Use of peptide

专利摘要:
Ligand polypeptide of the present invention has a CRH secretion effect, and thus, as a CRH secretion regulator, hypoaldosteroneism, hypocortisolemia, secondary or chronic adrenal insufficiency, Addison disease (boredom, nausea, pigmentation, hypogonadism, hair loss, hypotension) ), As well as useful for improving, preventing, and treating various diseases related to CRH secretion such as adrenal insufficiency and obesity, and also as analgesics.
公开号:KR20020058024A
申请号:KR1020027006387
申请日:2000-11-17
公开日:2002-07-12
发明作者:기따다찌에꼬；마쯔모또히로까즈；히누마슈지
申请人:다케다 야쿠힌 고교 가부시키가이샤；
IPC主号:

专利说明:

Use of Peptides {USE OF PEPTIDE}
[2] Most hormones and neurotransmitters regulate the function of living organisms through specific receptors on the cell membrane. Most of these receptors carry out intracellular signal transduction through activation of conjugated guanine nucleotide-binding proteins (hereinafter sometimes abbreviated as G proteins). These receptors are collectively referred to as G protein conjugated receptors or seven transmembrane receptors (7TMR) because they have a common structure with seven cell membrane penetration regions.
[3] Examples of such G protein conjugated receptor proteins include humanoid receptor proteins encoded by the phGR3 (or sometimes referred to as GPR10) gene [Genomics, Vol. 29, p. 335 (1995)], and corresponding rats. Type receptor protein UHR-1 [Biochem. Biophy. Res. Commun., Vol. 209, p. 606 (1995).
[4] In addition, PrRP [Nature, Vol. 393, pages 272-276 (1998)] is known as a bioactive peptide that functions as a ligand for phGR3 and UHR-1.
[5] PrRP has been observed to specifically release prolactin in the anterior lobe of the pituitary gland [Nature, 393, pages 272-276 (1998), Biochem. Biophy. Res. Commun., Vol. 259 pp. 321-324 (1999)], the action of PrRP in the brain is not clear. In addition, endogenous regulatory hormones that regulate the hypothalamic hormone, corticosteroid-releasing hormone (CRH), are unclear at this time.
[6] Summary of the Invention
[7] As a result of intensive studies to solve the above problems, the present inventors first produced two kinds of monoclonal antibodies specific for PrRP having different recognition sites, and produced a highly sensitive measuring system (Sandwich-EIA system) of PrRP ( WO 99-60112). In this study, the distribution of PrRP in rats was examined, and as reported [Biochemical and Biophysical Research Communications, Vol. 257, p. 264 (1999)], it was distributed in high concentrations in the hypothalamus and widely in the brain. The presence of distributed PrRP was confirmed. As a result of further studies, the inventors unexpectedly found that PrRP was detected in cerebrospinal fluid at a concentration of 5 to 6 times higher than the concentration in blood, thereby controlling the release of CRH, thereby completing the present invention.
[8] That is, the present invention relates to the following (1) to (10):
[9] (1) Ligands for G Protein Conjugated Receptor Proteins
[10] Corticosteroid-releasing hormone (CRH) secretion regulators containing peptides or amides or esters or salts thereof,
[11] (2) a polypeptide in which a ligand peptide or an amide or ester thereof or salt thereof for a G protein conjugated receptor protein contains an amino acid sequence identical or substantially identical to an amino acid sequence represented by SEQ ID NO: 44 or SEQ ID NO: 45 Or an amide or ester thereof or a salt thereof, a CRH secretion regulator according to the above (1),
[12] (3) the CRH secretion regulator according to the above (2), wherein the amino acid sequence represented by SEQ ID NO: 44 is an amino acid sequence represented by SEQ ID NO: 3, 18, or 32;
[13] (4) the CRH secretion regulator according to the above (2), wherein the amino acid sequence represented by SEQ ID NO: 45 is the amino acid sequence represented by SEQ ID NO: 6, 21, or 35;
[14] (5) the CRH secretion regulator according to the above (1), wherein the G protein conjugate receptor protein contains an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 46,
[15] (6) the CRH secretion regulator according to the above (1), which is a CRH secretion promoter;
[16] (7) the CRH secretion controlling agent according to the above (1), which is a drug for preventing or treating hypoaldosteroneism, hypocortisemia, secondary or chronic adrenal insufficiency, Addison's disease, adrenal insufficiency or obesity,
[17] (8) a CRH secretion modulator containing a compound or salt thereof that changes the binding of a G protein conjugated receptor protein to a ligand peptide to the G protein conjugated receptor protein,
[18] (9) the use of a ligand peptide or amide or ester thereof or salt thereof for a G protein conjugated receptor protein for the manufacture of a medicament having a CRH secretory action, and
[19] (10) A method for regulating CRH secretion, comprising administering a ligand peptide or an amide thereof or an ester thereof or a salt thereof to a G protein conjugated receptor protein to a mammal.
[1] The present invention relates to the use of bioactive peptides. More specifically, the present invention relates to an adrenal cortical stimulating hormone releasing hormone (CRH) secretion regulator containing a ligand polypeptide for a G protein conjugated receptor (hereinafter sometimes referred to as a receptor) protein.
[20] Fig. 1 shows the fluctuation of ACTH concentration in blood when PrRP-31 (the amide body of the peptide consisting of the amino acid sequence represented by SEQ ID NO: 32. The same below) is administered to the ventricles of rats. In the figure,-○-silver control (phosphate buffered saline),-●-silver PrRP-31 (1 nmol),-△-silver PrRP-31 (3 nmol),-■-silver PrRP-31 (10 nmol) Indicates. ACTH concentrations are expressed as measured values ± standard error. ** indicates a significant difference (n = 6-8) at a significance level of 1%, and * indicates a significant difference (n = 6-8) at a significance level of 5%.
[21] Fig. 2 shows the fluctuation of blood β-endorphin concentrations when PrRP-31 was administered to rat ventricle. In the figure,-○-silver control (phosphate buffered saline),-●-silver PrRP-31 (1 nmol),-△-silver PrRP-31 (3 nmol),-■-silver PrRP-31 (10 nmol) Indicates. [beta] -Endorphin concentration was expressed as measured value ± standard error. * Indicates that there is a significant difference (n = 6-8) with a significance level of 5%.
[22] Figure 3 shows the change in ACTH concentration in blood when anti-CRF IgG and normal rabbit IgG were administered in the jugular vein and PrRP-31 was administered intraratically after 30 minutes. In the figure,-○-is PrRP-31 (10 mmol) administration after control (phosphate buffered saline) administration,-△-is administration of PrRP-31 (10 mmol) after normal rabbit IgG administration,-●-is anti PrRP-31 (10 mmol) administration is shown after -CRF IgG administration.
[23] Fig. 4 shows the fluctuations in the β-endorphin concentrations in blood when anti-CRF IgG and normal rabbit IgG were administered in the jugular vein and PrRP-31 was administered intraratically after 30 minutes. In the figure,-○-is PrRP-31 (10 mmol) administration after control (phosphate buffered saline) administration,-△-is administration of PrRP-31 (10 mmol) after normal rabbit IgG administration,-●-is anti PrRP-31 (10 mmol) administration is shown after -CRF IgG administration. [beta] -Endorphin concentration was expressed as measured value ± standard error. * Indicates that there is a significant difference (n = 6-8) with a significance level of 5%.
[24] Figure 5 shows the variation of PrRP-31 concentration in cerebrospinal fluid at the time of immersion stress load.
[25] Figure 6 shows the variation of ACTH concentration in blood under immersion stress load. ACTH concentrations are expressed as measured values ± standard error. ** indicates that there is a significant difference (n = 7-8) with a significance level of 1%.
[26] Fig. 7 shows fluctuations in blood ACTH concentrations when α-helical CRF and saline were administered to the jugular vein, and PrRP-31 was administered intraventricularly 15 minutes later. In the figure,- -Indicates PrRP-31 (10 mmol) administration after physiological saline administration, and- -Shows administration of PrRP-31 (10 mmol) after -Helical CRH administration. ACTH concentrations are expressed as measured values ± standard error. ** indicates that there is a significant difference at the significance level of 1% (n = 7-8), and * indicates that there is a significant difference at the significance level of 5% (n = 7-8).
[27] In the present specification and the drawings, bases, amino acids, and the like are denoted by the abbreviations, based on the abbreviations according to [IUPAC-IUB Commission on Biochemical Nomenclature] or the common abbreviations in the art, and the examples are shown below. In addition, when an optical isomer exists about an amino acid, unless otherwise indicated, it shall show L body.
[28] DNA: deoxyribonucleic acid
[29] cDNA: complementary deoxyribonucleic acid
[30] A: Adenine
[31] T: Thymine
[32] G: guanine
[33] C: cytosine
[34] RNA: ribonucleic acid
[35] mRNA: messenger ribonucleic acid
[36] ATP: Adenosine Triphosphate
[37] EDTA: Ethylenediaminetetraacetic acid
[38] SDS: Sodium Dodecyl Sulfate
[39] EIA: Enzyme Immunoassay
[40] Gly or G: glycine
[41] Ala or A: Alanine
[42] Val or V: valine
[43] Leu or L: Leucine
[44] Ile or I: Isoleucine
[45] Ser or S: Serine
[46] Thr or T: threonine
[47] Cys or C: cysteine
[48] Met or M: methionine
[49] Glu or E: glutamic acid
[50] Asp or D: Aspartic Acid
[51] Lys or K: Lysine
[52] Arg or R: Arginine
[53] His or H: histidine
[54] Phe or F: Phenylalanine
[55] Tyr or Y: tyrosine
[56] Trp or W: Tryptophan
[57] Pro or P: Proline
[58] Asn or N: Asparagine
[59] Gln or Q: Glutamine
[60] pGlu: pyroglutamic acid
[61] Me: methyl
[62] Et: ethyl group
[63] Bu: Butyl
[64] Ph: phenyl group
[65] In addition, substituents, protecting groups, and reagents frequently used in the present specification are denoted by the following symbols.
[66] BHA: Benzhydrylamine
[67] pMBHA: p-methylbenzhydrylamine
[68] Tos: p-toluenesulfonyl
[69] CHO: Formyl
[70] HONB: N-hydroxy-5-norbornene-2,3-dicarboxyimide
[71] OcHex: cyclohexyl ester
[72] Bzl: Benzyl
[73] Cl ₂ -Bzl: dichlorobenzyl
[74] Bom: Benzyloxymethyl
[75] Z: benzyloxycarbonyl
[76] Br-Z: 2-bromobenzyloxycarbonyl
[77] Boc: t-butyloxycarbonyl
[78] DCM: Dichloromethane
[79] HOBt: 1-hydroxybenztriazole
[80] DCC: N, N'-dicyclohexylcarbodiimide
[81] TFA: trifluoroacetic acid
[82] DIEA: diisopropylethylamine
[83] Fmoc: N-9-fluorenylmethoxycarbonyl
[84] DNP: dinitrophenyl
[85] Bum: t-butoxymethyl
[86] Trt: Trityl
[87] As used herein, "substantially the same" means that the activity of the polypeptide (eg, binding activity of a ligand and a receptor) or the CRH secretion regulating activity (eg, CRH secretion promoting action, CRH secretion inhibiting action, etc.) of the polypeptide is substantially the same. It means. Therefore, the amino acid sequence "substantially identical" means that the activity of the polypeptide (e.g., binding activity of a ligand and a receptor), the CRH secretion regulating activity (e.g., CRH secretion promoting action, CRH secretion inhibiting action, etc.) As used herein, it refers to an amino acid sequence which may have variations as long as the same (not causing significant change) is maintained.
[88] In general, variations, substitutions, deletions, or additions of amino acids in amino acid sequences encoding polypeptides often do not result in significant (significant) changes in the physiological or chemical properties of the polypeptide. This is a well known fact. Examples of this substitution include those in which an amino acid is substituted with another amino acid having similar properties (characteristics), and in general, the substitution is carried out to the original polypeptide before substitution as the substitution is performed between amino acids having strong similarity in characteristics. The change in the characteristic is considered to be small.
[89] Amino acids are classified, for example, into the following classes based on the similarity of their properties as one criterion. (Iii) Examples of nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, methionine, and the like. (Ii) As a polar (neutral) amino acid, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, etc. are mentioned. (Iii) Arginine, lysine, histidine, etc. are mentioned as a (basic) amino acid which has a positive charge. (I) Aspartic acid, glutamic acid, etc. are mentioned as a (acidic) amino acid which has a negative charge.
[90] Substituents that are "substantially identical" in the amino acid sequence of interest in the present specification are often selected from other amino acids having similar characteristics in the class to which the amino acid belongs.
[91] Polypeptides obtained as a result of mutations in amino acid sequences such as substitutions, deletions or insertions that do not result in significant (significant) changes in the physiological or chemical properties of the original (unmodified) polypeptide. ) Is considered to be substantially the same as the original (unmodified) polypeptide that does not have such mutation, and the amino acid sequence of the variant polypeptide is substantially identical to the amino acid sequence of the original (unmodified) polypeptide. Considered to be the same.
[92] In addition, although the constituent amino acid in the polypeptide of this invention may be either D form or L form, L form is usually preferable unless there is particular notice.
[93] Polypeptides in the present invention are ligand peptides for G protein conjugated receptor proteins, or amides or esters thereof or salts thereof, ie ligand polypeptides capable of binding to G protein conjugated receptor proteins, or amides or esters thereof Salts, specifically, a polypeptide containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 44 or SEQ ID NO: 45 or an amide or ester thereof or salt thereof (hereinafter, simply Ligand polypeptide or the polypeptide of the present invention may be abbreviated).
[94] Here, the G protein conjugated receptor protein is a receptor protein having a common structure having seven cell membrane penetrating regions, most of which perform intracellular signal transduction through activation of the conjugated guanine nucleotide binding protein.
[95] The amino acid sequence represented by SEQ ID NO: 44 is preferably an amino acid sequence represented by SEQ ID NO: 3, 18 or 32, and particularly the amino acid sequence represented by the amino acid sequence represented by SEQ ID NO: 32. have.
[96] The amino acid sequence represented by SEQ ID NO: 45 is preferably an amino acid sequence represented by SEQ ID NO: 6, 21 or 35, and particularly the amino acid sequence represented by the amino acid sequence represented by SEQ ID NO: 35. have.
[97] Polypeptides of the invention include, but are not limited to, all tissues (eg, the pituitary gland, pancreas, brain, kidney, liver, gonad, thyroid gland) of warm-blooded animals (eg, humans, morphotes, rats, mice, pigs, sheep, cattle, monkeys, etc.) Gallbladder, bone marrow, adrenal gland, skin, muscle, lungs, digestive tract, blood vessels, heart, etc.) or a cell derived from a cell, and the like, and specifically, the amino acid sequence represented by SEQ ID NO: 44 or SEQ ID NO: 45, for example. Or an amino acid sequence that is substantially the same or substantially the same as an amino acid sequence represented by SEQ ID NO: 3, 18 or 32 or SEQ ID NO: 6, 21 or 35.
[98] For example, the polypeptide of the present invention may be an amino acid sequence represented by SEQ ID NO: 44 or SEQ ID NO: 45, preferably an amino acid sequence represented by SEQ ID NO: 3, 18 or 32 or SEQ ID NO: 6, 21 or 35. In addition to the containing polypeptide and the like, the amino acid sequence represented by SEQ ID NO: 44 or SEQ ID NO: 45, preferably 3, 18 or 32 or the amino acid sequence represented by SEQ ID NO: 6, 21 or 35 and about 50 to 99.9% ( Preferably 70 to 99.9%, more preferably 80 to 99.9%, even more preferably 90 to 99.9%), or the amino acid sequence having the homology, or represented by SEQ ID NO: 44 or SEQ ID NO: 45 Polypeptides having substantially the same activity as the polypeptide containing the amino acid sequence, preferably the amino acid sequence represented by SEQ ID NO: 3, 18 or 32 or SEQ ID NO: 6, 21 or 35, and the like. . Examples of this activity include activity of the ligand polypeptide, such as receptor binding activity, signal transduction activity, CRH secretion regulating activity (for example, CRH secretion promoting activity, CRH secretion inhibiting effect, etc.) of the polypeptide. The term "substantially homogeneous" indicates that the characteristics of the receptor binding activity and the polypeptide's CRH secretion regulating activity (for example, CRH secretion promoting action, CRH secretion inhibiting effect, etc.) are homogeneous. Therefore, the strength or weakness which is not remarkable about this receptor binding activity may be observed, and the molecular weight of this ligand polypeptide may differ. In some cases, differences in non-essential amino acid sequences of the peptides may be observed due to differences in species from which substantially identical peptides derived from the same genus of humans or warm-blooded animals are derived. These peptides due to differences in phases are also included.
[99] The ligand polypeptides of the present invention, their preparation and use are described in more detail below.
[100] Specific examples of the ligand polypeptides of the present invention include polypeptides derived from rats, cows, humans or mice containing the amino acid sequence represented by SEQ ID NO: 44 or SEQ ID NO: 45 (see also SEQ ID NO: 44). The third Xaa is Thr or Ala, the fifth Xaa is Arg or Gln, the tenth Xaa is Ile or Thr, the twenty-first Xaa is Thr or Ala, the twenty-second Xaa is Gly or Ser In SEQ ID NO: 45, the tenth Xaa represents Thr or Ala, the eleventh Xaa represents Gly or Ser).
[101] In addition, the ligand polypeptide of the present invention,
[102] (Iii) one or more than 15 amino acids in the amino acid sequence represented by SEQ ID NO: 44, preferably one or more than 10 or less, more preferably one or more than five or less amino acids are substituted with another amino acid sequence Amino Acid Sequence,
[103] (Ii) an amino acid sequence in which at least one amino acid sequence of amino acid sequence represented by SEQ ID NO: 44 is deleted, preferably at least one amino acid at most 10, more preferably at least one amino acid at least 5 amino acids,
[104] (Iii) an amino acid sequence having one or more than 15 amino acids, preferably one or more than 10 amino acids, more preferably one or more than five amino acids added to the amino acid sequence represented by SEQ ID NO: 44 (inserted) Sequence, and
[105] (Iii) an amino acid sequence in which the constituent amino acid (particularly its side chain) in the polypeptide of (iii), (ii) or (iii) is modified;
[106] Also included are polypeptides containing amides thereof or esters thereof or salts thereof.
[107] In addition, the ligand polypeptide of the present invention,
[108] (Iii) an amino acid sequence in which at least one amino acid sequence of the amino acid sequence represented by SEQ ID NO: 45 is substituted with another amino acid, preferably at least one amino acid at most five,
[109] (Iii) an amino acid sequence in which at least one amino acid sequence of amino acid sequence represented by SEQ ID NO: 45 is deleted, preferably at least one amino acid and at most five amino acids are deleted;
[110] (Iii) an (inserted) amino acid sequence in which at least one amino acid sequence, preferably at least one amino acid, and at most five amino acids are added to the amino acid sequence represented by SEQ ID NO: 45, and
[111] (Iii) an amino acid sequence in which the constituent amino acid (particularly its side chain) in the polypeptide of (iii), (iii) or (iii) is modified;
[112] Also included are polypeptides containing amides thereof or esters thereof or salts thereof.
[113] The ligand polypeptide of the present invention is a stable ligand polypeptide against the heat or protease or the physiology of the ligand polypeptide by intentionally or accidentally performing the substitution, deletion, addition, modification, etc. of (i) to (iii) in the amino acid sequence. It can be mutated (converted) into highly active ligand polypeptide with enhanced activity. These ligand polypeptides or amides, esters or salts thereof in the present invention also include these variant ligand polypeptides.
[114] In the present specification, the notation of a peptide is described as the N terminus (amino terminus) and the C terminus (carboxy terminus) at the right end according to the convention.
[115] As an example of modification in the constituent amino acid in the polypeptide of this invention, the N single side of Gln was cut | disconnected in vivo, and this Gln was converted into pyroglutamic acid, etc. are mentioned, for example.
[116] Polypeptide, for example SEQ ID NO: in the present invention: 44 or SEQ ID NO: Polypeptide represented by 45, C-terminal carboxyl group of the α- amino acid residue is normally carboxyl group (-COOH) or carboxylate (-COO ^-), but, C This carboxyl group of the terminal amino acid residue may be an amide (-CONH ₂ ) or an ester (-COOR). Examples of R of this ester represented by -COOR include C _1-6 alkyl groups such as methyl, ethyl, n-propyl, isopropyl or n-butyl, C _3-8 cycloalkyl groups such as cyclopentyl and cyclohexyl, phenyl, α- naphthyl such as C _6-12 aryl group, a benzyl, phenethyl, phenyl C _1-2 alkyl group, a benzhydryl such as diphenyl -C _1-2 alkyl, and α- naphthylmethyl, etc. of α- Not only C _7-14 aralkyl groups such as naphthyl-C _1-2 alkyl, but also pivaloyloxymethyl groups which are commonly used as oral esters.
[117] In addition, when the polypeptide of the present invention, for example, the polypeptide represented by SEQ ID NO: 44 or SEQ ID NO: 45 has a carboxyl group or carboxylate in addition to the C terminus, the polypeptide in which these groups are amidated or esterified is also the present invention. It is included in the polypeptide of the. As ester in this case, it is the same as the ester of the C-terminal amino acid residue mentioned above, for example.
[118] The ligand polypeptide of the present invention is particularly preferably a peptide in which a carboxyl group of the C-terminal amino acid residue is amidated. A polypeptide having an amino acid sequence represented by SEQ ID NO: 44 or SEQ ID NO: 45, particularly a polypeptide having an amino acid sequence represented by SEQ ID NO: 3, 6, 18, 21, 32 or 35 (particularly SEQ ID NO: 32 or 35) Polypeptides in which the carboxyl group of the C-terminal amino acid residue of the polypeptide having an amino acid sequence represented by
[119] As the salt of the polypeptide of the present invention, a salt with a physiologically acceptable base (such as an alkali metal) or an acid (organic acid, inorganic acid) is used, and a physiologically acceptable acid addition salt is particularly preferable. Such salts include, for example, salts with inorganic acids (such as hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid), or organic acids (such as acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid). And salts with methanesulfonic acid, benzenesulfonic acid).
[120] The ligand polypeptide of the present invention may be prepared by (i) purifying the polypeptide from tissues or cells of humans or warm-blooded animals, or (ii) in accordance with known polypeptide synthesis methods. Moreover, (iii) it can also manufacture by the method of culturing the transformant containing the DNA which codes this polypeptide (it mentions later).
[121] (Iii) When the ligand polypeptide is prepared from tissues or cells of humans or warm blooded animals, the tissues or cells of humans or warm blooded animals are homogenized and extracted with an acid or the like, and the extract is subjected to reverse phase chromatography, ion exchange chromatography, Purification and isolation can be carried out by combining chromatography such as affinity chromatography.
[122] (Ii) This ligand polypeptide can also be produced according to the synthesis method of a polypeptide known per se. As a method for synthesizing the peptide, any of a solid phase synthesis method and a liquid phase synthesis method may be used. In other words, the desired peptide can be prepared by condensing a partial peptide or amino acid and the remaining moiety which can constitute a ligand polypeptide, and leaving the protecting group when the product has a protecting group. As a well-known condensation method in this case, and the detachment method of a protecting group, the method of following (1)-(5) is mentioned, for example.
[123] ① [M. Bodanszky and M.A. Ondetti: Peptide Synthesis, Interscience Publishers, New York (1966)]
[124] ② [Schroeder and Luebke: The Peptide, Academic Press, New York (1965)]
[125] ③ [Nobuo Izumiya et al .: Fundamentals and Experiments in Peptide Synthesis, Maruzen, (1975)]
[126] ④ [Haru'aki Yajima and Shunpei Sakakibara: Biochemical Experiment Series 1:
[127] Protein Chemistry Ⅳ, 205, (1977)]
[128] ⑤ [Supervised by Haru'aki Yajima, Development of Drugs-Continued, Vol. 14, Peptide Synthesis, Hirokawa Shoten]
[129] Specifically as a synthesis method of this ligand polypeptide of said (ii), the following method etc. are mentioned, for example.
[130] In order to synthesize | combine the amide body of this ligand polypeptide, the peptide synthesis resin suitable for amide formation may be used. Such resins include, for example, chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxy Methylmethylphenylacetamide methyl resin, polyacrylamide resin, 4- (2 ', 4'-dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4- (2', 4'-dimethoxyphenyl-Fmoc aminoethyl) Phenoxy resins. Using such a resin, amino acids protected with an appropriate protecting group known by the α-amino group and the side chain functional group are condensed on this resin with the sequence of the desired peptide according to various condensation methods known per se. At the end of the reaction, the peptide can be cleaved from the resin, and various protecting groups can be removed to obtain the desired polypeptide.
[131] In order to condense the above-mentioned protected amino acids, various activating reagents which can be used for peptide synthesis can be used, but carbodiimides are particularly preferred. As carbodiimides, DCC, N, N'-diisopropylcarr are preferred. Bodyimide, N-ethyl-N '-(3-dimethylaminoprolyl) carbodiimide, and the like.
[132] Activation by these may include the addition of a protected amino acid with a racemization inhibitor (eg HOBt) directly to the resin, or after activation of a previously protected amino acid as a symmetric acid anhydride or HOBt ester or HOOBt ester. Can be. What is necessary is just to select suitably from the well-known solvent which can be used for peptide condensation reaction as a solvent used for activation of a protected amino acid and condensation with resin. Examples of the solvent include acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide and N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform and trifluoroethanol. Alcohols, sulfoxides such as dimethyl sulfoxide, tertiary amines such as pyridine, ethers such as dioxane and tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate Or a suitable mixture thereof. What is necessary is just to select suitably the reaction temperature in this condensation reaction from the temperature range which can be used for a well-known peptide bond formation reaction, and the range of about -20 degreeC-50 degreeC is mentioned normally. This activated amino acid derivative is usually used in 1.5 to 4 times excess. Condensation degree can be confirmed using a well-known ninhydrin reaction, As a result, when condensation is inadequate, sufficient condensation can be performed by repeating a condensation reaction, without removing a protecting group. When sufficient condensation cannot be obtained even if the reaction is repeated, acetylation of the unreacted amino acid with acetic anhydride or acetylimidazole may be performed so as not to affect subsequent reactions.
[133] Examples of protecting groups for amino groups of amino acids that are used as starting materials for the peptide synthesis include Z, Boc, tert-amyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br- Z, adamantyloxycarbonyl, trifluoroacetyl, phthalyl, formyl, 2-nitrophenylsulphenyl, diphenylphosphinothioyl, Fmoc, and the like. Examples of the protecting group for the carboxyl group include, for example, the aforementioned C _1-6 alkyl group, C _3-8 cycloalkyl group, C _7-14 aralkyl group, as well as 2-adamantyl, 4-nitrobenzyl, 4-methoxybenzyl, 4- Chlorobenzyl, phenacyl group and benzyloxycarbonyl hydrazide, tert-butoxycarbonyl hydrazide, trityl hydrazide and the like.
[134] The hydroxyl groups of serine and threonine can be protected, for example, by esterification or etherification. Suitable groups for this esterification include, for example, lower (C _1-6 ) alkanoyl groups such as acetyl groups, aroyl groups such as benzoyl groups, groups derived from carbonic acid such as benzyloxycarbonyl groups and ethoxycarbonyl groups. Moreover, as a group suitable for etherification, a benzyl group, tetrahydropyranyl group, tert- butyl group etc. are mentioned, for example.
[135] A protective group of tyrosine of a phenolic hydroxyl group is, for example, there may be mentioned Bzl, Cl ₂ -Bzl, such as 2-nitrobenzyl, Br-Z, tert- butyl.
[136] Examples of the protecting group for histidine imidazole include Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, and Fmoc.
[137] Ligand polypeptides or amides or esters thereof of the present invention, for example, exhibit the same CRH secretion regulating activity as polypeptides containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 44 or SEQ ID NO: 45. Any peptide having a mutation in the amino acid sequence may be used as long as it has it. As such a peptide, the peptide which has the amino acid sequence by which one or more 20 amino acids were deleted from the peptide which has the amino acid sequence shown by SEQ ID NO: 44, for example is mentioned. Specifically, for example, (a) a peptide having the second to thirty-first amino acid sequence of the amino acid sequence represented by SEQ ID NO: 44, (b) the third to the third amino acid sequence of the amino acid sequence represented by SEQ ID NO: 44 A peptide having the 31st amino acid sequence, (c) a peptide having the fourth to 31st amino acid sequences of the amino acid sequence represented by SEQ ID NO: 44, (d) a peptide of the amino acid sequence represented by SEQ ID NO: 44 A peptide having the fifth to 31st amino acid sequences, (e) a peptide having the sixth to 31st amino acid sequences of the amino acid sequence represented by SEQ ID NO: 44, (f) represented by SEQ ID NO: 44 A peptide having an amino acid sequence of seventh to thirty-first amino acid sequence, (g) a peptide having an amino acid sequence of eighth to 31st amino acid sequence of amino acid sequence represented by SEQ ID NO: 44, (h) a sequence Arc: a peptide having the 9th to 31st amino acid sequences of the amino acid sequence represented by 44, (i) a peptide having the 10th to 31st amino acid sequences of the amino acid sequence represented by SEQ ID NO: 44, (j) a peptide having the 11th to 31st amino acid sequence of the amino acid sequence represented by SEQ ID NO: 44, (k) the 12th to 31st amino acid sequence of the amino acid sequence represented by SEQ ID NO: 44 A peptide having the amino acid sequence represented by SEQ ID NO: 44, (l) a peptide having the 13th to 31st amino acid sequence represented by SEQ ID NO: 44, (m) the 14th to 31st amino acid sequence represented by SEQ ID NO: 44 A peptide having the amino acid sequence of the first amino acid, (n) a peptide having the amino acid sequence of the 15th to 31st amino acids of the amino acid sequence represented by SEQ ID NO: 44, and (o) an amino acid represented by SEQ ID NO: 44 (P) a peptide having the amino acid sequence of the 16th to 31st amino acids of the nucleotide sequence, (p) a peptide having the 17th to 31st amino acid sequence of the amino acid sequence of SEQ ID NO: 44, (q) SEQ ID NO: A peptide having the 18th to 31st amino acid sequence of the amino acid sequence represented by 44, (r) a peptide having the 19th to 31st amino acid sequence of the amino acid sequence represented by SEQ ID NO: 44, (s A peptide having the amino acid sequence of the 20th to 31st amino acids of the amino acid sequence represented by SEQ ID NO: 44, (t) having the 21st to 31st amino acid sequence of the amino acid sequence represented by SEQ ID NO: 44 Peptides and the like are preferred.
[138] As for the amino acid sequence represented by SEQ ID NO: 44 for SEQ ID NO: 3, 18, or 32, which is more preferable as the amino acid sequence represented by SEQ ID NO: 44, the amino acid sequence may have a mutation.
[139] Moreover, the peptide which has the amino acid sequence by which one or more 10 amino acids were deleted from the peptide which has the amino acid sequence shown by SEQ ID NO: 45 is mentioned. Specifically, for example, (a) a peptide having the second to twentieth amino acid sequence of the amino acid sequence represented by SEQ ID NO: 45, (b) the third to the third amino acid sequence of the amino acid sequence represented by SEQ ID NO: 45 A peptide having the 20th amino acid sequence, (c) a peptide having the fourth to 20th amino acid sequences of the amino acid sequence represented by SEQ ID NO: 45, (d) a peptide of the amino acid sequence represented by SEQ ID NO: 45 A peptide having the fifth to twentieth amino acid sequences, (e) a peptide having the sixth to twentieth amino acid sequences of the amino acid sequence represented by SEQ ID NO: 45, and (f) the SEQ ID NO: 45 A peptide having an amino acid sequence of the seventh to twentieth amino acids of the amino acid sequence, (g) a peptide having an amino acid sequence of the eighth to twentieth amino acids of the amino acid sequence represented by SEQ ID NO: 45, and (h) a sequence Arc: a peptide having the 9th to 20th amino acid sequence of the amino acid sequence represented by 45, (i) a peptide having the 10th to 20th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 45, (j) Preferred are peptides having the 11th to 20th amino acid sequences of the amino acid sequence represented by SEQ ID NO: 45.
[140] As the ligand polypeptide of the present invention, a rat whole brain or a bovine hypothalamus-derived polypeptide containing an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 15 may be cited. As illustrated with respect to the amino acid sequence represented by the number: 45, the amino acid sequence may have a mutation. These polypeptides may also be mentioned as precursors of polypeptides having an amino acid sequence represented by SEQ ID NO: 44 or SEQ ID NO: 45.
[141] In addition, the ligand polypeptide of the present invention, in addition to the above, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: And polypeptides having an amino acid sequence represented by SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 36 or SEQ ID NO: 37, and the like. As illustrated, the amino acid sequence may have a mutation.
[142] As for the amino acid sequence represented by SEQ ID NO: 45 for SEQ ID NO: 6, 21, or 35, which is more preferable as the amino acid sequence represented by SEQ ID NO: 45, the amino acid sequence may have a mutation.
[143] Ligand polypeptides of the invention may also be fusion proteins with other proteins (eg, known proteins whose functions or properties are well known).
[144] The DNA encoding the ligand polypeptide of the present invention may contain a nucleotide sequence encoding a polypeptide containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 44 or SEQ ID NO: 45 in the present invention. Any may be sufficient. The genomic DNA, genomic DNA library, cDNA derived from tissue or cells, cDNA library derived from tissue or cells, or synthetic DNA may be used. The vector used for a library may be any of bacteriophage, plasmid, cosmid, and phagemid. In addition, amplified by direct RT-PCR (reverse transcription PCR) using an RNA fraction prepared from tissues and cells.
[145] More specifically, DNA encoding the polypeptide derived from whole rat brain or bovine hypothalamus containing the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 15 includes DNA having a nucleotide sequence represented by SEQ ID NO: 2, or the like. Used.
[146] Here, in SEQ ID NO: 2, the 129th R represents G or A, and the 179th and 240th Y represent C or T. When the 179th Y is C, the amino acid sequence represented by SEQ ID NO: 1 is encoded. When the 179th Y is T, the amino acid sequence represented by SEQ ID NO: 15 is encoded.
[147] In addition, the DNA encoding the bovine-derived polypeptide containing the amino acid sequence represented by SEQ ID NO: 3, 4, 5, 6, 7 or 8 is represented by SEQ ID NO: 9, 10, 11, 12, 13 or 14, respectively. DNA having a nucleotide sequence to be used is used. Here, the 63rd R of SEQ ID NOs: 9, 10, 11 and the 30th R of SEQ ID NOs: 12, 13, 14 represent G or A.
[148] Moreover, as DNA which codes the rat derived polypeptide represented by SEQ ID NO: 16, 18, 19, 20, 21, 22, or 23, it is represented by SEQ ID NO: 17, 24, 25, 26, 27, 28 or 29, respectively. DNA having a nucleotide sequence is used.
[149] In addition, as DNA encoding the human-derived polypeptide represented by SEQ ID NO: 30, 32, 33, 34, 35, 36 or 37, respectively, represented by SEQ ID NO: 31, 38, 39, 40, 41, 42 or 43 DNA having a nucleotide sequence is used.
[150] In the present invention, a small polypeptide containing an amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 15, a rat polypeptide containing an amino acid sequence represented by SEQ ID NO: 16, or represented by SEQ ID NO: 30 In the DNA encoding the human-type polypeptide containing the amino acid sequence, for example, 6 or more and 90 or less (preferably 6 or more and 60 or less, more preferably 9 or more and 30 or less, even more preferably 12 or more 30) DNA fragments containing partial base sequences of up to) are preferably used as DNA detection probes.
[151] (Iii) The polypeptide of the present invention can also be produced by a method of culturing a transformant containing a DNA encoding the polypeptide as described below.
[152] Cloning (cloning) of the DNA encoding the polypeptide of the present invention can be performed according to the following method. That is, (1) a DNA having a partial base sequence of this polypeptide is synthesized, and this is used as a primer to amplify DNA encoding the polypeptide completely by PCR method, or (2) cDNA or genomic DNA, or DNA thereof. The DNA library obtained by inserting the fragment into a suitable vector may be selected by hybridization using, for example, a labeled DNA fragment or synthetic DNA having a partial or entire region of the ligand polypeptide. This hybridization method may be performed according to the method described in, for example, Molecular Cloning (2nd ed .; J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). In addition, when using a commercially available thing as a DNA library, what is necessary is just to carry out according to the method described in the attached instruction manual.
[153] DNA sequence conversion is performed using ODA-LA using PCR or a known kit such as Mutan ^TM- super Express Km (Takara Shuzo Co., Ltd.), Mutan ^TM -K (Takara Shuzo Co., Ltd.), or the like. It can carry out according to well-known methods, such as the PCR method, the Gapped duplex method, the Kunkel method, or the like, or a method similar thereto.
[154] The DNA encoding the cloned polypeptide may be used as it is, or may be used after cleavage with restriction enzymes or after addition of linker DNA. This DNA may be used to provide ATG as a translation initiation codon at its 5 'end. Moreover, you may have TAA, TGA, or TAG as a translation termination codon on the 3 'terminal side. These translation initiation codons or translation end codons can be added using a suitable synthetic DNA adapter.
[155] An expression vector containing DNA having a nucleotide sequence encoding the polypeptide is, for example, (a) cleaving a DNA fragment of interest from DNA containing a DNA encoding a polypeptide of the present invention, and (b) It can be prepared by ligation downstream of a promoter sequence in a known suitable expression vector.
[156] Examples of the vector include plasmids derived from E. coli (e.g. pBR322, pBR325, pUC12, pUC13), plasmids derived from Bacillus subtilis (e.g. pUB110, pTP5, pC194), yeast derived plasmids (e.g. pSH19, pSH15), bacteriophage such as lambda phage, animal viruses such as retrovirus, vaccinia virus, baculovirus and the like. The promoter may be any promoter that functions appropriately in a host used for expression of a gene encoding a polypeptide of interest.
[157] When the host at the time of transformation is Escherichia spp, trp promoter, lac promoter, recA promoter, λPL promoter, lpp promoter and the like are preferred, and when the host is Bacillus sp., The SPOl promoter, SPO2 promoter, penP promoter Etc., and when the host is a yeast, a PHO5 promoter, a PGK promoter, a GAP promoter, an ADH promoter, and the like are preferable. When the host is an animal cell, a SV40-derived promoter, a retrovirus promoter, a metallothionein promoter, a heat shock promoter, a cytomegalovirus promoter, an SPα promoter, and the like are preferably mentioned. In addition, it is preferable to use an enhancer to efficiently express the gene encoding the target polypeptide.
[158] Moreover, the signal sequence suitable for a host is added to the N terminal side of a polypeptide as needed. If the host is Escherichia spp., The alkaline phosphatase / signal sequence, OmpA / signal sequence, etc., if the host is Bacillus sp., The α-amylase / signal sequence, subtilisin signal sequence, the host is yeast In the case of MFα-signal sequence, invertase-signal sequence, and the host is an animal cell, insulin-signal sequence, α-interferon signal sequence, antibody molecule / signal sequence, etc. can be used, respectively. Thus, a transformant is prepared using a vector containing DNA encoding the constructed polypeptide.
[159] Examples of the host for transformation include known Escherichia bacteria, Bacillus bacteria, yeasts, insects and animal cells.
[160] As a specific example of the Escherichia genus, Escherichia coli K12.DH1 [Proc. Natl. Acad. Sci., USA, Vol. 60, p. 160 (1968)], JM103 [Nucleic Acids Research, vol. 9, p. 309 (1981)], JA221 [Journal of Molecular Biology, vol. 120, p. 517 (1978) ), HB101 [Journal of Molecular Biology, Vol. 41, p. 459 (1969)], C600 [Genetics, Vol. 39, p. 440 (1954)].
[161] Specific examples of Bacillus bacteria include Bacillus subtilis MI114 [Gene, Vol. 24, p. 255 (1983)], 207-21 [Journal of Biochemistry, vol. 95, p. 87 (1984)], and the like. Can be.
[162] As the yeast, for example, a saccharide in MRS three Levy Jia (Saccharomyces cerevisiae) AH22, AH22R ^-, and the like, NA87-11A, DKD-5D, 20B -12.
[163] Examples of insects include silkworm larvae and the like (Maeda et al., Nature, Vol. 315, p. 592 (1985)).
[164] Examples of the animal cells include monkey cells COS-7, Vero, Chinese hamster cell CHO, DHFR gene deletion Chinese hamster cell CHO (dhfr ^- CHO cells), mouse L cells, mouse myeloma cells, human FL cells and the like. .
[165] To transform Escherichia spp., For example, [Proc. Natl. Acad. Sci. USA, Vol. 69, page 2110 (1972)] or Gene, Vol. 17, page 107 (1982), or the like.
[166] In order to transform Bacillus bacteria, for example, it can be carried out according to the method described in [Molecular & General Genetics, Vol. 168, p. 111 (1979)].
[167] To transform yeast, for example, Proc. Natl. Acad. Sci. USA, Vol. 75, p. 1929 (1978)] or the like.
[168] In order to transform an insect cell, it may carry out according to the method described in [Bio / Technology, Vol. 6, p. 47 (1988)] etc., for example.
[169] In order to transform an animal cell, for example, it can be carried out according to the method described in [Virology, Vol. 52, p. 456 (1973)].
[170] In this way, a transformant transformed with an expression vector containing DNA encoding the polypeptide can be obtained.
[171] As a medium when the host cultures transformants of Escherichia spp. And Bacillus spp., A liquid medium is preferable, and the medium is prepared so as to contain a carbon source, nitrogen source, minerals and the like necessary for the growth of the transformant. Examples of carbon sources include glucose, dextrin, soluble starch, sucrose, and the like. Examples of nitrogen sources include ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean meal, potato extract, and the like. Examples of the mineral include calcium chloride, sodium dihydrogen phosphate, magnesium chloride and the like. In this medium, yeast extracts, vitamins, growth promoting factors and the like may be added as necessary. Although the pH of this medium may be to some extent as long as it is a pH at which a transformant grows, the pH is usually about 5 to 8 is preferable.
[172] As a medium for culturing Escherichia spp., For example, M9 medium containing glucose and kazamino acid (Miller, Journal of Experiments in Molecular Genetics, p. 431, Cold Spring Harbor Laboratory, New York 1972) is preferable. In order to operate a promoter efficiently as needed, you may incubate, for example by adding a chemical | medical agent, such as 3 (beta) -indolyl acrylic acid. When the host is Escherichia spp., The culture is usually performed at about 15 ° C. to 43 ° C. for about 3 to 24 hours, and aeration or agitation may be performed if necessary.
[173] When the host is Bacillus bacteria, the culture is usually carried out at about 30 ° C to 40 ° C for about 6 to 24 hours, and aeration or agitation may be carried out as necessary.
[174] As a medium for culturing a transformant whose host is a yeast, for example, the Burgholder minimal medium [Bostian, K.L. et al., Proc. Natl. Acad. Sci. USA, Vol. 77, p. 4505 (1980)] or SD medium containing 0.5% kazamino acid [Bitter, G.A. Et al., Proc. Natl. Acad. Sci. USA, Vol. 81, pp. 5330 (1984)].
[175] The pH of the medium is preferably adjusted to about 5-8. Cultivation is normally performed at about 20 degreeC-35 degreeC for about 24 to 72 hours, and can also be vented or stirred as needed.
[176] When culturing a transformant whose host is an insect, the medium includes 10% bovine serum inactivated in Grace's Insect Medium (Grace, TCC, Nature, Vol. 195, page 788 (1962)). What suitably added the additive etc. are used. The pH of the medium is preferably adjusted to about 6.2 to 6.4. Cultivation is normally performed at about 27 degreeC for about 3-5 days, and it can also ventilate and stir as needed.
[177] When culturing a transformant in which the host is an animal cell, the medium is, for example, MEM medium containing about 5-20% of fetal bovine serum [Science, Vol. 122, p. 501 (1952)], DMEM medium [Virology, Volume 8, page 396 (1959)], RPMI 1640 Badge [The Journal of the American Medical Association Volume 199, page 519 (1967)], Badge 199 [Proceeding of the Society for the Biological Medicine, Volume 73] , Page 1 (1950)]. It is preferable that pH is about 6-8. Cultivation is normally performed at about 30 degreeC-40 degreeC for about 15 to 60 hours, and can also be ventilated or stirred as needed.
[178] In order to isolate and purify a polypeptide from the culture (culture medium and cultured cells or cultured cells), for example, the following method may be performed.
[179] When the polypeptide accumulates in the cultured cells or cultured cells, the cells or cells are collected after the culture and suspended in a suitable buffer, and then the cells or cells are known by sonication, lysozyme treatment and / or lyolysis. The target polypeptide can be obtained as an extract by disrupting the cells and then subjecting to known centrifugation or filtration. In this buffer, a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 (registered trademark of Wako Pure Chemical Industries, Ltd., may be omitted hereinafter) may be used as necessary. do.
[180] On the other hand, when the polypeptide is secreted in the culture, after culturing, the cultured cells or cultured cells and the culture supernatant can be separated by a known method to obtain the culture supernatant.
[181] Purification of the polypeptide contained in the resulting culture supernatant or extract can be carried out by appropriately combining separation and purification methods known per se. These known separation and purification methods include mainly (1) methods using solubility such as salting out and solvent precipitation, and (2) mainly molecular weights such as dialysis, ultrafiltration, gel filtration and SDS-polyacrylamide gel electrophoresis. Method of using the difference of (3) method of using the difference of charge such as ion exchange chromatography, (4) method of using specific affinity such as affinity chromatography, (5) hydrophobicity such as reversed phase high performance liquid chromatography The method of using a difference, (6) The method of using a difference of isoelectric points, such as an isoelectric point electrophoresis method and chromatographic focusing, etc. are mentioned.
[182] When the polypeptide is obtained in the free form, it can be converted into a salt by a method known per se or a method equivalent thereto, and when the polypeptide is obtained as a salt, it can be converted into a vitreous or another salt by a method known per se or a method equivalent thereto.
[183] In addition, prior to or after purification of the polypeptide, the polypeptide may be optionally modified or partially removed from the polypeptide by applying an appropriate protein modifying enzyme according to a method known thereto. Examples of this protein modifying enzyme include trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like. The activity of the mutant polypeptide thus obtained can be measured by binding experiments with receptors or by enzyme immunoassay using specific antibodies.
[184] Ligand polypeptides of the present invention have a regulating action of CRH secretion, that is, promoting and inhibiting CRH secretion. That is, since the ligand polypeptide of the present invention has a promoting action of CRH secretion, as can be seen in the examples described below, it can be used for prophylaxis and treatment of various diseases related to CRH secretion failure (medicine and animal medicine). . On the other hand, since the ligand polypeptide of the present invention has a strong affinity with the receptor protein, when the dosage is increased, desensitization occurs to the CRH secretion, which also has a function of inhibiting the CRH secretion. In this case, it can be used for the prophylaxis and treatment of various diseases related to CRH oversecretion.
[185] Thus, the ligand polypeptide of the present invention is a CRH secretion promoter, hypoaldosteroneism, hypocortisolemia, secondary and chronic adrenal insufficiency, Addison's disease (boredom, nausea, pigmentation, hypogonadism, hair loss, hypotension), adrenal insufficiency and It is useful not only as an agent for improving, preventing and treating various diseases related to CRH secretion such as obesity, but also as an analgesic drug.
[186] In addition, the ligand polypeptide of the present invention is a CRH secretion inhibitor, Cushing's disease, Cushing's syndrome (central obesity, full moon eye, hypertension, red purple skin progenitor, hairy), adrenal cortex stimulating hormone (ACTH) production pituitary tumor, CRH production Tumors, aldosterone producing tumors, aldosteroneism (primary, secondary), primary cortisol resistance, congenital adrenal deficiency, antistress, congestion, anorexia nervosa, insomnia, gastric and duodenal ulcers, and irritable bowel syndrome It is useful as a drug for improving, preventing and treating diseases.
[187] The ligand polypeptide of the present invention is also useful as a test agent for investigating CRH secretion function.
[188] What is necessary is just to formulate according to a conventional method, when using the polypeptide of this invention for the above-mentioned medicine or animal medicine. Injections such as sterile solutions or suspensions, orally or as water or other pharmaceutically acceptable liquids, such as sugar-coated tablets, capsules, elixirs, microcapsules, etc., if necessary. It may be used parenterally in the form or as a nasal agent. For example, the polypeptide or salt thereof may be prepared by blending with a physiologically acceptable carrier, flavor, excipient, vehicle, preservative, stabilizer, binder, and the like into the unit dosage form required for generally accepted pharmaceutical practice. The amount of active ingredient in these formulations is such that adequate dosages in the ranges indicated are obtained.
[189] As additives that can be blended into tablets, capsules and the like, for example, gelatin, corn starch, tragant gum, binders such as gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid and the like, swelling agents, magnesium stearate and the like Such lubricants, sweeteners as sucrose, lactose or saccharin, flavoring agents such as peppermint, acamonoyl or cherry, and the like are used. When the production unit form is a capsule, the above type of material may further contain a liquid carrier such as oil or fat. Injectable sterile compositions may be prescribed in accordance with conventional formulation practice, such as dissolving or suspending active substances, sesame oil, naturally occurring vegetable oils such as palm oil, etc. in a vehicle such as water for injection.
[190] Aqueous injectable solutions include, for example, isotonic solutions containing physiological saline, glucose or other adjuvants (e.g., D-sorbitol, D-mannitol, sodium chloride, and the like), and the like. , etc.), polyalcohol may be used in combination as (e. g., propylene glycol, polyethylene glycol), nonionic surfactants (e.g., polysorbate ^{80 (TM), HCO-50} ). Sesame oil, soybean oil, etc. are mentioned as an oil-based liquid, You may use together with benzyl benzoate, a benzyl alcohol, etc. as a dissolution supplement preparation. In addition, buffers (e.g., phosphate buffers, sodium acetate buffers), analgesics (e.g. benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (e.g. human serum albumin, polyethylene glycol, etc.), preservatives (e.g. benzyl alcohol, phenol) Etc.), antioxidant, etc. may be mix | blended. The prepared injection solution is usually aseptically filled into a suitable ampoule.
[191] Since the preparation thus obtained is safe and low toxicity, it can be administered, for example, to mammals (eg, humans, mice, rats, morphotes, rabbits, sheep, pigs, cattle, cats, dogs, monkeys, chimpanzees, etc.).
[192] The dosage of the CRH secretion modulator containing the polypeptide of the present invention varies depending on the symptoms and the like. However, when orally administered, the polypeptide of the present invention is usually used once per patient for hypoaldosteroneism (for 60 kg of body weight). 0.1 mg-100 mg, Preferably it is about 1.0 mg-50 mg, More preferably, about 1.0 mg-20 mg. In the case of parenteral administration, this single dose varies depending on the administration target, symptoms, administration method, and the like, but for example, in the form of an injection, a patient with hypoaldosteroneism (for 60 kg body weight) Usually, about 0.01 mg to 30 mg, preferably about 0.1 mg to 20 mg, and more preferably about 0.1 mg to 10 mg may be administered by intravenous injection. For other animals, the amount converted to 60 kg body weight may be administered.
[193] The G protein conjugated receptor protein (hereinafter, sometimes simply referred to as the receptor protein of the present invention) or ligand polypeptide used in the present invention can be prepared based on the description of WO96 / 05302 or WO97 / 24436, for example.
[194] Specific examples of the receptor protein include a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 46 or a salt thereof. An amino acid sequence that is "substantially identical" may have variations as long as the activity of the protein such as the binding activity between the receptor and the ligand (ligand polypeptide of the present invention) is substantially the same (not causing significant change). Amino acid sequence, which has the same meaning as the receptor protein as defined in WO96 / 05302 or WO97 / 24436.
[195] As an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 46, for example, the amino acid sequence represented by SEQ ID NO: 48, etc. may be mentioned.
[196] As the nucleotide sequence of the DNA encoding the amino acid sequence represented by SEQ ID NO: 46, for example, the nucleotide sequence represented by SEQ ID NO: 47 may be mentioned, and the DNA encoding the amino acid sequence represented by SEQ ID NO: 48 may be used. Examples of the nucleotide sequence include a nucleotide sequence represented by SEQ ID NO: 49 and the like.
[197] A method for obtaining a CRH secretion regulator containing a compound or a salt thereof which changes the binding property of the ligand polypeptide of the present invention to the receptor protein of the present invention or a salt thereof and the use thereof will be described below.
[198] Compounds or salts thereof that change the binding of the ligand polypeptide of the present invention to the receptor protein of the present invention, or salts thereof, include compounds or salts thereof that promote the function (eg, CRH secretion, etc.) of the ligand polypeptide of the present invention, and Compounds or salts thereof that inhibit the function (eg, CRH secretion, etc.) of the ligand polypeptides of the present invention are included.
[199] Since the ligand polypeptide of the present invention has CRH secretion regulating action (CRH secretion promoting action, CRH secretion inhibiting action, etc.), the compound or salt thereof that promotes the function of the ligand polypeptide of the present invention (eg CRH secretion action, etc.) As a CRH secretion accelerator, it is related to CRH secretion such as hypoaldosteroneism, hypocortisemia, secondary and chronic adrenal insufficiency, Addison's disease (boredom, nausea, pigmentation, hypogonadism, alopecia, hypotension), adrenal insufficiency and obesity It is not only useful as a medicine for improving, preventing and treating various diseases, but also as an analgesic medicine.
[200] On the other hand, compounds that inhibit the CRH secretion of the ligand polypeptide of the present invention or salts thereof Cushing's disease, Cushing's syndrome (central obesity, full moon, high blood pressure, reddish purple progenitor, hairy), adrenal cortex stimulating hormone (ACTH) production Pituitary tumors, CRH producing tumors, aldosterone producing tumors, aldosteroneism (primary, secondary), primary cortisol resistance, congenital adrenal enzyme deficiency, antistress, congestion, anorexia nervosa, insomnia, gastroduodenal ulcers and irritable bowel syndrome It can be used as an agent for improving, preventing and treating various diseases related to CRH secretion.
[201] Thus, the ligand polypeptides of the present invention are useful as reagents for screening compounds or salts thereof that promote or inhibit the function (eg CRH secretion, etc.) of the ligand polypeptides of the present invention.
[202] Compounds or salts thereof that promote or inhibit the function of the ligand polypeptides of the invention include, for example, G protein conjugated receptor proteins (eg, phGR3, UHR-1, etc. (WO96 / 05302, WO97 / 24436)) and It can be obtained by screening for compounds that change the binding to a ligand polypeptide.
[203] This screening method is described below.
[204] By constructing a G protein conjugated receptor protein (e.g., phGR3, UHR-1, etc. (WO96 / 05302, WO97 / 24436)) expression system and using a receptor binding assay system using the expression system, Compounds that change binding to G protein conjugated receptor proteins (eg, phGR3, UHR-1, etc. (WO96 / 05302, WO97 / 24436), such as peptides, proteins, non-peptidic compounds, synthetic compounds, Fermentation products, etc.) or salts thereof can be screened efficiently.
[205] Such compounds include (a) cell stimulatory activity (eg, arachidonic acid free, acetylcholine free, intracellular Ca ²⁺ free, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, via G protein conjugated receptors, Compounds having cell membrane potential change, phosphorylation of intracellular proteins, activation of c-fos, activity that promotes lowering pH, etc., or (b) compounds having no cell stimulatory activity (so-called receptor proteins) Antagonists), (c) compounds that reduce the avidity of binding of the ligand polypeptide of the present invention to the G protein conjugated receptor protein and the like.
[206] That is, the compound which changes the binding property of the ligand peptide of this invention and this receptor protein with respect to the receptor protein of this invention, or its salt is a case where (i) contacting a receptor protein and the ligand polypeptide of this invention, and (ii) Obtained by a method for screening a compound or a salt thereof which changes the binding property between the ligand polypeptide of the present invention and the receptor protein, characterized in that a comparison is made between the receptor protein and the ligand polypeptide and the test compound of the present invention. Can be.
[207] In this screening method, the binding amount of the ligand to the receptor protein, cell stimulatory activity, etc. in the cases of (i) and (ii) are measured and compared.
[208] More specifically as this screening method, the following ①, ②, ③, etc. are mentioned:
[209] (1) The labeled ligand of the present invention when the labeled ligand polypeptide of the present invention is contacted with the receptor protein of the present invention, and when the labeled ligand polypeptide and the test compound are contacted with the receptor protein of the present invention. A method for screening a compound or a salt thereof for changing the binding property of the ligand polypeptide of the present invention to the receptor protein of the present invention, characterized by measuring and comparing the binding amount of the polypeptide to the receptor protein,
[210] (2) When the labeled ligand polypeptide of the present invention is contacted with a cell containing the receptor protein of the present invention or a membrane fraction of the cell, the labeled ligand polypeptide and the test compound of the present invention contain the receptor protein of the present invention. When a cell or a membrane fraction of the cell is contacted, the ligand polypeptide of the present invention and the ligand polypeptide of the present invention are characterized in that the amount of binding of the labeled ligand polypeptide of the present invention to this cell or the membrane fraction is measured and compared. Methods of screening for compounds or salts thereof that change the binding to receptor proteins,
[211] (3) When the labeled ligand polypeptide of the present invention is conjugated to the receptor protein expressed on the cell membrane by culturing a transformant containing a DNA encoding the receptor protein of the present invention, the labeled ligand polypeptide and the test of the present invention. When the compound is contacted with a receptor protein expressed on a cell membrane by culturing a transformant containing a DNA encoding the receptor protein of the present invention, the amount of binding of the labeled ligand polypeptide of the present invention to this receptor protein is determined. A method for screening a compound or salt thereof for changing the binding property of a ligand polypeptide of the invention to a receptor protein of the invention, characterized in that it is measured and compared.
[212] Below, the said screening method is demonstrated more concretely.
[213] First, the receptor protein of the present invention used in the above screening method may be any one containing the above-described receptor protein of the present invention, but cell membrane fractions of mammalian organs containing the receptor protein are preferable. However, in particular, human-derived organs are very difficult to obtain, and therefore, a receptor-derived protein derived from humans, which is expressed in large quantities using recombinants, is suitable for screening.
[214] In order to manufacture the receptor protein of the present invention, it is preferable to carry out by expressing the DNA encoding the receptor protein in mammalian cells or insect cells. Although cDNA is normally used for the DNA fragment which codes the target protein part, it is not necessarily limited to this. For example, gene fragments or synthetic DNA may be used. In order to introduce DNA fragments encoding receptor proteins into host animal cells and express them efficiently, the polyhedrin promoter of nuclear polyhedrosis virus (NPV), which belongs to baculoviruses that host insects, is used. And a promoter derived from SV40, a retrovirus promoter, a metallothionein promoter, a human heat shock promoter, a cytomegalovirus promoter, or an SRα promoter. Examination of the amount and quality of the expressed receptor can be performed by a method known per se. See, eg, Nambi, P. et al., J. Biol. Chem., Vol. 267, pages 19555 to 19559, 1992].
[215] Therefore, in the screening method described above, the receptor protein of the present invention may be a receptor protein purified according to a method known per se, or a cell containing the receptor protein may be used, and the receptor protein may be contained. Membrane fractions of cells may be used.
[216] When using the cell containing the receptor protein of this invention in the said screening method, you may fix this cell with glutaraldehyde, formalin, etc. The immobilization method may be carried out according to a method known per se.
[217] The cell containing the receptor protein of the present invention refers to a host cell expressing the receptor protein. As the host cell, Escherichia coli, Bacillus subtilis, yeast, insect cells, animal cells and the like are preferable.
[218] The cell membrane fraction refers to a fraction containing a large amount of cell membranes obtained by the methods known per se after the cells are disrupted. Examples of cell crushing methods include pulverizing cells with a Potter-Elvehjem type homogenizer, crushing by Waring blender or Polytron (Kinematica), ultrasonic crushing, Crushing etc. which blow out a cell from a thin nozzle, pressurizing with a french press etc. are mentioned. For the cell membrane fractionation, centrifugal fractionation such as fractionation centrifugation or density gradient centrifugation is mainly used. For example, the precipitate obtained by centrifuging the cell lysate at a low speed (500 rpm to 3000 rpm) for a short time (usually about 1 minute to 10 minutes) and centrifuging the supernatant again at a high speed (15000 rpm to 30000 rpm) for 30 minutes to 2 hours is used to prevent the precipitate. It is made into fractions. The membrane fraction contains a large amount of membrane components such as expressed receptor protein of the present invention and cell-derived phospholipids and membrane proteins.
[219] The amount of the receptor protein in the cell or membrane fraction containing the receptor protein is preferably 10 ³ to 10 ⁸ molecules, more preferably 10 ⁵ to 10 ⁷ molecules per cell. In addition, the greater the expression amount, the higher the ligand binding activity (inactivity) per membrane fraction, which makes it possible to construct a highly sensitive screening system and to measure a large number of samples in the same lot.
[220] For screening compounds that change the binding of the ligand polypeptide of the invention to the receptor protein of the invention, for example, the appropriate receptor protein fraction and labeled ligand polypeptide of the invention are used.
[221] As the receptor protein fraction, a recombinant receptor protein fraction or the like which is a natural receptor protein fraction or has an equivalent activity is preferable. Here, equivalent activity means equivalent ligand binding activity, signal information transfer activity, and the like.
[222] As the labeled ligand, a labeled ligand, a labeled ligand analog compound, or the like is used. For example, ligands labeled with [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S] and the like are used.
[223] Specifically, in order to screen compounds which change the binding property between the ligand polypeptide of the present invention and the receptor protein of the present invention, first, a cell or cell fraction containing the receptor protein of the present invention is subjected to a buffer suitable for screening. Receptor protein standards are prepared by suspending in. The buffer may be any buffer as long as it does not inhibit the binding of the ligand to a receptor protein such as a phosphate buffer of pH 4 to 10 (preferably pH 6 to 8), a tris-hydrochloric acid buffer, or the like. The purpose of reducing non-specific binding as CHAPS, Tween-80 ^TM (Kao-Atlas Inc.) may be added surface active agents such as, digi tonin, deoxycholate in a buffer. Moreover, protease inhibitors, such as PMSF, a leupeptin, E-64 (peptide gene shock show), pepstatin, can be added in order to suppress degradation of a receptor and ligand by a protease. A fixed amount (5000 cpm-500000 cpm) of labeled ligand is added to 0.01 ml-10 ml of this receptor solution, and 10 ^-4 M-10 ^-10 M test compound coexists simultaneously. A reaction tube is also prepared in which excess unlabeled ligand is added to determine the nonspecific binding amount (NSB). The reaction is carried out at about 0 ° C to 50 ° C, preferably at about 4 ° C to 37 ° C for about 20 minutes to 24 hours, preferably about 30 minutes to 3 hours. After the reaction, the resultant is filtered with a glass fiber filter paper or the like, washed with an appropriate amount of the same buffer solution, and the radioactivity remaining on the glass fiber filter paper is measured by a liquid scintillation counter or a γ-counter. When the count (B ₀ -NSB) obtained by subtracting the nonspecific binding amount (NSB) from the count (B ₀ ) without antagonist is 100%, the specific binding amount (B-NSB) is, for example, 50% or less. The test compound may be selected as a candidate having an antagonistic inhibitory ability, that is, a compound or a salt thereof that inhibits the function (eg CRH secretion) of the ligand polypeptide of the present invention.
[224] In addition, in order to perform a method for screening a compound that changes the binding property between the ligand polypeptide of the present invention and the receptor protein of the present invention, for example, cell stimulatory activity through the receptor protein (eg, arachidonic acid free, acetylcholine free, cells intracellular Ca ²⁺ glass, intracellular cAMP production, intracellular cGMP produced, inositol phosphate generation, cell membrane potential change, phosphorylation of cellular protein, c-fos activation, the activity of active or suppressed to facilitate the lowering of pH, etc., etc.) Can be measured using known methods or commercially available measurement kits.
[225] Specifically, first, cells containing the receptor protein of the present invention are cultured in a multi-well plate or the like. In screening, the cells are exchanged with an appropriate buffer solution which is not toxic to fresh medium or cells in advance, incubated with a test compound or the like for a predetermined time, and then the cells are extracted or the supernatant is recovered and the resulting products are respectively recovered. Quantify according to the method. If the production of a substance (eg, arachidonic acid, etc.) as an indicator of cell stimulation activity is difficult due to the enzymes contained in the cells, the assay may be performed by adding an inhibitor to this enzyme. In addition, the activity of cAMP production inhibition and the like can be detected by a production inhibitory effect on cells in which the basal production of cells has been increased with forskolin.
[226] In order to perform screening by measuring cell stimulatory activity, cells expressing an appropriate receptor protein are used. As the cell expressing the receptor protein of the present invention, a cell line having the naturally occurring receptor protein of the present invention, a cell line expressing the aforementioned recombinant receptor protein, or the like is preferable.
[227] As the test compound, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermented products, cell extracts, plant extracts, animal tissue extracts and the like are used. These compounds may be novel compounds or known compounds.
[228] A kit for screening a compound or salt thereof for changing the binding property of a ligand polypeptide of the present invention to a receptor protein of the present invention contains a receptor protein of the present invention, a cell containing the receptor protein of the present invention, or a receptor protein of the present invention. And the like, which contain a membrane fraction of cells.
[229] Examples of the screening kit include the following.
[230] 1. Reagent for screening
[231] ① Buffer for measurement and Buffer for washing
[232] What added 0.05% bovine serum albumin (made by Sigma) to Hank's Balanced Salt Solution (made by Gibco Co., Ltd.).
[233] It may be sterilized by filtration with a filter having a pore diameter of 0.45 µm and stored at 4 ° C, or may be produced during use.
[234] ② G-protein conjugated receptor standard
[235] CHO cells expressing the receptor protein of the present invention were passaged at 5 × 10 ⁵ cells / well in 12 well plates and incubated in 37 ° C., 5% CO ₂ , 95% air for 2 days.
[236] ③ labeled ligand
[237] Ligand polypeptides of the invention labeled with commercially available [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S] and the like.
[238] The aqueous solution is stored at 4 ° C or -20 ° C and diluted to 1 µM with a buffer for measurement at the time of use.
[239] ④ Ligand Standard
[240] The ligand polypeptide of the present invention is dissolved in PBS containing 0.1% bovine serum albumin (manufactured by Sigma Co., Ltd.) to 1 mM and stored at -20 ° C.
[241] 2. Measurement
[242] (1) After washing the receptor protein-expressing CHO cells of the present invention incubated in a 12 well tissue culture plate twice with 1 ml of the measurement buffer, 490 µl of the measurement buffer is added to each well.
[243] (2) After adding 5 µl of the test compound solution of 10 ^-3 to 10 ^-10 M, 5 µl of the labeled ligand is added and allowed to react at room temperature for 1 hour. To determine the nonspecific binding amount, 5 μl of 10 ⁻³ M ligand polypeptide is added instead of the test compound.
[244] ③ The reaction solution is removed and washed three times with 1 ml of washing buffer. The labeled ligand bound to the cells is dissolved in 0.2N NaOH-1% SDS and mixed with 4 ml of liquid scintillator A (manufactured by Wako Pure Chemical Industries, Ltd.).
[245] (4) The radioactivity is measured using a liquid scintillation counter (manufactured by Beckman), and the percentage of maximum binding (PMB) is obtained by the following equation.
[246] PMB = [(B-NSB) / (B ₀ -NSB)] × 100
[247] PMB: maximum join percentage
[248] B: Value when a sample is added
[249] NSB: nonspecific binding amount
[250] B ₀ : Maximum binding amount
[251] What is necessary is just to carry out according to a conventional method, when using the compound or its salt which changes the binding property of the ligand polypeptide of this invention with this receptor protein, or its salt to the receptor protein of this invention as mentioned above. Injections such as sterile solutions or suspensions orally or with water or other pharmaceutically acceptable liquids, such as tablets, capsules, elixirs, microcapsules, etc. It may be used parenterally in the form or as a nasal agent. For example, the compound or salt thereof may be prepared by blending with a physiologically acceptable carrier, flavor, excipient, vehicle, preservative, stabilizer, binder, and the like into the unit dosage form required for generally accepted pharmaceutical practice. The amount of active ingredient in these formulations is such that adequate dosages in the ranges indicated are obtained.
[252] As additives that can be blended into tablets, capsules and the like, for example, gelatin, corn starch, tragant gum, binders such as gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid and the like, swelling agents, magnesium stearate and the like Such lubricants, sweeteners as sucrose, lactose or saccharin, flavoring agents such as peppermint, acamonoyl or cherry, and the like are used. When the production unit form is a capsule, the above type of material may further contain a liquid carrier such as oil or fat. Aseptic compositions for injection may be prescribed according to conventional formulation practice, such as dissolving or suspending active substances, sesame oil, naturally occurring vegetable oils such as palm oil, and the like in a vehicle such as water for injection.
[253] Aqueous injectable solutions include, for example, isotonic solutions containing physiological saline, glucose or other adjuvants (e.g., D-sorbitol, D-mannitol, sodium chloride, and the like), and the like. Etc.), polyalcohol (for example, propylene glycol, polyethylene glycol), nonionic surfactant (for example, polysorbate 80 ( ^TM ), HCO-50), etc. may be used together. Sesame oil, soybean oil, etc. are mentioned as an oil-based liquid, You may use together with benzyl benzoate, a benzyl alcohol, etc. as a dissolution supplement preparation. In addition, buffers (e.g., phosphate buffers, sodium acetate buffers), analgesics (e.g. benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (e.g. human serum albumin, polyethylene glycol, etc.), preservatives (e.g. benzyl alcohol, phenol) Etc.), antioxidant, etc. may be mix | blended. The prepared injection solution is usually filled in a suitable ampoule.
[254] Since the preparation thus obtained is safe and low toxicity, it can be administered, for example, to mammals (eg, humans, mice, rats, morphotes, rabbits, sheep, pigs, cattle, cats, dogs, monkeys, chimpanzees, etc.).
[255] The dosage of the CRH secretion modulator containing a compound or salt thereof which changes the binding property of the ligand polypeptide of the present invention to the receptor protein of the present invention or a salt thereof varies depending on symptoms and the like. In general, about 0.1 mg to 100 mg, preferably about 1.0 mg to 50 mg, and more preferably about 1.0 mg to 20 mg of this compound or a salt thereof is given to a patient with hypoaldosteroneism per 60 kg body weight. do. In the case of parenteral administration, the single dose may vary depending on the administration target, symptoms, administration method, and the like.However, for example, in the form of an injection, the compound or a salt thereof is given to a patient with hypoaldosteroneism (for weight 60 kg). Usually, about 0.01 mg to 30 mg, preferably about 0.1 mg to 20 mg, and more preferably about 0.1 mg to 10 mg may be administered by intravenous injection. Other animals may be administered in an amount equivalent to 60 kg of body weight.
[256] SEQ ID NOs in the Sequence Listing of the present specification represent the following sequences.
[257] [SEQ ID NO: 1]
[258] The full length amino acid sequence of the bovine hypothalamic derived ligand polypeptide is shown.
[259] [SEQ ID NO: 2]
[260] The entire nucleotide sequence of the bovine hypothalamic-derived ligand polypeptide cDNA is shown.
[261] [SEQ ID NO: 3]
[262] The amino acid sequence of the bovine hypothalamus-derived ligand polypeptide is shown. It corresponds to the 23rd-53rd amino acid sequence of SEQ ID NO: 1.
[263] [SEQ ID NO: 4]
[264] The amino acid sequence of the bovine hypothalamus-derived ligand polypeptide is shown. It corresponds to the 23rd-54th amino acid sequence of SEQ ID NO: 1.
[265] [SEQ ID NO: 5]
[266] The amino acid sequence of the bovine hypothalamus-derived ligand polypeptide is shown. It corresponds to the 23rd to 55th amino acid sequence of SEQ ID NO: 1.
[267] [SEQ ID NO: 6]
[268] The amino acid sequence of the bovine hypothalamus-derived ligand polypeptide is shown. It corresponds to the 34th to 53rd amino acid sequence of SEQ ID NO: 1.
[269] [SEQ ID NO: 7]
[270] The amino acid sequence of the bovine hypothalamus-derived ligand polypeptide is shown. It corresponds to the 34th to 54th amino acid sequence of SEQ ID NO: 1.
[271] [SEQ ID NO: 8]
[272] The amino acid sequence of the bovine hypothalamus-derived ligand polypeptide is shown. It corresponds to the 34th to 55th amino acid sequence of SEQ ID NO: 1.
[273] [SEQ ID NO: 9]
[274] The base sequence of the DNA encoding the bovine hypothalamic-derived ligand polypeptide (SEQ ID NO: 3) is shown.
[275] [SEQ ID NO: 10]
[276] The base sequence of the DNA encoding the bovine hypothalamic-derived ligand polypeptide (SEQ ID NO: 4) is shown.
[277] [SEQ ID NO: 11]
[278] The base sequence of the DNA encoding the bovine hypothalamic-derived ligand polypeptide (SEQ ID NO: 5) is shown.
[279] [SEQ ID NO: 12]
[280] The base sequence of the DNA encoding the bovine hypothalamic-derived ligand polypeptide (SEQ ID NO: 6) is shown.
[281] [SEQ ID NO: 13]
[282] The base sequence of the DNA encoding the bovine hypothalamic-derived ligand polypeptide (SEQ ID NO: 7) is shown.
[283] [SEQ ID NO: 14]
[284] The base sequence of the DNA encoding the bovine hypothalamic-derived ligand polypeptide (SEQ ID NO: 8) is shown.
[285] [SEQ ID NO: 15]
[286] The full length amino acid sequence of the ligand polypeptide found from the small genomic DNA is shown.
[287] [SEQ ID NO: 16]
[288] The full length amino acid sequence of the rat ligand polypeptide is shown.
[289] [SEQ ID NO: 17]
[290] The entire nucleotide sequence of the rat ligand polypeptide cDNA is shown.
[291] [SEQ ID NO: 18]
[292] The amino acid sequence of the rat ligand polypeptide is shown. It corresponds to the 22nd-52th amino acid sequence of SEQ ID NO: 16.
[293] [SEQ ID NO: 19]
[294] The amino acid sequence of the rat ligand polypeptide is shown. It corresponds to the 22nd-53rd amino acid sequence of SEQ ID NO: 16.
[295] [SEQ ID NO: 20]
[296] The amino acid sequence of the rat ligand polypeptide is shown. It corresponds to the 22nd-54th amino acid sequence of SEQ ID NO: 16.
[297] [SEQ ID NO: 21]
[298] The amino acid sequence of the rat ligand polypeptide is shown. It corresponds to the amino acid sequence of SEQ ID NO: 16 agenda from 33 to 52nd.
[299] [SEQ ID NO: 22]
[300] The amino acid sequence of the rat ligand polypeptide is shown. It corresponds to the 33rd-53rd amino acid sequence of SEQ ID NO: 16.
[301] [SEQ ID NO: 23]
[302] The amino acid sequence of the rat ligand polypeptide is shown. It corresponds to the 33rd-54th amino acid sequence of SEQ ID NO: 16.
[303] [SEQ ID NO: 24]
[304] The base sequence of the DNA encoding the rat ligand polypeptide (SEQ ID NO: 18) is shown.
[305] [SEQ ID NO: 25]
[306] The base sequence of the DNA encoding the rat ligand polypeptide (SEQ ID NO: 19) is shown.
[307] [SEQ ID NO: 26]
[308] The base sequence of the DNA encoding the rat ligand polypeptide (SEQ ID NO: 20) is shown.
[309] [SEQ ID NO: 27]
[310] The base sequence of the DNA encoding the rat ligand polypeptide (SEQ ID NO: 21) is shown.
[311] [SEQ ID NO: 28]
[312] The base sequence of the DNA encoding the rat ligand polypeptide (SEQ ID NO: 22) is shown.
[313] [SEQ ID NO: 29]
[314] The base sequence of the DNA encoding the rat ligand polypeptide (SEQ ID NO: 23) is shown.
[315] [SEQ ID NO: 30]
[316] The full length amino acid sequence of the humanized ligand polypeptide is shown.
[317] [SEQ ID NO: 31]
[318] The entire nucleotide sequence of the humanoid ligand polypeptide cDNA is shown.
[319] [SEQ ID NO: 32]
[320] The amino acid sequence of the human-type ligand polypeptide is shown. It corresponds to the 23rd-53rd amino acid sequence of SEQ ID NO: 30.
[321] [SEQ ID NO: 33]
[322] The amino acid sequence of the human-type ligand polypeptide is shown. It corresponds to the 23rd-54th amino acid sequence of SEQ ID NO: 30.
[323] [SEQ ID NO: 34]
[324] The amino acid sequence of the human-type ligand polypeptide is shown. It corresponds to the 23rd-55th amino acid sequence of SEQ ID NO: 30.
[325] [SEQ ID NO: 35]
[326] The amino acid sequence of the human-type ligand polypeptide is shown. It corresponds to the amino acid sequence of the 34th-53st agenda of SEQ ID NO: 30.
[327] [SEQ ID NO: 36]
[328] The amino acid sequence of the human-type ligand polypeptide is shown. It corresponds to the 34th-54th amino acid sequence of SEQ ID NO: 30.
[329] [SEQ ID NO: 37]
[330] The amino acid sequence of the human-type ligand polypeptide is shown. It corresponds to the 34th to 55th amino acid sequence of SEQ ID NO: 30.
[331] [SEQ ID NO: 38]
[332] The base sequence of the DNA encoding a humanoid ligand polypeptide (SEQ ID NO: 32) is shown.
[333] [SEQ ID NO: 39]
[334] The base sequence of the DNA encoding a humanoid ligand polypeptide (SEQ ID NO: 33) is shown.
[335] [SEQ ID NO: 40]
[336] Represents the nucleotide sequence of the DNA encoding the human-type ligand polypeptide (SEQ ID NO: 34)
[337] [SEQ ID NO: 41]
[338] The base sequence of the DNA encoding a humanoid ligand polypeptide (SEQ ID NO: 35) is shown.
[339] [SEQ ID NO: 42]
[340] The base sequence of the DNA encoding a humanoid ligand polypeptide (SEQ ID NO: 36) is shown.
[341] [SEQ ID NO: 43]
[342] The base sequence of the DNA encoding a humanoid ligand polypeptide (SEQ ID NO: 37) is shown.
[343] [SEQ ID NO: 44]
[344] The amino acid sequence of the ligand polypeptide of this invention is shown. In the sequence, the third Xaa is Thr or Ala, the fifth Xaa is Arg or Gln, the tenth Xaa is Ile or Thr, the twenty-first Xaa is Thr or Ala, and the twenty-second Xaa is Gly Or Ser.
[345] [SEQ ID NO: 45]
[346] The amino acid sequence of the ligand polypeptide of this invention is shown. In the sequence, the 10th Xaa represents Thr or Ala, and the 11th Xaa represents Gly or Ser.
[347] [SEQ ID NO: 46]
[348] The amino acid sequence of phGR3 which is a receptor of the ligand polypeptide of this invention is shown.
[349] [SEQ ID NO: 47]
[350] The base sequence of the DNA which codes for the amino acid sequence of phGR3 which is a receptor of the ligand polypeptide of this invention is shown.
[351] [SEQ ID NO: 48]
[352] The amino acid sequence of UHR-1 which is a receptor of the ligand polypeptide of this invention is shown.
[353] [SEQ ID NO: 49]
[354] The base sequence of DNA which codes for the amino acid sequence of UHR-1 which is a receptor of the ligand polypeptide of this invention is shown.
[355] E. coli JM109 / phGR3 transformant having the nucleotide sequence encoding the amino acid sequence of phGR3 represented by SEQ ID NO: 47 was 1-1-3 Higashi, Tsukubashi, Ibaragi, Japan, on September 27, 1994. It was deposited with the Institute of Biotechnology and Industrial Technology (NIBH) under the deposit number FERM BP-4807, and on September 22, 1994, with the Fermentation Research Institute (IFO), 2-17-85, Jusanbonmachi, Osaka, Osaka. Deposited as IFO 15748.
[356] Although an Example is given to the following and this invention is demonstrated in detail, these do not limit the scope of the present invention.
[357] Example 1 Third intraventricular administration of PrRP-31 (peptide amidated by a carboxyl group of the C-terminal amino acid residue of a peptide consisting of the amino acid sequence represented by SEQ ID NO: 32) was performed by corticosteroid hormone (ACTH) in plasma and Effect on β-Endorphin Concentration
[358] Adult Wistar male rats (weight 350-380 g at the time of surgery) were anesthetized by intraperitoneal administration of pentobarbital 50 mg / kg and fixed to the rat brain stereotactic fixation device. The incision bar was lowered 3.3 mm on the interaural line. A dental drill was used to expose the skull and plant the guide cannula AG-12 (0.4 mm inner diameter, 0.5 mm outer diameter, ACOM) in the third ventricle. I made a hole in the goal. In addition, anchor screws were planted around four places. A stainless guide cannula AG-12 was inserted with its tip positioned above the third ventricle. The stereotactic coordinates were AP: +7.1 mm, L: 0.0 mm, and H: +2.0 mm in the interoral line according to the Atlas of Paxinos and Watson (1986). The guide cannula was secured to the skull with instant adhesive, dental cement and anchor screws. A stainless steel dummy cannula, AD-12 (0.35 mm outer diameter, ACOM Corporation) was inserted into the guide cannula and fixed with a cap nut (ACOM Corporation). After surgery, rats were housed in individual cages.
[359] After the guide cannula was planted, it was reared for about 1 week to recover after surgery, and blood collection surgery was performed under free behavior. Rats subjected to the above surgery were anesthetized by intraperitoneal administration of pentobarbital 50 mg / kg. The left jugular vein was exposed by lying in a prone position on the dissection pad. Polyethylene tube SP35 (inner diameter 0.5 mm, outer diameter 0.9 mm, Natsumesei Sakusho) was cut into a length of about 30 cm, filled with saline containing 200 units / ml of heparin, and inserted into the jugular vein and fixed by 4.5 cm. . Another end of the tube was exposed at the neck (dorsal side) through the dorsal subcutaneous.
[360] One night after the operation, 400 µl of blood was collected using a 1 ml syringe and a 25 gauge needle (both Terumosa) 30 minutes before administration of PrRP-31. In order to prevent blood coagulation, 20 µl of saline containing 200 units / ml of heparin was previously put into the syringe. Remove the cap nut and dummy cannula attached to the rat's skull and instead use a stainless microinjection cannula (internal diameter 0.17 mm, external diameter) connected to a Teflon tube (length 50 cm, inner diameter 0.1 mm, outer diameter 0.35 mm, Accom). 0.35 mm, ACOM Corporation) was inserted. The length of the microinjection cannula was adjusted such that its tip 1 mm was exposed from the guide cannula. One side of the Teflon tube was connected to a microsyringe pump and phosphate buffered saline containing 0.5% bovine serum albumin (BSA) or phosphoric acid containing 0.5% BSA dissolved in PrRP-31 (1, 3, 10 nmol each). 10 µl of the buffered saline was injected into the lateral ventricle at a flow rate of 5 µl / min. 15 minutes after the end of injection, the microinjection cannula was removed and the dummy cannula was again secured with a cap nut. Blood samples were collected 700 μl from the jugular vein 15 minutes before the start of intraventricular administration and 0, 5, 15, 30, 45, and 60 minutes after the start of intraventricular administration, respectively. The collected blood was centrifuged (5,000 rmp, 10 minutes) using a micro-cooling centrifuge (MR-150, Tommy Seiko) to recover the supernatant (plasma). ACTH and β-endorphin contained in plasma were measured using a radioimmunoassay (ACTH; Mitsubishi Kagaku, β-endorphin; Peninsula). As shown in Figures 1 and 2, in the PrRP-31 administration group, the concentration of blood ACTH and β-endorphin concentration was observed with a peak 15 minutes after administration compared to the control group.
[361] Example 2 Inhibitory Activity of PrRP-31 on Blood ACTH and β-Endorphin Concentration by CRH Antibody
[362] Adult Wistar male rats treated in the same manner as Example 1 were treated with phosphoric acid containing 0.5% bovine serum albumin (BSA) of 400 μg of anti-CRH IgG (PHOENIX PHARMACEUTICAL) and normal rabbit IgG (Pepro Tech EC). It was dissolved in buffered saline and administered to the jugular vein, and 30 minutes later, PrRP-31 10 mmol was administered intraventricularly. After 0, 5, 15, 30, 45 and 60 minutes from the start of the intraventricular administration, 700 μl were drawn from the jugular vein, and the blood was centrifuged using a micro-cooling centrifuge (MR-150, Tommy Seiko). rmp, 10 minutes) to recover the supernatant (plasma). ACTH and β-endorphin contained in plasma were measured using a radioimmunoassay (ACTH; Mitsubishi Kagaku, β-endorphin; Peninsula). As shown in Fig. 3 and 4, the anti-CRH IgG-administered group was observed to suppress the increase of blood ACTH and β-endorphin concentration by PrRP-31 administration compared to the normal rabbit IgG-administered group.
[363] Example 3 Effect of Stress on PrRP-31 In Vivo
[364] The present inventors consider that PrRP-31 is secreted from PrRP-31 producing cells or PrRP-31-containing nerve endings due to stress load, thereby increasing the PrRP-31 content in vivo, and the concentration of PrRP-31 in cerebrospinal fluid during stress-induced stress load Was measured. The Ustar male male rats (200-220 g in weight) were placed in a stress cage (W265 × L95 × H200 mm, Natsume Seisakusho), and then submerged in water (25 ° C.) up to the neck to load the needle restraint stress. After 0, 0.5, 1, 2, 4, 6 and 8 hours of the start, the rats were taken out of water and anesthetized by intraperitoneal administration of pentobarbital 50 mg / kg. Cerebrospinal fluid is collected from Chou, R.B. And the control puncture method (J. Pharmaclo. Exp. Ther., 219; 42-48, 1981). That is, the rats were lying on their backs and the incisions were made on the scalp, and the muscle layer was cut again. A needle (25G, Terumo) attached to a 1 ml syringe (Teromo) is punctured at about 2 mm in the middle of the skull and punctures the syringe at the point where the needle tip is about 5 mm. I broke it. When the needle is inserted, transparent cerebrospinal fluid is introduced. After confirming the inflow of cerebrospinal fluid, the needle was fixed with the instant adhesive and the cerebrospinal fluid was collected for about 10 minutes. Sampling was stopped at the point of blood incorporation and the hemostatic forceps were stopped and the cerebrospinal fluid contained 2 mM EDTA, 300KIU / ml aprotinin (Behringer Mannheim Yamanouchi), and CHAPS containing 0.05% final concentration. Placed in one Eppendorf tube. The recovered cerebrospinal fluid was stored under ice-cooling and centrifuged four times at 13,000 rpm x 5 minutes to recover and freeze the supernatant. PrRP concentration in cerebrospinal fluid was measured by a high sensitivity measuring system (WO99-60112) produced using two types of specific monoclonal antibodies against PrRP-31 having different recognition sites. PrRP-31 concentration (mean ± standard error) in cerebrospinal fluid was 2.90 ± 0.25 fmol / ml and 2.60 ± 0.25 fmol / ml, respectively, for control values 0.92 ± 0.10 fmol / ml Remarkably high values (p <0.01, n = 7) were shown, and then decreased as time passed, as shown in FIG. 5. In addition, as a result of measuring the ACTH concentration of blood by immersion stress stress by radioimmunoassay (Mitsubishi Kagaku), as shown in FIG. 6, the peak decreases with time after peaking at 0.5 hours and PrRP- in cerebrospinal fluid. The same variation as the 31 concentration was shown.
[365] Example 4 Inhibitory Activity of PrRP-31 on the ACTH Concentration of Blood by CRH Antagonist
[366] The adult Wistar male rats treated in the same manner as in Example 1 were dissolved in 0.5 ml of physiological saline and 2 mg of α-helical CRF (Peninsula Laboratories Europe), a CRH receptor antagonist, was administered to the jugular vein. 0.5 ml of saline was administered to the jugular vein. In both groups, 10 min of PrRP-31 was administered intraventricularly 15 minutes later. At 0, 10, 20, 30, 45, 60 minutes after the start of intraventricular administration, 400 μl were drawn from the jugular vein, and the blood was centrifuged using a micro high-speed cooling centrifuge (MR-150, Tommy Seiko). rmp, 10 minutes) to recover the supernatant (plasma). ACTH contained in plasma was measured by radioimmunoassay (ACTH; Mitsubishi Kagaku). As shown in FIG. 7, in the α-helical CRF-administered group, significant suppression of an increase in blood ACTH concentration by PrRP-31 intraventricular administration was observed as compared to the physiological saline-administered group.
[367] Formulation Example 1
[368] After dissolving 50 mg of the ligand polypeptide of the present invention in 50 ml of Japanese Pharmacopoeia distilled water, distilled water for Japanese Pharmacopoeia is added to 100 ml. The solution was filtered under sterile conditions, and then 1 ml of this solution was taken, filled into an injection vial under sterile conditions, and sealed by lyophilization.
[369] Formulation Example 2
[370] After dissolving 100 mg of the ligand polypeptide of the present invention in 50 ml of distilled water for Japanese Pharmacopoeia injection, distilled water for Japanese Pharmacopoeia is added to 100 ml. The solution was filtered under sterile conditions, and then 1 ml of this solution was taken, filled into an injection vial under sterile conditions, and sealed by lyophilization.
[371] Ligand polypeptides of the present invention have a regulating action of CRH secretion, that is, promoting and inhibiting CRH secretion. That is, since the ligand polypeptide of the present invention has a function of promoting CRH secretion, it can be used for the prophylaxis and treatment of various diseases related to CRH secretion failure. On the other hand, since the ligand polypeptide of the present invention has a strong affinity with the receptor protein, when the dose is increased, desensitization occurs to the CRH secretion, which also has a function of inhibiting the CRH secretion. In this case, it can be used for the prophylaxis and treatment of various diseases related to CRH oversecretion.
[372] Thus, the ligand polypeptide of the present invention is a CRH secretion promoter, hypoaldosteroneism, hypocortisolemia, secondary and chronic adrenal insufficiency, Addison's disease (boredom, nausea, pigmentation, hypogonadism, hair loss, hypotension), adrenal insufficiency and It is useful not only as an agent for improving, preventing and treating various diseases related to CRH secretion such as obesity, but also as an analgesic drug.
[373] In addition, the ligand polypeptide of the present invention is a CRH secretion inhibitor, Cushing's disease, Cushing's syndrome (central obesity, full moon eye, hypertension, red purple skin progenitor, hairy), adrenal cortex stimulating hormone (ACTH) production pituitary tumor, CRH production Tumors, aldosterone producing tumors, aldosteroneism (primary, secondary), primary cortisol resistance, congenital adrenal deficiency, antistress, congestion, anorexia nervosa, insomnia, gastric and duodenal ulcers and irritable bowel syndrome It is useful as a drug for improving, preventing and treating diseases.
[374] In addition, the ligand polypeptide of the present invention is useful as a test agent for investigating CRH secretion function.

权利要求:
Claims (10)
[1" claim-type="Currently amended] A corticosteroid-releasing hormone secretion regulator containing a ligand peptide or an amide thereof or an ester thereof or a salt thereof against a G protein conjugated receptor protein.
[2" claim-type="Currently amended] The amino acid sequence of claim 1, wherein the ligand peptide or amide thereof or ester thereof or salt thereof for the G protein conjugated receptor protein is identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 44 or SEQ ID NO: 45. An adrenal cortex stimulating hormone-releasing hormone secretion agent, which is a polypeptide or an amide or ester thereof or a salt thereof.
[3" claim-type="Currently amended] The corticosteroid-releasing hormone secretion regulator of claim 2, wherein the amino acid sequence represented by SEQ ID NO: 44 is an amino acid sequence represented by SEQ ID NO: 3, 18 or 32.
[4" claim-type="Currently amended] The corticosteroid-releasing hormone secretion regulator of claim 2, wherein the amino acid sequence represented by SEQ ID NO: 45 is an amino acid sequence represented by SEQ ID NO: 6, 21, or 35.
[5" claim-type="Currently amended] The corticosteroid-releasing hormone secretion regulator of claim 1, wherein the G-protein conjugated receptor is a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 46.
[6" claim-type="Currently amended] The corticosteroid-stimulating hormone secretion modulator according to claim 1, which is an adrenal cortex stimulating hormone-releasing hormone secretagogue.
[7" claim-type="Currently amended] The corticosteroid-releasing hormone secretion agent according to claim 1, which is a drug for preventing or treating hypoaldosteroneism, hypocortisolemia, secondary or chronic adrenal insufficiency, Addison's disease, adrenal insufficiency or obesity.
[8" claim-type="Currently amended] An adrenal cortical stimulating hormone-releasing hormone secretion agent containing a compound or a salt thereof that changes the binding of a G protein conjugated receptor protein to a ligand peptide to the G protein conjugated receptor protein.
[9" claim-type="Currently amended] Use of a ligand peptide or an amide or ester thereof or salt thereof for a G protein conjugated receptor protein for the manufacture of a medicament having an adrenal cortical stimulating hormone release hormone secretion action.
[10" claim-type="Currently amended] A method for regulating corticosteroid-releasing hormone secretion, characterized by administering a ligand peptide or an amide thereof or an ester thereof or a salt thereof to a G protein conjugated receptor protein.

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同族专利:

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公开号 | 公开日

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WO2001035984A1|2001-05-25|

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EP1230928A4|2004-01-28|

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CN1390131A|2003-01-08|

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US20050209151A1|2005-09-22|

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AU1415301A|2001-05-30|

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CA2391872A1|2001-05-25|

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EP1230928A1|2002-08-14|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
1999-11-18|Priority to JPJP-P-1999-00327900

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1999-11-18|Priority to JP32790099

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2000-09-26|Priority to JP2000297073

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2000-09-26|Priority to JPJP-P-2000-00297073

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2000-11-17|Application filed by 다케다 야쿠힌 고교 가부시키가이샤

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2000-11-17|Priority to PCT/JP2000/008119

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2002-07-12|Publication of KR20020058024A

优先权:

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申请号 | 申请日 | 专利标题

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JPJP-P-1999-00327900|1999-11-18|

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JP32790099|1999-11-18|

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JP2000297073|2000-09-26|

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JPJP-P-2000-00297073|2000-09-26|

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PCT/JP2000/008119|WO2001035984A1|1999-11-18|2000-11-17|Use of peptide|