Py: 4-Pyridyl, 2Thie: 2-Thienyl, 3Thie: 3-Thienyl, 2Fur: 2-Furyl, 3Fur: 3-Furyl, 1Naph: 1-Naphthyl, 2Naph: 2-Naphthyl, Ac: Acetyl, and Tf: Trifluoromethanesulfonyl.
A substituted phenyl group is represented as “numeral indicating the substituent position-abbreviation of the substituent-Ph” in the Tables. “di” before the substituent means that the group has two substituents. For example, 4-MeO-3,5-diMe-Ph- means 4-methoxy-3,5-dimethylphenyl group.
In the column “Syn” relative to the production method in the Tables below, the same Preparation Example Number is given to the compounds of which the salt forms differ but which were produced through the same type of reaction. Interconversion between a free base and its salt is a technical common sense of those skilled in the art.
Reference Preparation Example 1
5-Bromo-2-hydroxybenzonitrile, isobutyl bromide, and potassium carbonate were heated at 80° C. in DMF in the presence of tetra-n-butylammonium bromide to obtain 5-bromo-2-isobutoxybenzonitrile. F: 254, 256.
Reference Preparation Example 2
After 2,2-dimethyl-1-propanol and sodium hydride were stirred at 0° C. in DMF, 5-bromo-2-fluorobenzonitrile was added thereto, followed by reaction at room temperature to obtain 5-bromo-2-(2,2-dimethylpropoxy)benzonitrile. NMRC: 3.67 (2H, s), 6.83 (1H, d), 7.64 (1H, d).
Reference Preparation Example 3
5-Bromo-2-fluorobenzonitrile and piperidine were heated at 80° C. in DMSO in the presence of cesium carbonate to obtain 5-bromo-2-piperidin-1-ylbenzonitrile. F: 265.
Reference Preparation Example 4
5-Bromo-2-isobutoxybenzonitrile and triisopropyl borate were dissolved in a mixed solvent of THF and toluene and an n-butyllithium-hexane solution was added dropwise to the solution at a temperature below −60° C. After the temperature was elevated to −20° C., 1M hydrochloric acid was added, followed by stirring at room temperature to obtain (3-cyano-4-isobutoxyphenyl)boronic acid. F: 220.
Reference Preparation Example 5
[4-(Benzyloxy)-3-cyanophenyl]boric acid and methyl 2-chloroisonicotinate were dissolved in a mixture liquid of toluene and an aqueous 2 M sodium carbonate solution, and in the presence of tetrakis(triphenylphosphine)palladium, the mixture was stirred under heating in an argon atmosphere at 100° C. to obtain methyl 2-[4-(benzyloxy)-3-cyanophenyl]isonicotinate. F: 345.
Reference Preparation Example 6
Methyl 2-[4(benzyloxy)-3-cyanophenyl]isonicotinate and pentamethylbenzene were heated under reflux temperature in trifluoroacetic acid to obtain methyl 2-(3-cyano-4-hydroxyphenyl)isonicotinate. F: 255.
Reference Preparation Example 7
Methyl 3-fluoroisonicotinate was oxidized with 3-chloroperbenzoic acid, followed by heating in the presence of phosphoryl chloride. The product was separated by silica gel column chromatography to obtain methyl 2-chloro-5-fluoroisonicotinate (EI: 189) and methyl 2-chloro-3-fluoroisonicotinate (EI: 189).
Reference Preparation Example 8
Methyl 2-(3-cyano-4-hydroxyphenyl)isonicotinate and N-chlorosuccinimide were stirred at room temperature in acetonitrile to obtain methyl 2-(3-chloro-5-cyano-4-hydroxyphenyl)isonicotinate. ES: 289.
Reference Preparation Example 9
Methyl 2-(3-cyano-4-hydroxyphenyl)isonicotinate and N-bromosuccinimide were stirred at room temperature in acetonitrile to obtain methyl 2-(3-bromo-5-cyano-4-hydroxyphenyl) isonicotinate. FN: 333.
Reference Preparation Example 10
Sodium hydride was added to a DMF solution of 2,3-difluorobenzonitrile and 2-(methylsulfonyl)ethanol, followed by stirring at room temperature to obtain 3-fluoro-2-hydroxybenzonitrile. FN: 136.
3-Fluoro-2-hydroxybenzonitrile and N-bromosuccinimide were stirred at room temperature in acetonitrile to obtain 5-bromo-3-fluoro-2-hydroxybenzonitrile. EI: 215, 217.
Reference Preparation Example 11
(3-Cyano-4-benzyloxy-5-fluorophenyl)boronic acid and methyl 2-chloroisonicotinate were dissolved in a mixed solution of toluene and a 2M aqueous sodium carbonate solution, followed by heating under reflux for 3 hours in the presence of tetrakis(triphenylphosphine)palladium to obtain methyl 2-(3-cyano-4-benzyloxy-5-fluorophenyl)isonicotinate. F: 363.
Methyl 2-(3-cyano-4-benzyloxy-5-fluorophenyl)isonicotinate is stirred at room temperature in methanol-THF (1:1) under a hydrogen atmosphere at normal pressure in the presence of palladium-carbon to obtain methyl 2-(3-cyano-5-fluoro-4-hydroxyphenyl)isonicotinate. FN: 271.
Reference Preparation Example 12
Cesium fluoride and tetrakis(triphenylphosphine)palladium were added to a 1,2-dimethoxyethane solution of (3-cyano-4-fluorophenyl)boronic acid and methyl 2-chloroisonicotinate, and the mixture was heated under reflux under an argon atmosphere to obtain methyl 2-(3-cyano-4-fluorophenyl)isonicotinate. F: 257.
Reference Preparation Example 13
(4-Benzyloxy-3-cyanophenyl)boronic acid and methyl 4,5-dibromo-3-fluorothiophene-2-carboxylate were dissolved in a mixed solution of toluene and a 1M aqueous sodium carbonate solution and the whole was heated at 110° C. for 2.5 days in the presence of tetrakis(triphenylphosphine)palladium to obtain methyl 4-bromo-5-(4-benzyloxy-3-cyanophenyl)-3-fluorothiophene-2-carboxylate. Methyl 4-bromo-5-(4-benzyloxy-3-cyanophenyl)-3-fluorothiophene-2-carboxylate and triethylamine were stirred at room temperature in 1,4-dioxane under a hydrogen atmosphere at normal pressure in the presence of palladium-carbon to obtain methyl 5-(3-cyano-4-hydroxyphenyl)-3-fluorothiophene-2-carboxylate. FN: 276.
Reference Preparation Example 14
A 4M HCl/1,4-dioxane solution was added to a DMF solution of 4-(benzyloxy)isophthalonitrile and thioacetamide, followed by stirring at 60° C. to obtain 4-(benzyloxy)-3-cyanobenzenecarbothioamide. AP: 291 (Na).
Reference Preparation Example 15
4-(Benzyloxy)-3-cyanobenzenecarbothioamide and ethyl 2-chloroacetacetate were stirred in ethanol at 75° C. to obtain ethyl 2-[4-(benzyloxy)-3-cyanophenyl]-4-methyl-1,3-thiazole-5-carboxylate. AP: 401(Na).
Reference Preparation Example 16
4-(Benzyloxy)-3-cyanobenzenecarbothioamide and methyl 2-chloro-3-oxopropionate were heated under reflux in 1-butanol in the presence of Molecular Sieves 4A to obtain methyl 2-[4-(benzyloxy)-3-cyanophenyl]-1,3-thiazole-5-carboxylate. AP: 373(Na).
Reference Preparation Example 17
Ethyl 2-[4-(benzyloxy)-3-cyanophenyl]-4-methyl-1,3-thiazole-5-carboxylate was suspended in a mixture of THF and ethanol, then palladium-carbon was added thereto, and the mixture was stirred under a hydrogen atmosphere at room temperature to obtain ethyl 2-(3-cyano-4-hydroxyphenyl)-4-methyl-1,3-thiazole-5-carboxylate. APN: 287.
Reference Preparation Example 18
Methyl 2-(3-cyano-4-hydroxyphenyl)isonicotinate and trifluoromethanesulfonic anhydride were reacted at 0° C. in dichloromethane in the presence of diisopropylethylamine to obtain methyl 2-(3-cyano-4-{[(trifluoromethyl)sulfonyl]oxy}phenyl)isonicotinate. F: 387.
Reference Preparation Example 19
5-Bromo-2-iodobenzonitrile and 3-pyridylboric acid were dissolved in a mixture solution of aqueous 2 M sodium carbonate solution and toluene, then tetrakis(triphenylphosphine)palladium was added thereto, and the mixture was heated, stirred with heating in an argon atmosphere at 100° C. for 3 days to obtain 5-bromo-2-pyridin-3-ylbenzonitrile. EI: 258, 260.
Reference Preparation Example 20
Methyl 2-(3-cyano-4-fluorophenyl)isonicotinate and sodium azide were dissolved in a DMF solution, followed by stirring at 50° C. for 4 hours to obtain methyl 2-(4-azido-3-cyanophenyl)isonicotinate. NMRC: 7.38 (1H, d), 7.84 (1H, dd), 8.46 (1H, d)
Reference Preparation Example 21
5-Formyl-2-methoxybenzonitrile, sodium acetate and hydroxyamine were dissolved in ethanol, followed by stirring at 80° C. for 6 hours to obtain 5-[(hydroxyimino)methyl]-2-methoxybenzonitrile. APN: 175.
Reference Preparation Example 22
5-[(Hydroxyimino)methyl]-2-methoxybenzonitrile, 4 M hydrochloric acid and Oxon (registered trade name) were dissolved in a DMF solution, followed by stirring at room temperature for 12 hours to obtain 3-cyano-N-hydroxy-4-methoxybenzenecaboximidoyl chloride. NMRC: 7.01 (1H, d), 8.03 (1H, dd), 8.07 (1H, d).
Reference Preparation Example 23
3-Cyano-N-hydroxy-4-methoxybenzenecaboximidoyl chloride, ethyl propiolate and triethylamine were dissolved in a THF solution, followed by stirring at 40° C. to obtain ethyl 3-(3-cyano-4-methoxyphenyl)-5-isoxazolecarboxylate. AP: 295.
Reference Preparation Example 24
Ethyl 3-(3-cyano-4-methoxyphenyl)-5-isoxazolecarboxylate and tribromoborane were dissolved in a dichloromethane solution, followed by stirring for 2 hours under ice-cooling. Further, the mixture was stirred at 40° C. for 30 minutes to obtain ethyl 3-(3-cyano-4-hydroxyphenyl)-5-isoxazolecarboxylate. APN: 257.
Reference Preparation Example 25
Palladium-carbon was added to a methanol solution of methyl 2-(4-azido-3-cyanophenyl)isonicotinate, and the mixture was stirred in the presence of hydrogen gas at room temperature for 5 hours to obtain methyl 2-(4-amino-3-cyanophenyl)isonicotinate. AP: 254.
Reference Preparation Example 26
Methyl 5-(4-hydroxyphenyl)thiophene-2-carboxylate was obtained in accordance with the method of Reference Preparation Example 6 using methyl 5-(4-benzyloxyphenyl)thiophene-2-carboxylate and pentamethylbenzene. F: 233.
Sodium hydride and acetic anhydride were added to a THF solution of methyl 5-(4-hydroxyphenyl)thiophene-2-carboxylate and the whole was stirred at room temperature to obtain methyl 5-[4-(acetyloxy)phenyl]thiophene-2-carboxylate. ES: 277.
Aluminum chloride was added to a chlorobenzene solution of methyl 5-[4-(acetyloxy)phenyl]thiophene-2-carboxylate and the whole was stirred at 120° C. to obtain methyl 5-(3-acetyl-4-hydroxyphenyl)thiophene-2-carboxylate. F: 277.
Reference Preparation Example 27-65
Starting from the corresponding starting compounds, compounds of Reference Preparation Examples 27 to 31 were produced in the same manner as in Reference Preparation Example 1, compounds of Reference Preparation Examples 32 to 36 were produced in the same manner as in Reference Preparation Example 2, a compound of Reference Preparation Example 37 was produced in the same manner as in Reference Preparation Example 3, compounds of Reference Preparation Examples 38 to 50 were produced in the same manner as in Reference Preparation Example 4, a compound of Reference Preparation Example 51 was produced in the same manner as in Reference Preparation Example 5, a compound of Reference Preparation Example 52 was produced in the same manner as in Reference Preparation Example 6, a compound of Reference Preparation Example 53 was produced in the same manner as in Reference Preparation Example 11, a compound of Reference Preparation Example 54 was produced in the same manner as in Reference Preparation Example 12, a compound of Reference Preparation Example 55 was produced in the same manner as in Reference Preparation Example 15, compounds of Reference Preparation Examples 56 to 57 were produced in the same manner as in Reference Preparation Example 17, compounds of Reference Preparation Examples 58 to 65 were produced in the same manner as in Reference Preparation Example 18. As the starting compound in Reference Preparation Examples 62 and 64, used was the phenol compound described in Patent References 7 and 8. The structures and the physicochemical data of the compounds of Reference Preparation Examples 27 to 65 are shown in Table 1 to 2 below.
Reference Preparation Example 66
20% sodium ethoxide and isoamyl nitrite were added to and dissolved in an ethanol solution of 5-(cyanomethyl)-2-methoxybenzonitrile. Isopropyl alcohol was added, and the precipitate formed was collected by filtration. The resulting solid and 4-methylbenzenesulfonyl chloride were dissolved in ethanol, and the solution was refluxed for 5 hours to obtain 5-[cyano({[(4-methylphenyl)sulfonyl]oxy}imino)methyl]-2-methoxybenzonitrile. AP: 378.
Reference Preparation Example 67
Ethyl sulfanylacetate and triethylamine were dissolved in an ethanol solution of 5-[cyano({[(4-methylphenyl)sulfonyl]oxy}imino)methyl]-2-methoxybenzonitrile, followed by stirring for 5 hours under ice-cooling to obtain ethyl 4-amino-3-(3-cyano-4-methoxyphenyl)isothiazole-5-carboxylate. AP: 378.
Reference Preparation Example 68
3-Methylbutyl nitrate was dissolved in a tetrahydrofuran solution of ethyl 4-amino-3-(3-cyano-4-methoxyphenyl)isothiazole-5-carboxylate, followed by heating under reflux for 5 hours to obtain ethyl 3-(3-cyano-4-methoxyphenyl)isothiazole-5-carboxylate. AP: 311.
Reference Preparation Example 69
Under ice-cooling, boron tribromide was added to a dichloromethane solution of ethyl 3-(3-cyano-4-methoxyphenyl)isothiazole-5-carboxylate, followed by stirring for 1 hour and then stirring at 40° C. for 3 hours to obtain ethyl 3-(3-cyano-4-hydroxyphenyl)isothiazole-5-carboxylate. AP: 297.
Preparation Example 1
(1) In a mixed solution of 50 ml of toluene and 30 ml of a 2M aqueous sodium carbonate solution were dissolved 1.46 g of (3-cyano-4-isobutoxyphenyl)boronic acid and 1.86 g of methyl 2-chloroisonicotinate, and the resulting solution was heated at 100° C. for 1 hour in the presence of 0.49 g of tetrakis(triphenylphosphine)palladium. The reaction solution was extracted with ethyl acetate and the organic layer was washed with brine and then dried and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane:ethyl acetate:chloroform=70:15:15) to obtain 1.98 g of methyl 2-(3-cyano-4-isobutoxyphenyl)isonicotinate.
(2) Then, 1.98 g of the compound was dissolved in a mixed solution of 30 ml of methanol and 70 ml of THF, and 9 ml of a 1M aqueous sodium hydroxide solution was added thereto, followed by heating at 50° C. for 1 hour.
After cooling, the resulting solution was neutralized with 1M hydrochloric acid and then extracted with chloroform, followed by washing with brine. After the solution was dried, concentration was performed under reduced pressure and the resulting residue was recrystallized from a mixed solvent of ethanol and water to obtain 1.66 g of 2-(3-cyano-4-isobutoxyphenyl)isonicotinic acid.
Preparation Example 2
(1) In 5 ml of DMF were dissolved 82 mg of methyl 2-(3-cyano-4-hydroxyphenyl)isonicotinate and 66 mg of isopropyl iodide, and the resulting solution was heated at 80° C. for 3 hours in the presence of 72 mg of potassium carbonate and 10 mg of tetra-n-butylammonium bromide. The reaction solution was cooled and then diluted with water, followed by extraction with ethyl acetate. The organic layer was washed with brine and then dried and concentrated under reduced pressure. The resulting residue was washed with a mixed solvent (hexane:ethyl acetate=10:1) to obtain 91 mg of methyl 2-(3-cyano-4-isopropoxyphenyl)isonicotinate.
(2) Then, 86 mg of the compound was dissolved in a mixed solution of 3 ml of methanol and 3 ml of THF, and 0.35 ml of a 1M aqueous sodium hydroxide solution was added thereto, followed by heating at 60° C. for 1 hour. After being cooled to room temperature, the resulting solution was diluted with diisopropyl ether and water and an aqueous layer was separated. The aqueous layer was neutralized with 1M hydrochloric acid and then extracted with ethyl acetate. After being washed with water, the organic layer was dried and concentrated under reduced pressure to obtain 55 mg of 2-(3-cyano-4-isopropoxyphenyl)isonicotinic acid.
Preparation Example 3
(1) In 5 ml of THF were dissolved 63 mg of 3-(methylthio)-1-propanol and 100 mg of methyl 2-(3-cyano-4-hydroxyphenyl)isonicotinate, and the resulting solution was heated at 0° C. for 10 minutes in the presence of 0.15 ml of tributylphosphine and 149 mg of 1,1′-(azodicarbonyl)dipiperidine. Then, the reaction solution was warmed to room temperature and stirred all day and night. After removal of the solvent, water was added and extraction with ethyl acetate was performed. The resulting organic layer is washed with brine and then dried and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (chloroform:methanol=95:5) to obtain 92 mg of methyl 2-{3-cyano-4-[3-(methylthio)propoxy]phenyl}isonicotinate.
(2) Then, 92 mg of the compound was dissolved in a mixed solution of 3 ml of methanol and 3 ml of THF, and 0.32 ml of a 1M aqueous sodium hydroxide solution was added thereto, followed by heating at 60° C. for 1 hour. After being cooled, the reaction solution was diluted with diisopropyl ether and an aqueous layer was separated. The aqueous layer was neutralized with 1M hydrochloric acid and then extracted with ethyl acetate. After washing with brine, the organic layer was dried and concentrated under reduced pressure to obtain 81 mg of 2-{3-cyano-4-[3-(methylthio)propoxy]phenyl}isonicotinic acid.
Preparation Example 4
(1) In 7 ml of DMSO was dissolved 2.22 g of methyl 2-(3-cyano-4-fluorophenyl)isonicotinate, and 2.44 ml of hexamethyleneimine was added thereto, followed by heating at 50° C. for 5 hours. After cooling, the reaction solution was diluted with ethyl acetate and was washed with 1M hydrochloric acid, a saturated aqueous sodium hydrogen carbonate solution, and brine, successively. The organic layer was dried and then concentrated under reduced pressure and the resulting residue was dissolved in a mixed solvent of ethyl acetate and diisopropyl ether. Activated carbon was added thereto, followed by stirring for 1 hour. Then, the activated carbon was removed by filtration and washed with ethyl acetate. The resulting filtrate and washing liquid were combined and concentrated to obtain 2.58 g of methyl 2-(4-azepan-1-yl-3-cyanophenyl)isonicotinate.
(2) Then, 2.49 g of the compound was dissolved in a mixed solvent of 15 ml of methanol and 30 ml of THF, and 11 ml of a 1M aqueous sodium hydroxide solution was added thereto, followed by heating at 80° C. for 1 hour.
After cooling, the reaction solution was concentrated under reduced pressure. Then, water was added, followed by washing with diisopropyl ether. The resulting aqueous layer was filtered and then neutralized with 1M hydrochloric acid. The precipitated crystals were collected by filtration and washed with water and ethanol, successively. The crude crystals were recrystallized from a mixed solvent of DMSO and water to obtain 2.07 g of a salt free compound of 2-(4-azepan-1-yl-3-cyanophenyl)-isonicotinate. 295 mg of the free compound obtained in a similar manner was suspended in a mixed solvent of 4 ml of ethanol and 2 ml of THF, and 0.46 ml of a 4M hydrogen chloride-ethyl acetate solution was added thereto. After stirring at room temperature for 30 minutes, the precipitated crystals were collected by filtration to obtain 279 mg of 2-(4-azepan-1-yl-3-cyanophenyl)isonicotinic acid monohydrochloride.
Preparation Example 5
(1) In 0.4 ml of 1,4-dioxane were dissolved 237 mg of methyl 2-(3-cyano-4-{[(trifluoromethyl)sulfonyl]oxy}-phenyl)isonicotinate and 0.4 ml of heptamethyleneimine, followed by heating at 90° C. for 1 hour. After the reaction solution was cooled, purification by silica gel column chromatography (hexane:ethyl acetate:chloroform=80:10:10) was performed to obtain 23 mg of 2-(4-azocan-1-yl-3-cyanophenyl)isonicotinate.
(2) Then, 22 mg of the compound was dissolved in a mixed solution of 2 ml of methanol and 2 ml of THF, and 0.15 ml of a 1M aqueous sodium hydroxide solution was added thereto, followed by reaction at room temperature for 20 hours. To the reaction solution were added 0.15 ml of 1M hydrochloric acid and 20 ml of water, and the resulting precipitate was collected by filtration. The precipitate was washed with water and then dried to obtain 16 mg of 2-(4-azocan-1-yl-3-cyanophenyl)isonicotinic acid.
Preparation Example 6
(1) Under an argon atmosphere, 820 mg of 4-bromo-1-isobutoxy-2-nitrobenzene and 830 mg of bispinacolatodiboron were dissolved in toluene and the resulting solution was heated and refluxed for 15 hours in the presence of 60 mg of dichlorodi(triphenylphosphine)palladium, 50 mg of triphenylphosphine, and 350 mg of potassium acetate. After cooling to room temperature, 660 mg of methyl 5-bromothiophene-2-carboxylate, 150 mg of tetrakis(triphenylphosphine)palladium, and 7.5 ml of a 2M aqueous sodium carbonate solution were added to the solution, followed by heating at 100° C. for 6 hours. After cooling to room temperature, the solution was extracted with ethyl acetate and the resulting organic layer was dried and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane:ethyl acetate=90:10) to obtain 790 mg of methyl 5-(3-nitro-4-isobutoxyphenyl)thiophene-2-carboxylate.
(2) Then, 570 mg of the compound was dissolved in a mixed solution of 10 ml of ethanol and 10 ml of THF, and 3 ml of a 1M aqueous sodium hydroxide solution was added thereto, followed by heating at 60° C. for 18 hours. After cooling to room temperature, the resulting solution was diluted with water and ethyl acetate and an aqueous layer was separated. The aqueous layer was neutralized with 1M hydrochloric acid and then extracted with ethyl acetate. The resulting organic layer was dried and concentrated under reduced pressure to obtain 350 mg of 5-(3-nitro-4-isobutoxyphenyl)thiophene-2-carboxylic acid.
Preparation Example 7
(1) Using (3-cyano-4-isobutoxyphenyl)boronic acid and methyl 4,5-dibromo-3-fluorothiophene-2-carboxylate, methyl 4-bromo-5-(3-cyano-4-isobutoxyphenyl)-3-fluorothiophene-2-carboxylate was obtained in accordance with the method of Preparation Example 1(1). To an ethanol (60 ml) suspension of 1.75 g of the compound was added 1.00 g of 10% palladium-carbon, followed by stirring at room temperature for 4 hours under a hydrogen atmosphere at normal pressure. After insoluble matter was removed by filtration, the filtrate was concentrated under reduced pressure and the residue was purified by silica gel column chromatography (hexane:ethyl acetate=10:1 to 5:1) to obtain 0.39 g of methyl 5-(3-cyano-4-isobutoxyphenyl)-3-fluorothiophene-2-carboxylate.
(2) The compound was dissolved in 15 ml of THF, and 2.5 ml of a 1M aqueous sodium hydroxide solution was added thereto, followed by heating at 60° C. for 5 hours. After cooling to room temperature, 2.5 ml of 1M hydrochloric acid and water were added thereto, followed by extraction with ethyl acetate. The resulting organic layer was dried and concentrated under reduced pressure. The resulting solid was recrystallized from ethyl acetate and hexane to obtain 252 mg of 5-(3-cyano-4-isobutoxyphenyl)-3-fluorothiophene-2-carboxylic acid.
(3) Then, 215 mg of sodium hydroxide was added to an ethanol (60 ml) solution of 1.81 g of 5-(3-cyano-4-isobutoxyphenyl)-3-fluorothiophene-2-carboxylic acid, followed by stirring at 80° C. overnight. After the reaction solution was cooled to room temperature, the precipitate was collected by filtration to obtain 1.60 g of sodium 5-(3-cyano-4-isobutoxyphenyl)-3-fluorothiophene-2-carboxylate.
Preparation Example 8
(1) 87 mg of tetrakis(triphenylphosphine)palladium was added to a toluene (25 ml) suspension of 966 mg of methyl 2-(3-cyano-4-{[(trifluoromethyl)sulfonyl]oxy}phenyl)isonicotinate, 610 mg of phenylboronic acid and 518 mg of potassium carbonate, followed by heating at 100° C. in an argon atmosphere for 2 hours. Water was added to the reaction mixture, followed by extraction with ethyl acetate. The organic layer was washed with brine, dried and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane:ethyl acetate=95:5 to 70:30) to obtain 758 mg of methyl 2-(2-cyanobiphenyl-4-yl)isonicotinate.
(2) 758 mg of this compound was dissolved in a mixture of 10 ml of methanol and 10 ml of THF, and 7.2 ml of aqueous 1 M sodium hydroxide solution was added thereto, followed by heating at 60° C. for 13 hours. The reaction mixture was cooled to room temperature, neutralized with 1 M hydrochloric acid, and concentrated under reduced pressure. The residue was recrystallized from a mixture of ethanol and water to obtain 472 mg of 2-(2-cyanobiphenyl-4-yl)isonicotinic acid.
(3) 414 mg of this compound was dissolved in 15 ml of ethanol, and 1.5 ml of aqueous 1 M sodium hydroxide solution was added thereto, followed by stirring at room temperature for 30 minutes. The reaction solution was concentrated under reduced pressure to obtain 430 mg of sodium 2-(2-cyanobiphenyl-4-yl)isonicotinate.
Preparation Example 9
(1) 212 mg of methyl 2-(3-cyano-4-fluorophenyl)isonicotinate and 68 mg of pyrazole were dissolved in 4 ml of DMSO, and 102 mg of potassium tert-butoxide was added, followed by stirring at room temperature for 30 minutes. The reaction mixture was diluted with water, and extracted with ethyl acetate. The organic layer was washed with brine, dried and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane:ethyl acetate=67:33) to obtain 251 mg of methyl 2-[3-cyano-4-(1H-pyrazol-1-yl)phenyl]isonicotinate.
(2) 236 mg of this compound was dissolved in a mixture liquid of 10 ml of methanol and 5 ml of THF, and 1.16 ml of aqueous 1 M sodium hydroxide solution was added, followed by heating at 80° C. for 40 minutes. The reaction liquid was cooled to room temperature, washed with water, and the organic solvent was evaporated away under reduced pressure. The reaction liquid was washed with diethyl ether to obtain an aqueous layer. The aqueous layer was neutralized with 1 M hydrochloric acid, followed by extraction with ethyl acetate. The organic layer was washed with brine, dried and concentrated under reduced pressure, and the resulting residue was recrystallized from a mixture of ethanol and water to obtain 103 mg of 2-[3-cyano-4-(1H-pyrazol-1-yl)phenyl]isonicotinic acid.
(3) 92 mg of this compound was dissolved in ethanol, and 0.317 ml of aqueous 1 M sodium hydroxide solution was added thereto, followed by stirring at room temperature for 15 minutes. The reaction liquid was concentrated, the residue was suspended in 2-propanol, and the precipitate was collected by filtration to obtain 93 mg of sodium 2-[3-cyano-4-(1H-pyrazol-1-yl)phenyl]isonicotinate.
Preparation Example 10
(1) 1.32 g of methyl 2-[4′-(benzyloxy)-2,3′-dicyanobiphenyl-4-yl]isonicotinate, which had been obtained in the same manner as in Preparation Example 8(1) using 4-(benzyloxy)-3-cyanophenyl]boric acid and methyl 2-(3-cyano-4-{[(trifluoromethyl)sulfonyl]oxy}phenyl)isonicotinate, was dissolved in a mixture of 50 ml of THF and 50 ml of methanol, and 0.5 g of palladium-carbon was added, followed by stirring in a hydrogen atmosphere at room temperature for 12 hours. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain 0.5 g of methyl 2-(2,3′-dicyano-4′-hydroxybiphenyl-4-yl)isonicotinate.
(2) 230 mg of this compound was dissolved in DMF, and 50 μL of iodomethane and 108 mg of potassium carbonate were added, followed by stirring at room temperature for 2 hours. Water was added to the reaction mixture, followed by extraction with ethyl acetate. The organic layer was washed with brine, dried and concentrated under reduced pressure. Chloroform was added to the residue, and the precipitated crystal was collected by filtration, followed by washing with chloroform to obtain 73 mg of methyl 2-(2,3′-dicyano-4′-methoxybiphenyl-4-yl)isonicotinate.
(3) 73 mg of this compound was dissolved in 2 ml of methanol and 2 ml of THF, and 220 μL of an aqueous 1 M sodium hydroxide solution was added, followed by heating at 60° C. for 2 hours. After cooling, the solvent was removed under reduced pressure, and then water was added to the residue, followed by neutralization with 1 M hydrochloric acid. The precipitated crystal was collected by filtration and washed with a mixture of ethanol and water to obtain 64 mg of 2-(2,3′-dicyano-4′-methoxybiphenyl-4-yl)isonicotinic acid.
Preparation Example 11
(1) 58 mg of tetrakis(triphenylphosphine)palladium and 208 mg of potassium carbonate were added to a toluene (10 ml) solution of 386 mg of methyl 2-(3-cyano-4-{[(trifluoromethyl)sulfonyl]oxy}phenyl)isonicotinate and 534 mg of 1-(triisopropylsilyl)pyrrole-3-boronic acid, then this was irradiated with microwaves and heated at 130° C. in a nitrogen atmosphere for 1 hour. Water was added to the reaction mixture, followed by extraction with ethyl acetate. The organic layer was washed with brine, dried and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane:ethyl acetate=95:5 to 70:30) to obtain 24 mg of methyl 2-{3-cyano-4-[1-(triisopropylsilyl)-1H-pyrrol-3-yl]phenyl}isonicotinate.
(2) 24 mg of this compound was dissolved in 1 ml of THF, and 63 μL of 1 M tetrabutylammonium fluoride/THF solution was added thereto, followed by stirring at room temperature for 15 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (hexane:ethyl acetate=90:10 to 70:30) to obtain 6 mg of methyl 2-[3-cyano-4-(1H-pyrrol-3-yl)phenyl]isonicotinate.
(3) 6 mg of this compound was dissolved in a mixture of 0.5 ml of methanol and 0.5 ml of THF, and 22 μl of aqueous 1 M sodium hydroxide solution was added, followed by heating at 60° C. for 2 hours. The reaction liquid was cooled, and the solvent was removed under reduced pressure. Water was added to the residue, followed by neutralization with 1 M hydrochloric acid. The precipitated crystal was collected by filtration and washed with a mixture of ethanol and water to obtain 1.5 mg of 2-[3-cyano-4-(1H-pyrrol-3-yl)phenyl]isonicotinic acid.
Preparation Example 12
(1) 131 mg of methyl 2-(4-amino-3-cyanophenyl)isonicotinate and 67 μl of 2,5-dimethoxytetrahydrofuran were dissolved in 1.3 ml of acetic acid solution, followed by heating and stirring at 100° C. for 4 hours. The solution was poured into water, followed by extraction with ethyl acetate. The solvent of the organic layer was evaporated under reduced pressure, and the residue was purified by column chromatography (hexane:ethyl acetate=10:1 to 1:1) to obtain 100 mg of methyl 2-[3-cyano-4-(1H-pyrrol-1-yl)phenyl]isonicotinate.
(2) 100 mg of methyl 2-[3-cyano-4-(1H-pyrrol-1-yl)phenyl]isonicotinate was dissolved in a mixture of 2 ml of methanol and 3 ml of THF, and 66 μl of aqueous 1 M sodium hydroxide solution was added, followed by heating under reflux for 3 hours. The reaction mixture was cooled, then neutralized with 66 μl of 1 M hydrochloric acid, followed by extraction with a mixture of 2-propanol and chloroform (1:4). The organic layer was washed with brine. The solvent of the organic layer was evaporated under reduced pressure, and the resulting residue was recrystallized from a mixture of 2-propanol and chloroform (1:4) to obtain 95 mg of 2-[3-cyano-4-(1H-pyrrol-1-yl)phenyl]isonicotinic acid.
Preparation Example 13
(1) A toluene (0.5 ml) suspension of 20 mg of methyl 5-(3-cyano-4-{[(trifluoromethyl)sulfonyl]oxy}phenyl)thiophene-2-carboxylate and 10 mg of potassium carbonate was added to 15 mg of 3-aminobenzeneboronic acid monohydrate, and in an argon atmosphere, 8 mg of tetrakis(triphenylphosphine)palladium was added thereto. The mixture was stirred overnight at 100° C., then cooled to room temperature, and filtered through Celite. The solvent was evaporated under reduced pressure to obtain methyl 5-(3′-amino-2-cyanobiphenyl-4-yl)thiophene-2-carboxylate.
(2) 0.2 ml of aqueous 1 M sodium hydroxide solution was added to a methanol (0.25 ml)/tetrahydrofuran (0.25 ml) solution of methyl 5-(3′-amino-2-cyanobiphenyl-4-yl)thiophene-2-carboxylate, followed by stirring overnight at 60° C. 1 M hydrochloric acid was added to the reaction liquid to make it acidic, and the solvent was evaporated under reduced pressure. The residue was purified by HPLC [elution through column: SunFire (registered trademark) C18 5 μm, 19 mm×100 mm, solvent: MeOH/aqueous 0.1% formic acid solution=10/90 for 1 minute, ratio change to 95/5, taking 8 minutes, and further elution with 95/5 for 3 minutes, flow rate: 25 mL/min), thereby obtaining 2.5 mg of 5-(3′-amino-2-cyanobiphenyl-4-yl)thiophene-2-carboxylic acid.
Preparation Example 14
In the same manner as in Preparation Example 13 but using 25 mg of {4-[(tert-butoxycarbonyl)amino]-3-fluorophenyl}boronic acid in place of 3-aminobenzeneboronic acid monohydrate, 5-{4′-[(tert-butoxycarbonyl)amino]-2-cyano-3′-fluorobiphenyl-4-y;}thiophene-2-carboxylic acid was obtained. The compound was dissolved in a mixed solvent of 0.5 ml of dichloromethane and 0.5 ml of trifluoroacetic acid, followed by stirring at room temperature for 2 hours. The reaction liquid was evaporated under reduced pressure, and then purified in the same manner as that for the purification treatment in Example 13 to obtain 9.2 mg of 5-(4′-amino-2-cyano-3′-fluorobiphenyl-4-yl)thiophene-2-carboxylic acid.
Preparation Example 15
(1) 6 ml of aqueous 2 M sodium carbonate solution and 70 mg of tetrakistriphenylphosphine palladium were added to a toluene (15 ml) solution of 450 mg of (3-cyano-4-pyridin-3-ylphenyl)boronic acid and 412 mg of Methyl 2-chloroisonicotinate acid, and in an argon atmosphere, this was heated at 100° C. for 2 hours. 3 ml of ethanol was added, followed by further heating at 100° C. for 1 hour. Water was added to the reaction mixture, followed by extraction with chloroform. The organic layer was washed with brine, dried and concentrated under reduced pressure.
The residue was purified by silica gel column chromatography (chloroform:methanol=99:1 to 93:7) to obtain 127 mg of ethyl 2-(3-cyano-4-pyridin-3-ylphenyl)isonicotinate. F: 330.
(2) 100 mg of this compound was dissolved in a mixture of 10 ml of methanol and 3 ml THF, and 3 ml of aqueous 1 M sodium hydroxide solution was added thereto and heated at 60° C. for 1.5 hours. After cooled to room temperature, the reaction mixture was made to have pH of 3 to 4 with 1 M hydrochloric acid added thereto, and then concentrated under reduced pressure. The residue was washed with a mixture of ethanol and water to obtain 54 mg of 2-(3-cyano-4-pyridin-3-ylphenyl)isonicotinic acid 0.3 hydrochloride.
Preparation Example 16-269
In the same manner as in Preparation Examples 1 to 15 but starting from the corresponding starting compounds, the compounds of Preparation Examples 16 to 269 shown in Tables 3 to 20 below were produced. The structures and the physicochemical data of the compounds of Preparation Examples 1 to 269 are shown in Tables 3 to 20.
Thus produced, the compounds of formula (I) that are the active ingredient of the pharmaceutical composition of the present invention may be formed into pharmaceutical compositions according to the following formulation.
Formulation Example
100 g of an active ingredient is taken and mixed with 652 g of lactose and 163 g of corn starch. The mixture is, as combined with 300 g of an aqueous solution of 10% hydroxypropyl cellulose (for example, Nippon Soda's HPC-SL), granulated and dried using a fluidized layer granulator. The dried product is mixed with 50 g of low-substitution hydroxypropyl cellulose (for example, Shin-etsu Chemical's L-HPC), and further mixed with 5 g of magnesium stearate to prepare a mixture powder for tabletting. Using a rotary tabletting machine (for example, by Kikusui) with a tabletting mallet having a diameter of 8 mm and a mortar, the mixture powder is tableted into tablets of 200 mg each.
TABLE 1
REx
Str
Dat
27
EI: 287, 289
28
F: 298
29
EI: 215, 217
30
F: 252
31
F: 274, 276
32
EI: 269, 271
33
EI: 279, 281
34
F: 336, 338
35
F: 282, 284
36
EI: 279, 281
37
F: 251
38
ES: 254
39
FN: 232
40
FN: 261
41
F: 296 (G-2W)
42
F: 235
43
FN: 244
44
F: 358 (F-2W)
45
F: 302 (G-2W)
46
F: 304 (G-2W)
47
ES: 231
48
ES: 217
49
F: 328 (G-2W)
50
F: 281 (G-2W)
TABLE 2
REx
Str
Dat
51
F: 350
52
FN: 258
53
FN: 276
54
F: 262
55
AP: 401(Na)
56
APN: 287
57
APN: 259
58
F: 392
59
F: 421
60
AP: 443(Na)
61
AP: 415(Na)
62
AP: 412(Na)
63
AP: 413(Na)
64
AP: 412(Na)
65
AP: 429(Na)
TABLE 3
Ex
Syn
A—X—
R2
Dat
1
1
iBuO—
CN
F: 297; NMR: 4.00(2 H, d), 7.38(1 H, d), 7.77(1 H, dd)
2
2
iPrO—
CN
F: 283; NMR: 1.36(6 H, d), 7.41(1 H, d), 8.83(1 H, d)
3
3
MeS—(CH2)3—O—
CN
F: 329; NMR: 4.31(2 H, t), 7.40(1 H, d), 8.83(1 H, d)
16
1
BnO—
CN
F: 331; NMR: 5.34(2 H, s), 7.71(1 H, d), 8.39(1 H, d)
17
1
CN
F: 308; NMR: 3.20-3.28(4 H, m), 7.24(1 H, d), 7.75(1 H, dd)
18
1
CN
F: 294; NMR: 3.53-3.69(4 H, m), 6.88(1 H, d), 7.67(1 H, dd)
19
1
tBu—CH2—O—
CN
F: 311; NMR: 3.89(2 H, s), 7.37(1 H, d), 8.83(1 H, d)
20
1
CF3—CH2—O—
CN
F: 323; NMR: 5.09(2 H, q), 7.53(1 H, d), 7.80(1 H, dd)
12
1
CN
F: 325; NMR: 3.82-3.94(2 H, m), 7.49(1 H, d), 7.77(1 H, dd)
22
2
cPenO—
CN
F: 309; NMR: 5.10(1 H, m), 7.38(1 H, d), 8.83(1 H, d)
23
2
iPenO—
CN
ES: 311; NMR: 4.25(2 H, t), 7.41(1 H, d), 8.83(1 H, d)
24
2
EtO—
CN
F: 269; NMR: 1.41(3 H, t), 7.37(1 H, d), 8.83(1 H, d)
25
2
nBuO—
CN
F: 297; NMR: 4.23(2 H, t), 7.39(1 H, d), 8.83(1 H, d)
26
2
CN
F: 334; NMR: 6.01(2 H, t), 7.33(1 H, d), 8.83(1 H, d)
27
2
nPrO—
CN
F: 283; NMR: 4.19(2 H, t), 7.38(1 H, d), 7.77(1 H, dd)
28
2
2Py—CH2—O—
CN
F: 332; NMR: 5.45(2 H, s), 8.35(1 H, s), 8.82(1 H, d)
29
2
3Py—CH2—O—
CN
F: 332; NMR: 5.43(2 H, s), 8.36(1 H, s), 8.83(1 H, s)
30
2
iBuO—
CF3
F: 340; NMR: 3.98(2 H, d), 7.37(1 H, d), 7.76(1 H, dd)
31
2
MeO—
CN
F: 255; NMR: 4.00(3 H, s), 7.39(1 H, d), 7.77(1 H, dd)
32
2
nPenO—
CN
F: 311; NMR: 4.22(2 H, t), 7.38(1 H, d), 7.77(1 H, dd)
33
2
nHexO—
CN
F: 325; NMR: 4.21(2 H, t), 7.37(1 H, d), 7.77(1 H, dd)
34
2
(Et)2CHCH2O—
CN
F: 325; NMR: 4.13(2 H, d), 7.41(1 H, d), 7.77(1 H, dd)
35
2
MeO(CH2)3O—
CN
F: 313; NMR: 3.53(2 H, t), 7.39(1 H, d), 7.77(1 H, dd)
36
2
(Et)2CHO—
CN
F: 311; NMR: 0.95(6 H, t), 7.41(1 H, d), 7.77(1 H, dd)
37
2
PhOCH2CH2CH2O—
CN
F: 361; NMR: 4.34-4.43(2 H, m), 7.31(2 H, t), 7.78(1 H,
dd)
TABLE 4
38
2
MeOCH2CH2O—
CN
F: 299; NMR: 3.36(3 H, s), 7.41(1 H, d), 7.77(1 H, dd)
39
2
CN
F: 356; NMR: 7.46(1 H, d), 7.78(1 H, dd), 7.94(2 H, d)
40
2
CN
F: 435; NMR: 6.52(1 H, dd), 7.48(1 H, d), 7.78(1 H, dd)
41
2
NC—(CH2)3—O—
CN
F: 308; NMR: 2.70(2 H, t), 7.42(1 H, d), 7.78(1 H, dd)
42
2
cHex-CH2—O—
CN
F: 337; NMR: 4.03(2 H, d), 7.37(1 H, d), 7.77(1 H, dd)
43
2
HO2C—CH2—O—
CN
F: 299; NMR: 4.99(2 H, s), 7.29(1 H, d), 7.77(1 H, dd)
44
2
H2N(OC)CH2O—
CN
F: 298; NMR: 4.77(2 H, s), 7.21(1 H, d), 7.77(1 H, dd)
45
2
BnO—(CH2)3—O—
CN
F: 389; NMR: 4.51(2 H, s), 7.39(1 H, d), 7.77(1 H, dd)
46
2
CN
F: 325; NMR: 3.71(1 H, q), 7.40(1 H, d), 7.77(1 H, dd)
47
2
cHexO—
CN
F: 323; NMR: 4.65-4.74(1 H, m), 7.43(1 H, d), 7.75
(1 H, dd)
48
2
(Me)2N(CO)CH2O—
CN
F: 326; NMR: 2.86(3 H, s), 7.24(1 H, d), 7.77(1 H, d)
49
2
pHO—
CN
F: 317; NMR: 7.25(2 H, d), 7.52(2 H, t), 7.80(1 H, d)
50
2
CN
F: 356; NMR: 5.43(2 H, s), 7.46(1 H, d), 8.58(1 H, d)
51
2
CN
FN: 377; NMR: 4.86-4.96(1 H, m), 7.42(1 H, d), 8.83 (1 H, d)
TABLE 5
Ex
Syn
A—X—
Sal
Dat
4
4
HCl
F: 322; NMR: 3.71(4 H, dd), 7.09(1 H, d), 8.20(1 H, dd)
5
5
F: 336; NMR: 3.78(4 H, dd), 7.07(1 H, d), 7.68(1 H, dd)
52
4
cHex-CH2—NH—
Na
F: 358(Na);
NMR: 3.09(2 H, dd), 6.87(1 H, d), 7.58(1 H, dd)
53
1
iBuS—
Na
F: 335(Na);
NMR: 3.07(2 H, d), 7.65-7.70(2 H, m), 8.24(1 H, s)
54
2
cBu—CH2—O—
Na
F: 309;
NMR: 4.19(2 H, d), 7.36(1 H, d), 7.66(1 H, dd)
55
4
HCl
F: 322; NMR: 0.98(3 H, d), 7.78(1 H, dd), 8.43(1 H, d)
56
5
FN: 308; NMR: 3.79(4 H, dd), 7.28(1 H, d), 7.76(1 H, dd)
57
5
F: 407; NMR: 1.01(3 H, t), 7.27(1 H, d), 7.75(1 H, dd)
58
5
F: 338; NMR: 1.16(6 H, d), 7.26(1 H, d), 7.76(1 H, dd)
59
5
F: 306; NMR: 5.79-5.96(2 H, m), 7.24(1 H, d), 7.74(1 H, dd)
60
5
F: 381; NMR: 1.22(3 H, t), 7.28(1 H, d), 8.83(1 H, d)
61
4
Na
F: 326; NMR: 3.19-3.28(2 H, m), 7.28(1 H, d), 7.65(1 H, dd)
62
4
nPr—NH—
Na
F: 304(Na);
NMR: 3.20(2 H, dt), 6.37, (1 H, t), 6.88(1 H, d)
63
4
Na
F: 380(Na); NMR: 3.68(2 H, dd), 7.13(1 H, d), 7.60(1 H, d)
TABLE 6
Ex
Syn
A—X—
Sal
Dat
64
4
iBu-NH—
Na
F: 318 (Na); NMR: 3.07 (2H, dd), 6.39 (1H, t), 6.88 (1H, d)
65
4
cPen-NH—
Na
F: 330 (Na); NMR: 3.87-3.99 (1H, m), 5.96 (1H, d), 6.92
(1H, d)
66
4
nBu—NH—
Na
F: 318 (Na); NMR: 3.23 (2H, dt), 6.87 (1H, d), 7.58 (1H, d)
67
4
nBu—N(Me)—
Na
F: 332 (Na); NMR: 3.45 (2H, dd), 7.11 (1H, d), 7.60 (1H, dd)
68
4
Na
F: 346 (Na); NMR: 3.14 (2H, dd), 6.87 (1H, d), 7.58 (1H, dd)
69
4
Na
F: 322; NMR: 2.81 (1H, dt), 7.22 (1H, d), 7.64 (1H, dd)
70
4
nHex-N(Me)—
Na
F: 338; NMR: 3.44 (2H, dd), 7.09 (1H, d), 7.62 (1H, dd)
71
4
cOct-NH—
Na
F: 350; NMR: 3.65-3.76 (1H, m), 6.86 (1H, d), 7.56 (1H, dd)
72
4
cHex-NH—
Na
F: 322; NMR: 3.40-3.53 (1H, m), 6.93 (1H, d), 7.59 (1H, dd)
73
4
cHep-NH—
Na
F: 336; NMR: 3.61-3.72 (1H, m), 6.85 (1H, d), 7.58 (1H, d)
74
4
nPen-CH(Me)—NH—
Na
F: 338; NMR: 3.61-3.72 (1H, m), 6.89 (1H, d), 7.59 (1H, d)
75
4
nBu—N(Et)-
Na
F: 346 (Na); NMR: 3.47 (2H, q), 7.13 (1H, d), 7.61 (1H, dd)
76
4
APN: 403; NMR: 0.94 (3H, d), 7.31 (1H, d), 8.83 (1H, d)
77
4
APN: 418; NMR: 3.09 (3H, s), 7.74 (1H, dd), 8.43 (1H, d)
78
4
APN: 418; NMR: 3.26 (2H, d), 7.26 (1H, d), 8.44 (1H, d)
79
4
APN: 382; NMR: 3.79 (2H, d), 7.27 (1H, d), 8.83 (1H, dd)
80
4
APN: 340; NMR: 5.08 (4H, s), 7.03 (1H, d), 7.69 (1H, dd)
81
2
Na
F: 356; NMR: 5.51 (2H, s), 7.97 (1H, d), 8.57 (1H, d)
82
2
(Me)2C═CHCH2—O—
Na
F: 309; NMR: 5.49 (1H, t), 7.37 (1H, d), 7.66 (1H, d)
TABLE 7
Ex
Syn
Str
Dat
83
1
F: 315; NMR: 4.02 (2H, d), 7.42 (1H, d), 8.63 (1H, d)
84
1
F: 315; NMR: 4.00 (2H, d), 7.37 (1H, d), 8.31 (1H, d)
85
1
F: 331; NMR: 4.00 (2H, d), 7.37 (1H, d), 8.80 (1H, s)
86
2
FN: 375; NMR: 4.04 (2H, d), 7.83 (1H, dd), 8.46 (1H, s)
87
2
FN: 329; NMR: 4.05 (2H, d), 7.84 (1H, dd), 8.47 (1H, s)
88
2
F: 361; NMR: 4.20 (2H, t), 7.84 (1H, dd), 8.61 (1H, dd)
89
1
F: 331; NMR: 3.99 (2H, d), 7.13 (1H, d), 7.34 (1H, d)
90
2
F: 405; NMR: 4.12 (2H, d), 7.73 (1H, d), 8.45 (1H, d)
91
2
FN: 313; NMR: 4.13 (2H, d), 7.69 (1H, dd), 8.60 (1H, dd)
TABLE 8
Ex
Syn
A—X—
R1
R2
Sal
Dat
6
6
iBuO—
H
NO2
FN: 320;
NMR: 3.99 (2H, d), 7.43 (1H, d), 7.62 (1H, d)
92
2
nPrO—
H
CN
Na
F: 310 (Na);
NMR: 4.12 (2H, t), 7.16 (1H, d), 7.85 (1H, dd)
93
1
iBuO—
H
CN
FN: 300;
NMR: 3.98 (2H, d), 7.31 (1H, d), 7.59 (1H, d)
94
5
H
CN
Na
FN: 325; NMR: 1.51-1.57 (2H, m), 3.62 (2H, dd), 7.00 (1H, d)
95
4
nBu—N(Me)—
H
CN
Na
F: 337;
NMR: 2.96 (3H, s), 7.23 (1H, d), 7.79 (1H, d)
96
1
iBu—S—
H
CN
Na
F: 340 (Na);
NMR: 3.02 (1H, d), 7.22 (1H, d), 7.85 (1H, dd)
97
2
EtO—(CH2)2—O—
H
CN
F: 318;
NMR: 1.13 (3H, t), 7.92 (1H, dd), 8.07 (1H, d)
98
2
cBu—CH2—O—
H
CN
F: 314;
NMR: 4.17 (2H, d), 7.58 (1H, d), 8.15 (1H, d)
99
2
MeO—
H
CN
FN: 258;
NMR: 3.97 (3H, s), 7.60 (1H, d), 8.18 (1H, d)
100
2
EtO—
H
CN
FN: 272;
NMR: 1.39 (3H, t), 7.59 (1H, d), 8.16 (1H, d)
101
2
iPrO—
H
CN
F: 288;
NMR: 1.32 (6H, d), 7.59 (1H, d), 8.15 (1H, d)
102
2
iPenO—
H
CN
F: 316;
NMR: 0.96 (6H, d), 7.59 (1H, d), 8.16 (1H, d)
103
2
nBuO—
H
CN
F: 302;
NMR: 4.20 (2H, t), 7.60 (1H, d), 8.16 (1H, d)
104
2
cPenO—
H
CN
F: 314;
NMR: 5.05-5.08 (1H, m), 7.59 (1H, d), 8.15 (1H, d)
105
2
cPenO—
H
CN
Na
AP: 335 (Na);
NMR: 1.50-2.05 (8H, m), 7.12-7.40 (3H, m), 7.78-
8.03 (2H, m)
106
2
3Py—CH2—O—
H
CN
F:337;
NMR: 5.40 (2H, d), 7.60 (1H, d), 8.20 (1H, d)
107
2
4Py—CH2—O—
H
CN
ES: 337;
NMR: 5.44 (2H, s), 7.61 (1H, d), 8.23 (1H, d)
108
2
H
CN
F: 343; NMR: 5.48 (2H, s), 7.60 (1H, d), 8.18 (1H, d)
TABLE 9
109
2
H
CN
F: 339; NMR: 6.00 (2H, t), 7.59 (1H, d), 8.17 (1H, d)
110
2
NC—(CH2)3—O—
H
CN
FN: 311;
NMR: 4.27 (2H, t), 7.61 (1H, d), 8.19 (1H, d)
111
1
BnO—
H
CN
FN: 334;
NMR: 5.36 (2H, s), 7.71 (1H, d), 8.19 (1H, d)
112
1
tBu—CH2—O—
H
CN
F: 316;
NMR: 3.85 (2H, s), 7.60 (1H, d), 8.16 (1H, d)
113
1
H
CN
F: 313; NMR: 3.15-3.23 (4H, m), 7.18 (1H, d), 7.57 (1H, d)
114
1
H
CN
F: 299; NMR: 3.54-3.63 (4H, m), 6.84 (1H, d), 7.48 (1H, d)
115
6
cPr—CH2—O—
H
CN
F: 300;
NMR: 1.18-1.33 (1H, m), 7.59 (1H, d), 8.16 (1H, d)
116
5
H
CN
Na
FN: 325; NMR: 0.97 (3H, d), 2.74-2.85 (2H, m), 7.31 (1H, d)
117
2
cPenO—
H
Ac
FN: 329;
NMR: 2.55 (3H, s), 7.24 (1H, d), 7.50 (1H, d)
118
1
PhO—
H
CN
Na
FN: 320;
NMR: 6.97 (1H, d), 7.49 (2H, t), 7.87 (1H, dd)
119
2
cPen-O—
F
CN
FN: 330;
NMR: 5.02-5.06 (1H, m), 7.17 (1H, d), 7.89 (1H, dd)
TABLE 10
Ex
Syn
A—X—
Sal
Dat
7
7
iBuO—
Na
F: 320;
NMR: 1.10 (3H, d), 7.27 (1H, d), 7.31 (1H, s)
120
7
tBu—CH2—O—
Na
F: 356 (Na);
NMR: 3.83 (2H, s), 7.27 (1H, d), 7.29 (1H, s)
121
7
PhO—
Na
FN: 338;
NMR: 6.96 (1H, d), 7.38 (1H, s), 7.49 (2H, t)
122
7
cHexO—
Na
FN: 344;
NMR: 4.58-4.68 (1H, m), 7.30 (1H, s), 7.33 (1H, d)
123
2
nPrO—
F: 306;
NMR: 4.17 (2H, t), 7.34 (1H, d), 7.64 (1H, s)
124
2
cPenO—
F: 332;
NMR: 5.03-5.12 (1H, m), 7.34 (1H, d), 7.63 (1H, s)
125
2
cPenO—
Na
F: 332;
NMR: 5.00-5.07 (1H, m), 7.28 (1H, d), 7.30 (1H, s)
126
2
cPr-CH2—O—
F: 318; NMR: 4.07 (2H, d), 7.32 (1H, d), 7.64 (1H, s)
127
2
EtO—
F: 292; NMR: 4.26 (2H, q), 7.33 (1H, d), 7.64 (1H, s)
128
2
MeO—
F: 278; NMR: 3.98 (3H, s), 7.34 (1H, d), 7.64 (1H, s)
129
2
iPenO—
F: 334; NMR: 4.23 (2H, t), 7.36 (1H, d), 7.63 (1H, s)
130
2
cHex-CH2—O—
Na
FN: 358;
NMR: 3.98 (2H, d), 7.28 (1H, d), 7.31 (1H, s)
131
2
(Et)2CHCH2—O—
Na
F: 346;
NMR: 4.07 (2H, d), 7.30 (1H, s), 7.85 (1H, dd)
132
2
Na
F: 380 (Na);
NMR: 4.99 (2H, d), 7.65 (1H, s), 8.02 (1H, dd)
TABLE 11
Ex
Syn
A—X—
Sal
Dat
8
8
Ph—
Na
F: 301; NMR: 8.33 (1H, s), 8.55 (1H, dd), 8.64 (1H, d)
9
9
Na
FN: 289; NMR: 6.67 (1H, t), 7.95 (1H, d), 8.66 (1H, d)
10
10
4-MeO—3-CN—Ph—
APN: 354; NMR: 4.01 (3H, s), 8.47 (1H, s), 8.87 (1H, d)
11
11
APN: 288; NMR: 7.84 (1H, d), 8.49 (1H, d), 8.79 (1H, d)
12
12
APN: 288; NMR: 6.39 (2H, t), 7.76 (1H, d), 8.50 (1H, s)
15
15
3Py—
0.3
ES: 302; NMR: 8.52 (1H, s), 8.60 (1H, dd), 8.93 (1H, d)
HCl
133
8
4-F—Ph—
FN: 317; NMR: 8.50 (1H, s), 8.46 (1H, dd), 8.92 (1H, d)
134
8
3-MeO—Ph—
FN: 329; NMR: 3.85 (3H, s), 7.86 (1H, dd), 8.54 (1H, dd)
135
8
4-MeO—Ph—
Na
F: 331; NMR: 3.85 (3H, s), 7.12 (2H, d), 8.42 (1H, dd)
136
8
4-Cl—Ph—
FN: 333; NMR: 8.50(1H, s), 8.56 (1H, dd), 8.92 (1H, d)
137
8
4-CF3—Ph—
Na
FN: 367; NMR: 8.37 (1H, s), 8.51 (1H, dd), 8.67 (1H, d)
138
8
2-MeO—Ph—
F: 331; NMR: 3.80 (3H, s), 7.65 (1H, d), 8.91 (1H, dd)
139
8
4-Me—Ph—
Na
F: 315; NMR: 2.40 (3H, s), 7.37 (2H, d), 8.44 (1H, dd)
140
8
2Thie-
F: 307; NMR: 7.29 (1H, dd), 8.49 (1H, s), 8.51 (1H, dd)
141
8
3Thie-
Na
ES: 307; NMR: 7.57 (1H, dd), 8.34 (1H, s), 8.43 (1H, dd)
142
8
3Fur-
F: 291; NMR: 7.10 (1H, dd), 8.50 (1H, dd), 8.66 (1H, d)
143
8
4-NC—Ph—
FN: 324; NMR: 8.52 (1H, s), 8.59 (1H, dd), 8.92 (1H, d)
144
8
4-HOOC—Ph—
F: 345; NMR: 8.11 (2H, d), 8.59 (1H, dd), 8.92 (1H, d)
145
8
2Fur
FN: 289; NMR: 6.78 (1H, dd), 7.37 (1H, d), 8.57 (1H, dd)
146
8
0.3 HCl
FN: 289; NMR: 7.83 (1H, dd), 7.92 (1H, d), 8.89 (1H, d)
147
8
4-iBuO—Ph—
FN: 371; NMR: 1.01 (6H, s), 7.12 (2H, d), 8.51 (1H, dd)
148
8
4-Et—Ph—
FN: 327; NMR: 1.25 (3H, t), 7.41 (2H, d), 8.53 (1H, dd)
149
8
3-Me—Ph—
FN: 313; NMR: 2.42 (3H, s), 7.86 (1H, dd), 8.54 (1H, dd)
150
8
2-Me—Ph—
FN: 313; NMR: 2.19 (3H, s), 7.87 (1H, dd), 8.53 (1H, dd)
TABLE 12
151
8
3-Cl—Ph—
Anal: Calc. C; 68.17%, H; 3.31%, N; 8.37%, Cl; 10.59%
Found C; 67.90%, H; 3.51%, N; 8.23%, Cl; 10.43%;
NMR: 8.51 (1H, s), 8.56 (1H, d), 8.92 (1H, d)
152
8
2-Cl—Ph—
Na
FN: 333; NMR: 7.74 (1H, dd), 8.44 (1H, dd), 8.68 (1H, d)
153
8
4-tBu—Ph—
Na
FN: 355; NMR: 1.35 (9H, s), 7.86 (1H, dd), 8.69 (1H, d)
154
8
FN: 319; NMR: 2.17 (3H, s), 7.86 (1H, d), 8.92 (1H, d)
155
8
4-HO—Ph—
FN: 315; NMR: 6.94 (2H, d), 8.49 (1H, dd), 8.90 (1H, d)
156
8
Na
FN: 319; NMR: 2.54 (3H, s), 7.54 (1H, d), 8.64 (1H, d)
157
8
3,5-di(CF3)—Ph—
APN: 435; NMR: 8.30 (1H, s), 8.61 (1H, dd), 8.93 (1H, d)
158
8
2Naph-
APN: 349; NMR: 7.59-7.69 (2H, m), 8.61 (1H, dd), 8.93
(1H, d)
159
8
APN: 367; NMR: 8.03 (1H, s), 8.58 (1H, dd), 8.92 (1H, d)
160
8
APN: 375; NMR: 7.37-7.59 (2H, m), 8.57 (1H, dd), 8.92 (1H, d)
161
8
4-BnO—3-NC—Ph—
APN: 430; NMR: 5.40 (2H, s), 7.99 (1H, dd), 8.91 (1H, d)
162
8
2,4-diMeO—Ph—
APN: 359; NMR: 3.85 (3H, s), 7.85 (1H, dd), 8.90 (1H, d)
163
8
4-MeS—Ph—
APN: 345; NMR: 2.56 (3H, s), 8.53 (1H, dd), 8.91 (1H, d)
164
8
4-(CF3)O—Ph—
APN: 383; NMR: 7.58 (2H, d), 8.57 (1H, dd), 8.92 (1H, d)
165
8
4-EtO—Ph—
APN: 343; NMR: 4.12 (2H, q), 7.85 (1H, dd), 8.90 (1H, d)
166
8
4-PhO—Ph—
APN: 391; NMR: 7.12-7.18 (4H, m), 7.86 (1H, dd), 8.53
(1H, dd)
167
8
3,4-diMeO—Ph—
APN: 359; NMR: 3.85 (6H, d), 7.85 (1H, dd), 8.90 (1H, d)
168
8
4-Me2N—Ph—
APN: 342; NMR: 3.00 (6H, s), 7.83 (1H, dd), 8.89 (1H, d)
169
8
APN: 343; NMR: 6.14 (2H, s), 7.85 (1H, dd), 8.91 (1H, d)
170
8
3-Ph—Ph—
APN: 375; NMR: 7.35-7.60 (3H, m), 8.57 (1H, dd), 8.91
(1H, d)
171
8
4-MeO—3-Me—Ph—
APN: 343; NMR: 2.24 (3H, s), 7.85 (1H, dd), 8.90 (1H, d)
172
8
4-MeO—3,5-diMe—Ph—
APN: 357; NMR: 3.74 (3H, s), 7.85 (1H, dd), 8.90 (1H, d)
173
8
3-Me-4-(CF3)O—Ph—
APN: 397; NMR: 2.39 (3H, s), 8.56 (1H, dd), 8.92 (1H, d)
174
8
4Py—
APN: 300; NMR: 8.51 (1H, s), 8.60 (1H, dd), 8.91 (1H, d)
175
8
4-MeO—2,5-diMe—Ph—
APN: 357; NMR: 2.16 (6H, s), 7.86 (1H, dd), 8.91 (1H, d)
TABLE 13
176
8
4-nBuO—Ph—
APN: 371; NMR: 0.96 (3H, t), 7.85 (1H, dd), 8.90 (1H, d)
177
8
APN: 341; NMR: 4.63 (2H, t), 7.72 (1H, d), 8.90 (1H, dd)
178
9
Na
F: 363 (Na); NMR: 7.25-7.43 (2H, m), 7.94 (1H, d), 8.68 (1H, s)
179
9
Na
F: 341; NMR: 7.35 (1H, t), 8.41 (1H, s), 8.69 (1H, d)
180
9
APN: 303; NMR: 7.74 (1H, s), 8.71 (1H, d), 8.88 (1H, d)
181
9
AP: 328; NMR: 7.26 (1H, s), 7.98 (1H, s), 8.60 (1H, d)
182
9
APN: 313; NMR: 7.69 (1H, dd), 8.56 (1H, s), 8.69 (1H, dd)
TABLE 14
Ex
Syn
A—X—
Sal
Dat
13
13
3-H2N—Ph—
ES: 321; H: 1.93
14
14
4-H2N—3-F—Ph—
ES: 339; H: 2.51
183
8
Ph—
Na
FN: 304; NMR: 7.23 (1H, d), 7.99 (1H, dd), 8.21 (1H, d)
184
8
4-Me—Ph—
Na
FN: 318; NMR: 2.39 (3H, s), 7.24 (1H, d), 7.35 (2H, d)
185
8
4-MeO—Ph—
Na
FN: 334; NMR: 3.84 (3H, s), 7.22 (1H, d), 7.95 (1H, dd)
186
8
4-CF3—Ph—
Na
F: 374; NMR: 7.25 (1H, d), 8.03 (1H, dd), 8.27 (1H, d)
187
8
4-Cl—Ph—
Na
FN: 338; NMR: 7.20 (1H, d), 7.58-7.68 (5H, m), 8.22
(1H, d)
188
13
3Py—
ES: 307
189
13
3-Me—Ph—
ES: 320
190
13
2-Me—Ph—
ES: 320; H: 2.94
191
13
3-HO—Ph—
ES: 322; H: 2.50
192
13
2,3-diMe—Ph—
ES: 334
193
13
3-MeO—Ph—
ES: 336; H: 2.91
TABLE 15
194
13
2-MeO—Ph—
ES: 336
195
13
ES: 337
196
13
2-Cl—Ph—
ES: 340; H: 2.90
197
13
ES: 345; H: 2.80
198
13
4-Ac—Ph—
ES: 348; H: 2.68
199
13
4-Me2N—Ph—
ES: 349; H: 2.92
200
13
3-Me2N—Ph—
ES: 349; H: 2.49
201
13
3-HOOC—Ph—
ES: 350
202
13
ES: 354
203
13
1Naph-
ES: 356
204
13
2Naph-
ES: 356; H: 3.38
205
13
ES: 357
206
13
ES: 359; H: 3.15
207
13
ES: 362; H: 3.67
208
13
ES: 362; H: 3.15
209
13
4-tBu—Ph—
ES: 362; H: 3.54
210
13
3-AcNH—Ph—
ES: 363; H: 2.44
211
13
3,4-diMeO—Ph—
ES: 366
212
13
ES: 368
213
13
2-CF3—Ph—
ES: 374; H: 2.92
214
13
ES: 375
215
13
3-[Me2N(CO)]—Ph—
ES: 377
216
13
ES: 378; H: 2.96
TABLE 16
217
13
3-Ph—Ph—
ES: 382; H: 3.46
218
13
3-[MeS(O)2]—Ph—
ES: 384
219
13
4-cHex-Ph—
ES: 388; H: 3.84
220
13
2-(CF3)O—Ph—
ES: 390
221
13
ES: 391; H: 2.96
222
13
ES: 358
223
13
ES: 396; H: 3.37
224
13
4-PhO—Ph—
ES: 398; H: 3.46
225
13
ES: 412; H: 3.46
226
13
ES: 389
227
14
4-H2N—3-MeO—Ph—
ES: 351; H: 3.19
228
13
ES: 554; H: 3.02
229
13
4-AcNH—Ph—
ES: 363; H: 2.43
230
13
ES: 386; H: 3.43
231
13
2-PhO—Ph—
ES: 398
232
13
4-[Ph(CO)]—Ph—
ES: 410; H: 3.20
233
13
ES: 485
234
13
4-iPrO—Ph—
ES: 364; H: 3.26
235
13
4-BnO—Ph—
ES: 412; H: 3.51
236
13
ES: 357; H: 2.07
TABLE 17
Ex
Syn
A—X—
Rb—
Sal
Dat
237
8
Ph—
Me—
AP: 321; NMR: 2.71 (3H, s), 7.78 (1H, d), 8.49 (1H,
d)
238
8
4-Me—Ph—
Me—
Na
F: 335; NMR: 2.40 (3H, s), 7.37 (2H, d), 8.20 (1H, dd)
239
8
4-Et—Ph—
Me—
AP: 349; NMR: 2.65-2.76 (5H, m), 7.41 (2H, d),
8.47 (1H, d)
240
8
4-(CF3)O—Ph—
Me—
AP: 405; NMRC: 2.70 (3H, s), 7.58 (2H, d), 8.51 (1H,
d)
241
8
4-MeO—Ph—
Me—
AP: 351; NMR: 2.70 (3H, s), 3.85 (3H, s), 8.44 (1H,
d)
242
8
3-MeO—Ph—
Me—
AP: 373 (Na); NMR: 2.71 (3H, s), 3.84 (3H, s), 8.48
(1H, d)
243
8
3-Me—Ph—
Me—
AP: 335; NMR: 2.41 (3H, s), 2.70 (3H, s), 8.32 (1H,
dd)
244
8
4-tBu—Ph—
Me—
APN: 375; NMR: 1.35 (9H, s), 2.71 (3H, s), 7.77 (1H,
d)
245
8
3Fur-
Me—
APN: 309; NMR: 2.70 (3H, s), 7.10 (1H, dd), 8.29
(1H, dd)
246
8
3Thie-
Me—
APN: 325; NMR: 2.70 (3H, s), 7.57 (1H, dd), 7.87
(1H, d)
247
8
4-Me2N—Ph—
Me—
APN: 362; NMR: 2.70 (3H, s), 3.00 (6H, s), 7.71 (1H,
d)
248
8
Ph—
Et—
AP: 357 (Na); NMR: 3.14 (2H, q), 7.52-7.70 (5H, m),
7.78 (1H, d)
249
8
4-Me—Ph—
Et—
AP: 371 (Na); NMR: 1.29 (3H, t), 2.40 (3H, s), 7.75
(1H, d)
250
8
Ph—
H—
APN: 305; NMR: 7.52-7.61 (3H, m), 7.81 (1H, d),
8.50 (1H, s)
TABLE 18
Ex
Syn
A—X—
Dat
251
8
Ph—
APN: 288; NMR: 7.46-7.69 (5H, m), 8.55 (1H, d), 9.23 (1H, s)
252
8
4-Me—Ph—
APN: 302; NMR: 2.40 (3H, s), 8.52 (1H, d), 9.22 (1H, s)
253
8
2Thie-
APN: 294; NMR: 7.28 (1H, dd), 7.90 (1H, d), 8.16 (1H, s)
254
8
4-MeO—Ph—
APN: 318; NMR: 3.84 (3H, s), 7.75 (1H, d), 8.51 (1H, d)
255
8
3-Me—Ph—
APN: 302; NMR: 2.41 (3H, s), 7.77 (1H, d), 8.17 (1H, s)
256
8
4-tBu—Ph—
APN: 344; NMR: 1.35 (9H, s), 7.58 (4H, s), 8.16 (1H, s)
257
8
3Fur-
APN: 278; NMR: 7.08 (1H, dd), 7.90 (1H, d), 9.21 (1H, s)
258
8
APN: 330; NMR: 4.62 (2H, t), 7.72 (1H, d), 8.19 (1H, s)
259
8
3Thie-
APN:294; NMR: 7.55 (1H, dd), 7.88 (1H, d), 9.22 (1H, s)
TABLE 19
Ex
Syn
A—X—
Z
Dat
260
8
Ph—
NMe
AP: 326 (Na); NMR: 4.16 (3H, s), 8.25 (1H, dd), 8.39 (1H, d)
261
8
Ph—
O
APN: 289; NMR: 7.81 (1H, d), 7.97 (1H, s), 8.57 (1H, d)
262
8
4-Me—Ph—
O
APN: 303; NMR: 2.40 (3H, s), 7.78 (1H, d), 7.91 (1H, s)
263
8
4-tBu—Ph—
O
APN: 345; NMR: 1.35 (9H, s), 7.80 (1H, d), 7.97 (1H, s)
264
8
3Thie-
O
APN: 295; NMR: 7.67 (1H, dd), 7.98 (1H, d), 8.03 (1H, s)
265
8
3Fur-
O
APN: 279; NMR: 7.91 (2H, s), 7.93 (1H, s), 8.50 (1H, d)
TABLE 20
Ex
Syn
Str
Dat
266
8
APN: 367; NMR: 8.03 (1H, s), 8.58 (1H, dd), 8.92 (1H, d)
267
8
APN: 375; NMR: 7.37-7.59 (2H, m), 8.57 (1H, dd), 8.92 (1H, d)
268
8
APN: 305; NMR: 7.78 (1H, d), 8.63 (1H, s), 8.68 (1H, d)
269
8
APN: 361; NMR: 1.35 (9H, s), 7.77 (1H, d), 8.62 (1H, s)
Examples
The pharmacological effect of the non-purine xanthine oxidase inhibitor of the present invention is described with reference to the following Examples. The test results of the compounds of formula (I) for their xanthine oxidase-inhibiting effect are shown as Reference Examples.
Reference Example
Xanthine Oxidase Inhibiting Activity
(1) Preparation of Test Compound:
A test compound was dissolved in DMSO (by Nacalai) to have a concentration of 10 mM, and then just before use, its concentration was adjusted to a desired one.
(2) Measurement Method:
The xanthine oxidase inhibitory activity of the compound of the present invention was evaluated according to a partly modified method of a method described in a reference “Free Radic. Biol. Med. 6, 607-615, 1992”. Concretely, xanthine oxidase (derived from butter milk, by Sigma) was mixed with 50 mM phosphate buffer to be 0.03 units/ml, and applied to a 96-well plate in an amount of 50 μl/well. The test compound diluted to have a final concentration was added to the plate in an amount of 2 μl/well, and processed at room temperature for 20 minutes. Pterin (by Sigma) was added to it to have a final concentration of 5 μM in an amount of 50 μl/well, and reacted at room temperature for 10 minutes. Under a condition of excitation at 345 nm and emission at 390 nm (pterin is oxidized by xanthine oxidase to give isoxanthopterin, and under the condition it emits light), the sample was analyzed using a microplate reader sapphire (by Tacan).
The light emission by isoxanthopterin in the presence or absence of xanthine oxidase was defined as 0% inhibition and 100% inhibition, respectively, and the concentration (IC50) of the test compound for 50% inhibition was computed.
The compounds of formula (I) had good xanthine oxidase inhibiting activity. IC50 of typical compounds in Preparation Examples are shown in the following Table 21.
TABLE 21
Prepara-
tion
Prepara-
Prepara-
Prepara-
Exam-
IC50
tion
IC50
tion
IC50
tion
IC50
ple
(nM)
Example
(nM)
Example
(nM)
Example
(nM)
1
3.6
3
5.0
4
1.2
6
6.9
7
2.2
16
2.6
20
10
21
4.1
30
6.3
37
4.2
40
7.3
51
4.3
53
2.5
55
1.3
59
2.8
67
3.1
73
1.1
77
2.2
82
3.1
83
3.2
85
4.0
86
5.8
92
3.2
95
4.1
96
2.9
106
2.8
109
5.1
110
13
111
2.5
113
1.8
116
2.0
117
5.9
118
6.3
132
3.7
119
1.5
209
0.5
IC50 of the compounds of Preparation Examples 8 to 14, 133, 138, 141, 143, 145, 153, 158 to 164, 166, 169, 172, 179, 197, 199, 216, 219, 221, 226, 237, 240, 243, 250, 251 and 252 was at most 20 nM.
Patent Reference 1 says that IC50 for xanthine oxidase inhibiting activity of the compound A (Example 77 in Patent Reference 1) referred to in the following Example is 1.8 nM. A reference says that IC50 for xanthine oxidase inhibiting activity of the compound B (Example 12 in Patent Reference 7) is 5.8 nM (Bioorganic Medicinal Chemistry Letters, 11, 2001, 879-882, Compound No. 5e).
Compound A: 2-(3-cyano-4-isobutyloxyphenyl)-4-methyl-1,3-thiazole-5-carboxylic acid.
Compound B: 1-(3-cyano-4-neopentyloxyphenyl)-1H-pyrazole-4-carboxylic acid.
The above tests confirm that the compounds as the active ingredient of the pharmaceutical composition of the present invention has a strong xanthine oxidase inhibiting activity.
The compounds as the active ingredient of the pharmaceutical composition of the present invention were tested and evaluated for the therapeutical effect for gastric ulcer and small intestine ulcer, according to the following test methods.
Example 1
Pharmacological Effect in Gastric Ulcer Model
A test compound was suspended in a 0.5% methyl cellulose (MC) solution to prepare a chemical liquid having a concentration 5 ml/kg; and this was orally administered to SD rats (n=12 in each group). The test compound was controlled to be 10 mg/kg or 30 mg/kg. A control group was separately prepared, in which 5 ml/kg of the 0.5% MC solution was orally administered to the rats. After 1 hour, 40 mg/kg of NSAID, diclofenac (by Sigma) was orally administered to all rats of all groups, as a suspension in 0.5% MC solution (5 ml/kg). In 6 hours after the diclofenac administration, the length of the ulcer formed in the gastric corpus was measured using a stereomicroscope (by Nikon) and a micrometer (by Olympus), and the data of 12 cases were averaged. The antiulceration potency (%) of the test compound was computed according to the following formula:
Antiulceration Potency(%): [1−(mean ulcer length of compound-administered group/mean ulcer length of 0.5% MC-administered group)]×100.
As a result, the compounds as the active ingredient of the pharmaceutical composition of the present invention showed a significant (Student's t-test, p<0.05) inhibitory potency, as compared with the control group. The inhibitory potency of each compound is shown in the parenthesis after the Preparation Example Number.
Preparation Example 8 (72%: 30 mg/kg), 183 (27%: 30 mg/kg), 237 (32%: 10 mg/kg), 251 (43%: 30 mg/kg), 254 (32%: 30 mg/kg), Compound A (36%: 30 mg/kg), Compound B (43%: 30 mg/kg).
On the other hand, in the group administered with 30 mg/kg of allopurinol, no inhibitory potency was confirmed. The above results confirm that the compounds as the active ingredient of the pharmaceutical composition of the present invention have an excellent inhibitory effect for gastric ulcer.
Example 2
Pharmacological Effect in Small Intestine Ulcer Model
A test compound was suspended in a 0.5% methyl cellulose (MC) solution to prepare a chemical liquid having a concentration 5 ml/kg; and this was orally administered to SD rats (n=10 in each group). The test compound was controlled to be 10 mg/kg in every case. A control group was separately prepared, in which 5 ml/kg of the 0.5% MC solution was orally administered to the rats. After 1 hour, 10 mg/kg of NSAID, indomethacin (by Sigma) was orally administered to the rats, as a suspension in 0.5% MC solution (5 ml/kg). In 9 hours after the indomethacin administration, the test compound was again orally administered to every rat in the same amount as previously; and after 24 hours, the area of the ulcer formed in the small intestine was measured using a stereomicroscope (by Nikon) and a micrometer (by Olympus), and the data of 10 cases were averaged. The antiulceration potency (%) of the test compound was computed according to the following formula:
Antiulceration Potency(%): [1−(mean ulcer area of compound-administered group/mean ulcer area of 0.5% MC-administered group)]×100.
As a result, all the compounds showed a significant (Student's t-test, p<0.05) inhibitory potency, as compared with the control group. The inhibitory potency of each compound is shown in the parenthesis after the Preparation Example Number.
Preparation Example 8 (29%), 183 (30%), 237 (59%), 250 (73%), 251 (41%), 254 (46%).
On the other hand, in the group administered with 10 mg/kg of allopurinol, no inhibitory potency was confirmed. A proton pump inhibitor used as an agent for treating gastric ulcer, omeprazole (by Sigma) was ineffective in this test. The above results confirm that the compounds as the active ingredient of the pharmaceutical composition of the present invention have an excellent inhibitory effect for small intestine ulcer.
As in the above, the compounds of the present invention showed a remedial effect both in the gastric ulcer model and the small intestine ulcer model, and no conventional remedies could have the effect.
The test results shown in the above-mentioned Examples confirm that the compounds as the active ingredient of the pharmaceutical composition of the present invention have a strong potency of xanthine oxidase inhibition, and in animal tests, the compounds show an excellent effect of curing digestive ulcer. Accordingly, these compounds are expected as an agent for treating or preventing ulcer that forms in digestive tracts by the attack thereto such as gastric acid, pepsin, stress, Helicobacter pylori bacteria or NSAID. Further, the compounds as the active ingredient of the pharmaceutical composition of the present invention do not have a purine structure and their toxicity is low, and accordingly, they are superior to allopurinol in the efficaciousness as so mentioned in the above.
INDUSTRIAL APPLICABILITY
The pharmaceutical composition of the present invention is useful as an agent for treating or preventing ulcer that forms in digestive tracts by the attack of gastric acid, pepsin, stress, Helicobacter pylori, NSAID, etc. In particular, the pharmaceutical composition of the present invention is effective also for ulcer in small intestine, for which remedies for gastric/duodenal ulcer that inhibit gastric acid secretion such as proton pump inhibitors are ineffective, and is therefore useful as an ulcer-treating agent heretofore unknown in the art. In addition, it is superior to allopurinol in the efficaciousness and the safety.
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12280668
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astellas pharma inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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514/354
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Apr 1st, 2022 05:13PM
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Apr 1st, 2022 05:13PM
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Astellas Pharma
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tyo:4503
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Astellas Pharma
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Aug 23rd, 2011 12:00AM
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Jun 29th, 2009 12:00AM
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https://www.uspto.gov?id=US08003105-20110823
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Method of treating cancer by co-administration of anticancer agents
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The present invention relates to a method of treating cancer by co-administration of an effective amount of 1-(2-methoxyethyl)-2-methyl-4,9-dioxo-3-(pyrazin-2-ylmethyl)-4,9-dihydro-1H-naphtho[2,3-d]imidazol-3-ium bromide and an effective amount of one or more anticancer agents selected from the group consisting of carboplatin, cisplatin, paclitaxel, vinorelbine, gemcitabine, irinotecan, docetaxel, doxorubicin, dacarbazine and rituximab, or a retuximab-containing combination therapy selected from R-ICE and R-DHAP. The treatment method of the present invention is useful for the treatment for all solid tumors and lymphomas, preferably skin cancer, bladder cancer, breast cancer, uterine cancer, ovary cancer, prostate cancer, lung cancer, colon cancer, pancreas cancer, renal cancer, gastric cancer and the like. Particularly, they are expected as therapeutic agents for tumor types which show resistance against existing anticancer agents.
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8003105
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1. A method for treatment of a cancer patient, which comprises administering an effective amount of 1-(2-methoxyethyl)-2-methyl-4,9-dioxo-3-(pyrazin-2-ylmethyl)-4,9-dihydro-1H-naphtho[2,3-d]imidazol-3-ium bromide to said patient, combined with
i) rituximab,
ii) a combination therapy R-ICE consisting of rituximab, ifosfamide, carboplatin and etoposide, or
iii) a combination therapy R-DHAP consisting of rituximab, cytarabine and cisplatin as anticancer agents.
2. The method as claimed in claim 1, wherein rituximab is combined with said effective amount of 1-(2-methoxyethyl)-2-methyl-4,9-dioxo-3-(pyrazin-2-ylmethyl)-4,9-dihydro-1H-naphtho[2,3-d]imidazol-3-ium bromide.
3. The method as claimed in claim 1, wherein a combination therapy selected from the group consisting of R-ICE and R-DHAP is combined with said effective amount of 1-(2-methoxyethyl)-2-methyl-4,9-dioxo-3-(pyrazin-2-ylmethyl)-4,9-dihydro-1H-naphtho[2,3-d]imidazol-3-ium bromide.
4. The method as claimed in claim 3, wherein R-ICE is combined with said effective amount of 1-(2-methoxyethyl)-2-methyl-4,9-dioxo-3-(pyrazin-2-ylmethyl)-4,9-dihydro-1H-naphtho[2,3-d]imidazol-3-ium bromide.
5. The method as claimed in claim 1, wherein the patient has lymphoma.
6. The method as claimed in claim 1, wherein said 1-(2-methoxyethyl)-2-methyl-4,9-dioxo-3-(pyrazin-2-ylmethyl)-4,9-dihydro-1H-naphtho[2,3-d]imidazol-3-ium bromide is administered intravenously by infusion at a dose of 1-10 mg/m2/day continuously for a period of 4-14 days.
7. A method as claimed in claim 6, wherein said 1-(2-methoxyethyl)-2-methyl-4,9-dioxo-3-(pyrazin-2-ylmethyl)-4,9-dihydro-1H-naphtho[2,3-d]imidazol-3-ium bromide is administered intravenously by infusion at a dose of 3-8 mg/m2/day continuously for 7 days followed by 14 days of no 1-(2-methoxyethyl)-2-methyl-4,9-dioxo-3-(pyrazin-2-ylmethyl)-4,9-dihydro-1H-naphtho[2,3-d]imidazol-3-ium bromide, after which the entire administration cycle can be repeated depending on the patient's conditions.
8. A kit comprising at least one cycle dosage of 1-(2-methoxyethyl)-2-methyl-4,9-dioxo-3-(pyrazin-2-ylmethyl)-4,9-dihydro-1H-naphtho[2,3-d]imidazol-3-ium bromide and
i) rituximab,
ii) anticancer agents for a combination therapy R-ICE consisting of rituximab, ifosfamide, carboplatin and etoposide, or
iii) anticancer agents for a combination therapy R-DHAP consisting of rituximab, cytarabine and cisplatin as anticancer agents.
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8
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CROSS-REFERENCE TO RELATED APPLICATION
This is a divisional of application Ser. No. 11/965,476 filed Dec. 27, 2007, now U.S. Pat. No. 7,618,992, which claims benefit of Provisional Application No. 60/882,809 filed Dec. 29, 2006, and Provisional Application No. 60/950,771 filed Jul. 19, 2007. The entire disclosures of the prior applications, U.S. patent application Ser. No. 11/965,476 and U.S. Provisional Application Nos. 60/882,809 and 60/950,771 are considered part of the disclosure and are hereby incorporated by reference.
TECHNICAL FIELD
The present invention relates to a method of treating cancer by co-administration of 1-(2-methoxyethyl)-2-methyl-4,9-dioxo-3-(pyrazin-2-ylmethyl)-4,9-dihydro-1H-naphtho[2,3-d]imidazol-3-ium bromide and one or more other anticancer agents. The present invention also relates to the use of 1-(2-methoxyethyl)-2-methyl-4,9-dioxo-3-(pyrazin-2-ylmethyl)-4,9-dihydro-1H-naphtho[2,3-d]imidazol-3-ium bromide for production of medicaments for treating cancer by administration with one or more other anticancer agents.
BACKGROUND ART
Survivin is a member of the IAP (Inhibitor of Apoptosis) family of proteins, and is highly expressed in all primary tumor types. Survivin is undetectable in most normal differentiated tissues, but is present in normal placenta, testes, and rapidly dividing cells such as CD34+ bone marrow stem cells. High expression of survivin in tumors is correlated with poor survival among patients with non-small cell lung cancer (NSCLC) [Ref. 1]. Suppression of survivin induces tumor cell death and renders the cells sensitive to normal cell cycle regulation [Ref. 2-Ref. 4]. Given its preferential expression in tumor cells, its ability to block apoptosis and regulate cancer cell proliferation, and its correlation with poor survival, survivin stands out as a putative novel target for cancer therapy. It has been reported that the down-regulation of survivin expression by the antisense or siRNA of survivin was shown to sensitize tumor cells to various anticancer drugs [Ref. 7-Ref. 13].
Fused condensed imidazolium derivatives which are expected to be candidates for anti-tumor agents having good anti-tumor activity, low toxicity and wide broad safety margins are disclosed in International Publications [Ref. 5 and Ref. 6]. Particularly, 1-(2-methoxyethyl)-2-methyl-4,9-dioxo-3-(pyrazin-2-ylmethyl)-4,9-dihydro-1H-naphtho[2,3-d]imidazol-3-ium bromide (hereinafter YM155), shown by the following formula, is a compound which is expected to be useful as an anti-tumor agent, because it has good in vivo tumor growth inhibitory activity and low toxicity.
YM155 is the first compound found to specifically suppress survivin and it has potent anti-tumor activities in experimental human hormone refractory prostate cancer (HRPC) xenograft models [Ref. 14], human NSCLC xenograft models [Ref. 15], and in patients with advanced solid tumors and non-Hodgkin's lymphoma (NHL) [Ref. 16 and Ref. 17]. YM155 showed time-dependent anti-tumor activity, and 7-day continuous infusion of YM155 induced tumor regression in the NSCLC xenograft model [Ref. 15]. YM155 caused fewer side effects, e.g., body weight decrease and hematological toxicities which are frequently observed with paclitaxel, cisplatin and doxorubicin treatment [Ref. 16 and Ref. 17].
In general, there is a limit to efficacy of chemotherapy for tumors, particularly malignant tumors, when an anticancer agent is administered alone, in view of adverse reactions, and it is quite rare to attain a sufficient anticancer effect. In a clinical practice, accordingly, a multi-drug combination therapy in which 2 or more species of drugs having different mechanisms of action are used in combination has been employed. Through combination therapy, reduction of adverse drug reaction and potentiation of the anticancer activity are intended by combination of anticancer agents having different mechanisms of action, including 1) reduction of the non-sensitive cell population, 2) prevention or delaying of occurrence of drug resistance, and 3) dispersing of toxicity by means of a combination of drugs having different toxicities. With combination therapy, however, a random combination of anticancer agents having different mechanisms of action does not necessarily yield a potentiation effect of anticancer activities. Thus, studies have been conducted to obtain a combination of anticancer agents having a much higher anticancer action.
Representative combinational drugs include, for example, cisplatin and gemcitabine as well as carboplatin and paclitaxel, which are known as standard combination therapy for lung cancer [Ref. 18 and Ref. 19]. A randomized phase III study of combination therapies, R-ICE (rituximab, ifosfamide, carboplatin and etoposide) and R-DHAP (rituximab, cisplatin, Cytarabine (ara-C) and dexamethasone) is in progress in patients with lymphoma (DLBCL) [Ref. 20, 21 and 22].
When the combinational drugs are used together, there are a lot of cases where the side effects increase as well as the anti-tumor effects. Therefore there are many difficulties in combining two or more anti-tumor agents. Especially, a combination of anticancer agents with a synergistic anticancer action resulting in marked reduction of cancer cells and further complete removal of the cancer cells, leading to a complete cure of cancer without an increase of side effects, is eagerly desired.
REFERENCES
1. Monzo M, et al., J Clin Oncol 1999; 17:2100-4.
2. Giodini A, et al., Cancer Res 2002; 62:2462-7.
3. Mesri M, et al., Am J Pathol 2001; 158:1757-65.
4. Yamamoto T, et al., Med Electron Microsc 2001; 34:207-12.
5. Pamphlets of International Publication 01/60803
6. Pamphlets of International Publication 2004/092160
7. Zhonghua Wei Chang Wai Ke Za Zhi. 2005 September; 8(5):455-8
8. Cancer Lett. 2006 Feb. 8; 232(2):243-54
9. Blood. 2006 Feb. 15; 107(4):1555-63. Epub 2005 Oct. 27
10. Int. J. Cancer: 118, 812-20 (2006)
11. J Clin Invest. 2001 October; 108(7):981-90.
12. Prostate 65, 10-19 (2005)
13. The Prostate 64:293-302 (2005)
14. Proceedings AACR-NCI-EORTC 2005, Abstract #B203.
15. Proc Amer Assoc Cancer Res 2006; 47:[Abstract #5671]
16. Annals of Oncology, 2006; 17(Suppl. 3):23, Abstract #O.403.
17. Journal of Clinical Oncoclogy, 2006:ASCO Annual Meeting Proceedings Part I. Vol. 24, No. 18S (June 20 Supplement), Abstract #3014.
18. Cancer, 1996, 77:2458-63.
19. Cancer, 2000, 89:1714-9.
20. Annals of Oncology, 2006; 17(Suppl. 4):iv31-2.
21. Blood. 2006 May 15; 103(10):3684-8. Epub 2004 Jan. 22.
22. Leukemia & Lymphoma 2007 May; 48(5):897-904.
DISCLOSURE OF THE INVENTION
The present inventors have found that a combinational use of 1-(2-methoxyethyl)-2-methyl-4,9-dioxo-3-(pyrazin-2-ylmethyl)-4,9-dihydro-1H-naphtho[2,3-d]imidazol-3-ium bromide (YM155) with an existing anticancer agent or agents, particularly, docetaxel or rituximab, unexpectedly potentiates the anticancer effect. Thus, the present invention was completed.
That is, the present invention relates to a method for treatment of a cancer patient, which comprises administering an effective amount of 1-(2-methoxyethyl)-2-methyl-4,9-dioxo-3-(pyrazin-2-ylmethyl)-4,9-dihydro-1H-naphtho[2,3-d]imidazol-3-ium bromide to said patient, combined with
i) an effective amount of one or more anticancer agents selected from the group consisting of carboplatin (CBDCA), cisplatin (CDDP), paclitaxel (TXL), vinorelbine (VIN), gemcitabine (GEM), irinotecan (CPT-11), docetaxel (TXT), doxorubicin (DXR), dacarbazine (DTIC) and rituximab (RTX), or
ii) a rituximab-containing combination therapy selected from R-ICE (consisting of rituximab, ifosfamide (IFM), carboplatin and etoposide (ETP)), and R-DHAP (consisting of rituximab, cytarabine (ara-C) and cisplatin as anticancer agents).
In the invention, the preferable methods are as follows:
(1) The method as described above, wherein docetaxel or paclitaxel is combined.
(2) The method as described in (1), wherein docetaxel is combined.
(3) The method as described in (2), wherein the patient has hormone-resistant prostate cancer.
(4) The method as described above, wherein the patient has lung cancer and one or more agents selected from docetaxel, paclitaxel, carboplatin, cisplatin and gemcitabine are combined.
(5) The method as described above, wherein rituximab is combined.
(6) The method as described above, wherein a combination therapy selected from R-ICE and R-DHAP is combined.
(7) The method as described in (6), wherein R-ICE is combined.
(8) The method as described in (5) or (6), wherein the patient has lymphoma.
(9) The method as described above, wherein 1-(2-methoxyethyl)-2-methyl-4,9-dioxo-3-(pyrazin-2-ylmethyl)-4,9-dihydro-1H-naphtho[2,3-d]imidazol-3-ium bromide is administered intravenously by infusion at a dose of 1-10 mg/m2/day continuously for a period of 4-14 days.
(10) The method described in (9), wherein 1-(2-methoxyethyl)-2-methyl-4,9-dioxo-3-(pyrazin-2-ylmethyl)-4,9-dihydro-1H-naphtho[2,3-d]imidazol-3-ium bromide is administered intravenously by infusion at a dose of 3-8 mg/m2/day continuously for 7 days, followed by drug holidays of 14 days, which administration cycle as one cycle is repeated depending on the conditions.
The present invention also relates to the following inventions:
(11) Use of 1-(2-methoxyethyl)-2-methyl-4,9-dioxo-3-(pyrazin-2-ylmethyl)-4,9-dihydro-1H-naphtho[2,3-d]imidazol-3-ium bromide for production of a medicament for treating cancer, which is characterized by combinational administration with
i) one or more anticancer agents selected from the group consisting of carboplatin, cisplatin, paclitaxel, vinorelbine, gemcitabine, irinotecan, docetaxel, doxorubicin, dacarbazine and rituximab, or
ii) a combination therapy selected from R-ICE and R-DHAP.
(12) An agent for cancer treatment comprising as an active ingredient 1-(2-methoxyethyl)-2-methyl-4,9-dioxo-3-(pyrazin-2-ylmethyl)-4,9-dihydro-1H-naphtho[2,3-d]imidazol-3-ium bromide, which is characterized by combinational administration with
i) one or more anticancer agents selected from the group consisting of carboplatin, cisplatin, paclitaxel, vinorelbine, gemcitabine, irinotecan, docetaxel, doxorubicin, dacarbazine and rituximab, or
ii) a combination therapy selected from R-ICE and R-DHAP.
(13) A potentiator of an anticancer effect of i) one or more anticancer agents selected from the group consisting of carboplatin, cisplatin, paclitaxel, vinorelbine, gemcitabine, irinotecan, docetaxel, doxorubicin, dacarbazine and rituximab, or ii) a combination therapy selected from R-ICE and R-DHAP, which comprises as an active ingredient 1-(2-methoxyethyl)-2-methyl-4,9-dioxo-3-(pyrazin-2-ylmethyl)-4,9-dihydro-1H-naphtho[2,3-d]imidazol-3-ium bromide.
(14) A kit comprising at least one cycle dosage of 1-(2-methoxyethyl)-2-methyl-4,9-dioxo-3-(pyrazin-2-ylmethyl)-4,9-dihydro-1H-naphtho[2,3-d]imidazol-3-ium bromide and i) one or more anticancer agents selected from the group consisting of carboplatin, cisplatin, paclitaxel, vinorelbine, gemcitabine, irinotecan, docetaxel, doxorubicin, dacarbazine and rituximab, or ii) anticancer agents for a combination therapy selected from R-ICE and R-DHAP. Usually, R-DHAP contains dexamethasone as an agent for treatment of chemotherapy-induced nausea and vomiting in addition to the above-mentioned anti-cancer drugs.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 contains graphs showing the results of Test Examples 1 and 2 in which YM155 was administered in combination with docetaxel. (a)-(c) are graphs respectively showing the tumor volume (mm3) in Test Example 1, Test Example 2B) and Test Example 2A); (d)-(f) are graphs respectively showing body weight (g) in Test Example 1, Test Example 2B) and Test Example 2A).
FIG. 2, (a) shows the mean tumor volume (mm3) from co-administration of YM155 and carboplatin (CBDCA) in Test Example 3, and (b) shows the mean body weight (g), respectively. (c) shows the mean tumor volume (mm3) from co-administration of YM155 and cisplatin (CDDP) in Test Example 3, and (d) shows the mean body weight (g), respectively.
FIG. 3, (a) shows the mean tumor volume (mm3) from co-administration of YM155 and gemicitabine (GEM) in Test Example 3, and (b) shows the mean body weight (g), respectively. (c) shows the mean tumor volume (mm3) from co-administration of YM155 and vinorelbine (VIN) in Test Example 3, and (d) shows the mean body weight (g), respectively.
FIG. 4, (a) shows the mean tumor volume (mm3) from co-administration of YM155 and doxorubicin (DXR) in Test Example 3, and (b) shows the mean body weight (g), respectively. (c) shows the mean tumor volume (mm3) from co-administration of YM155 and irinotecan (CPT-11) in Test Example 3, and (d) shows the mean body weight (g), respectively.
FIG. 5, (a) shows the mean tumor volume (mm3) from co-administration of YM155 and paclitaxel (TXL) in Test Example 3, and (b) shows the mean body weight (g), respectively.
FIG. 6, (a) shows the mean tumor volume (mm3) from co-administration of YM155 and Dacarbazine (DTIC) in Test Example 4, and (b) shows the mean body weight (g), respectively.
FIG. 7, (a) shows the mean tumor volume (mm3) from co-administration of YM155 and R-ICE (RICE) in Test Example 5, and (b) shows the mean body weight (g), respectively. And (c) shows the mean tumor volume (mm3) from co-administration of YM155 and Rituximab (RTX) in Test Example 5, and (d) shows the mean body weight (g), respectively.
BEST MODE FOR CARRYING OUT OF THE INVENTION
The following describes the invention in detail.
YM155 used in the invention is readily available according to the processes of production as disclosed in International Publication 01/60803 and International Publication 2004/092160.
YM155 may be administered orally or parenterally, and preferably intravenously. In this connection, the injection preparation for intravenous administration includes those containing sterile aqueous or non-aqueous solutions, suspensions, and emulsions. The aqueous solvent includes, for example, distilled water for injection and physiological saline. The non-aqueous solvent includes, for example, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol, polysorbate 80 (trade name), and the like. Such compositions may contain further tonicity adjusting agents, antiseptics, moistening agents, emulsifying agents, dispersing agents, stabilizers, and solubilizing agents. These may be sterilized, for example, by filtration through a bacterial filter, blending of sterilizers or irradiation. Alternatively, it is possible to prepare a germ-free solid composition and dissolve or suspend it in sterile water or a sterile solvent for injection immediately before use.
In intravenous administration, YM155 may be administered usually at 0.1-20 mg/m2/day, preferably at 1-10 mg/m2/day, once a day or divided in plural doses, or continuously by infusion (continuous instillation). Preferably, it may be infused at 3-10 mg/m2/day continuously for a period of 4 days to 20 days, more preferably 4 days to 14 days, or 5 days, 7 days, 10 days or 14 days, and yet more preferably infused continuously for 7 days. When the administration is further continued, it is preferable to employ a medication cycle comprising a term of drug holidays of 1 day to 2 months, preferably 7 days to 21 days, more preferably 14 days, after termination of the above term of medication. In a particularly preferred embodiment, it may be administered continuously by infusion at a dose of 3-8 mg/m2/day for 7 days, followed by drug holidays of 14 days; this cycle as one cycle is repeated depending on the conditions. When an anticancer agent used in combination has a particular medication cycle, it is preferable to establish a medication cycle for YM155 and said anticancer agent so that potentiation is attained. Specifically, the frequency of administration, dosage, time of infusion, medication cycle, and the like, may be determined properly according to individual cases considering the kind of anticancer agent, state of the patients, age, gender, etc.
The following table lists existing anticancer agents suitable for combinational use with YM155 of the invention, together with the main indications of cancer. The cancers indicated for these anticancer agents in the combination therapy of the invention is not limited to these cancers.
TABLE 1
Anticancer Agent
Main Indications of Cancer
Carboplatin
CBDCA
colon cancer, lung cancer
Cisplatin
CDDP
colon cancer, lung cancer
Paclitaxel
TXL
lung cancer, ovary cancer
Vinorelbine
VNR
lung cancer
Gemcitabine
GEM
lung cancer, pancreas cancer,
colon cancer
Irinotecan
CPT-11
colon cancer
Docetaxel
TXT
prostate cancer, lung cancer
Doxorubicin
DXR
bladder cancer
Dacarbazine
DTIC
malignant melanoma
Rituximab
RTX
CD20-positive B-cell non-Hodgkin's
lymphoma
Ifosfamide
IFM
Third line chemotherapy of germ
cell testicular cancer when used
in combination with certain other
approved antineoplastic agents
Etoposide
ETP
In combination with other approved
chemotherapeutic agents as first
line treatment in patients with
small cell lung cancer.
Cytarabine
ara-C
Leukemia
The above-mentioned anticancer agents have already been used clinically, and their administration route, administration cycle, and dose are apparent for a person skilled in the art. The appropriate dosage and administration are different depending on the species of cancer, condition and combinational agents. Detailed information is available from a variety of databases provided by the FDA, for example, “Approved Oncology Drugs” (http://www.fda.gov/cder/cancer/approved.htm)“Orange Book” (http://www.fda.gov/cder/orange/default.htm), or from the information on the home pages provided by Pharmaceuticals and Medical Devices Agency of Japan (http://www.info.pmda.go.jp/). The information about the anticancer agents stored in these databases is incorporated by reference herein in the present invention.
For example, according to the home pages provided by Pharmaceuticals and Medical Devices Agency of Japan, the package insert of TAXOTERE injection (registered trade mark; trade name of docetaxel) describes the dosage and administration of docetaxel as follows: “1. breast cancer, non-small cell lung cancer, gastric cancer, head and neck cancer; dosage and administration for each indication; 1. usually, intravenously injected by infusion at a dose of 60 mg/m2 as docetaxel for an adult over 1 hour once a day at intervals of 3-4 weeks. The dosage may be increased or decreased depending on the condition, but the highest dose is up to 70 mg/m2 at a time.” And the package insert of RITUXAN Injection (registered trade mark; trade name of rituximab) describes as follows: “the dosage and administration: CD20-positive B-cell non-Hodgkin's lymphoma; dosage and administration 1. the recommended dosage for an adult of rituximab (genetical recombination) (JAN) is 375 mg/m2 per one dose, given as an IV infusion once weekly. The maximum administering frequency is assumed to be eight times.”
The following is partially extracted Dosage and Administration regarding each anticancer agent disclosed in “Approved Oncology Drugs”. For practical administration, it will be appreciated naturally that the complete information has to be taken into consideration.
Docetaxel: “Breast Cancer: The recommended dose of TAXOTERE is 60-100 mg/m2 administered intravenously over 1 hour every 3 weeks. Non Small Cell Lung Cancer: The recommended dose of TAXOTERE is 75 mg/m2 administered intravenously over 1 hour every 3 weeks.”
Carboplatin: “Single agent therapy: PARAPLATIN (trade name of carboplatin), as a single agent, has been shown to be effective in patients with recurrent ovarian carcinoma at a dosage of 360 mg/m2 I.V. on day 1 every 4 weeks.”
Cisplatin: “Advanced Bladder Cancer: PLATINOL-AQ (trade name of cisplatin injection) should be administered as a single agent at a dose of 50-70 mg/m2 I.V. per cycle once every 3 to 4 weeks depending on the extent of prior exposure to radiation therapy and/or prior chemotherapy.”
Paclitaxel: “In patients previously treated with chemotherapy for ovarian cancer, the recommended regimen is TAXOL (trade name of paclitaxel) 135 mg/m2 or 175 mg/m2 administered intravenously over 3 hours every 3 weeks. For patients with carcinoma of the breast, TAXOL at a dose of 175 mg/m2 administered intravenously over 3 hours every 3 weeks has been shown to be effective after failure of chemotherapy for metastatic disease or relapse within 6 months of adjuvant chemotherapy.”
Vinorelbine: “The usual initial dose of NAVELBINE (trade name of vinorelbine) is 30 mg/m2 administered weekly. The recommended method of administration is an intravenous injection over 6 to 10 minutes.”
Gemcitabine: “GEMZAR (trade name of gemcitabine) is for intravenous use only. Adults Single-Agent Use: Pancreatic Cancer—should be administered by intravenous infusion at a dose of 1000 mg/m2 over 30 minutes once weekly for up to 7 weeks (or until toxicity necessitates reducing or holding a dose), followed by a week of rest from treatment. Subsequent cycles should consist of infusions once weekly for 3 consecutive weeks out of every 4 weeks.”
Irinotecan: “Starting Dose and Dose Modifications Weekly Dosage Schedule: The usual recommended starting dose of CAMPTOSAR Injection (trade name of Irinotecan injection) is 125 mg/m2. In patients with a combined history of prior pelvic/abdominal irradiation and modestly elevated serum total bilirubin levels (1.0 to 2.0 mg/dL) prior to treatment with CAMPTOSAR, there may be a substantially increased likelihood of grade 3 or 4 neutropenia. All doses should be administered as an intravenous infusion over 90 minutes. The recommended treatment regimen (one treatment course) is once weekly treatment for 4 weeks, followed by a 2-week rest period.”
Doxorubicin: “Ovarian Cancer Patients Doxil (trade name of doxorubicin HCl liposome injection) should be administered intravenously at a dose of 50 mg/m2 (doxorubicin HCl equivalent) at an initial rate of 1 mg/min to minimize the risk of infusion reactions. If no infusion-related AEs are observed, the rate of infusion can be increased to complete administration of the drug over one hour. The patient should be dosed once every 4 weeks, for as long as the patient does not progress, shows no evidence of cardiotoxicity, and continues to tolerate treatment. A minimum of 4 courses is recommended because median time to response in clinical trials was 4 months.”
Dacarbazine: “Malignant Melanoma: The recommended dosage is 2 to 4.5 mg/kg/day for 10 days. Treatment may be repeated at 4 week intervals. 2 An alternate recommended dosage is 250 mg/square meter body surface/day I.V. for 5 days. Treatment may be repeated every 3 weeks.”
Rituximab: The recommended dosage of RITUXAN (trade name of rituximab) is 375 mg/m2 given as an IV infusion once weekly for four doses (days 1, 8, 15, and 22).
Ifosfamide: IFEX (trade name of ifosfamide) should be administered intravenously at a dose of 1.2 g/m2 per day for 5 consecutive days.
Etoposide: In small cell lung cancer, the VePesid For Injection dose in combination with other approved chemotherapeutic drugs ranges from 35 mg/m2/day for 4 days to 50 mg/m2/day for 5 days.
Cytarabine: In the induction therapy of acute non-lymphocytic leukemia, the usual cytarabine dose in combination with other anticancer drugs is 100 mg/m2/day by continuous IV infusion (days 1 to 7) or 100 mg/m2 IV every 12 hours (days 1 to 7). Regarding R-ICE and R-DHAP, detailed information is available from references on clinical trials as follows:
R-ICE (Blood. 2006 May 15; 103(10):3684-8): Rituximab (375 mg/m2) is administered on day 1 of each cycle and 48 hours before the initiation of the first cycle of ICE. ICE chemotherapy is administered on an inpatient basis beginning on day 3 of each cycle. Etoposide (100 mg/m2) is administered as an intravenous bolus daily for 3 days, from days 3 to 5. Carboplatin capped at 800 mg is administered as a bolus infusion on day 4. Ifosfamide (5000 mg/m2), mixed with an equal amount of mesna, was administered as a continuous intravenous infusion over 24 hours beginning on day 4. R-DHAP (Blood. 1988 January; 71:117-22, Leukemia & Lymphoma 2007 May; 48(5):897-904): Rituximab (375 mg/m2) is administered on day 1 of each cycle and 48 hours before the initiation of the first cycle of DHAP. Cisplatin (100 mg) is administered as a continuous intravenous infusion over 24 hours beginning on day 1. Cytarabine (2000 mg/m2) is administered as an intravenous bolus twice daily on day 2. Additionally, in order to control chemotherapy-induced nausea and vomiting, dexamethasone (40 mg/m2) is administered as an intravenous bolus daily for 3 days, from days 1 to 4.
In using the combination therapy of the present invention, the same dose as that usually given as a single agent or a slightly reduced dose (for example, 0.10-0.99 times the highest dose as a single agent) may be given through a normal administration route.
In the case of an anticancer agent which is administered in a certain administration cycle, the cycle may properly be adjusted so as to be suitable for combinational use with YM155. Specifically, the frequency of administration, dosage, time of infusion, medication cycle, and the like may be determined properly according to individual cases considering the state of patients, age, gender, etc.
The mode of medication in combinational administration of YM155 and an anticancer agent is not particularly limited as long as a suitable administration route, frequency of administration and dosage are properly selected. For example, (1) administration of a composition containing YM155 and an anticancer agent, i.e., as a single preparation; (2) combinational administration of 2 species of preparations separately prepared from YM155 and an anticancer agent, respectively, through the same administration route; (3) time interval difference administration of 2 species of preparations separately prepared from YM155 and an anticancer agent, respectively, through the same administration route (for example, YM155 is administered first, and then an anticancer agent, or vice versa); (4) combinational administration of 2 species of preparations separately prepared from YM155 and an anticancer agent, respectively, through different administration routes; and (5) time interval difference administration of 2 species of preparations separately prepared from YM155 and an anticancer agent, respectively, through different administration routes (for example, YM155 is administered first, and then an anticancer agent, or vice versa).
In a preferred embodiment of combinational administration of the present invention, YM155 and another anticancer agent are separately formulated, and the resulting 2 types of preparations are administered concomitantly (including partially concomitant) or at a time interval through an administration route suitable to each preparation and in a proper frequency of administration. In the time interval difference administration, it is necessary to administer the preparations at a time interval which satisfies the potentiation of the anticancer effect. Preferably, the 2nd preparation is administered within 2 weeks, more preferably 7 days, yet more preferably 3 days, after administration of the first preparation. When there is concern about a drug-drug interaction between YM155 and another anticancer agent, it is appropriate to administer them at an interval required for avoidance of such an interaction.
As shown in Test Examples, it was found that the therapeutic effect on cancer was potentiated by combination therapy of YM155 and an existing anticancer agent. The effect was confirmed by significant reduction of the tumor volume in comparison with that by the respective single application of YM155 or an existing anticancer agent. On the other hand, the body weight was equal to that at the administration of an existing anticancer agent as a single agent, indicating that possibly there is no change in adverse reaction in the combinational use. Therefore, it was found that the combinational use of YM155 and an existing anticancer agent potentiates an anticancer action with no change of adverse reaction, resulting in a satisfactory therapeutic effect for cancer.
Surprisingly, it was elucidated that a combinational use of YM155 and a taxane-type anticancer agent, particularly docetaxel, exhibited a much better cancer-regression action. In an animal model as shown in the Test Examples, an anticancer effect was confirmed which was so potent that disappearance of cancer was attained. In a combinational use with docetaxel, disappearance of cancer was observed in all or part of the animals tested, and totally the tumor volume was reduced to almost 0 (zero) after the lapse of 35 days; no tendency was observed toward the growth of cancer cells again. From this finding, it was estimated that the inhibitory effect of YM155 on expression of survivin and the anticancer effect of docetaxel worked synergistically to attain a particularly excellent therapeutic effect for cancer.
In a preliminary test using an animal model to which an other type of cancer was transplanted, e.g., prostate cancer (PC-3), a taxane-type anticancer agent, docetaxel or paclitaxel, was administered combined with YM155. As a result, the anticancer action was potentiated in comparison with administration of the respective single agents and the tumor volume was reduced well. This suggests that the combinational use of YM155 potentiates the anticancer action of existing anticancer agents and induces a good cancer therapeutic effect not limited by kinds of cancers. In addition, it has been disclosed in the above-cited references No. 14 to 17 that YM155 per se exhibits a potent anticancer action on a variety of cancers in which survivin is involved. These findings show that the method of the invention for treatment of cancers by co-administration of YM155 and an existing anticancer agent, in which the inhibitory effect of YM155 on expression of survivin and the anticancer effect of the existing anticancer agent work synergistically, affords a high therapeutic effect for cancers and can be applied to treatment of a variety of cancers. In particular, since YM155 has a new mechanism of action working to inhibit the expression of survivin, it is useful in the treatment of kinds of cancers which are resistant to existing anticancer agents.
The anticancer agents used in combination with YM155 include, preferably, those usually used against an objective kind of cancers, particularly those used in a standard therapy. For example, docetaxel for hormone-resistant prostate cancer, dacarbazine for melanoma, one or more agents selected from docetaxel, paclitaxel, carboplatin, cisplatin and gemcitabine for lung cancer, or R-ICE and R-DHAP for lymphoma, are included. The combinational use of YM155 in addition to these anticancer agents used in a standard therapy, is expected to potentiate the therapeutic effect for cancers in the objective kind of cancers.
EXAMPLES
The followings show the results of pharmacological tests indicating the usefulness of the combinational use of the invention.
Test Example 1
1) Test Compounds
The dose levels are expressed in terms of YM155, the cationic moiety of the drug substance. Docetaxel hydrate (Taxotere® INJECTION) was purchased from Sanofi-Aventis Pharma Ltd. (West Lavel, FRA) and used as docetaxel.
2) Preparation of Test Compounds
YM155 was dissolved in and diluted with physiological saline in order to prepare dosing solutions (concentrations calculated using the following formulation: dose×mean body weight for each group/daily release volume). These solutions were prepared immediately before the implantation of an osmotic pump (Alzet® model 1007D Micro-Osmotic Pump, DURECT Co., CA, USA) into the test subjects. The solutions were then loaded into the pump. Docetaxel was diluted with physiological saline to prepare 2 mg/mL solutions immediately before administration.
3) Cells
The human lung carcinoma cell line Calu 6 (HTB-56, Lot No. 208280) was obtained from American Type Culture Collection (VA, USA). The cells were cultured at 37° C. in a 5% CO2 atmosphere in RPMI1640 medium supplemented with 10% heat-inactivated fetal bovine serum. The cells were collected with trypsin, suspended in PBS at 6×107 cells/mL, and then mixed with an equivalent volume of Matrigel® Basement Membrane Matrix (Becton Dickinson Co., Bedford, Mass., USA).
4) Animals
Five-week-old male nude mice (CAnN.Cg-Foxn1nu/CrlCrlj(nu/nu)) were purchased from Charles River Laboratories Japan, Inc (Kanagawa, Japan). They were maintained on a standard diet and water throughout the experiments under specific pathogen-free conditions. The cells were cultured in vitro and 3×106 cells/0.1 mL/mouse were grafted subcutaneously into the flank of 6-week-old nude mice. The mice with tumor volumes (length×width2×0.5) of 97.3 to 182.7 mm3 were divided into groups. This was done using SAS software in order to minimize intragroup and intergroup tumor volume variation.
5) Administration and Measurement
The first day of administration was designated day 0, and observation continued until day 35. Each group (n=8) was treated as follows.
Vehicle control group
Vehicle
YM155 group
YM155 2 mg/kg/day (7-day sc continuous
infusion)
Docetaxel group
Docetaxel 20 mg/kg/day (iv bolus on day 0,
4, and 8)
Combination group
YM155 2 mg/kg/day + Docetaxel 20 mg/kg/day
For 7-day sc continuous infusion, an osmotic pump containing either YM155 or physiological saline, was implanted in the dorsum of each animal while under anesthesia. The vehicle control group received a 7-day sc continuous infusion of physiological saline starting on day 0, and iv bolus injection of physiological saline on days 0, 4 and 8. The YM155 group received a 7-day sc continuous infusion of YM155 at 2 mg/kg/day starting on day 0, and iv bolus injection of physiological saline on days 0, 4 and 8. The docetaxel group received iv bolus injection of docetaxel at 20 mg/10 mL/kg on days 0, 4 and 8, as well as a 7-day sc continuous infusion of physiological saline starting on day 0. The combination group received both compounds instead of the vehicle, similarly to the YM155 and docetaxel groups.
Body weight and tumor diameter were measured every 3-4 days using calipers, and tumor volume was determined by calculating the volume of an ellipsoid using the formula: length×width2×0.5. Antitumor activities are expressed as percent inhibition of tumor growth (% inh) and percent regression of the tumor (% reg). The percent inhibition of tumor growth on day 35 was calculated for each group using the following formula: 100×[1−{(mean tumor volume of each group on day 35)−(mean tumor volume of each group on day 0)}/{(mean tumor volume of the control group on day 35)−(mean tumor volume of the control group on day 0)}]. The percent regression of the tumor was calculated for all groups with observed tumor regression, using the following formula: 100×{1−(mean tumor volume of each group on day 35)/(mean tumor volume of each group on day 0)}. The number of Complete Regressions (CRs) in all groups were checked during the experimental period. The CR is defined by instances in which the tumor burden falls bellow the limit of palpation.
6) Statistical Analysis
Values are expressed as the mean± the standard error of the mean (SEM). Tumor volume and body weight on day 35 were compared between each single compound group and combination group using the Student's t-test. P values of less than 5% are considered significant. SAS software was used for data processing.
7) Results
When the anti-tumor activity of YM155 in combination with docetaxel was examined, the combination of YM155 at 2 mg/kg/day with docetaxel at 20 mg/kg/day completely inhibited tumor growth (>100%), and induced complete regression by 100% (Table 2 and FIG. 1-(a)). YM155 treatment alone inhibited tumor growth by 99% on day 35. Docetaxel also exhibited complete inhibition (>100%) of tumor growth, and induced tumor regression by 52% on day 35. During the 5 weeks of observation, YM155 or docetaxel treatment group showed tumor regression only during the first few weeks which was followed by successive tumor regrowth during the last few weeks. On the other hand, YM155 in combination with docetaxel showed complete regression of the tumors in all cases on day 35. No statistically significant decrease in body weight was observed in the combination group as compared to docetaxel group (FIG. 1-(d)).
TABLE 2
Number of
Tumor Volume (mm3)
Body Weight (g)
Antitumor Activity
Complete
Treatment Groups
day 0
day 35
day 35
(% inh/% reg)
Regressions
Vehicle Control
120.9 ± 7.0
2848.0 ± 185.1
26.56 ± 0.56
—
0/8
YM155
121.4 ± 6.9
142.3 ± 16.9
26.13 ± 0.56
99%
0/8
Docetaxel
121.4 ± 7.0
58.0 ± 20.4
24.95 ± 0.54
>100%/52%
0/8
YM155 + Docetaxel
121.1 ± 7.2
0.0 ± 0.0¶##
24.11 ± 0.66#,N.S.
>100%/100%
8/8
Values are expressed as the mean ± SEM (n = 8).
¶P < 0.05 versus Docetaxel group,
##P < 0.01 versus YM155 group,
#P < 0.05 versus YM155 group,
N.S.no significance from Docetaxel group (Student's t-test).
8) Discussion
Results of this study indicate that YM155 significantly potentiate the anti-tumor activity of docetaxel without an increase in systemic toxicity, which is evidenced by overt symptoms such as body weight loss. The results also suggest that YM155 in combination with docetaxel is tolerated by mice, and that makes a strong combination for the treatment of cancer.
Test Example 2
The following study was conducted in the same way as Test Example 1 except for the administration timing.
A) The first day of administration was designated day 0, and observation continued until day 35. Each group (n=8) was treated as follows.
Vehicle control group
Vehicle
YM155 group
YM155 2 mg/kg/day (7-day sc continuous
infusion)
Docetaxel group
Docetaxel 20 mg/kg/day (iv bolus on day 7,
11, and 15)
Combination group
YM155 2 mg/kg/day → Docetaxel 20 mg/kg/day
For 7-day sc continuous infusion, an osmotic pump containing either YM155 or physiological saline, was implanted in the dorsum of each animal while under anesthesia. The vehicle control group received a 7-day sc continuous infusion of physiological saline starting on day 0, and iv bolus injection of physiological saline on days 7, 11 and 15. The YM155 group received a 7-day sc continuous infusion of YM155 at 2 mg/kg/day starting on day 0 and iv bolus injection of physiological saline on days 7, 11 and 15. The docetaxel group received iv bolus injection of docetaxel at 20 mg/10 mL/kg on days 7, 11 and 15, as well as a 7-day sc continuous infusion of physiological saline starting on day 0. The combination group received both compounds instead of the vehicle, similarly to the YM155 and docetaxel groups.
Results
When the anti-tumor activity of YM155 in sequential combination with docetaxel was examined, the combination of YM155 at 2 mg/kg/day with docetaxel at 20 mg/kg/day completely inhibited tumor growth (>100%), and induced complete regression by 99% (Table 3 and FIG. 1-(c)). YM155 treatment alone inhibited tumor growth by 99% on day 35. Docetaxel also exhibited complete inhibition (>100%) of tumor growth, and induced tumor regression by 10% on day 35. During the 5 weeks of observation, YM155 or docetaxel treatment group showed tumor regression only during the first few weeks which was followed by successive tumor regrowth during the last few weeks. On the other hand, YM155 in combination with docetaxel showed complete regression of the tumors in all cases, and tumor volume was significantly (P<0.01) reduced in mice treated with YM155 in combination with docetaxel as compared to each single compound treatment on day 35. No statistically significant decrease in body weight was observed in the combination group as compared to Docetaxel group (FIG. 1-(f)).
TABLE 3
Tumor Volume (mm3)
Body Weight (g)
Antitumor Activity
Treatment Groups
day 0
day 35
day 35
(% inh/% reg)
Number of CRs
Vehicle Control
208.5 ± 18.6
4237.0 ± 545.7
25.88 ± 0.70
—
0/8
YM155
208.5 ± 18.6
251.8 ± 50.7
26.25 ± 0.25
99%/—
0/8
Docetaxel
209.1 ± 18.3
188.8 ± 29.1
21.03 ± 0.94
>100%/10%
0/8
YM155→Docetaxel
208.6 ± 18.1
1.3 ± 1.3¶¶##
24.13 ± 0.78¶#
>100%/99%
7/8
Values are expressed as the mean ± SEM (n = 8).
¶¶P < 0.01 versus Docetaxel group,
##P < 0.01 versus YM155 group,
#P < 0.05 versus YM155 group,
¶P < 0.05 versus Docetaxel group (Student's t-test).
B) The first day of administration was designated day 0, and observation continued until day 35. Each group (n=8) was treated as follows.
Vehicle control group
Vehicle
YM155 group
YM155 2 mg/kg/day (7-day sc continuous
infusion)
Docetaxel group
Docetaxel 20 mg/kg/day (iv bolus on day 0,
4, and 8)
Combination group
Docetaxel 20 mg/kg/day → YM155 2 mg/kg/day
For 7-day sc continuous infusion, an osmotic pump containing either YM155 or physiological saline, was implanted in the dorsum of each animal while under anesthesia. The vehicle control group received a 7-day sc continuous infusion of physiological saline starting on day 8, and iv bolus injection of physiological saline on days 0, 4 and 8. The YM155 group received a 7-day sc continuous infusion of YM155 at 2 mg/kg/day starting on day 8, and iv bolus injection of physiological saline on days 0, 4 and 8. The docetaxel group received iv bolus injection of docetaxel at 20 mg/10 mL/kg on days 0, 4 and 8, as well as a 7-day sc continuous infusion of physiological saline starting on day 8. The combination group received both compounds instead of the vehicle, similarly to the YM155 and docetaxel groups.
Results
When the anti-tumor activity of YM155 in sequential combination with docetaxel was examined, the combination of YM155 at 2 mg/kg/day with docetaxel at 20 mg/kg/day completely inhibited tumor growth (>100%), and induced complete regression by 97% (Table 4 and FIG. 1-(b)). YM155 treatment alone inhibited tumor growth by 61% on day 35. Docetaxel also exhibited complete inhibition (>100%) of tumor growth, and induced tumor regression by 68% on day 35. During the 5 weeks of observation, YM155 or docetaxel treatment group showed tumor regression only during the first few weeks which was followed by successive tumor regrowth during the last few weeks. On the other hand, YM155 in combination with docetaxel showed complete regression of the tumors in three cases, and tumor volume was significantly (P<0.01) reduced in mice treated with YM155 in combination with docetaxel as compared to each single compound treatment on day 35. No statistically significant decrease in body weight was observed in the combination group as compared to docetaxel group (FIG. 1-(e)).
TABLE 4
Tumor Volume (mm3)
Body Weight (g)
Antitumor Activity
Treatment Groups
day 0
day 35
day 35
(% inh/% reg)
Number of CRs
Vehicle Control
207.7 ± 23.5
3380.8 ± 325.4
24.33 ± 0.85
—
0/8
YM155
206.9 ± 21.8
1437.5 ± 225.5
24.80 ± 1.01
61%/—
0/8
Docetaxel
210.0 ± 22.6
67.2 ± 15.1
23.84 ± 1.01
>100%/68%
0/8
Docetaxel →YM155
210.1 ± 20.4
5.5 ± 1.7¶¶##
25.74 ± 1.13N.S.,N.S.
>100%/97%
3/8
Values are expressed as the mean ± SEM (n = 8).
¶¶P < 0.01 versus Docetaxel group,
##P < 0.01 versus YM155 group,
N.S.no significance from YM155 group and Docetaxel group (Student's t-test).
Test Example 3
The study was conducted in the same way as Test Example 1, except that the listed agents in Table 5 were used instead of Docetaxel according to the following dosage regimen.
TABLE 5
Anticancer Agent
Dosage Regimen
Result
Carboplatin
CBDCA
60
mg/kg/day i.v. (day 0, 1)
FIG. 2-(a) & (b)
Cisplatin
CDDP
3
mg/kg/day i.v. (day 0-4, 7-11)
FIG. 2-(c) & (d)
Gemcitabine
GEM
200
mg/kg/day i.v. (day 0, 3, 6)
FIG. 3-(a) & (b)
Vinorelbine
VNR
10
mg/kg/day i.v. (day 0, 7)
FIG. 3-(c) & (d)
Doxorubicin
DXR
10
mg/kg/day i.v. (day 0, 7)
FIG. 4-(a) & (b)
Irinotecan
CPT-11
60
mg/kg/day i.v. (day 0-4)
FIG. 4-(c) & (d)
Paclitaxel
TXL
10-15
mg/kg/day i.v. (day 0-4)
FIG. 5-(a) & (b)
(In the Table 5, i.v. means intravenous administration, for example, (day 0, 1) means once-a-day administration at the initial day of administration (day 0) and at the 1st day (day 1); (day 0-4) means once-a-day administration from the initial day of administration (day 0) to the 4th day (day 4), respectively.)
Results were shown in FIGS. 2 to 5. In the figures, values are expressed as the mean±SEM (n=8). ¶¶: P<0.01 versus an anticancer agent group, ##: P<0.01 versus YM155 group, #: P<0.05 versus YM155 group, N.S.: no significance from YM155 group and the anticancer agent group (Student's t-test).
The respective anticancer agents were purchased as commercially available products, and prepared according to the procedure directed in package inserts to use in the tests.
Test Example 4
The study was conducted in the same way as Test Example 1, except that Dacarbazine was used instead of Docetaxel and Melanoma A375 cells were used instead of Calu 6 cells according to the following dosage regimen (n=8). Results were shown in FIG. 6.
YM155 group
YM155 3 mg/kg/day (7-day sc continuous infusion)
Dacarbazine
Dacarbazine 200 mg/kg, 5 times weekly, i.v.
group
Combination
YM155 3 mg/kg/day (7-day sc continuous infusion) +
group
Dacarbazine 200 mg/kg, 5 times weekly, i.v.
Dacarbazine was purchased as commercially available products, and prepared according to the procedure directed in package inserts to use in the tests.
Test Example 5
The study was conducted in the same way as Test Example 1, except that R-ICE(RICE) or Rituximab(RTX) was used instead of Docetaxel, and lymphoma WSU-DLCL-2 cells were used instead of Calu 6 cells according to the following dosage regimen.
YM155 group YM155 1 mg/kg/day (7-day sc continuous infusion)
RICE group RTX 50 mg/kg, i.v. (day 0)+(IFM 200 mg/kg+ETP 10 mg/kg), i.v. (day 1, 2 and 3)+CBDCA 30 mg/kg, i.v. (day 1)
YM155+RICE group YM155 1 mg/kg/day (7-day sc continuous infusion)+RTX 50 mg/kg, i.v. (day 0)+(IFM 200 mg/kg+ETP 10 mg/kg), i.v. (day 1, 2 and 3)+CBDCA 30 mg/kg, i.v (day 1).
YM155+RTX group YM155 1 mg/kg/day (7-day sc continuous infusion)+RTX 50 mg/kg, i.v. (day 0).
RTX, IFM and ETP were purchased as commercially available products, and prepared according to the procedure directed in package inserts to use in the tests. Results were shown in FIG. 7. In the figures, values are expressed as the mean±SEM (n=6). ¶¶: P<0.01 versus an anticancer agent group, ¶: P<0.05 versus an anticancer agent group, ##: P<0.01 versus YM155 group, N.S.: no significance from YM155 group and the anticancer agent group (Student's t-test). YM155 in combination with R-ICE showed complete regression of the tumors in one case, and YM155 in combination with RTX showed complete regression of the tumors in two cases.
The preliminary study was done by using the method similar to the above-mentioned YM155+RICE group, but R-DHAP was used instead of R-ICE, wherein R-DHAP was consisting of rituximab (50 mg/kg i.v., day 0 and 2), cisplatin (5 mg/kg, i.v., day 2), cytarabin (70 mg/kg, i.v., day 4). The tumor volume was significantly (P<0.01) reduced in the combination group as compared to R-DHAP group on day 18.
Test Example 6
The study was conducted in the same way as Test Example 5 according to the following dosage regimen.
YM155 group YM155 2 mg/kg/day (7-day sc continuous infusion)
RICE group RTX 50 mg/kg, i.v. (day 6 and 8)+(IFM 400 mg/kg+CBDCA 30 mg/kg), i.v (day 9)+ETP 10 mg/kg, i.v (day 8, 9 and 10)
YM155→RICE group YM155 2 mg/kg/day (7-day sc continuous infusion)+RTX 50 mg/kg, i.v. (day 6 and 8)+(IFM 400 mg/kg+CBDCA 30 mg/kg), i.v. (day 9)+ETP 10 mg/kg, i.v. (day 8, 9 and 10)
Results were shown in table 6. In the table, values are expressed as the mean±SEM (n=8, RICE group: n=6). ¶¶: P<0.01 versus an RICE group, ##: P<0.01 versus YM155 group, N.S.: no significance from RICE group (Student's t-test). YM155 in combination with R-ICE showed complete regression of the tumors in six cases.
TABLE 6
Tumor Volume (mm3)
Body Weight (g)
Antitumor Activity
Number of
Treatment Groups
day 0
day 28
day 28
(% inh/% reg)
CRs
Vehicle Control
404.4 ± 14.5
2131.5 ± 276.6
29.02 ± 0.38
—
0/8
YM155
405.1 ± 13.8
1181.3 ± 174.7
28.81 ± 0.23
45%
0/8
RICE
404.2 ± 13.0
1356.0 ± 371.7
29.54 ± 0.51
56%
0/6
YM155 → RICE
404.7 ± 12.6
17.6 ± 11.5¶¶##
28.55 ± 0.26N.S.
>100%/96%
6/8
INDUSTRIAL APPLICABILITY
The method of the invention for treatment of cancer by co-administration of YM155 and an existing anticancer agent, in which the inhibitory action of YM155 on expression of survivin works synergistically with the anticancer effect of the existing anticancer agent, affords a high therapeutic effect for cancers and is useful in treatment of a variety of cancers to which existing anticancer agents are applied, accordingly. Thus, the method of the invention for treatment of cancer by co-administration of YM155 and an existing anticancer agent is useful in treatment of cancers, preferably all solid cancers and lymphomas, particularly skin cancer, bladder cancer, breast cancer, uterine cancer, ovary cancer, prostate cancer, lung cancer, colon cancer, pancreas cancer, renal cancer, gastric cancer, and the like. Particularly, they are expected as therapeutic agents for some kinds of cancers which show resistance against existing anticancer agents.
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12493577
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astellas pharma inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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424/155.1
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Apr 1st, 2022 05:13PM
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Apr 1st, 2022 05:13PM
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Astellas Pharma
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