Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4721—Lipocortins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P5/00—Drugs for disorders of the endocrine system
  • A61P5/24—Drugs for disorders of the endocrine system of the sex hormones
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
  • G01N33/76—Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates to a physiologically active inhibitor of annexin 5 and a physiologically active inhibitor of annexin 5 containing the physiologically active inhibitor as an active ingredient, in particular, a polypeptide that suppresses the biological activity of annexin 5, and a polypeptide derivative of the polypeptide. And modified polypeptides, DNAs encoding the polypeptides, antibodies that specifically bind to the polypeptides, and uses thereof. Background art
• Annexin has attracted attention as a new calcium-binding protein (Cell 55, 1-3, 1988, Biochim. Biophys. Acta 1197, 63-93, 1994).
• Annexin has a highly homologous repeating structure consisting of about 70 amino acid residues in the molecule (four times for Annexin 6), and this site shows the ability to bind to calcium and phospholipids. (Cell 55, 1-3, 1988, Biochemistry 26, 8067-8092, 1987).
• Belongs to the so-called EF band super family, such as calmodulin and troponin Proteins are a new category of proteins with different modes of calcium binding.
• Annexin is presumed to be a protein that plays an important physiological function because it has a function of binding phospholipids depending on calcium concentration and has extremely high homology between animal species.
• Annexin 5 is an annexin that is abundant in various tissues. In addition to the above functions, Annexin 5 inhibits protein kinase C activity and blood clotting (J. Biol. Chem. 265, 17420-17423, 1990. Biochemistry 31, 1886-1891, 1992). High affinity for collagen has been reported, suggesting a role in cell attachment to the basement membrane (FEBS Lett 310, 143-147, 1992). Annexin 5 is widely distributed among tissues, and its distribution is cell type-specific U. Histochem. 39, 1189-1198, 1991, Endocrine 5, 9-14, 1996).
• Annexin 5 is available as a marker protein for ovarian tumors (Gynecol Obstet Invest 32, 107-111, 1991), and annexin 5 autoantibodies are detected in some patients with lupus erythematosus (LE). (Am. J. Hematol. 47, 56-58, 1994). These observations suggest that Annexin 5 is involved in several important cellular functions.
• Annexin 5 is expressed in the anterior pituitary gland (Biochem. Biophys. Res. Commun. 186, 894-898, 1992).
• stimulating hormone-producing cells Cell Tissue Res. 292, 85-89, 1998.
• Annexin 5 mRNA expression in the anterior pituitary gland was increased after ovariectomy, and this increase was suppressed by estrogen administration (Mol. Cell Endocrnol. 141, 73-78, 1998).
• the present inventors have proposed the role of annexin 5 in gonadotropin secretion, and investigated the role of annexin 5 in pituitary gonadotropin-producing cells.
• gonadotropin-releasing hormone (Gn (RH) to stimulate the release of LH and follicle-stimulating hormone (FSH) at the in vivo mouth, demonstrating the expression of the mRNA for annexin 5 by gonadotropin-producing cells under the stimulatory effect of (RH).
• Annexin 5 action peptide sequence consisting of 15 amino acid residues on the amino terminal side of Annexin 5, and completed the present invention.
• This sequence is a sequence specific to Annexin 5, and by constructing a polypeptide derivative or a modified polypeptide containing this sequence, the action of Annexin 5 is antagonistically inhibited, Physiological activity can be suppressed.
• annexin 5 secretes gonadotropin (luteinizing hormone and follicle-stimulating hormone) in the anterior pituitary gland. It has been found to stimulate remarkably, and has been confirmed to regulate the synthesis and release of the hormone in gonadotropin-producing cells.
• gonadotropin-releasing hormone G n RH
• an antibody that specifically binds to the polypeptide sequence is prepared by utilizing the action-specific sequence in annexin 5 of the present invention, whereby the detection of annexin 5 can be performed using the antibody. Measurements can be made. Further, by using the DNA sequence encoding the action-specific sequence in Annexin 5 to prepare an antisense oligonucleotide having the sequence, the expression of the Annexin 5 gene can be suppressed, and the Annexin 5 gene can be detected. Can be used as a probe. Disclosure of the invention
• the present invention relates to a physiologically active inhibitor of Annexin 5 comprising the polypeptide of SEQ ID NO: 1 in the Sequence Listing or the polypeptide of SEQ ID NO: 1 in the Sequence Listing (Claim 1);
• a physiologically active inhibitor of Annexin 5 containing a polypeptide sequence in which one to several amino acids of the polypeptide have been deleted, substituted or added, and having an inhibitory activity on Annexin 5 (claim 2);
• Stated Annexin 5 bioactivity inhibitor (Claim 4) or a substance containing the polypeptide sequence of SEQ ID NO: 1 in the sequence listing is a polybeptide obtained by modifying the amino terminal of the polypeptide of SEQ ID NO: 1 with an acetyl group.
• the present invention provides a DNA sequence encoding the polypeptide of SEQ ID NO: 1 in the Sequence Listing (Claim 7), or a DNA sequence encoding the polypeptide of SEQ ID NO: 1 in the Sequence Listing, 9.
• an antisense oligonucleotide that specifically suppresses the expression of annexin 5 gene and the physiological activity function of annexin 5 (claim 9) And a probe for detecting the gene of Annexin 5 comprising all or a part of the antisense strand of the DNA sequence according to claim 7 or 8, or a polypeptide of SEQ ID NO: 1 in the sequence listing.
• an antibody characterized in that it specifically binds to the polypeptide (claim 11) or the antibody according to claim 11 in which the antibody is a monoclonal antibody (claim 11). Item 12) and the antibody according to claim 11 (claim 13), wherein the antibody is a polyclonal antibody.
• the present invention provides a physiologically active inhibitor of annexin 5 comprising the annexin 5 physiological activity inhibitor according to claim 1 or 2 as an active ingredient (claim 14), and an annexin 5 according to claim 3.
• a physiological activity inhibitor of annexin 5 (claim 15), characterized in that the physiologically active inhibitor of annexin 5 is used as an active ingredient, and a physiologically active inhibitor of annexin 5 according to claim 4 or 5 as an active ingredient.
• the inhibitor of the physiological activity of annexin 5 (claim 16) and the inhibition of the physiological activity of annexin 5 are suppression of the physiological activity regulating the synthesis and release of annexin 5 gonadotropin.
• FIG. 1 is a diagram showing the results of a mass spectrum analysis of a biological activity inhibitor AN-14 of annexin 5 of the present invention prepared in an example of the present invention.
• FIG. 2 is a view showing the results of HPLC of the annexin 5 physiological activity inhibitor AN-14 of the present invention prepared in the examples of the present invention.
• UV wavelength 210 nm
• Elution A solution: 0.1% TF ⁇ ⁇ ⁇
• B solution 0.1% TF ⁇ C ⁇
• gradient 10% to 60% ⁇ solution for 30 minutes
• flow rate 1.0 ml / min
• FIG. 3 shows the results of time-resolved fluorescence immunoassay measuring the effect of AN-1 on the amount of GnRH released from LH in primary cultured rat pituitary cells.
• FIG. 4 is a graph showing the antagonistic effect of AN-14 on the inhibitory effect of GnRH on the growth of L ⁇ 2 cells, which are pituitary gonadotrophy cell lines.
• FIG. 5 is a diagram showing an LH3 promoter reporter vector prepared by introducing the LH] 3 promoter region ( ⁇ 797 to 15) into pGL3, a luciferase gene expression vector. .
• FIG. 6 is a graph showing the results of analyzing the antagonistic effect of AN-14 on the effect of promoting 011 shaku 11 on the expression of 1 ⁇ 1/3 subunit gene by measuring luciferase activity. .
• the present invention relates to a polypeptide of SEQ ID NO: 1 comprising the polypeptide of SEQ ID NO: 1 in the sequence listing or the biological activity inhibitor of Annexin 5 comprising the polypeptide sequence of SEQ ID NO: 1 in the sequence listing.
• the terminal side corresponds to 15 amino acid residues.
• This sequence is a specific sequence having a function as an action peptide sequence of Annexin 5, and contains this sequence and constructs a polypeptide derivative or modified polypeptide different from the subsequent sequence of Annexin 5. By doing so, the action of Annexin 5 can be antagonistically inhibited and the physiological activity of Annexin 5 can be suppressed
• polypeptide derivative of the present invention several to several tens of amino acids are added following the 15 amino acid residues of SEQ ID NO: 1 for ease of function, synthesis or handling. It is preferable to use a polypeptide derivative to which an appropriate amino acid is bonded.
• polypeptide modified with an acetyl group or the like a polypeptide whose amino terminal is modified with an acetyl group is particularly preferable.
• the present invention also includes a polypeptide sequence in which one to several amino acids of the polypeptide of SEQ ID NO: 1 have been deleted, substituted or added, and has an action to inhibit the physiological activity of annexin 5, It consists of a variant of the polypeptide of SEQ ID NO: 1 in the sequence listing, Annexin 5, which is a physiologically active inhibitor.
• the annexin 5 physiological activity inhibitor of the present invention can be used as an annexin 5 physiological activity inhibitor.
• the substance inhibiting the physiological activity of annexin 5 of the present invention competitively inhibits the physiological function of annexin 5 and suppresses the physiological activity of annexin 5.
• annexin 5 is known to mediate the action of gonadotropin-releasing hormone (GnRH) secreted by the hypothalamus at the pituitary gonadotrophy.
• GnRH gonadotropin-releasing hormone
• the administration can antagonistically inhibit the physiological function of Annexin 5 and suppress the synthesis and release of gonadotropin (LH and FSH).
• the physiological activity inhibitor can be administered in the form of an injection.
• the physiologically active substance is prepared by dissolving the substance in water, physiological saline, glucose solution or the like, and if necessary, contains a pharmaceutically acceptable buffer, preservative or stabilizer.
• a pharmaceutically acceptable buffer, preservative or stabilizer can be administered.
• the annexin 5 physiological activity inhibitor of the present invention can be administered to organs such as the ovary, adrenal gland, heart, lung, and thyroid where annexin 5 is expressed to suppress the physiological functions related to annexin 5. it can.
• the polypeptide of the annexin 5 physiological activity inhibitor of the present invention can be synthesized by a known polypeptide synthesis method. Alternatively, by genetic engineering using a DNA sequence encoding the polypeptide. It can also be manufactured.
• the DNA sequence encoding the polypeptide of SEQ ID NO: 1 in the Sequence Listing, which is a physiological activity inhibitor of Annexin 5 of the present invention, is represented by the sequence of SEQ ID NO: 2 in the Sequence Listing. This sequence shows the specific sequence of Annexin 5.
• the conditions under which the antisense oligonucleotide of the present invention “hybridizes with the DNA sequence under stringent conditions” include, for example, hybridization at 42 ° C., and 1 XSSC, 0 Washing at 42 ° C with buffer containing 1% SDS can be mentioned, hybridization at 65 ° C, and 0.1 XSSC, containing 0.1% SDS A washing treatment with a buffer solution at 65 ° C. can be more preferably mentioned.
• antisense oligonucleotides can be administered directly to cells. Further, if necessary, it can be administered together with a pharmaceutically acceptable reagent for introduction into a cell, for example, a lipofectin reagent, a lipofectamine reagent, a DOTAP reagent and the like.
• a pharmaceutically acceptable reagent for introduction into a cell for example, a lipofectin reagent, a lipofectamine reagent, a DOTAP reagent and the like.
• a sequence consisting of all or part of the antisense strand of the DNA sequence can be prepared and used as a probe for detecting the gene of Annexin 5.
• the sequence of the sequence listing which is the polypeptide of the present invention
• an antibody can be induced to produce an antibody that specifically binds to the polypeptide.
• the antibody can be produced as a monoclonal antibody or as a polyclonal antibody using the polypeptide of the present invention as an antigen by an ordinary method.
• annexin 5 can be detected and measured by a well-known immunoassay.
• Mature female cells from Wistar Imamichi system rats pituitary release min (Endocrinology 107, 1095-1104, 1980) , 1 with 1 0 5 culture tray cells 9 6 Ueru per Ueru 0
• the cells were pre-cultured in DMEM medium containing 2% fetal bovine serum for 2 days. On the day of the experiment, the cells were pre-cultured in a culture solution without fetal calf serum for 3 hours, and gonadotropin-releasing hormone (GnRH:
• Gonadotropin hormone-producing cells of the pituitary gland were passaged tumor reduction to a tree stand the cell lines in L ⁇ ⁇ 2 cells (donor than the force Riforunia University of San Diego School of! 5. Mellon PhD), of Konfuruento Before reaching the state, the cells were separated, seeded with 200,000 cells in a 35 mm diameter dish, and cultured in a DMEM medium containing 10% fetal bovine serum.
• the LH iS promoter overnight region (1977 to +5) was introduced into pGL3 (Promega), a luciferase gene expression vector, and firefly 'Lucif was introduced.
• the physiological activity of Annexin 5 can be adjusted by providing a physiological activity inhibitor of Annexin 5 and a physiological activity inhibitor of Annexin 5 containing the physiological activity inhibitor as an active ingredient.
• the annexin 5 bioactivity inhibitor of the present invention is administered to an annexin 5-expressing ovary, adrenal gland, heart, lung, thyroid, or other organ to suppress annexin 5-related physiological functions. be able to.
• the secretion of sex hormones is suppressed, and the prevention or treatment of sex hormone-dependent diseases becomes possible.
• a DNA encoding a polypeptide which is a specific sequence of Annexin 5, an antisense oligonucleotide thereof, and an antibody which specifically binds to the polypeptide of the present invention it can regulate the expression of 5 genes, or detect and measure Annexin 5. These are useful for elucidating the physiological function of Annexin 5 and also for regulating the physiological activity of Annexin 5, and are useful for preventing and treating functional disorders related to Annexin 5.

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• 2002
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