WO2002085399A1 - Peptide-containing preparations - Google Patents

Peptide-containing preparations Download PDF

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Publication number
WO2002085399A1
WO2002085399A1 PCT/JP2002/003898 JP0203898W WO02085399A1 WO 2002085399 A1 WO2002085399 A1 WO 2002085399A1 JP 0203898 W JP0203898 W JP 0203898W WO 02085399 A1 WO02085399 A1 WO 02085399A1
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WO
WIPO (PCT)
Prior art keywords
sustained
release preparation
metastin
preparation according
peptide
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PCT/JP2002/003898
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French (fr)
Japanese (ja)
Inventor
Shigeyuki Takada
Tetsuya Ohtaki
Yoshihiro Omachi
Takao Yamada
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Takeda Chemical Industries, Ltd.
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Publication of WO2002085399A1 publication Critical patent/WO2002085399A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to a preparation containing metastin (a peptide encoded by the KiSS-1 gene: hereinafter sometimes referred to as a KiSS-1 peptide) or a derivative thereof or a salt thereof.
  • metastin a peptide encoded by the KiSS-1 gene: hereinafter sometimes referred to as a KiSS-1 peptide
  • KiSS-1 peptide is a C-terminal partial peptide of the protein encoded by the cancer metastasis suppressor gene KiSS-1 (Genomics ⁇ 54, pp.145-148, 1998). Since the transposition ability of the KiSS-1 gene is reduced when human chromosome 6 is transferred to C8161 melanoma, the subtractive hybridizer method was used from the low-transferred strain obtained by transferring chromosome 6 and the parent strain ( It has been found that transfection of the KiSS-1 gene into C8161 melanoma results in a decrease in the metastatic potential according to the expression level (Subtractive Hybridization Technique) (J. Natl. Cancer Inst. , 88, 1731-1737, 1996). It has also been reported that the translocation of the KiSS-1 gene reduces the metastatic potential of human breast cancer cell MDA-MB-435 (Cancer Research, 57, 2384-2387, 1997).
  • Metastin a peptide cut out from the KiSS-1 gene product, is expected to have cancer metastasis-suppressing activity because its gene is a cancer metastasis-suppressing gene.
  • metastin inhibits chemotaxis and invasion of CH0 cells that express the human receptor for metastin ⁇ 7 ⁇ 5, and in vivo to the lung of B16-BL6 malignant melanoma that expresses hOT7T175 Weakens the metastatic properties (Nature, vol. 411, p. 613, 2001, etc.). Furthermore, that the present gene is expressed in a large amount in the placenta (J. Natl. Cancer Inst., Vol. 88, pp.
  • T7T 175 which is a human receptor of the peptide, Due to its high expression in the placenta and relatively high expression in the knee (eg, Nature 411, 613-617, 2001, etc.), the peptide plays an important role in the placenta. Alternatively, it is expected that this peptide also exerts some physiological functions in the knee.
  • metastin or its derivatives or salts thereof should be administered at high doses for a long period of time to obtain sufficient pharmacological effects. Is required.
  • an aqueous solution of metastin or a derivative thereof or a salt thereof is administered subcutaneously, there is a concern about the occurrence of side effects and the inconvenience and pain of the patient in actual administration for treatment or prevention. Therefore, development of a useful preparation containing metastin or a derivative thereof or a salt thereof capable of solving these problems is desired. Disclosure of the invention
  • the present inventors have conducted intensive studies and as a result, when metastin, its derivative or its salt is administered as a sustained-release preparation, it acts on highly metastatic cancer cells and suppresses metastasis In addition, the sustained release of metastin or its derivatives or its salts over a long period of time does not require high doses to achieve medicinal effects, so that side effects can be reduced and daily administration is not required.
  • the present inventors have found that the preparation of the present invention has excellent properties as a clinical medicine, such as reduced inconvenience and pain, and completed the present invention.
  • sustained-release preparation containing metastin or a derivative or salt thereof
  • a peptide wherein metastin or a derivative thereof contains the amino acid sequence at the 47th to 54th position from the N-terminus of the amino acid sequence represented by SEQ ID NO: 1 and comprises 8 to 54 amino acid residues Or a sustained-release preparation according to the above (1), which is a derivative thereof,
  • sustained-release preparation according to (3) wherein the peptide is a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5.
  • the sustained-release preparation according to the above (1) which is a capsule,
  • sustained-release preparation which comprises metastin or a derivative thereof or a salt thereof, and any other pharmaceutically active substance.
  • FIG. 1 is a graph showing the time course of blood metastin (1-54) concentration (pmol / ml) after subcutaneous administration of sustained-release metastin (1-54) -containing microcapsules in rats.
  • the “metastin” used in the present invention includes all 54 consecutive amino acids from the N-terminal to the first to 54th amino acids in the amino acid sequence represented by SEQ ID NO: 1.
  • the term “metastin derivative” used in the present invention refers to a peptide having an amino acid residue and having an amide (one C ⁇ NH 2 ) at the C-terminus.
  • the physiological activity of metastin for example, metastin and its receptor
  • SEQ ID NO: 1 cell stimulating activity of receptor-expressing cells caused by metastin, etc.
  • the C-terminus may be an amide (one-CO NH 2 ), or may be any of a carboxylate group (—CO OH), a carboxylate group (COO—), and an ester (one COOR). It is preferable that the C-terminal carbonyl group is an amidated amide.
  • the metastin derivative has substantially the same activity as the metastin physiological activity, and in the amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence at the 47th to 54th amino acid sequence from the N-terminal ( The length is not particularly limited as long as it has (8 essential amino acid residues), but a peptide consisting of 8 to 54 amino acid residues (preferably, 8 to 15 amino acid residues) Alternatively, a derivative thereof is preferably used.
  • amino acid sequence represented by SEQ ID NO: 1 a peptide or a derivative thereof containing the 47th to 54th amino acid sequence from the N-terminal and consisting of 8 to 54 amino acid residues
  • amino acid sequence represented by SEQ ID NO: 1 a peptide comprising the amino acid sequence at the 47th to 54th position from the N-terminus and consisting of 8 to 15 amino acid residues, or a peptide thereof.
  • Derivatives are preferred.
  • the arrangement of eight essential amino acid residues is as follows: May be located at any site, as long as the derivative has substantially the same activity as the metastin bioactivity, but the eight essential amino acid residues are located at the C-terminus. Is desirable. That is, in the amino acid sequence represented by SEQ ID NO: 1, the 47th to 54th amino acid sequence from the N-terminal is A peptide or derivative thereof having 8 to 54 amino acid residues at the C-terminus is preferable. In particular, in the amino acid sequence represented by SEQ ID NO: 1, the 47th to 54th amino acid sequence from the N-terminal is Peptides having 8 to 15 amino acid residues at the C-terminus or derivatives thereof are preferred.
  • the C-terminal of a peptide represented by a specific amino acid sequence has an amide (-CONH 2 ), a carboxyl group (—COOH), a carboxylate group (COCT) and an ester (1-COH). COOR).
  • a peptide derivative means that the C-terminus has a carboxy group (one COOH) or a carboxylate group (COO_ ) And an ester (a COOR), each of which includes a lipoxyl form, a carboxylate form, and an ester form, and the C-terminal of the peptide represented by the specific amino acid sequence has a lipoxyl group
  • (COOH) a peptide derivative is an amide in which the C-terminus has been converted to any of an amide (-CONH 2 ), a carboxylate group (COO-) and an ester (-COOR) , Carboxylates and esters.
  • an amide having an amide (one CONH 2 ) at the C-terminus is preferably used.
  • the metastin or a derivative thereof may be, for example, any of a fermentation product, a synthetic compound, a synthetic peptide, and the like, or may be of natural origin, and may be a recombinant peptide produced by a genetic engineering technique. May be.
  • metastin or a derivative thereof examples include, for example, metastin or a derivative thereof derived from all mammals such as humans, monkeys, baboons, chimpanzees, bushus, sea lions, higgins, horses, mice, rats, and the like. Among them, those derived from humans are preferred. When the preparation of the present invention is applied to humans, it is particularly preferable to use metastin derived from human or a derivative thereof.
  • the metastin or a derivative thereof used in the present invention preferably, for example, in the amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence at the 47th to 54th position from the N-terminus is located at the C-terminus, and 8 to 15 amino acids Petit consisting of amino acid sequence And so on.
  • a peptide comprising the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 (especially an amide thereof), and the like can be mentioned.
  • a mouse or rat-derived peptide (Kiss-1 gene product) containing the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 described in WO 01/751 04 is used. Is also good.
  • the C-terminus of metastin or a derivative thereof is usually an amide (one CONH 2 ), but may be an ester (one COOR), a hydroxyl group (one COOH), or a hydroxyl group (COO—).
  • an amide in which the C-terminal carbonyl group is amidated is particularly preferable.
  • R in the ester e.g., methyl, Echiru, n- propyl, C E, such as isopropyl or n- butyl - 6 alkyl group, for example, C 3 of cyclopentyl Le, cyclohexane, etc.
  • cyclohexyl - 8 cycloalkyl group for example, C 7 such as phenyl, alpha-naphthyl C 6 ⁇ 2 Ariru group such as for example, benzyl, phenylene route C DOO 2 alkyl or a- naphthylmethyl etc.
  • a- Nafuchiru 2 alkyl Le group such phenethyl —
  • pivaloyloxymethyl groups commonly used as oral esters are used.
  • the metastin or its derivatives used in the present invention N-terminal of the Amino group protecting groups such as Mechionin residues (e.g., C 2, such as a formyl group, Asechiru - 6 Ashiru group - C E such as 6 Arukanoiru group ),
  • the N-terminal side is cleaved in vivo, the dalminyl mill group is oxidized with glutamine, and the substituent on the side chain of the amino acid in the molecule (for example, mono-OH, one SH, -COOH, amino group, Imida zone Ichiru group, indole group, Guanijino group, etc.) a suitable protecting group (e.g., formyl group, C 2 such Asechiru - such as 6 Arukanoiru group (: Bok 6 Ashiru group ) Or complex peptides such as so-called glycopeptides to which sugar chains are bound.
  • Mechionin residues e.g.,
  • the sugar chains include, for example, D-mannose And neutral sugars such as D-galactose and L-fructose; amino sugars such as D-darcosamine and D-galactosamine; and sialic acid.
  • Metastin or a derivative thereof may form a salt, and as a salt of metastin or a derivative thereof, a pharmacologically acceptable salt is particularly preferable.
  • salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.), and organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, conodic acid) , Tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc.), salts with inorganic bases (eg, alkali metal salts such as sodium salt, potassium salt, etc.); calcium salts And alkaline earth metal salts such as magnesium salts; aluminum salts and ammonium salts; salts with organic bases (eg, trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, trietano, etc.). .
  • inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc
  • Metastin or a derivative thereof used in the present invention can be obtained by a known method (for example, a method described in WO 00/24890, J. Chem. Soc., Perkin Trans. 1, 1748, 2001) and the like. Alternatively, it can be produced according to a method analogous thereto, for example, according to a known peptide synthesis method, or can be produced by cleaving a known peptide or precursor with an appropriate peptide.
  • the method for synthesizing the peptide may be, for example, either a solid phase synthesis method or a liquid phase synthesis method.
  • the target peptide can be produced by condensing a partial peptide or amino acid capable of constituting the peptide or the precursor with the remaining portion, and removing the protecting group when the product has a protecting group. Can be done. Examples of known condensation methods and elimination of protecting groups include the methods described in the following [1] to [5].
  • the sustained-release preparation containing metastin or a derivative thereof or a salt thereof of the present invention preferably contains one carrier (carrier or base).
  • the carrier include proteins, polysaccharides, polymers (biodegradable polymers and non-biodegradable polymers), ribosomes, and inorganic substances.
  • Proteins include, for example, collagen, gelatin, fibrin, serum albumin and the like.
  • polysaccharide examples include starch, hyaluronic acid, chitosan, chitin, alginate, agarose, dextran, and cellulose derivatives.
  • biodegradable polymer examples include aliphatic polyesters, polyphosphazenes, polyorthoesters, and the like.
  • non-degradable polymer in vivo examples include polyglycerin fatty acid ester, silicon, ethyl vinyl acetate copolymer, polyhydroxyethyl methyl methacrylate, polyvinyl alcohol, and polyvinylpyrrolidone.
  • ribosomes include, in addition to ordinary ribosomes, for example, polyethylene glycol (PEG) -modified ribosomes, sugar-modified ribosomes, and the like.
  • PEG polyethylene glycol
  • Examples of the inorganic substance include hydroxyapatite, tricalcium phosphate, calcium phosphate, calcium sulfate, and the like.
  • a polymer for example, a biodegradable polymer, a non-biodegradable polymer, etc.
  • a biodegradable polymer is, for example, an aliphatic polymer.
  • the non-degradable polymer in vivo such as polyester, for example, polyglycerin fatty acid ester and the like are preferable.
  • a lactic acid / daricholic acid polymer or the like is used as the aliphatic polyester.
  • the composition ratio of lactic acid / daricholic acid polymer (lactic acid / daricholic acid; LZG ratio) (mol%) is preferably about 100Z0 to about 40Z60, more preferably about 100Z0 to about 50/50, and particularly preferably about 75/50.
  • the weight average molecular weight of the lactic acid / daricholic acid polymer having a weight average molecular weight of about 3,000 to about 80,000, preferably about 5,000 to about 25,000, and more preferably about 5,000 to about 25,000, Preferably, it is about 7,000 to about 20,000.
  • the dispersity (weight average molecular weight / number average molecular weight) of the lactic acid / daricholic acid polymer is preferably about 1.2 to about 4.0, more preferably about 1.5 to about 3.5.
  • polyglycerin fatty acid esters include tetraglycerol monopalmitate (TGMP), tetraglycerol monopalmitate (TGDP), tetraglyceryl tripalmitate (TGTP), tetraglycerol hexapalmitate (TGHP), and tetraglycerol monopalmitate.
  • TGMS Tetradari Serol Distearate
  • TGTS Tetraglycerol Tristearate
  • TGHS Tetraglycerol Hexstearate
  • HGPS Hexaglycerose-Lpentastearate
  • TGHP Tetra Laglycerol hexapalmitate
  • the sustained-release preparation of the present invention is produced according to a known method.
  • the sustained-release preparation of the present invention is prepared by mixing metastin or a derivative thereof or a salt thereof with a carrier (for example, the above-mentioned carrier) usually used in the production of a sustained-release preparation, and molding as necessary.
  • a carrier for example, the above-mentioned carrier
  • the amount of metastin or a derivative or salt thereof used is about 0.01 to about 50% (wZw), preferably about 0.1 to about 30% (w / w) based on the carrier. Used as
  • sustained-release preparation of the present invention examples include, for example, (A) a sustained-release preparation containing metastin or a derivative thereof or a salt thereof using polyglycerol fatty acid ester, which is a non-degradable polymer in vivo, as a carrier. [Hereinafter, KiSS-1 peptide containing sustained release And (B) a method for producing a KiSS-1 peptide-containing sustained-release preparation using a biodegradable lactic acid / glycolic acid polymer as a carrier.
  • KiSS-1 peptide-containing sustained-release preparation using polyglycerin fatty acid ester as carrier
  • the amount of metastin or a derivative thereof or a salt thereof to be used varies depending on the type of metastin or a derivative thereof or a salt thereof, a desired pharmacological effect and duration of the effect, and the like. To about 50% (w / w), preferably about 0.1 to about 30% (w / w).
  • the lactate noglycolic acid polymer is dissolved in an organic solvent (preferably dichloromethane) and emulsified after adding an aqueous solution of metastin or a derivative or salt thereof. This is vacuum-dried to obtain lactic acid / glycolic acid polymer powder in which metastin or a derivative thereof or a salt thereof is uniformly dispersed. This is heated and cooled to form a disk, film, rod, etc. In this way, it is possible to obtain a sustained-release preparation of lactic acid z-daricholic acid polymer containing a predetermined amount of metastin or a derivative thereof or a salt thereof.
  • the heating temperature is about 50 to about 100 ° C
  • the cooling temperature is about 0 to about 40 ° C.
  • the amount of metastin or a derivative thereof or a salt thereof varies depending on the type of metastatin or a derivative thereof or a salt thereof, a desired pharmacological effect and a duration of the effect. 0 1 to about 5 0% (w / w), preferably about 0.1 to about 30% (w / w), particularly preferably about 1 to about 20% (w / w).
  • the lactic acid / glycolic acid polymer is dissolved in an organic solvent (preferably, dichloromethane, etc.), and the powder of metastin or a derivative thereof or a salt thereof is added and then uniformly dispersed.
  • the resulting dispersion is dried under vacuum to obtain lactic acid / daricholic acid polymer powder in which metastin or a derivative thereof or a salt thereof is uniformly dispersed.
  • This is heated and cooled to form a disc, film, rod, etc.
  • a sustained-release preparation of a polymer of lactic acid-Z-glycolic acid containing a predetermined amount of metastin or a derivative thereof or a salt thereof can be obtained.
  • the heating temperature, the cooling temperature, and the amount of metastin or a derivative or salt thereof used are the same as described above.
  • W / OZW emulsions and O / W emulsions respectively consist of (i) an aqueous solution, dispersion or suspension of metastin or a derivative or salt thereof as an internal aqueous phase, and an organic solvent solution of lactic acid / daricholic acid polymer as an oil phase. Or (ii) dissolving or suspending metastin or a derivative or salt thereof in an organic solvent solution of lactic acid-Z-glycolic acid polymer to obtain an oil phase, and converting the U) or ( ⁇ ) into water (Outer aqueous phase), and dispersed and emulsified.
  • metastin or a derivative thereof or a salt thereof is dissolved, dispersed or suspended in water to produce an internal aqueous phase.
  • concentration of metastin or a derivative or salt thereof in an aqueous solution, dispersion or suspension is, for example, 0.01 to 90% (w / w), preferably 0.01 to 80% (w / w). / w).
  • the amount of the above-mentioned metastin or its derivative or its salt used depends on the type of metastin or its derivative or its salt, the desired pharmacological effect and the duration of the effect. However, based on the lactic acid / glycolic acid polymer, about 0.01 to about 50% (w / w), preferably about 0.1 to about 30% (w / w), particularly preferably about 0.1 to about 30% (w / w) 1 to about 20% (w / w).
  • gelatin agar, sodium alginate, polyvinyl alcohol or basic amino acids (eg, arginine, histidine, lysine, etc.) should be added to the internal aqueous phase to increase the incorporation of metastin or its derivatives or its salts into microcapsules. May be added (eg, excipients, auxiliaries, etc.).
  • the amount of the drug-retaining substance to be added is generally about 0.01 to about 10 times the weight of metastin or a derivative or salt thereof.
  • the inner aqueous phase may be used after being freeze-dried to a powder state and then dissolved by adding water to an appropriate concentration.
  • a lactic acid Z-glycolic acid polymer is dissolved in an organic solvent to produce an oil phase.
  • organic solvent examples include halogenated hydrocarbons (eg, dichloromethane, chloroform, chloroethane, trichloroethane, carbon tetrachloride, etc.), fatty acid esters (eg, ethyl acetate, butyl acetate, etc.), aromatic hydrocarbons (eg, benzene, Toluene, xylene, etc.), of which dichloromethane is preferred.
  • halogenated hydrocarbons eg, dichloromethane, chloroform, chloroethane, trichloroethane, carbon tetrachloride, etc.
  • fatty acid esters eg, ethyl acetate, butyl acetate, etc.
  • aromatic hydrocarbons eg, benzene, Toluene, xylene, etc.
  • the concentration of the lactic acid Z-daricholic acid polymer in the organic solvent varies depending on the type and molecular weight of the lactic acid Z-daricholic acid polymer and the type of the organic solvent, but the weight of citric acid / daricholic acid polymer / (weight of organic solvent + (Weight of lactic acid / daricholic acid polymer)] (X100%) is usually about 0.01 to about 90% (w / w), preferably about 0.01 to about 70% (w / w). / w). It is better to dissolve so that there is no undissolved matter.
  • aqueous solution, dispersion or suspension (inner aqueous phase) of the above-mentioned metastin or a derivative thereof or a salt thereof is added to an organic solvent solution (oil phase) of the lactic acid noglycolic acid polymer thus obtained, and the mixture is homogenized. Disperse and emulsify with a mixer etc. to produce W / ⁇ emulsion.
  • the above (ii) that is, an oil phase obtained by dissolving or suspending metastin or a derivative thereof or a salt thereof in an organic solvent solution of a lactic acid / glycolic acid polymer is produced as follows. First, an organic solvent solution of a lactic acid / daricholic acid polymer is produced. As the organic solvent, those similar to the organic solvent used in producing the above wzo emulsion are used.
  • the concentration of the lactic acid / daricholic acid polymer in the organic solvent solution varies depending on the molecular weight of the lactic acid / daricholic acid polymer and the type of the organic solvent; however, [weight of lactic acid z glycolic acid polymer Z (weight of organic solvent + weight of lactic acid (X100%) is usually about 0.01 to about 70% (w / w), preferably about 1 to about 60% (w / w). is there.
  • metastin or a derivative thereof or a salt thereof is dissolved or suspended in an organic solvent solution of lactic acid-Z-glycolic acid polymer to produce an oil phase.
  • the amount of metastin or a derivative thereof or a salt thereof used is such that the ratio of the metastin or a derivative thereof or a salt thereof to the lactic acid-z-glycolic acid polymer is the same as that in the case of producing the above w / o emulsion (i). Just choose.
  • the amount of the outer aqueous phase used is usually about 1 to about 100,000 times as much as the above (i) or (ii), preferably about 10 to about 5,000 times, particularly preferably about 10 to about 5,000 times. It is 50 to about 1, 000 times the volume.
  • An emulsifier is usually added to the external aqueous phase.
  • the emulsifier generally stable
  • any substance that can form an O / W emulsion or a zero-w emulsion can be used.
  • Examples include anionic surfactants, nonionic surfactants, polyoxyethylene castor oil derivatives, polyvinylpyrrolidone, polyvinyl alcohol, and carboxymethyl. Cellulose, lecithin, gelatin, hyaluronic acid and the like can be mentioned, among which polyvinyl alcohol is preferable.
  • the concentration of the emulsifier in the external aqueous phase is usually about 0.001 to about 20% (w / w), preferably about 0.01 to about 10% (wZw), and particularly preferably about 0. 0 5 to about 5% (w / w).
  • the WZO ZW emulsion or O / W emulsion thus obtained (hereinafter These are sometimes simply abbreviated as emulsions) by subjecting them to an underwater drying method, whereby microcapsules can be produced by removing the organic solvent contained in these emulsions.
  • microcapsules obtained in this manner are collected by centrifugation or a sieve or the like, and, if desired, a sugar or sugar alcohol, an inorganic salt or the like, preferably a coagulation inhibitor such as mannitol or sorbitol in order to prevent aggregation of the microcapsules. After adding lyophilized.
  • the mixing ratio (weight ratio) of the microcapsules and the anti-agglomeration agent is about 50: 1 to about 1: 1, preferably about 20: 1 to about 1: 1, and more preferably about 10: 1 to about 5: 1. : 1.
  • the method of adding the anti-agglomeration agent is not particularly limited as long as the microcapsules and the anti-agglomeration agent are uniformly mixed, and examples thereof include a method of dispersing the microcapsules in an aqueous solution of the anti-agglomeration agent.
  • micro-force capsule thus obtained is heated and dried under reduced pressure, if necessary, to more completely remove the water and the solvent in the microcapsules.
  • a powder of metastin or a derivative thereof or a salt thereof may be used in an organic solvent solution in which a lactic acid / z-dalicholate polymer is dissolved (phase 0). It can also be produced by a method (for example, the s / ozw method or the like) by removing the solvent from the szo-type dispersion liquid dispersed in water.
  • a polymer of lactic acid “glycolic acid” is dissolved in an organic solvent, and powder (S phase) of metastin or a derivative or salt thereof is added and dispersed in the organic solvent.
  • the mixing ratio (weight ratio) of metastin or a derivative thereof or a salt thereof and a lactic acid / daricholic acid polymer is, for example, about 1: 10,000 to about 1: 1, preferably about 1: 1. 1,000 to about 2: 5, more preferably about 1: 100 to about 1: 3
  • external physical energy for example, ultrasonic irradiation, a one-bottle type stirrer, a homogenizer and the like are used.
  • the organic solvent dispersion (SZO type dispersion) thus prepared is further added to an aqueous solvent (W phase), and the same external physical energy as described above, for example, ultrasonic irradiation, turbine An szozw emulsion is formed using a stirrer or a homogenizer. Thereafter, the oil phase solvent is evaporated to produce microcapsules.
  • the volume of the aqueous phase is generally selected from about 1 to about 100,000 times the volume of the oil phase. More preferably, it is selected from about 10-fold to about 5,000-fold, particularly preferably from about 50-fold to about 1,000-fold.
  • An emulsifier may be added to the outer aqueous phase.
  • the emulsifier may be any as long as it can form a generally stable s / ozw emulsion.
  • examples of the emulsifier include anionic surfactants, nonionic surfactants, polyoxyethylene castor oil derivatives, polyvinylpyrrolidone, polyvinyl alcohol, carboxymethylcellulose, lecithin, gelatin, hyaluronic acid and the like. These may be used in appropriate combinations.
  • the concentration of the emulsifier in the external aqueous phase is preferably from about 0.001% to about 20% (w / w). More preferably, it is about 0.01% to about 10% (w / w), particularly preferably about 0.05% to about 5% (w / w).
  • microcapsules obtained in this manner are separated by centrifugation or filtration, and then the emulsifier and the like adhering to the surface of the microcapsules are removed by washing with distilled water, and dispersed again in distilled water or the like. Lyophilize. Thereafter, if necessary, the mixture is heated to further remove water and organic solvents in the micro force cell. It may be heated under reduced pressure.
  • the heating conditions are heating and drying at a temperature not lower than the glass transition temperature of the lactic acid / daricholic acid polymer used, and at such a level that the particles of the microcapsules do not adhere to each other.
  • the lactic acid / daricholic acid polymer is heated and dried in a temperature range from the glass transition temperature to about 30 ° C higher than the glass transition temperature.
  • the glass transition temperature refers to an intermediate point obtained when the temperature is raised at a heating rate of 10 to 20 ° C. per minute using a differential scanning calorimeter.
  • the microcapsules thus obtained may be added with an anticoagulant in order to prevent coagulation of the particles.
  • the anticoagulant examples include water-soluble polysaccharides such as mannitol, lactose, glucose, starches (eg, corn starch), hyaluronic acid and alkali metal salts thereof, proteins such as glycine, fibrin, collagen, sodium chloride, and phosphoric acid Inorganic salts such as sodium hydrogen and the like are appropriately used.
  • the microcapsules can be formed into a disc shape, a film shape, a rod shape (rod shape) or the like by heating and then cooling as in the case of the above (B_l-a).
  • a polyvalent metal compound such as zinc oxide may be added to the organic solvent.
  • the amount of zinc oxide to be used is, for example, about 0.01 to about 100 parts by weight, preferably about 0.1 to about 20 parts by weight, based on 100 parts by weight of the lactic acid / daricholic acid polymer.
  • the particle size of zinc oxide is usually about 0.001 to about 10 ⁇ m, preferably about 0.05 to about 1 m.
  • a sustained-release preparation obtained using a polyvalent metal compound such as zinc oxide has excellent properties such as “high drug uptake rate” and “sustained drug release over a long period of time”. Have.
  • metastin or a derivative thereof or a salt thereof may be dissolved in an aqueous solution of a volatile salt such as ammonium acetate and freeze-dried before use.
  • the freeze-dried product of metastin or a derivative thereof or a salt thereof obtained by treating with ammonium acetate as described above has a small particle size and has excellent operability, and thus is advantageous in producing a sustained-release preparation. .
  • the sustained-release preparation of the present invention can be produced in various forms as it is or using pharmaceutically acceptable additives (eg, stabilizers, preservatives, soothing agents, etc.) as desired.
  • pharmaceutically acceptable additives eg, stabilizers, preservatives, soothing agents, etc.
  • preparations include parenteral preparations (eg, injections, implants, suppositories, etc.), oral preparations (eg, solid preparations such as capsules, tablets, granules, powders, etc., syrups, emulsions, suspensions) And the like).
  • Made Stabilizers as pharmaceutically acceptable additives include, for example, human serum albumin and polyethylene glycol; preservatives such as benzyl alcohol and phenol; and soothing agents such as benzalkonium chloride. And pro-hydrochloric acid.
  • the content of metastin or a derivative thereof or a salt thereof in the preparation of the present invention can be appropriately selected usually from the range of about 0.01
  • the sustained-release preparation of the present invention may be prepared by adding metastin or a derivative thereof or a salt thereof to an aqueous solvent (eg, distilled water, physiological saline, Ringer's solution, etc.) or an oily solvent (eg, olive oil, sesame oil, cottonseed oil, corn oil) Or vegetable oils such as propylene glycol, etc.), and then dissolved in a suspension or emulsified, and then sold in a container for a sustained-release preparation [eg, Duros (trade name, manufactured by Alza).
  • an aqueous solvent eg, distilled water, physiological saline, Ringer's solution, etc.
  • an oily solvent eg, olive oil, sesame oil, cottonseed oil, corn oil
  • vegetable oils such as propylene glycol, etc.
  • the sustained-release preparation of the present invention may be a parenteral preparation (eg, intramuscular, intraperitoneal, subcutaneous, organ, etc.) as a microcapsule or a preparation prepared by using microcapsules as a raw material in various dosage forms.
  • Solids such as injections or implants for intranasal preparations, transmucosal preparations for the nasal cavity, rectum, uterus, etc., oral preparations (eg, capsules (eg, hard capsules, soft capsules, etc.), granules, powders, etc.)
  • the preparations of the present invention are particularly preferably for injection.
  • the sustained-release preparation is a microcapsule
  • the microcapsule may be used as a dispersant (eg, a surfactant such as Tween80, HC0-60, or a polysaccharide such as carboxymethylcellulose, sodium alginate, or hyaluronic acid).
  • a dispersant eg, a surfactant such as Tween80, HC0-60, or a polysaccharide such as carboxymethylcellulose, sodium alginate, or hyaluronic acid.
  • a practical sustained-release injection can be obtained by using an aqueous suspension with a preservative (eg, methyl paraben, propyl paraben, etc.), an isotonic agent (eg, sodium chloride, mannitol, sorbitol, glucose, etc.).
  • a preservative eg, methyl paraben, propyl paraben, etc.
  • an isotonic agent eg, sodium chloride, manni
  • vegetable oils such as sesame oil and corn oil, or mixtures of these with phospholipids such as lecithin, or dispersed with medium-chain fatty acid triglycerides (eg, Migliol 812), are used as oily suspending agents. It should be a sustained-release injection.
  • the particle size of the microphone mouth capsule is within a range that satisfies the degree of dispersion and needle penetration for use as a suspension injection.
  • the average particle diameter may be, for example, in the range of about 0.1 to about 300 / ⁇ .
  • the average particle size is preferably in the range from about 1 to about 150 m, particularly preferably in the range from about 2 to about 100 m.
  • Aseptic treatment of the above microcapsules includes, but is not particularly limited to, a method of sterilizing the entire production process, a method of sterilizing with a gamma ray, and a method of adding a preservative.
  • the preparation of the present invention is safe and has low toxicity, it can be used, for example, for human mammals (for example, monkeys, baboons, chimpanzees, pigs, sea lions, sheep, sheep, dogs, dogs, cats, mice, rats, etc.). Can be administered.
  • human mammals for example, monkeys, baboons, chimpanzees, pigs, sea lions, sheep, sheep, dogs, dogs, cats, mice, rats, etc.
  • the preparation of the present invention can be used for treating or preventing all diseases in which the physiological activity of metastin is involved.
  • the preparation of the present invention since the preparation of the present invention has a cancer metastasis inhibitory activity, it can be used for any cancer (eg, lung cancer, stomach cancer, liver cancer, knee cancer, colon cancer, rectal cancer, colon cancer, prostate cancer, ovarian cancer, cervical cancer). , Breast cancer, kidney cancer, bladder cancer, brain tumor, etc.).
  • the preparation of the present invention has a function of regulating knee function, it can be effectively used for treatment or prevention of various knee diseases (for example, acute or chronic knee inflammation, knee cancer, etc.).
  • the preparation of the present invention since the preparation of the present invention has a placental function regulating action, it is useful for treating or preventing choriocarcinoma, hydatidiform mole, invasive mole, miscarriage, fetal growth deficiency, abnormal glucose metabolism, abnormal lipid metabolism or parturition. It can be used effectively.
  • the dosage of the preparation of the present invention depends on the type and content of the active ingredient metastin or a derivative thereof or a salt thereof, dosage form, duration of release, administration subject, administration route, administration purpose, target disease, symptoms, etc.
  • the amount of the active ingredient may be appropriately maintained in the body at a pharmaceutically effective concentration for a desired duration.
  • one dose of metastin or a derivative or salt thereof may be, for example, about 0.1 to 0.1 mg / day.
  • An amount in the range of about 10 O mg Z kg body weight, preferably in the range of about 1 to 5 O mg / kg body weight is used.
  • the administration frequency is once a week, once every two weeks, once every 1 month, once every 2 months, once every 6 months, etc., the type and content of the KiSS-1 peptide, dosage form, release Can be appropriately selected depending on the duration of the disease, the target disease, the target animal, and the like.
  • Preferred is a one-week to two-month sustained-release preparation, more preferably a one-week to one-month sustained-release preparation.
  • the preparation of the present invention may be used as a medicament for various diseases in which metastin or a derivative thereof or a salt thereof is pharmaceutically effective, particularly a chemotherapeutic agent for cancer treatment, a hormonal therapeutic agent, and an immunotherapeutic agent.
  • concomitant drug a chemotherapeutic agent for cancer treatment
  • a hormonal therapeutic agent e.g., a hormone that is administered to cancer treatment
  • an immunotherapeutic agent e.g., an immunotherapeutic agent for cancer treatment
  • concomitant drug e.g., chemotherapeutic agent for cancer treatment, a hormonal therapeutic agent, and an immunotherapeutic agent.
  • the timing of administration of the preparation of the present invention and the concomitant drug is not limited, and these may be administered to the subject simultaneously or with a time lag.
  • the dose can be appropriately selected based on the clinically used dose.
  • the mixing ratio of the preparation of the present invention and the concomitant drug can be appropriately selected according to the administration subject, administration route, target disease,
  • Chemotherapeutic agents include, for example, alkylating agents (eg, cyclophosphamide, diphosphamide, dimustine, laemustine, carpocon, etc.), antimetabolites (eg, methotrexate, 5-fluorouracil, tegafur, carmofur, UFT, doxyfluridine, Cytarabine, enocitabine, mercaptopurine, mercaptoprin liposide, thioguanine, etc., anticancer antibiotics (for example, mitomycin, adriamycin, daunorubicin, epirubicin, pyrarubicin, idarubicin, bleomycin, pebromycin, clitincin-derived anticancer agents, etc.) , Vinblastine, vindesine, etoposide, camptothecin, irinotecan, etc.), cisplatin, carpoplatin, neda Platinum, paclitaxel
  • hormonal therapies include corticosteroids (eg, prednisolone, prednisone, dexamethasone, cortisone acetate, etc.), estrogen drugs (eg, estradiol, ethinylestradiol, phosphfestrol, chlorotriacene, etc.), Anti-estrogen drugs (such as epithios-nol, mepithiostan, evening moxifen, clomiphene, etc.), luteinizing hormones (such as hydroxyprogesterone haplonate, dydrogesterone, medroxyprogesterone, norethisterone, norletindrone, etc.), LHRH derivatives (such as leuprorelin acetate) Etc.) I can do it.
  • corticosteroids eg, prednisolone, prednisone, dexamethasone, cortisone acetate, etc.
  • estrogen drugs eg, estradi
  • immunotherapeutic agent examples include microorganisms or bacterial components (eg, muramyl dipeptide derivative, picibanil, etc.), polysaccharides having immunopotentiating activity (eg, lentinan, schizophyllan, krestin, etc.), and genetic engineering techniques.
  • Cytokinin eg, interferon, interleukin 2 (IL-2), interleukin 12 (IL-12), tumor necrosis factor (TNF), etc.
  • colony stimulating factor eg, granulocyte colony stimulating factor, erythropoietin Etc.
  • drugs that have been shown to improve cachexia in animal models and clinically, such as cyclooxygenase inhibitors (e.g., indomethacin) (Cancer Reseach, Vol. 49, pp. 5935-5939, 1989], progesterone derivatives (eg, megestrol acetate) [Journal of Clinical Oncology, Journal of Clinical Oncology, Vol.
  • sugar Steroids eg, dexamethasone
  • metoclobramides e.g., metoclobramides
  • tetrahydrocannabinols all of which are the same as described above
  • fat metabolism improvers eg, eicosapentaenoic acid, etc.
  • growth hormone IGF-1
  • cachexia Is a factor which induces TNF- a, L I F, IL one 6, antibody against Oncostatin M can also be used in combination with the present invention formulation.
  • general drugs used for treatment or prevention of diseases of the placenta and the knee are also used as concomitant drugs.
  • agents include anti-inflammatory agents, antipyretic analgesics, antibacterial agents, antiviral agents, hormonal agents, etc., which are commonly used in clinical practice.
  • SEQ ID NO: 7 shows the nucleotide sequence of DNA encoding the amino acid sequence represented by SEQ ID NO: 1.
  • SEQ ID NO: 8 shows the nucleotide sequence of DNA encoding the amino acid sequence represented by SEQ ID NO: 2.
  • SEQ ID NO: 3 shows the nucleotide sequence of DNA encoding the amino acid sequence represented by SEQ ID NO: 3. (SEQ ID NO: 9)
  • SEQ ID NO: 10 shows the nucleotide sequence of DNA encoding the amino acid sequence represented by SEQ ID NO: 4. (SEQ ID NO: 10)
  • Example Example 1 shows the nucleotide sequence of DNA encoding the amino acid sequence represented by SEQ ID NO: 5.
  • This SZO dispersion is added to 800 ml of a 0.1% aqueous solution of polyvinyl alcohol, and the mixture is stirred and emulsified using a homomixer. Stir at room temperature for 3 hours to evaporate dichloromethane, and centrifuge (about 2,000 rpm) to collect microcapsules. After washing twice with 400 ml of distilled water, add 0.2 g of D-mannitol and freeze-dry. Further, in order to remove the residual solvent, vacuum drying is performed at 46 ° C for 3 days to obtain microcapsules containing sustained-release metastin (1-54).
  • This S / II dispersion was added to 80% of a 0.1% aqueous solution of polyvinyl alcohol, and the mixture was stirred and emulsified using a homomixer. After stirring at room temperature for 3 hours to evaporate dichloromethane, microcapsules were collected by centrifugation (about 2,000 rpm). Then, after washing twice with 40 Oinl of distilled water, 0.2 g of D-mannitol was added and freeze-dried. Furthermore, in order to remove the residual solvent, vacuum drying was performed at 46 ° C for 3 days to obtain microcapsules containing sustained-release metastin (1-54).
  • HexaGlycerol PentaStearate one of the polyglycerol fatty acid esters, is heated and melted at 70 ° C after heating and melting at 156 Omg.
  • Add 4 Omg of the freeze-dried powder of (1-54)) mix with stirring, aspirate into a 2.0-millimeter implanted needle, and cool and solidify at room temperature.
  • the obtained cylindrical pellets are cut into 10 orchids to obtain a sustained-release metastin (1-154) containing 2.0 mm in length and 10 m in length and containing HGPS.
  • 1 Omg of zinc oxide are dissolved in 2.7 ml of dichloromethane.
  • 30 Omg of a freeze-dried powder of human-derived KiSS-1 peptide (metastin (45-54)) represented by SEQ ID NO: 3 was added and atomized using Polytron (Kinemachi Rikisha). I do.
  • This SZO dispersion is added to a 0.1% aqueous solution of polyvinyl alcohol (80 Offl 1), and the mixture is stirred and emulsified using a homomixer.
  • 30 Omg of a freeze-dried powder of human-derived KiSS-1 peptide (metastin (40-54)) represented by SEQ ID NO: 2 was added and atomized using a Polytron (Kinemachi Rikisha). I do.
  • This S / 0 dispersion is added to a 0.1% aqueous solution of polyvinyl alcohol 80 On 1 and stirred and emulsified using a homomixer.
  • 1 Omg of zinc oxide were dissolved in 2.7 ml of dichloromethane.
  • organic solvent solution was added 1.2 ml of an aqueous solution of human-derived KiSS-1 peptide (metastin (40-54)) represented by SEQ ID NO: 2 at a concentration of 25 Omg / ml, and Polytron (Kinematica) was added.
  • zinc oxide 10 mg
  • 2 Omg of a freeze-dried powder of human KiSS-1 peptide (metastin (1-54)) represented by SEQ ID NO: 1 was added to this organic solvent solution, and the mixture was atomized using Polytron (Kinemachi Rikisha).
  • This SZO dispersion was added to 800 ml of a 0.1% aqueous solution of polyvinyl alcohol, and the mixture was stirred and emulsified using a homomixer. After stirring at room temperature for 3 hours to evaporate dichloromethane, microcapsules were collected by centrifugation (about 2,000 rpm). Then, after washing twice with 400 ml of distilled water, 0.2 g of D-mannitol was added and freeze-dried. Furthermore, in order to remove the residual solvent, vacuum drying was performed at 46 ° C for 3 days to obtain microcapsules containing sustained-release metastin (1-54).
  • 240 fflg of a freeze-dried powder of human-derived KiSS-1 peptide (metastin (1-54)) represented by SEQ ID NO: 1 was added, and the mixture was pulverized using Polytron (Kinemachi Rikisha).
  • This SZO dispersion was added to a 0.1% aqueous polyvinyl alcohol solution (80 Oml) and stirred and emulsified using a homomixer. After stirring at room temperature for 3 hours to evaporate dichloromethane, microcapsules were collected by centrifugation (about 2,000 rpm). Then, after washing twice with 400 ml of distilled water, 0.24 g of D-mannitol was added and freeze-dried. Further, in order to remove the residual solvent, the microcapsules containing sustained-release metastin (1-54) were obtained by vacuum drying at 46 ° C for 3 days.
  • the S / O dispersion was added to 800 ml of a 0.1% aqueous solution of polyvinyl alcohol, and the mixture was stirred and emulsified using a homomixer. After stirring at room temperature for 3 hours to evaporate dichloromethane, microcapsules were collected by centrifugation (about 2,000 rpm). Then, after washing twice with 40 Oml of distilled water, 0.24 g of D-mannitol was added and freeze-dried. Furthermore, in order to remove the residual solvent, vacuum drying was performed at 46 ° C for 3 days to obtain a microcapsule containing sustained-release metastin (1-54).
  • Experimental example 1
  • the microcapsules containing sustained release metastin (1-54) prepared in Example 10 were The metastin (1-54) was administered subcutaneously at a dose of 6 mg equivalent, and the time-dependent change in blood metastin (1-54) concentration was measured over 2 weeks. The results are shown in Figure 1. As shown in FIG. 1, blood metastin (1-154) concentration was detected over a two-week period. It is apparent from this that the sustained-release preparation of the present invention has excellent sustained-release properties.
  • hOT7T175 cDNA (SEQ ID NO: 6 described in WO 00/24890) was ligated to a pAKKO-neo mammalian cell expression plasmid having neo f as a dominant selectable marker using a standard method, and the ⁇ 0T7T175 expression construct was ligated. Obtained.
  • the 1 ⁇ 0 ⁇ 7 ⁇ 75 expression construct was introduced into B16 cells using Eiiectene (QIAGEN) transfection reagent.
  • the B16 cells were selectively cultured in RPMI 1640 selection medium containing G418 at a final concentration of 1.2 mg / ml, and the formed colonies were isolated using a cloning ring to obtain 17 clones of G418-resistant ⁇ 16 / hl75 cells. The strain was isolated.
  • the 17 transgenic ⁇ 16 ⁇ -175 cell lines were cultured in T25 flasks using RPMI 1640 selection medium containing G418 at a final concentration of 1.2 mg / ml, and total RNA was extracted using RNeasy (QIAGEN).
  • CDNA synthesis was performed using Superscript II (Gibco BRL) using 1 g of RNA extracted from each clone as type III.
  • TadMan PCR was performed using TadMan primer set and TaqMan probe set for T7T175, and several receptor expression strains including cl. 4 were selected.
  • the responsiveness to metastin and metastin (45-54) of the selected 5 clones (cl.4, cl.17, cl.19, cl.2, cl.20) was determined by intracellular Ca using FLIPR. It was examined by detecting changes in 2+ concentration. As a result, for three clones (cl. 4, cl. 17, cl. 19) with high expression levels detected by TaQMan PCR (ca. 1000-7000 copies / ng total thigh), the final concentration was 1 (each). It was confirmed to respond to ⁇ of 6 Micromax metastin or 10- 8 Micromax of metastin (4 5 5 4).
  • Metastin or a derivative thereof or a salt thereof exhibits a more effective pharmacological effect by forming a sustained-release preparation.
  • the sustained-release preparation of the present invention containing metastin or a derivative thereof or a salt thereof is useful for treating or preventing all diseases caused by impaired metastin bioactivity.
  • the preparation of the present invention since the preparation of the present invention has a cancer metastasis inhibitory activity, it is useful for treating or preventing any cancer.
  • the preparation of the present invention since the preparation of the present invention has a placental function regulating action, it is useful for treating or preventing choriocarcinoma, hydatidiform mole, invasive mole, miscarriage, fetal growth deficiency, glucose metabolism disorder, lipid metabolism disorder or delivery abnormality. Useful. Further, since the preparation of the present invention has a knee function-regulating action, it is also useful for treating or preventing knee disease.

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Abstract

Metastin-containing preparations which have an activity of inhibiting cancer metastasis, are useful in treating or preventing any cancers, have and effect of controlling placental functions, are useful in treating or preventing villous cancer, hydatid mole, invasive mole, abortion, fetal hypoplasia, sugar metabolic error, lipid metabolic error or abnormalities in delivery, can exert the drug effect only in a small dose, can relieve side effects, are not necessarily administered everyday, and can relieve inconvenience and pain of patients. Sustained-release preparations which are useful in treating or preventing any diseases caused by disorders in physiological activity of metastin. Because of having an activity of inhibiting cancer metastasis, these preparations are particularly useful in treating or preventing any cancers. Because of having an effect of controlling placental functions, these preparations are useful in treating or preventing villous cancer, hydatid mole, invasive mole, abortion, fetal hypoplasia, sugar metabolic error, lipid metabolic error or abnormalities in delivery. Because of having an effect of controlling pancreatic functions, the preparations are also useful in treating or preventing pancreatic diseases.

Description

明細書  Specification
ペプチド含有製剤 技術分野  Peptide-containing preparations Technical field
本発明は、 メタスチン （metast in) 〔KiSS- 1遺伝子にコードされるペプチド：以 下、 KiSS-1ペプチドと称することがある〕 もしくはその誘導体またはその塩を含有 する製剤に関する。 背景技術  The present invention relates to a preparation containing metastin (a peptide encoded by the KiSS-1 gene: hereinafter sometimes referred to as a KiSS-1 peptide) or a derivative thereof or a salt thereof. Background art
KiSS- 1ペプチドは、 ガン転移抑制遺伝子 KiSS - 1 (Genomics^ 54巻、 145頁- 148頁 、 1998年） にコードされるタンパク質の C端部分ペプチドである。 KiSS- 1遺伝子は 、 ヒト 6番染色体を C8161メラノ一マに移入すると転移能が低下することから、 6 番染色体を移入して得た低転移株と親株からサブトラクティブハイプリダイザ一 シヨン法 (Subtract ive Hybridizat ion Technique) により取得され、 KiSS- 1遺伝 子を C8161メラノーマにトランスフエクションするとその発現量に応じて転移能が 低下することが見出されている （J. Nat l. Cancer Inst.、 88巻、 1731頁- 1737頁、 1996年） 。 また、 KiSS- 1遺伝子のトランスフエクシヨンによりヒト乳ガン細胞 MDA -MB- 435の転移能も低下することが報告されている （Cancer Research, 57巻、 238 4頁 -2387頁、 1997年） 。  KiSS-1 peptide is a C-terminal partial peptide of the protein encoded by the cancer metastasis suppressor gene KiSS-1 (Genomics ^ 54, pp.145-148, 1998). Since the transposition ability of the KiSS-1 gene is reduced when human chromosome 6 is transferred to C8161 melanoma, the subtractive hybridizer method was used from the low-transferred strain obtained by transferring chromosome 6 and the parent strain ( It has been found that transfection of the KiSS-1 gene into C8161 melanoma results in a decrease in the metastatic potential according to the expression level (Subtractive Hybridization Technique) (J. Natl. Cancer Inst. , 88, 1731-1737, 1996). It has also been reported that the translocation of the KiSS-1 gene reduces the metastatic potential of human breast cancer cell MDA-MB-435 (Cancer Research, 57, 2384-2387, 1997).
KiSS- 1遺伝子産物から切り出されて生成するペプチド、 メタスチンには、その遺 伝子がガン転移抑制遺伝子であることから、ガン転移抑制活性を有することが期待 される。 メタスチンは in vi troでは、 メタスチンのヒト型受容体である Τ7ΤΠ5 を発現させた CH0細胞の走化性と浸潤を阻害し、 in vivoでは hOT7T175を発現させた B16—BL6悪性黒色細胞腫の肺への転移性を弱める （Nature, 411卷， 613頁， 2001 年等） 。 さらに、 本遺伝子が胎盤に多量に発現されていること （J. Nat l. Cancer Inst.、 88巻、 1731頁- 1737頁、 1996年） ゃ該ペプチドのヒト型受容体である T7T 175が、 胎盤に多量に、 膝臓にも比較的高く発現 （例、 Nature 411卷, 613頁- 617 頁、 2001年等） していることより、 該ペプチドが胎盤で重要な機能を担っている、 または膝臓においても本ペプチドは何らかの生理機能を発揮しているものと期待 される。 Metastin, a peptide cut out from the KiSS-1 gene product, is expected to have cancer metastasis-suppressing activity because its gene is a cancer metastasis-suppressing gene. In vitro, metastin inhibits chemotaxis and invasion of CH0 cells that express the human receptor for metastin Τ7ΤΠ5, and in vivo to the lung of B16-BL6 malignant melanoma that expresses hOT7T175 Weakens the metastatic properties (Nature, vol. 411, p. 613, 2001, etc.). Furthermore, that the present gene is expressed in a large amount in the placenta (J. Natl. Cancer Inst., Vol. 88, pp. 1731-1737, 1996) ゃ T7T 175, which is a human receptor of the peptide, Due to its high expression in the placenta and relatively high expression in the knee (eg, Nature 411, 613-617, 2001, etc.), the peptide plays an important role in the placenta. Alternatively, it is expected that this peptide also exerts some physiological functions in the knee.
生理活性ポリぺプチドは一般的に生体内での半減期が短いために、メタスチンも しくはその誘導体またはその塩についても、充分な薬理効果を得るためには高投与 量で長期間投与することが必要となる。 しかしながら、例えばメタスチンもしくは その誘導体またはその塩の水溶液を皮下投与する場合には、副作用の発現や、治療 又は予防のための実際の投与において患者の不便性や苦痛が懸念される。 従って、 これらの問題を解決できるメタスチンもしくはその誘導体またはその塩を含有す る有用な製剤の開発が望まれている。 発明の開示  Since bioactive polypeptides generally have a short half-life in vivo, metastin or its derivatives or salts thereof should be administered at high doses for a long period of time to obtain sufficient pharmacological effects. Is required. However, for example, when an aqueous solution of metastin or a derivative thereof or a salt thereof is administered subcutaneously, there is a concern about the occurrence of side effects and the inconvenience and pain of the patient in actual administration for treatment or prevention. Therefore, development of a useful preparation containing metastin or a derivative thereof or a salt thereof capable of solving these problems is desired. Disclosure of the invention
本発明者らは、 上記の課題に鑑み、 鋭意研究を重ねた結果、 メタスチン、 その誘 導体またはその塩を徐放性製剤として投与すると、高転移性ガン細胞に作用して転 移を抑制するほか、長期間にわたりメタスチンもしくはその誘導体またはその塩が 徐放され、 したがって薬効を得るために高投与量を必要としないので、副作用の軽 減が達成でき、かつ連日投与することがないため患者の不便性や苦痛も軽減される 等の臨床上の医薬として優れた性質を本発明の製剤が有していることを見出し、本 発明を完成するに至った。  In view of the above problems, the present inventors have conducted intensive studies and as a result, when metastin, its derivative or its salt is administered as a sustained-release preparation, it acts on highly metastatic cancer cells and suppresses metastasis In addition, the sustained release of metastin or its derivatives or its salts over a long period of time does not require high doses to achieve medicinal effects, so that side effects can be reduced and daily administration is not required. The present inventors have found that the preparation of the present invention has excellent properties as a clinical medicine, such as reduced inconvenience and pain, and completed the present invention.
即ち、 本発明は、  That is, the present invention
( 1 ) メタスチンもしくはその誘導体またはその塩を含有する徐放性製剤、 (1) a sustained-release preparation containing metastin or a derivative or salt thereof,
( 2 ) メタスチンを含有する上記 （1 ) 記載の徐放性製剤、 (2) The sustained-release preparation according to the above (1), which comprises metastin,
( 3 ) メタスチンまたはその誘導体が、 配列番号： 1で表わされるアミノ酸配列の N末端から 4 7ないし 5 4番目のアミノ酸配列を含有し、かつ 8ないし 5 4個のァ ミノ酸残基からなるペプチドもしくはその誘導体である上記（1 )記載の徐放性製 剤、  (3) a peptide wherein metastin or a derivative thereof contains the amino acid sequence at the 47th to 54th position from the N-terminus of the amino acid sequence represented by SEQ ID NO: 1 and comprises 8 to 54 amino acid residues Or a sustained-release preparation according to the above (1), which is a derivative thereof,
( 4 ) ペプチドが、 配列番号： 1で表わされるアミノ酸配列における N末端から 4 7ないし 5 4番目のアミノ酸配列を C末端に有するペプチドである上記（3 )記載 の徐放性製剤、 (5) ペプチドが、 8ないし 15個のアミノ酸残基からなるペプチドである上記 （ 3) または （4) 記載の徐放性製剤、 (4) The sustained-release preparation according to (3), wherein the peptide is a peptide having an amino acid sequence of 47 to 54th from the N-terminus in the amino acid sequence represented by SEQ ID NO: 1 at the C-terminus. (5) The sustained-release preparation according to (3) or (4), wherein the peptide is a peptide consisting of 8 to 15 amino acid residues.
(6) ペプチドが、 配列番号： 2、 配列番号： 3、 配列番号： 4または配列番号： 5で表わされるアミノ酸配列からなるペプチドである上記（3)記載の徐放性製剤  (6) The sustained-release preparation according to the above (3), wherein the peptide is a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5.
(7)メタスチンもしくはその誘導体またはその塩及びキヤリァ一を含有する上記(7) The above containing metastin or a derivative thereof or a salt thereof and a carrier
(I) 記載の徐放性製剤、 (I) described sustained release formulation,
(8) キャリア一がポリマ一である上記 （7) 記載の徐放性製剤、  (8) The sustained-release preparation according to the above (7), wherein the carrier is a polymer,
(9) ポリマーが生体内分解性ポリマーである上記 （8) 記載の徐放性製剤、 (10) 生体内分解性ポリマーが脂肪族ポリエステルである上記 （9)記載の徐放 性製剤、  (9) the sustained release preparation according to (8), wherein the polymer is a biodegradable polymer; (10) the sustained release preparation according to (9), wherein the biodegradable polymer is an aliphatic polyester;
(I I)脂肪族ポリエステルが乳酸 Zグリコール酸重合体である上記（10) 記載 の徐放性製剤、  (II) The sustained release preparation according to the above (10), wherein the aliphatic polyester is a lactic acid Z glycolic acid polymer,
(12)乳酸ノグリコール酸重合体の乳酸 Zグリコール酸組成比が約 100Z0な いし約 40/60である上記 （11) 記載の徐放性製剤、  (12) The sustained-release preparation according to the above (11), wherein the composition ratio of the lactic acid-noglycolic acid polymer to the lactic acid-Z glycolic acid is about 100Z0 or about 40/60.
(13)乳酸/ダリコール酸重合体の重量平均分子量が約 3， 000ないし約 80 , 000である上記 （11) 記載の徐放性製剤、  (13) The sustained-release preparation according to the above (11), wherein the weight average molecular weight of the lactic acid / daricholic acid polymer is about 3,000 to about 80,000.
(14) ポリマーが生体内非分解性ポリマーである上記 （8) 記載の徐放性製剤、 (14) The sustained-release preparation according to the above (8), wherein the polymer is a non-degradable polymer in vivo.
(15)生体内非分解性ポリマーがポリグリセリン脂肪酸エステルである上記（1 4) 記載の徐放性製剤、 (15) The sustained-release preparation according to the above (14), wherein the non-degradable polymer in vivo is a polyglycerin fatty acid ester,
(16) 癌の治療又は予防剤である上記 （1) 記載の徐放性製剤、  (16) The sustained-release preparation according to the above (1), which is an agent for treating or preventing cancer,
(17) 胎盤機能改善剤である上記 （1) 記載の徐放性製剤、  (17) The sustained release preparation according to the above (1), which is a placental function improving agent,
(18) 非経口投与剤である上記 （1) 記載の徐放性製剤、  (18) The sustained release preparation according to the above (1), which is a parenteral administration preparation,
(19) 皮下投与剤である上記 （1) 記載の徐放性製剤、  (19) The sustained-release preparation according to the above (1), which is a subcutaneous preparation,
(20) 筋肉内投与剤である上記 （1) 記載の徐放性製剤、  (20) The sustained-release preparation according to the above (1), which is an intramuscular preparation,
(21) 腹腔内投与剤である上記 （1) 記載の徐放性製剤、  (21) The sustained-release preparation according to the above (1), which is an intraperitoneal agent,
(22)徐放性製剤を製造するためのメタスヂンもしくはその誘導体またはその塩 の使用、 (23) 哺乳動物に対して上記 （1) 記載の徐放性製剤の有効量を投与することを 特徴とする癌の治療又は予防方法等に関する。 (22) Use of metaspine or a derivative or salt thereof for producing a sustained-release preparation, (23) A method for treating or preventing cancer, which comprises administering to a mammal an effective amount of the sustained-release preparation described in (1) above.
さらに本発明は、  Furthermore, the present invention
(24) ペプチドが、 配列番号： 1で表わされるアミノ酸配列の N末端から 47な いし 54番目のアミノ酸配列を C末端に有し、かつ 8ないし 54個のアミノ酸残基 からなるペプチドである上記 （4) 記載の徐放性製剤、  (24) The above-mentioned peptide, wherein the peptide has an amino acid sequence at the 47th to 54th position from the N-terminus of the amino acid sequence represented by SEQ ID NO: 1 at the C-terminus, and comprises 8 to 54 amino acid residues. 4) The sustained-release preparation according to the above,
(25) ペプチドが、 配列番号： 2、 配列番号： 3、 配列番号： 4または配列番号 ： 5で表されるアミノ酸配列からなるペプチドである上記 （3) 記載の徐放性製剤 (26) マイクロカプセル剤である上記 （1) 記載の徐放性製剤、  (25) The sustained-release preparation according to (3), wherein the peptide is a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5. The sustained-release preparation according to the above (1), which is a capsule,
(27) 経口投与剤である上記 （1) 記載の徐放性製剤、  (27) The sustained release preparation according to the above (1), which is an orally administered drug,
(28) 注射剤または点滴剤である上記 （1) 記載の徐放性製剤、  (28) The sustained-release preparation according to the above (1), which is an injection or infusion.
(29) キャリア一がタンパク質、 ポリサッカライド、 リボソーム及びノ又は無機 物である上記 （7) 記載の徐放性製剤、  (29) The sustained-release preparation according to the above (7), wherein the carrier is a protein, a polysaccharide, a ribosome, or a mineral.
(30) メタスチンもしくはその誘導体またはその塩の使用量が、 キャリアーに対 して約 0.01ないし約 50% (w/w) である上記 （7) 記載の徐放性製剤、 (31) メタスチンもしくはその誘導体またはその塩の使用量が、 キャリアーに対 して約 0. 1ないし約 50% (w/w) である上記 （7) 記載の徐放性製剤、 (32) メタスチンもしくはその誘導体またはその塩の使用量が、 キャリアーに対 して約 0. 1ないし約 30% (w/w) である上記 （7) 記載の徐放性製剤、 (30) The sustained-release preparation according to (7), wherein the amount of metastin or a derivative thereof or a salt thereof used is about 0.01 to about 50% (w / w) with respect to the carrier, (31) metastin or a salt thereof. The sustained release preparation according to the above (7), wherein the amount of the derivative or a salt thereof used is about 0.1 to about 50% (w / w) with respect to the carrier, (32) metastin or a derivative thereof or a salt thereof. The sustained release preparation according to the above (7), wherein the amount of the drug used is about 0.1 to about 30% (w / w) with respect to the carrier.
(33)乳酸 Zグリコール酸重合体の乳酸 Zグリコール酸組成比が約 100/0な いし約 50/50である上記 （11) 記載の徐放性製剤、 (33) The sustained-release preparation according to the above (11), wherein the lactic acid-Z-glycolic acid polymer has a lactic acid-Z-glycolic acid composition ratio of about 100/0 to about 50/50.
(34)乳酸/ダリコール酸重合体の乳酸/ダリコール酸組成比が約 75/25な いし約 50Z50である上記 （11) 記載の徐放性製剤、  (34) The sustained-release preparation according to (11), wherein the lactic acid / daricholic acid polymer has a lactic acid / daricholic acid composition ratio of about 75/25 to about 50Z50.
( 35)乳酸 Zダリコール酸重合体の重量平均分子量が約 5, 000ないし約 25, 000である上記 （11) 記載の徐放性製剤、  (35) The sustained release preparation according to the above (11), wherein the weight average molecular weight of the lactic acid Z dalicholate polymer is from about 5,000 to about 25,000.
(36)乳酸ノグリコール酸重合体の重量平均分子量が約 7, 000ないし約 20， 000である上記 （11) 記載の徐放性製剤、 (37)メタスチンもしくはその誘導体またはその塩の使用量が、 乳酸 Zグリコ一 ル酸重合体に対して約 0. 1ないし約 50% (w/w) である上記 （11)記載の 徐放性製剤、 (36) The sustained-release preparation according to the above (11), wherein the weight average molecular weight of the lactic acid noglycolic acid polymer is about 7,000 to about 20,000. (37) The sustained release according to the above (11), wherein the amount of metastin or a derivative thereof or a salt thereof is about 0.1 to about 50% (w / w) based on the lactic acid Z-glycolic acid polymer. Formulation,
(38) さらに多価金属化合物を含有する上記 （11) 記載の徐放性製剤、 (39) 多価金属化合物が酸化亜鉛である上記 （38) 記載の徐放性製剤、 (40)酸化亜鉛の使用量が、 乳酸 Zグリコール酸重合体 100重量部に対して約 (38) The sustained release preparation according to (11), further comprising a polyvalent metal compound, (39) the sustained release preparation according to (38), wherein the polyvalent metal compound is zinc oxide, (40) zinc oxide Amount of lactic acid-Z glycolic acid polymer is about 100 parts by weight
0. 01ないし約 100重量部である上記 （39) 記載の徐放性製剤、 0.01 to about 100 parts by weight of the sustained release preparation according to the above (39),
(41) メタスチンもしくはその誘導体またはその塩、及びその他の任意の医薬活 性物質を含有する上記 （1) 記載の徐放性製剤、  (41) The sustained-release preparation according to the above (1), which comprises metastin or a derivative thereof or a salt thereof, and any other pharmaceutically active substance.
(42)その他の任意の医薬活性物質が抗癌剤である上記 （41)記載の徐放性製 剤、  (42) The sustained-release preparation according to the above (41), wherein the other arbitrary pharmaceutically active substance is an anticancer agent,
(43)哺乳動物に対して上記 （1)記載の徐放性製剤の有効量を投与することを 特徴とする癌の転移を抑制する方法、  (43) a method for suppressing metastasis of cancer, which comprises administering to a mammal an effective amount of the sustained-release preparation according to (1),
(44)哺乳動物に対して上記 （1)記載の徐放性製剤の有効量を投与することを 特徴とする胎盤機能不全が関与する疾患の治療又は予防方法、  (44) a method for treating or preventing a disease associated with placental dysfunction, which comprises administering to a mammal an effective amount of the sustained-release preparation according to (1),
(45)哺乳動物に対して上記 （1)記載の徐放性製剤の有効量を投与することを 特徴とする膝臓機能不全が関与する疾患の治療又は予防方法、  (45) a method for treating or preventing a disease involving knee dysfunction, which comprises administering to a mammal an effective amount of the sustained-release preparation according to (1),
(46) 任意の医薬を併用する、 上記 （43) 〜 （45) のいずれかに記載の治療 又は予防方法等も提供する。 図面の簡単な説明  (46) The therapeutic or preventive method according to any one of (43) to (45) above, which further comprises using any drug in combination. BRIEF DESCRIPTION OF THE FIGURES
図 1は、 ラットにおける徐放性メタスチン （1-54) 含有マイクロカプセルの 皮下投与後の血中メタスチン （1-54)濃度 （pmol/ml) の経時変化を示すグラフ である。 発明を実施するための最良の形態  FIG. 1 is a graph showing the time course of blood metastin (1-54) concentration (pmol / ml) after subcutaneous administration of sustained-release metastin (1-54) -containing microcapsules in rats. BEST MODE FOR CARRYING OUT THE INVENTION
本発明で用いられる 「メタスチン」 は、 配列番号： 1で表されるアミノ酸配列に おける N末端から 1番目ないし 54番目のアミノ酸の連続した 54個全てのアミ ノ酸残基を有し、 かつ C末端がアミド （一 C〇NH 2) であるペプチドを意味する 本発明で用いられる 「メタスチンの誘導体」 は、 メタスチンの生理活性 （例えば 、 メタスチンとその受容体 （例えば、 WOOO/24890などに記載の T7T175など） の結 合活性、 メタスチンによって引き起こされる受容体発現細胞の細胞刺激活性など） と実質的に同等の活性を有し、配列番号： 1で表されるアミノ酸配列における N末 端から 4 7番目ないし 5 4番目の連続した 8個全てのアミノ酸残基（配列番号： 1 で表わされるアミノ酸配列において、 N末端から 4 7ないし 5 4番目のアミノ酸配 列） を含有するペプチドまたはその誘導体を意味する。 その C末端は、 アミド （一 C O NH 2) であってもよく、 また、 力ルポキシル基 （― C O OH) 、 カルポキシ レート基 （C O O— ) 及びエステル （一 C O O R) の何れであってもよいが、 C末 端の力ルポキシル基がアミド化されたアミド体であることが好ましい。 また、 メタ スチンの誘導体は、 メタスチンの生理活性と実質的に同等の活性を有し、 かつ、 配 列番号： 1で表わされるアミノ酸配列において、 N末端から 4 7ないし 5 4番目の アミノ酸配列 （8個の必須アミノ酸残基） を有するものであれば、 その長さは特に 限定されないが、 8ないし 5 4個のアミノ酸残基（好ましくは、 8ないし 1 5個の アミノ酸残基）からなるペプチドもしくはその誘導体が好ましく用いられる。すな わち、 配列番号： 1で表わされるアミノ酸配列において、 N末端から 4 7ないし 5 4番目のアミノ酸配列を含有し、かつ 8ないし 5 4個のアミノ酸残基からなるぺプ チドもしくはその誘導体が好ましく、 なかでも、 配列番号： 1で表わされるァミノ 酸配列において、 N末端から 4 7ないし 5 4番目のアミノ酸配列を含有し、かつ 8 ないし 1 5個のアミノ酸残基からなるぺプチドもしくはその誘導体が好ましい。 また、 前記したメタスチンの誘導体において、 8個の必須アミノ酸残基 （配列番 号： 1で表わされるアミノ酸配列において、 N末端から 4 7ないし 5 4番目のアミ ノ酸配列） の配置は、 該メタスチンの誘導体がメタスチンの生理活性と実質的に同 等の活性を有するのであれば、何れの部位に配置されていてもよいが、該 8個の必 須アミノ酸残基は C末端に配置されるのが望ましい。 すなわち、 配列番号： 1で表 わされるアミノ酸配列において、 N末端から 4 7ないし 5 4番目のアミノ酸配列を C末端に有し、 8ないし 54個のアミノ酸残基からなるペプチドもしくはその誘導 体が好ましく、 なかでも、 配列番号： 1で表わされるアミノ酸配列において、 N末 端から 47ないし 54番目のアミノ酸配列を C末端に有し、 8ないし 15個のアミ ノ酸残基からなるぺプチドもしくはその誘導体が好ましい。 The “metastin” used in the present invention includes all 54 consecutive amino acids from the N-terminal to the first to 54th amino acids in the amino acid sequence represented by SEQ ID NO: 1. The term “metastin derivative” used in the present invention refers to a peptide having an amino acid residue and having an amide (one C 一 NH 2 ) at the C-terminus. The physiological activity of metastin (for example, metastin and its receptor) (Eg, T7T175 described in WOOO / 24890, etc.), cell stimulating activity of receptor-expressing cells caused by metastin, etc.) and represented by SEQ ID NO: 1. All 47 consecutive amino acids from the N-terminus to the 47th to 54th amino acid residue in the amino acid sequence (47 to 54th amino acid sequence from the N-terminus in the amino acid sequence represented by SEQ ID NO: 1) ) Or a derivative thereof. The C-terminus may be an amide (one-CO NH 2 ), or may be any of a carboxylate group (—CO OH), a carboxylate group (COO—), and an ester (one COOR). It is preferable that the C-terminal carbonyl group is an amidated amide. In addition, the metastin derivative has substantially the same activity as the metastin physiological activity, and in the amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence at the 47th to 54th amino acid sequence from the N-terminal ( The length is not particularly limited as long as it has (8 essential amino acid residues), but a peptide consisting of 8 to 54 amino acid residues (preferably, 8 to 15 amino acid residues) Alternatively, a derivative thereof is preferably used. That is, in the amino acid sequence represented by SEQ ID NO: 1, a peptide or a derivative thereof containing the 47th to 54th amino acid sequence from the N-terminal and consisting of 8 to 54 amino acid residues In particular, in the amino acid sequence represented by SEQ ID NO: 1, a peptide comprising the amino acid sequence at the 47th to 54th position from the N-terminus and consisting of 8 to 15 amino acid residues, or a peptide thereof. Derivatives are preferred. In the metastin derivative described above, the arrangement of eight essential amino acid residues (amino acid sequence at positions 47 to 54 from the N-terminus in the amino acid sequence represented by SEQ ID NO: 1) is as follows: May be located at any site, as long as the derivative has substantially the same activity as the metastin bioactivity, but the eight essential amino acid residues are located at the C-terminus. Is desirable. That is, in the amino acid sequence represented by SEQ ID NO: 1, the 47th to 54th amino acid sequence from the N-terminal is A peptide or derivative thereof having 8 to 54 amino acid residues at the C-terminus is preferable. In particular, in the amino acid sequence represented by SEQ ID NO: 1, the 47th to 54th amino acid sequence from the N-terminal is Peptides having 8 to 15 amino acid residues at the C-terminus or derivatives thereof are preferred.
本発明で用いられる 「ペプチドの誘導体」 は、 特定のアミノ酸配列で示されるぺ プチドの C末端が、 アミド （一 CONH2) 、 カルボキシル基 （― COOH) 、 力 ルポキシレート基 （COCT) 及びエステル （一 COOR) の何れかに変換された ものを包含する。例えば、 特定のアミノ酸配列で示されるペプチドの C末端がアミ ド （一 CONH2) である場合には、 ペプチドの誘導体とは、 C末端がカルボキシ ル基 （一 COOH) 、 カルポキシレート基 （COO_) 及びエステル （一 COOR ) の何れかに変換された力ルポキシル体、 カルボキシレ一ト体及びエステル体をそ れぞれ包含し、特定のアミノ酸配列で示されるペプチドの C末端が力ルポキシル基 (一 COOH) である場合には、 ペプチドの誘導体とは、 C末端が、 アミド （一 C ONH2) 、 カルポキシレート基 （COO— ) 及びエステル （—COOR) の何れか に変換されたアミド体、 カルボキシレ一ト体及びエステル体をそれぞれ包含する。 ぺプチドもしくはその誘導体としては、 例えば C末端がアミド （一 CONH2) で あるアミド体が好ましく用いられる。 In the “peptide derivative” used in the present invention, the C-terminal of a peptide represented by a specific amino acid sequence has an amide (-CONH 2 ), a carboxyl group (—COOH), a carboxylate group (COCT) and an ester (1-COH). COOR). For example, when the C-terminus of a peptide represented by a specific amino acid sequence is an amide (one CONH 2 ), a peptide derivative means that the C-terminus has a carboxy group (one COOH) or a carboxylate group (COO_ ) And an ester (a COOR), each of which includes a lipoxyl form, a carboxylate form, and an ester form, and the C-terminal of the peptide represented by the specific amino acid sequence has a lipoxyl group ( In the case of (COOH), a peptide derivative is an amide in which the C-terminus has been converted to any of an amide (-CONH 2 ), a carboxylate group (COO-) and an ester (-COOR) , Carboxylates and esters. As the peptide or a derivative thereof, for example, an amide having an amide (one CONH 2 ) at the C-terminus is preferably used.
上記メタスチンもしくはその誘導体は、 例えば発酵生産物、 合成化合物、 合成べ プチドなどの何れであってもよく、 また、 天然由来のものでもよく、遺伝子工学的 手法によつて製造された組み換え型べプチドでもよい。  The metastin or a derivative thereof may be, for example, any of a fermentation product, a synthetic compound, a synthetic peptide, and the like, or may be of natural origin, and may be a recombinant peptide produced by a genetic engineering technique. May be.
天然由来のメタスチンもしくはその誘導体としては、 例えばヒト、 サル、 マント ヒヒ、 チンパンジー、 ブ夕、 ゥシ、 ヒッジ、 ゥマ、 マウス、 ラッ卜などのあらゆる 哺乳動物由来のメタスチンもしくはその誘導体が用いられ、中でもヒト由来のもの が好ましい。本発明の製剤をヒトに適用する場合は、 とりわけヒト由来のメタスチ ンもしくはその誘導体を用いるのが好ましい。  Examples of the naturally occurring metastin or a derivative thereof include, for example, metastin or a derivative thereof derived from all mammals such as humans, monkeys, baboons, chimpanzees, bushus, sea lions, higgins, horses, mice, rats, and the like. Among them, those derived from humans are preferred. When the preparation of the present invention is applied to humans, it is particularly preferable to use metastin derived from human or a derivative thereof.
本発明に用いられるメタスチンもしくはその誘導体として好ましくは、 例えば、 配列番号： 1で表されるアミノ酸配列において、 N末端から 47ないし 54番目の アミノ酸配列を C末端に有し、かつ 8ないし 15個のアミノ酸配列からなるぺプチ ドなどがあげられる。 As the metastin or a derivative thereof used in the present invention, preferably, for example, in the amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence at the 47th to 54th position from the N-terminus is located at the C-terminus, and 8 to 15 amino acids Petit consisting of amino acid sequence And so on.
さらに好ましくは、 配列番号： 2、 配列番号： 3、 配列番号： 4または配列番号 ： 5で表されるアミノ酸配列からなるペプチド （特にこれらのアミド体） などがあ げられる。 あるいは、 WO 01/751 04に記載の配列番号： 1、 配列番号 ： 2または配列番号： 3で表されるアミノ酸配列を含有するマウスまたはラット由 来のペプチド （Kiss- 1遺伝子産物） を用いてもよい。  More preferably, a peptide comprising the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 (especially an amide thereof), and the like can be mentioned. Alternatively, a mouse or rat-derived peptide (Kiss-1 gene product) containing the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 described in WO 01/751 04 is used. Is also good.
また、 メタスチンもしくはその誘導体の C末端は通常アミド （一 CONH2) で あるが、 エステル （一 COOR) あるいは力ルポキシル基 （一 COOH) または力 ルポキシレート基 （COO— ) であってもよい。 中でも C末端の力ルポキシル基が アミド化されたアミド体であるものが特に好ましい。 The C-terminus of metastin or a derivative thereof is usually an amide (one CONH 2 ), but may be an ester (one COOR), a hydroxyl group (one COOH), or a hydroxyl group (COO—). Among them, an amide in which the C-terminal carbonyl group is amidated is particularly preferable.
ここでエステルにおける Rとしては、 例えば、 メチル、 ェチル、 n—プロピル、 イソプロピルもしくは n—ブチルなどの Cェ— 6アルキル基、 例えば、 シクロペンチ ル、 シクロへキシルなどの C3— 8シクロアルキル基、 例えば、 フエニル、 α—ナフ チルなどの C 6^ 2ァリール基、 例えば、 ベンジル、 フエネチルなどのフエ二ルー Cト 2アルキル基もしくは a—ナフチルメチルなどの a—ナフチルー 2アルキ ル基などの C 7— 4ァラルキル基などのほか、 経口用エステルとして汎用されるピ バロィルォキシメチル基などが用いられる。 Here, as R in the ester, e.g., methyl, Echiru, n- propyl, C E, such as isopropyl or n- butyl - 6 alkyl group, for example, C 3 of cyclopentyl Le, cyclohexane, etc. cyclohexyl - 8 cycloalkyl group, for example, C 7 such as phenyl, alpha-naphthyl C 6 ^ 2 Ariru group such as for example, benzyl, phenylene route C DOO 2 alkyl or a- naphthylmethyl etc. a- Nafuchiru 2 alkyl Le group such phenethyl — In addition to 4- aralkyl groups, pivaloyloxymethyl groups commonly used as oral esters are used.
さらに、本発明で用いられるメタスチンもしくはその誘導体には、 メチォニン残 基などの N末端のァミノ基が保護基 （例えば、 ホルミル基、 ァセチルなどの C2— 6 アルカノィル基などの Cェ—6ァシル基など） で保護されているもの、 N端側が生体 内で切断され生成したダル夕ミル基がピ口グルタミン酸化したもの、分子内のアミ ノ酸の側鎖上の置換基 （例えば、 一 OH、 一 SH、 -COOH, アミノ基、 イミダ ゾ一ル基、 インドール基、 グァニジノ基など） が適当な保護基 （例えば、 ホルミル 基、 ァセチルなどの C2— 6アルカノィル基などの (：卜 6ァシル基など） で保護され ているもの、あるいは糖鎖が結合したいわゆる糖ペプチドなどの複合ペプチドなど も含まれる。 糖タンパク質である場合、 糖鎖としては、 例えば D—マンノース、 D —ガラクト一ス、 L一フルクトース等の中性糖、 D—ダルコサミン、 D—ガラクト サミン等のアミノ糖、 及びシアル酸等が挙げられる。 メタスチンもしくはその誘導体は塩を形成していてもよく、メタスチンもしくは その誘導体の塩としては、 とりわけ薬理学的に許容される塩が好ましい。 この様な 塩としては、 例えば無機酸 （例えば塩酸、 りん酸、 臭化水素酸、 硫酸等） との塩、 有機酸 （例えば酢酸、 ギ酸、 プロピオン酸、 フマル酸、 マレイン酸、 コノヽク酸、 酒 石酸、 クェン酸、 リンゴ酸、 蓚酸、 安息香酸、 メタンスルホン酸、 ベンゼンスルホ ン酸等） との塩、 無機塩基との塩 （例えばナトリウム塩、 カリウム塩等のアルカリ 金属塩；カルシウム塩、 マグネシウム塩等のアルカリ土類金属塩；アルミニウム塩 、 アンモニゥム塩等） 、 有機塩基との塩 （例えばトリメチルァミン、 トリェチルァ ミン、 ピリジン、 ピコリン、 エタノールァミン、 ジエタノールァミン、 トリェタノ 等が用いられる。 Furthermore, the metastin or its derivatives used in the present invention, N-terminal of the Amino group protecting groups such as Mechionin residues (e.g., C 2, such as a formyl group, Asechiru - 6 Ashiru group - C E such as 6 Arukanoiru group ), The N-terminal side is cleaved in vivo, the dalminyl mill group is oxidized with glutamine, and the substituent on the side chain of the amino acid in the molecule (for example, mono-OH, one SH, -COOH, amino group, Imida zone Ichiru group, indole group, Guanijino group, etc.) a suitable protecting group (e.g., formyl group, C 2 such Asechiru - such as 6 Arukanoiru group (: Bok 6 Ashiru group ) Or complex peptides such as so-called glycopeptides to which sugar chains are bound. In the case of glycoproteins, the sugar chains include, for example, D-mannose And neutral sugars such as D-galactose and L-fructose; amino sugars such as D-darcosamine and D-galactosamine; and sialic acid. Metastin or a derivative thereof may form a salt, and as a salt of metastin or a derivative thereof, a pharmacologically acceptable salt is particularly preferable. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.), and organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, conodic acid) , Tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc.), salts with inorganic bases (eg, alkali metal salts such as sodium salt, potassium salt, etc.); calcium salts And alkaline earth metal salts such as magnesium salts; aluminum salts and ammonium salts; salts with organic bases (eg, trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, trietano, etc.). .
本発明に用いられるメタスチンもしくはその誘導体は、 公知の方法 （例えば、 W 0 00/24890, J. Chem. Soc. , Perkin Trans. 1巻， 1748頁， 2001年)等に記載の方法な ど） あるいはそれに準じる方法に従って製造することができ、例えば公知のぺプチ ドの合成法に従って、あるいは公知のペプチド又は前駆体を適当なぺプチダーゼで 切断することによって製造することができる。ペプチドの合成方法は、 例えば固相 合成法、 液相合成法の何れでもよい。 即ち、 目的とするペプチドは、 そのペプチド 又は前駆体を構成し得る部分べプチドもしくはアミノ酸と残余部分とを縮合させ、 生成物が保護基を有する場合は保護基を脱離することにより製造することができ る。 公知の縮合方法や保護基の脱離としては、 例えば以下の [1]ないし [5]に記載さ れた方法が挙げられる。  Metastin or a derivative thereof used in the present invention can be obtained by a known method (for example, a method described in WO 00/24890, J. Chem. Soc., Perkin Trans. 1, 1748, 2001) and the like. Alternatively, it can be produced according to a method analogous thereto, for example, according to a known peptide synthesis method, or can be produced by cleaving a known peptide or precursor with an appropriate peptide. The method for synthesizing the peptide may be, for example, either a solid phase synthesis method or a liquid phase synthesis method. That is, the target peptide can be produced by condensing a partial peptide or amino acid capable of constituting the peptide or the precursor with the remaining portion, and removing the protecting group when the product has a protecting group. Can be done. Examples of known condensation methods and elimination of protecting groups include the methods described in the following [1] to [5].
[1] M. Bodanszky及び M. A. Ondet t i, ペプチド シンセシス （Pept ide Synthe s is) ， Interscience Publ ishers, New York (1966年)  [1] M. Bodanszky and M. A. Ondet ti, Peptide Synthes is, Interscience Publ ishers, New York (1966)
[2] Schroeder及び Luebke, ザペプチド (The Pept ide) , Academic Press, N ew York (1965年）  [2] Schroeder and Luebke, The Peptide, Academic Press, New York (1965)
[3] 泉屋信夫他、 ペプチド合成の基礎と実験、 丸善 （株） （1975年）  [3] Nobuo Izumiya et al. Basics and experiments on peptide synthesis, Maruzen Co., Ltd. (1975)
[4] 矢島治明及び榊原俊平、 生化学実験講座 1、 ペプチド又は前駆体の化学 IV、 2 [4] Haruaki Yajima and Shunpei Sakakibara, Laboratory of Biochemistry 1, Chemistry of Peptides or Precursors IV, 2
05、 （1977年） [5] 矢島治明監修、 続医薬品の開発第 14巻ペプチド合成広川書店 また、 反応後は通常の精製法、 例えば溶媒抽出 ·蒸留-カラムクロマトグラフィ —·液体クロマトグラフィー ·再結晶等を組み合わせて目的とするペプチド又は前 駆体を精製単離することができる。上記方法で得られるぺプチド又は前駆体は遊離 体である場合は、公知の方法によって適当な塩に変換することができるし、逆に塩 で得られた場合は、 公知の方法によって遊離体に変換することができる。 05, (1977) [5] Supervised by Haruaki Yajima, Development of Pharmaceuticals Volume 14 Peptide Synthesis Hirokawa Shoten After the reaction, the objective is a combination of ordinary purification methods such as solvent extraction, distillation-column chromatography, liquid chromatography, and recrystallization. Can be purified and isolated. When the peptide or precursor obtained by the above method is in a free form, it can be converted to an appropriate salt by a known method, and conversely, when it is obtained in a salt form, it can be converted into a free form by a known method. Can be converted.
本発明のメタスチンもしくはその誘導体またはその塩を含有する徐放性製剤は、 キャリア一 （担体または基剤） を含有しているのが好ましい。 キャリア一としては 、 例えば、 タンパク質、 ポリサッカライド、 ポリマ一 （生体内分解性ポリマー、 生 体内非分解性ポリマー） 、 リボソーム、 無機物等が挙げられる。  The sustained-release preparation containing metastin or a derivative thereof or a salt thereof of the present invention preferably contains one carrier (carrier or base). Examples of the carrier include proteins, polysaccharides, polymers (biodegradable polymers and non-biodegradable polymers), ribosomes, and inorganic substances.
タンパク質としては、 例えばコラーゲン、 ゼラチン、 フイブリン、 血清アルプミ ン等が挙げられる。  Proteins include, for example, collagen, gelatin, fibrin, serum albumin and the like.
ポリサッカライドとしては、 例えばデンプン、 ヒアルロン酸、 キトサン、 キチン 、 アルギン酸塩、 ァガロース、 デキストラン、 セルロース誘導体等が挙げられる。 生体内分解性ポリマ一としては、 例えば脂肪族ポリエステル、ポリフォスファゼ ン、 ボリオルソエステル等が挙げられる。  Examples of the polysaccharide include starch, hyaluronic acid, chitosan, chitin, alginate, agarose, dextran, and cellulose derivatives. Examples of the biodegradable polymer include aliphatic polyesters, polyphosphazenes, polyorthoesters, and the like.
生体内非分解性ポリマーとしては、例えばポリグリセリン脂肪酸エステル、 シリ コン、 ェチルビニルァセテー卜共重合体、 ポリヒドロキシェチルメチルメ夕ァクリ レート、 ポリビニルアルコール、 ポリビニルピロリドン等が挙げられる。  Examples of the non-degradable polymer in vivo include polyglycerin fatty acid ester, silicon, ethyl vinyl acetate copolymer, polyhydroxyethyl methyl methacrylate, polyvinyl alcohol, and polyvinylpyrrolidone.
リボソームとしては、通常のリボソームに加えて例えばポリエチレングリコ一ル ( P E G) 修飾リボソーム、 糖修飾リボソーム等が挙げられる。  Examples of ribosomes include, in addition to ordinary ribosomes, for example, polyethylene glycol (PEG) -modified ribosomes, sugar-modified ribosomes, and the like.
無機物としては、例えばハイドロキシァパタイト、 トリカルシウムフォスフェイ 卜、 カルシウム力ルポネート、 カルシウムサルフェイト等が挙げられる。  Examples of the inorganic substance include hydroxyapatite, tricalcium phosphate, calcium phosphate, calcium sulfate, and the like.
本発明の徐放性製剤に用いるキャリア一としては、例えばポリマー（例えば生体 内分解性ポリマー、 生体内非分解性ポリマ一等） 等が好ましく、 特に生体内分解性 ポリマ一としては、例えば脂肪族ポリエステル等、 生体内非分解性ポリマーとして は、 例えばポリグリセリン脂肪酸エステル等が好ましい。  As the carrier used in the sustained-release preparation of the present invention, for example, a polymer (for example, a biodegradable polymer, a non-biodegradable polymer, etc.) is preferable, and particularly, a biodegradable polymer is, for example, an aliphatic polymer. As the non-degradable polymer in vivo such as polyester, for example, polyglycerin fatty acid ester and the like are preferable.
脂肪族ポリエステルとしては、例えば乳酸/ダリコール酸重合体等が用いられる 乳酸/ダリコール酸重合体の組成比 （乳酸/ダリコール酸； LZG比） （モル％ ) は約 100Z0ないし約 40Z60が好ましく、更に好ましくは約 100Z0な いし約 50/50であり、特に好ましくは約 75/25ないし約 50/50である 乳酸/ダリコール酸重合体の重量平均分子量は約 3, 000ないし約 80, 00. 0が好ましく、更に好ましくは約 5, 000ないし約 25， 000であり、特に好ま しくは約 7， 000ないし約 20, 000である。 As the aliphatic polyester, for example, a lactic acid / daricholic acid polymer or the like is used. The composition ratio of lactic acid / daricholic acid polymer (lactic acid / daricholic acid; LZG ratio) (mol%) is preferably about 100Z0 to about 40Z60, more preferably about 100Z0 to about 50/50, and particularly preferably about 75/50. The weight average molecular weight of the lactic acid / daricholic acid polymer having a weight average molecular weight of about 3,000 to about 80,000, preferably about 5,000 to about 25,000, and more preferably about 5,000 to about 25,000, Preferably, it is about 7,000 to about 20,000.
乳酸/ダリコール酸重合体の分散度 （重量平均分子量/数平均分子量） は、 好ま しくは約 1.2ないし約 4.0、 更に好ましくは約 1. 5ないし約 3. 5である。 ポリグリセリン脂肪酸エステルとしては、例えばテトラグリセロールモノパルミ テート （TGMP) 、 テ卜ラグリセ口一ルジパルミテート （TGDP) 、 テ卜ラグ リセロールトリパルミテート （TGTP) 、 テトラグリセロールへキサパルミテー 卜 (TGHP) 、 テトラグリセロールモノステアレー卜 (TGMS) 、 テトラダリ セロールジステアレート (TGDS) 、 テトラグリセロールトリステアレート (T GTS) 、 テトラグリセロールへキサステアレート (TGHS) 、 へキサグリセ口 —ルペンタステアレート （HGPS) 、 テ卜ラグリセロールへキサパルミテート ( TGHP) 等が挙げられ、 これらを単独であるいは混合して用いることができる。 本発明の徐放性製剤は、 公知の方法にしたがって製造される。  The dispersity (weight average molecular weight / number average molecular weight) of the lactic acid / daricholic acid polymer is preferably about 1.2 to about 4.0, more preferably about 1.5 to about 3.5. Examples of polyglycerin fatty acid esters include tetraglycerol monopalmitate (TGMP), tetraglycerol monopalmitate (TGDP), tetraglyceryl tripalmitate (TGTP), tetraglycerol hexapalmitate (TGHP), and tetraglycerol monopalmitate. Stearate (TGMS), Tetradari Serol Distearate (TGDS), Tetraglycerol Tristearate (TGTS), Tetraglycerol Hexstearate (TGHS), Hexaglycerose-Lpentastearate (HGPS), Tetra Laglycerol hexapalmitate (TGHP) and the like can be used, and these can be used alone or in combination. The sustained-release preparation of the present invention is produced according to a known method.
本発明の徐放性製剤は、 具体的には、 メタスチンもしくはその誘導体またはその 塩と、 徐放性製剤の製造時に通常用いられるキャリアー （例えば前記キャリアー） とを混合し、 必要に応じて成形することによって製造される。 該キャリア一は、 例 えばメタスチンもしくはその誘導体またはその塩の使用量が、キャリアーに対して 約 0.01ないし約 50 % (wZw)、 好ましくは約 0. 1ないし約 30% (w/w ) となるように使用される。  Specifically, the sustained-release preparation of the present invention is prepared by mixing metastin or a derivative thereof or a salt thereof with a carrier (for example, the above-mentioned carrier) usually used in the production of a sustained-release preparation, and molding as necessary. Manufactured by In the carrier 1, for example, the amount of metastin or a derivative or salt thereof used is about 0.01 to about 50% (wZw), preferably about 0.1 to about 30% (w / w) based on the carrier. Used as
以下に本発明の徐放性製剤として、 例えば（A) 生体内非分解性ポリマ一である ポリグリセリン脂肪酸エステルをキャリア一として用いた「メタスチンもしくはそ の誘導体またはその塩を含有する徐放性製剤」 〔以下、 KiSS- 1ペプチド含有徐放性 製剤と称することがある〕 、 及び（B ) 生体内分解性ポリマーである乳酸/グリコ —ル酸重合体をキャリア一として用いた KiSS-1ペプチド含有徐放性製剤の製造法 について例示する。 Examples of the sustained-release preparation of the present invention include, for example, (A) a sustained-release preparation containing metastin or a derivative thereof or a salt thereof using polyglycerol fatty acid ester, which is a non-degradable polymer in vivo, as a carrier. [Hereinafter, KiSS-1 peptide containing sustained release And (B) a method for producing a KiSS-1 peptide-containing sustained-release preparation using a biodegradable lactic acid / glycolic acid polymer as a carrier.
(A)ポリグリセリン脂肪酸エステルをキヤリァ一として用いた KiSS- 1ぺプチド含 有徐放性製剤  (A) KiSS-1 peptide-containing sustained-release preparation using polyglycerin fatty acid ester as carrier
ポリグリセリン脂肪酸エステルを加温 ·融解し、 メタスチンもしくはその誘導体 またはその塩の粉末を添加し、 攪拌等により均等に分散させる。 その後冷却し、 円 盤状、 フィルム状、 棒状等に成形する。 このようにして所定量のメタスチンもしく はその誘導体またはその塩を含有するポリグリセリン脂肪酸エステルの徐放性製 剤を得ることができる。加温温度は約 5 0ないし約 1 0 0 °C、冷却温度は約 0ない し約 4 0 °Cである。 メタスチンもしくはその誘導体またはその塩の使用量は、 メタ スチンもしくはその誘導体またはその塩の種類、所望の薬理効果及び効果の持続期 間等により異なるが、 ポリグリセリン脂肪酸エステルに対して約 0. 0 1ないし約 5 0 % (w/w) 、 好ましくは約 0. 1ないし約 3 0 % (w/w) である。  Heat and melt the polyglycerin fatty acid ester, add the powder of metastin or a derivative thereof or a salt thereof, and uniformly disperse the mixture by stirring or the like. Then, it is cooled and formed into a disk, film, rod, etc. In this way, a sustained-release preparation of polyglycerin fatty acid ester containing a predetermined amount of metastin or a derivative thereof or a salt thereof can be obtained. The heating temperature is about 50 to about 100 ° C, and the cooling temperature is about 0 to about 40 ° C. The amount of metastin or a derivative thereof or a salt thereof to be used varies depending on the type of metastin or a derivative thereof or a salt thereof, a desired pharmacological effect and duration of the effect, and the like. To about 50% (w / w), preferably about 0.1 to about 30% (w / w).
(B)乳酸/ダリコール酸重合体をキヤリァ一として用いた KiSS_lぺプチド含有徐 放性製剤  (B) KiSS_l peptide-containing sustained-release preparation using lactic acid / daricholic acid polymer as carrier
(B— 1 ) 棒状成形物等の製造法について詳述する。  (B-1) A method for producing a rod-shaped molded product will be described in detail.
(B - 1 - a )  (B-1-a)
乳酸ノグリコール酸重合体を有機溶媒（好ましくはジクロルメタン等）で溶解し 、 メタスチンもしくはその誘導体またはその塩の水溶液を添加後乳化する。 これを 真空乾燥し、メタスチンもしくはその誘導体またはその塩が均等に分散した乳酸/ グリコール酸重合体の粉末を得る。 これを加温し、 冷却することにより、 円盤状、 フィルム状、 棒状 （ロッド状） 等に成形する。 このようにして所定量のメタスチン もしくはその誘導体またはその塩を含有する乳酸 Zダリコール酸重合体の徐放性 製剤を得ることができる。加温温度は約 5 0ないし約 1 0 0 °C、冷却温度は約 0な いし約 4 0 °Cである。 メタスチンもしくはその誘導体またはその塩の使用量は、 メ タスチンもしくはその誘導体またはその塩の種類、所望の薬理効果及び効果の持続 期間等により異なるが、'乳酸/ダリコール酸重合体に対して約 0. 0 1ないし約 5 0 % (w/w) 、 好ましくは約 0. 1ないし約 3 0 % (w/w) 、 特に好ましくは約 1ないし約 2 0 % (w/w) である。 The lactate noglycolic acid polymer is dissolved in an organic solvent (preferably dichloromethane) and emulsified after adding an aqueous solution of metastin or a derivative or salt thereof. This is vacuum-dried to obtain lactic acid / glycolic acid polymer powder in which metastin or a derivative thereof or a salt thereof is uniformly dispersed. This is heated and cooled to form a disk, film, rod, etc. In this way, it is possible to obtain a sustained-release preparation of lactic acid z-daricholic acid polymer containing a predetermined amount of metastin or a derivative thereof or a salt thereof. The heating temperature is about 50 to about 100 ° C, and the cooling temperature is about 0 to about 40 ° C. The amount of metastin or a derivative thereof or a salt thereof varies depending on the type of metastatin or a derivative thereof or a salt thereof, a desired pharmacological effect and a duration of the effect. 0 1 to about 5 0% (w / w), preferably about 0.1 to about 30% (w / w), particularly preferably about 1 to about 20% (w / w).
(Β - 1 - b)  (Β-1-b)
乳酸 グリコール酸重合体を有機溶媒（好ましくはジクロルメタン等） で溶解し 、 メタスチンもしくはその誘導体またはその塩の粉末を添加後、 均一に分散させる 。得られる分散系を真空乾燥し、 メタスチンもしくはその誘導体またはその塩が均 等に分散した乳酸/ダリコール酸重合体の粉末を得る。 これを加温し、冷却するこ とにより、 円盤状、 フィルム状、 棒状 （ロッド状） 等に成形する。 このようにして 所定量のメタスチンもしくはその誘導体またはその塩を含有する乳酸 Zグリコー ル酸重合体の徐放性製剤を得ることができる。加温温度、 冷却温度、 およびメタス チンもしくはその誘導体またはその塩の使用量は、 前記と同様である。  The lactic acid / glycolic acid polymer is dissolved in an organic solvent (preferably, dichloromethane, etc.), and the powder of metastin or a derivative thereof or a salt thereof is added and then uniformly dispersed. The resulting dispersion is dried under vacuum to obtain lactic acid / daricholic acid polymer powder in which metastin or a derivative thereof or a salt thereof is uniformly dispersed. This is heated and cooled to form a disc, film, rod, etc. In this manner, a sustained-release preparation of a polymer of lactic acid-Z-glycolic acid containing a predetermined amount of metastin or a derivative thereof or a salt thereof can be obtained. The heating temperature, the cooling temperature, and the amount of metastin or a derivative or salt thereof used are the same as described above.
(B - 2 ) マイクロカプセル （マイクロスフェアとも称する） の製造法について詳 述する。  (B-2) The method of manufacturing microcapsules (also called microspheres) will be described in detail.
W/OZWエマルシヨン及び O/Wエマルシヨンは、 それぞれ（ i ) メタスチン もしくはその誘導体またはその塩の水溶液、分散液又は懸濁液を内水相とし、 乳酸 /ダリコール酸重合体の有機溶媒溶液を油相とする WZOエマルシヨンを得るか、 又は（i i) メタスチンもしくはその誘導体またはその塩を乳酸 Zグリコール酸重合 体の有機溶媒溶液に溶解あるいは懸濁して油相を得、 この U ) 又は （Π) を水 （ 外水相） に添加し、 分散、 乳化することによって製造される。  W / OZW emulsions and O / W emulsions respectively consist of (i) an aqueous solution, dispersion or suspension of metastin or a derivative or salt thereof as an internal aqueous phase, and an organic solvent solution of lactic acid / daricholic acid polymer as an oil phase. Or (ii) dissolving or suspending metastin or a derivative or salt thereof in an organic solvent solution of lactic acid-Z-glycolic acid polymer to obtain an oil phase, and converting the U) or (Π) into water (Outer aqueous phase), and dispersed and emulsified.
上記 （i) 、 即ち、 メタスチンもしくはその誘導体またはその'塩の水溶液、 分散 液又は懸濁液を内水相とし、乳酸 Zダリコール酸重合体の有機溶媒溶液を油相とす る wzoエマルシヨンは、 以下のようにして製造される。  The above-mentioned (i), that is, wzo emulsion in which an aqueous solution, dispersion or suspension of metastin or a derivative thereof or a salt thereof is used as an internal aqueous phase, and an organic solvent solution of lactic acid z-daricholic acid polymer is used as an oil phase, It is manufactured as follows.
まず、 メタスチンもしくはその誘導体またはその塩を水に溶解、 分散又は懸濁し 、 内水相を製造する。 メタスチン.もしくはその誘導体またはその塩の水溶液、 分散 液又は懸濁液中の濃度は、 例えば 0. 0 0 1ないし 9 0 % (w/w) 、 好ましくは 0. 0 1ないし 8 0 % (w/w) である。  First, metastin or a derivative thereof or a salt thereof is dissolved, dispersed or suspended in water to produce an internal aqueous phase. The concentration of metastin or a derivative or salt thereof in an aqueous solution, dispersion or suspension is, for example, 0.01 to 90% (w / w), preferably 0.01 to 80% (w / w). / w).
上記メタスチンもしくはその誘導体またはその塩の使用量は、メタスチンもしく はその誘導体またはその塩の種類、所望の薬理効果及び効果の持続期間等により異 なるが、 乳酸/グリコール酸重合体に対して、 約 0. 0 1ないし約 5 0 % (w/w ) 、 好ましくは約 0. 1ないし約 3 0 % (w/w) 、 特に好ましくは約 1ないし約 2 0 % (w/w) である。 The amount of the above-mentioned metastin or its derivative or its salt used depends on the type of metastin or its derivative or its salt, the desired pharmacological effect and the duration of the effect. However, based on the lactic acid / glycolic acid polymer, about 0.01 to about 50% (w / w), preferably about 0.1 to about 30% (w / w), particularly preferably about 0.1 to about 30% (w / w) 1 to about 20% (w / w).
必要であれば、メタスチンもしくはその誘導体またはその塩のマイクロカプセル への取り込みを上げるために、 内水相にゼラチン、 寒天、 アルギン酸ナトリウム、 ポリビニルアルコールあるいは塩基性アミノ酸 （例えばアルギニン、 ヒスチジン、 リジン等） 等の薬物保持物質 （例えば、 賦形剤、 助剤など） を加えてもよい。 該薬 物保持物質の添加量は、 メタスチンもしくはその誘導体またはその塩に対し、 通常 約 0. 0 1ないし約 1 0重量倍である。  If necessary, gelatin, agar, sodium alginate, polyvinyl alcohol or basic amino acids (eg, arginine, histidine, lysine, etc.) should be added to the internal aqueous phase to increase the incorporation of metastin or its derivatives or its salts into microcapsules. May be added (eg, excipients, auxiliaries, etc.). The amount of the drug-retaining substance to be added is generally about 0.01 to about 10 times the weight of metastin or a derivative or salt thereof.
内水相は、 一旦凍結乾燥して粉末状態とした後、 適当な濃度となるように水を添 加して溶解して用いてもよい。  The inner aqueous phase may be used after being freeze-dried to a powder state and then dissolved by adding water to an appropriate concentration.
別に、 乳酸 Zグリコール酸重合体を有機溶媒に溶解し、 油相を製造する。  Separately, a lactic acid Z-glycolic acid polymer is dissolved in an organic solvent to produce an oil phase.
前記有機溶媒としては、 ハロゲン化炭化水素 （例えばジクロロメタン、 クロロホ ルム、 クロロェタン、 トリクロロェタン、 四塩化炭素等） 、 脂肪酸エステル （例え ば酢酸ェチル、 酢酸ブチル等） 、 芳香族炭化水素 （例えばベンゼン、 トルエン、 キ シレン等) が挙げられ、 中でもジクロロメ夕ンが好ましい。  Examples of the organic solvent include halogenated hydrocarbons (eg, dichloromethane, chloroform, chloroethane, trichloroethane, carbon tetrachloride, etc.), fatty acid esters (eg, ethyl acetate, butyl acetate, etc.), aromatic hydrocarbons (eg, benzene, Toluene, xylene, etc.), of which dichloromethane is preferred.
有機溶媒中の乳酸 Zダリコール酸重合体の濃度は、該乳酸 Zダリコール酸重合体 の種類、 分子量、 有機溶媒の種類により異なるが、 し酸/ダリコール酸重合体の 重量/ (有機溶媒の重量 +乳酸/ダリコール酸重合体の重量） ] ( X 1 0 0 %) は 、 通常約 0. 0 1ないし約 9 0 % (w/w) 、 好ましくは約 0. 0 1ないし約 7 0 % (w/w) である。 未溶解物がないように溶解するのがよい。  The concentration of the lactic acid Z-daricholic acid polymer in the organic solvent varies depending on the type and molecular weight of the lactic acid Z-daricholic acid polymer and the type of the organic solvent, but the weight of citric acid / daricholic acid polymer / (weight of organic solvent + (Weight of lactic acid / daricholic acid polymer)] (X100%) is usually about 0.01 to about 90% (w / w), preferably about 0.01 to about 70% (w / w). / w). It is better to dissolve so that there is no undissolved matter.
このようにして得られる乳酸ノグリコール酸重合体の有機溶媒溶液 （油相） に、 上記したメタスチンもしくはその誘導体またはその塩の水溶液、分散液又は懸濁液 (内水相) を添加し、 ホモミキサー等で分散、 乳化し、 W/〇エマルシヨンを製造 する。  An aqueous solution, dispersion or suspension (inner aqueous phase) of the above-mentioned metastin or a derivative thereof or a salt thereof is added to an organic solvent solution (oil phase) of the lactic acid noglycolic acid polymer thus obtained, and the mixture is homogenized. Disperse and emulsify with a mixer etc. to produce W / 〇 emulsion.
一方、 上記 （i i) 、 即ち、 メタスチンもしくはその誘導体またはその塩を乳酸/ グリコール酸重合体の有機溶媒溶液に溶解あるいは懸濁して得られる油相は、以下 のようにして製造される。 まず、乳酸/ダリコール酸重合体の有機溶媒溶液を製造する。該有機溶媒として は、上記 wzoエマルションを製造する際に用いた有機溶媒と同様のものが用いら れる。 On the other hand, the above (ii), that is, an oil phase obtained by dissolving or suspending metastin or a derivative thereof or a salt thereof in an organic solvent solution of a lactic acid / glycolic acid polymer is produced as follows. First, an organic solvent solution of a lactic acid / daricholic acid polymer is produced. As the organic solvent, those similar to the organic solvent used in producing the above wzo emulsion are used.
有機溶媒溶液中の乳酸/ダリコール酸重合体の濃度は、該乳酸/ダリコール酸重 合体の分子量、有機溶媒の種類によって異なるが、 [乳酸 zグリコール酸重合体の 重量 Z (有機溶媒の重量 +乳酸 Zダリコール酸重合体の重量） ] (X 1 0 0 %) は 、 通常約 0. 0 1ないし約 7 0 % (w/w) 、 好ましくは約 1ないし約 6 0 % (w /w) である。  The concentration of the lactic acid / daricholic acid polymer in the organic solvent solution varies depending on the molecular weight of the lactic acid / daricholic acid polymer and the type of the organic solvent; however, [weight of lactic acid z glycolic acid polymer Z (weight of organic solvent + weight of lactic acid (X100%) is usually about 0.01 to about 70% (w / w), preferably about 1 to about 60% (w / w). is there.
次に、メタスチンもしくはその誘導体またはその塩を乳酸 Zグリコール酸重合体 の有機溶媒溶液に溶解あるいは懸濁して油相を製造する。  Next, metastin or a derivative thereof or a salt thereof is dissolved or suspended in an organic solvent solution of lactic acid-Z-glycolic acid polymer to produce an oil phase.
メタスチンもしくはその誘導体またはその塩の使用量は、該メタスチンもしくは その誘導体またはその塩の乳酸 zグリコ一ル酸重合体に対する割合が上記 w/o エマルシヨン （i ) を製造する場合と同様になるように選択すればよい。  The amount of metastin or a derivative thereof or a salt thereof used is such that the ratio of the metastin or a derivative thereof or a salt thereof to the lactic acid-z-glycolic acid polymer is the same as that in the case of producing the above w / o emulsion (i). Just choose.
ついで上記した （i) WZOエマルシヨン又は （i i) 油相を、 外水相に添加し、 ホモミキサー等を用いて分散、乳化し、それぞれ W/OZWエマルシヨン又は OZ Wエマルシヨンを製造する。  Then, the above-mentioned (i) WZO emulsion or (ii) the oil phase is added to the external aqueous phase, and dispersed and emulsified using a homomixer or the like to produce a W / OZW emulsion or an OZW emulsion, respectively.
外水相の使用量は、 通常上記 （i) 又は （i i) の約 1ないし約 1 0 , 0 0 0容量 倍、 好ましくは約 1 0ないし約 5， 0 0 0容量倍、 特に好ましくは約 5 0ないし約 1 , 0 0 0容量倍である。  The amount of the outer aqueous phase used is usually about 1 to about 100,000 times as much as the above (i) or (ii), preferably about 10 to about 5,000 times, particularly preferably about 10 to about 5,000 times. It is 50 to about 1, 000 times the volume.
外水相中には、 通常乳化剤を添加する。 該乳化剤としては、 一般的に安定な An emulsifier is usually added to the external aqueous phase. As the emulsifier, generally stable
O/Wエマルション又は 0ノ Wエマルシヨンを形成し得るものであればよく、例え ばァニオン性界面活性剤、 非イオン性界面活性剤、 ポリオキシエチレンヒマシ油誘 導体、 ポリビニルピロリドン、 ポリビニルアルコール、 カルボキシメチルセルロー ス、 レシチン、 ゼラチン、 ヒアルロン酸等が挙げられるが、 中でもポリビニルアル コールが好ましい。 外水相中の乳化剤の濃度は、 通常約 0. 0 0 1ないし約 2 0 % (w/w) 、 好ましくは約 0. 0 1ないし約 1 0 % (wZw) 、 特に好ましくは約 0. 0 5ないし約 5 % (w/w) である。 Any substance that can form an O / W emulsion or a zero-w emulsion can be used.Examples include anionic surfactants, nonionic surfactants, polyoxyethylene castor oil derivatives, polyvinylpyrrolidone, polyvinyl alcohol, and carboxymethyl. Cellulose, lecithin, gelatin, hyaluronic acid and the like can be mentioned, among which polyvinyl alcohol is preferable. The concentration of the emulsifier in the external aqueous phase is usually about 0.001 to about 20% (w / w), preferably about 0.01 to about 10% (wZw), and particularly preferably about 0. 0 5 to about 5% (w / w).
このようにして得られる WZO ZWェマルション又は O/Wエマルション (以下 、 これらを単にエマルシヨンと略記する場合がある） を水中乾燥法に付すことによ り、 これらエマルシヨンに含まれる有機溶媒を除去してマイクロカプセルを製造す ることができる。 The WZO ZW emulsion or O / W emulsion thus obtained (hereinafter These are sometimes simply abbreviated as emulsions) by subjecting them to an underwater drying method, whereby microcapsules can be produced by removing the organic solvent contained in these emulsions.
このようにして得られるマイクロカプセルは、 遠心分離あるいは篩等で回収し、 所望により、マイクロカプセル同士の凝集を防止するため糖あるいは糖アルコール 、 無機塩等、 好ましくはマンニトール、 ソルビトール等の凝集防止剤を添加した後 、 凍結乾燥に付す。  The microcapsules obtained in this manner are collected by centrifugation or a sieve or the like, and, if desired, a sugar or sugar alcohol, an inorganic salt or the like, preferably a coagulation inhibitor such as mannitol or sorbitol in order to prevent aggregation of the microcapsules. After adding lyophilized.
マイクロカプセルと凝集防止剤の混合割合 （重量比） は、 約 5 0 ： 1ないし約 1 ： 1、 好ましくは約 2 0 ： 1ないし約 1 ： 1、 更に好ましくは約 1 0 ： 1ないし約 5 ： 1である。  The mixing ratio (weight ratio) of the microcapsules and the anti-agglomeration agent is about 50: 1 to about 1: 1, preferably about 20: 1 to about 1: 1, and more preferably about 10: 1 to about 5: 1. : 1.
凝集防止剤の添加方法は、マイクロカプセルと凝集防止剤とが均一に混合される 方法であれば特に限定されないが、例えば凝集防止剤の水溶液にマイク口カプセル を分散する方法等が挙げられる。  The method of adding the anti-agglomeration agent is not particularly limited as long as the microcapsules and the anti-agglomeration agent are uniformly mixed, and examples thereof include a method of dispersing the microcapsules in an aqueous solution of the anti-agglomeration agent.
メタスチンもしくはその誘導体またはその塩の徐放性マイクロカプセルの製造 法においては、水中乾燥法によりマイクロカプセル化するのが好適である場合が多 い。  In a method for producing sustained-release microcapsules of metastin or a derivative thereof or a salt thereof, it is often preferable to carry out microcapsulation by a water drying method.
このようにして得られたマイクロ力プセルは、必要があれば減圧下加温乾燥しマ イクロカプセル中の水分及び溶媒の除去をより完全に行う。  The micro-force capsule thus obtained is heated and dried under reduced pressure, if necessary, to more completely remove the water and the solvent in the microcapsules.
また、前記した WZO/Wエマルション又は OZWエマルシヨンを用いる方法の 他に、 メタスチンもしくはその誘導体またはその塩の粉末 （s相） を、 乳酸 zダリ コール酸重合体を溶解した有機溶媒液（0相） に分散させた、 s zo型分散液から 溶媒を除去することによる方法（例えば s /ozw法など） により製造することも できる。  In addition to the above-mentioned method using a WZO / W emulsion or an OZW emulsion, a powder of metastin or a derivative thereof or a salt thereof (s phase) may be used in an organic solvent solution in which a lactic acid / z-dalicholate polymer is dissolved (phase 0). It can also be produced by a method (for example, the s / ozw method or the like) by removing the solvent from the szo-type dispersion liquid dispersed in water.
本法は、 まず乳酸 "グリコール酸重合体を有機溶媒に溶解し、 この有機溶媒液中 にメタスチンもしくはその誘導体またはその塩の粉末（S相） を添加し分散させる In this method, first, a polymer of lactic acid “glycolic acid” is dissolved in an organic solvent, and powder (S phase) of metastin or a derivative or salt thereof is added and dispersed in the organic solvent.
。 この際、 メタスチンもしくはその誘導体またはその塩と乳酸/ダリコール酸重合 体との混合割合 （重量比） は、 例えば約 1 : 1 0， 0 0 0ないし約 1 ： 1、 好まし くは約 1 ： 1 , 0 0 0ないし約 2 ： 5、 更に好ましくは約 1 ： 1 0 0ないし約 1 ： 3である。 また、 メタスチンもしくはその誘導体またはその塩の粉末を有機溶媒液 中に均一に分散させるため、外部物理的エネルギーを加えることが好ましい。その 方法としては例えば超音波照射、 夕一ビン型撹拌器、 ホモジナイザー等が用いられ る。 . At this time, the mixing ratio (weight ratio) of metastin or a derivative thereof or a salt thereof and a lactic acid / daricholic acid polymer is, for example, about 1: 10,000 to about 1: 1, preferably about 1: 1. 1,000 to about 2: 5, more preferably about 1: 100 to about 1: 3 Further, in order to uniformly disperse the powder of metastin or a derivative thereof or a salt thereof in an organic solvent solution, it is preferable to apply external physical energy. As the method, for example, ultrasonic irradiation, a one-bottle type stirrer, a homogenizer and the like are used.
次いでこのようにして調製された有機溶媒分散液 （S ZO型分散液） を、 更に水 性溶媒 （W相） 中に添加して、 上記と同様の外部物理的エネルギー、 例えば超音波 照射、 タービン型撹拌器、 あるいはホモジナイザー等により szozw型エマルシ ヨンを形成させる。 以後、 油相溶媒を蒸発させマイクロカプセルを製造する。 この 際の水相体積は、 一般的には油相体積の約 1倍ないし約 1 0 , 0 0 0倍から選ばれ る。 更に好ましくは約 1 0倍ないし約 5， 0 0 0倍、 特に好ましくは約 5 0倍ない し約 1 , 0 0 0倍から選ばれる。  Next, the organic solvent dispersion (SZO type dispersion) thus prepared is further added to an aqueous solvent (W phase), and the same external physical energy as described above, for example, ultrasonic irradiation, turbine An szozw emulsion is formed using a stirrer or a homogenizer. Thereafter, the oil phase solvent is evaporated to produce microcapsules. In this case, the volume of the aqueous phase is generally selected from about 1 to about 100,000 times the volume of the oil phase. More preferably, it is selected from about 10-fold to about 5,000-fold, particularly preferably from about 50-fold to about 1,000-fold.
上記外水相中には、 乳化剤を加えてもよい。 該乳化剤としては、 一般的に安定な s /ozwエマルションを形成できるものであれば何れでもよい。乳化剤としては 、 例えばァニオン性界面活性剤、 非イオン性界面活性剤、 ポリオキシエチレンヒマ シ油誘導体、 ポリビニルピロリドン、 ポリビニルアルコール、 カルポキシメチルセ ルロース、 レシチン、 ゼラチン、 ヒアルロン酸等が挙げられる。 これらは適宜組み 合わせて使用してもよい。 外水相中の乳化剤の濃度は、 好ましくは約 0. 0 0 1 % ないし約 2 0 % (w/w) である。 更に好ましくは約 0. 0 1 %ないし約 1 0 % ( w/w) 、 特に好ましくは約 0. 0 5 %ないし約 5 % (w/w) である。  An emulsifier may be added to the outer aqueous phase. The emulsifier may be any as long as it can form a generally stable s / ozw emulsion. Examples of the emulsifier include anionic surfactants, nonionic surfactants, polyoxyethylene castor oil derivatives, polyvinylpyrrolidone, polyvinyl alcohol, carboxymethylcellulose, lecithin, gelatin, hyaluronic acid and the like. These may be used in appropriate combinations. The concentration of the emulsifier in the external aqueous phase is preferably from about 0.001% to about 20% (w / w). More preferably, it is about 0.01% to about 10% (w / w), particularly preferably about 0.05% to about 5% (w / w).
このようにして得られたマイクロカプセルは、遠心分離あるいは濾過操作により 分取した後、マイクロカプセルの表面に付着している乳化剤等を蒸留水による洗浄 で除去し、 再び蒸留水等に分散して凍結乾燥する。 その後必要であれば、 加温して マイクロ力プセル中の水分及び有機溶媒を更に除去する。減圧下に加温してもよい 。 加温条件としては、 用いた乳酸/ダリコール酸重合体のガラス転移温度以上で、 マイクロカプセルの各粒子が互いに付着しない程度の温度で加熱乾燥する。好まし くは、乳酸/ダリコール酸重合体のガラス転移温度からガラス転移温度より約 3 0 °C高い温度の範囲で加熱乾燥する。 ここでガラス転移温度とは、 示差走査熱量計を 用い、 加温速度毎分 1 0ないし 2 0 °Cで昇温した際に得られる中間点を云う。 このようにして得られるマイクロカプセルは、粒子同士の凝集を防ぐために凝集 防止剤を加えてもよい。 該凝集防止剤としては、 例えばマンニトール、 ラクトース 、 ブドウ糖、 デンプン類 （例えばコーンスターチ等） 、 ヒアルロン酸あるいはこの アルカリ金属塩等の水溶性多糖、 グリシン、 フイブリン、 コラーゲン等のタンパク 質、 塩化ナトリウム、 リン酸水素ナトリウム等の無機塩類等が適宜用いられる。 また、 マイクロカプセルは、 前記 （B _ l — a ) の場合と同様に、 加温後、 冷却 することにより、 円盤状、 フィルム状、 棒状 （ロッド状） 等に成形することもでき る。 The microcapsules obtained in this manner are separated by centrifugation or filtration, and then the emulsifier and the like adhering to the surface of the microcapsules are removed by washing with distilled water, and dispersed again in distilled water or the like. Lyophilize. Thereafter, if necessary, the mixture is heated to further remove water and organic solvents in the micro force cell. It may be heated under reduced pressure. The heating conditions are heating and drying at a temperature not lower than the glass transition temperature of the lactic acid / daricholic acid polymer used, and at such a level that the particles of the microcapsules do not adhere to each other. Preferably, the lactic acid / daricholic acid polymer is heated and dried in a temperature range from the glass transition temperature to about 30 ° C higher than the glass transition temperature. Here, the glass transition temperature refers to an intermediate point obtained when the temperature is raised at a heating rate of 10 to 20 ° C. per minute using a differential scanning calorimeter. The microcapsules thus obtained may be added with an anticoagulant in order to prevent coagulation of the particles. Examples of the anticoagulant include water-soluble polysaccharides such as mannitol, lactose, glucose, starches (eg, corn starch), hyaluronic acid and alkali metal salts thereof, proteins such as glycine, fibrin, collagen, sodium chloride, and phosphoric acid Inorganic salts such as sodium hydrogen and the like are appropriately used. Further, the microcapsules can be formed into a disc shape, a film shape, a rod shape (rod shape) or the like by heating and then cooling as in the case of the above (B_l-a).
前記した各種製造法において、有機溶媒に乳酸/ダリコール酸重合体を溶解する 際に、 該有機溶媒に酸化亜鉛などの多価金属化合物を添加してもよい。  In the various production methods described above, when dissolving the lactic acid / daricholic acid polymer in an organic solvent, a polyvalent metal compound such as zinc oxide may be added to the organic solvent.
酸化亜鉛の使用量は、乳酸/ダリコール酸重合体 1 0 0重量部に対し、 例えば約 0 . 0 1ないし約 1 0 0重量部、 好ましくは約 0 . 1ないし約 2 0重量部である。 また、 酸化亜鉛の粒子径は、 通常約 0 . 0 0 1ないし約 1 0 ^ m、 好ましくは約 0 . 0 0 5ないし約 1 mである。  The amount of zinc oxide to be used is, for example, about 0.01 to about 100 parts by weight, preferably about 0.1 to about 20 parts by weight, based on 100 parts by weight of the lactic acid / daricholic acid polymer. The particle size of zinc oxide is usually about 0.001 to about 10 ^ m, preferably about 0.05 to about 1 m.
このように、 酸化亜鉛などの多価金属化合物を使用して得られる徐放性製剤は、 「薬物取り込み率が高い」 、 「長期にわたって持続的に薬物を放出できる」 等の優 れた性質を有する。  Thus, a sustained-release preparation obtained using a polyvalent metal compound such as zinc oxide has excellent properties such as “high drug uptake rate” and “sustained drug release over a long period of time”. Have.
本発明の徐放性製剤を製造する際に、メタスチンもしくはその誘導体またはその 塩を、揮発性の塩類例えば酢酸アンモニゥムの水溶液に溶解し、 凍結乾燥して用い てもよい。  In producing the sustained-release preparation of the present invention, metastin or a derivative thereof or a salt thereof may be dissolved in an aqueous solution of a volatile salt such as ammonium acetate and freeze-dried before use.
このように酢酸ァンモニゥムで処理して得られるメタスチンもしくはその誘導 体またはその塩の凍結乾燥品は、 粒子径が小さく、 優れた操作性を有するので、 徐 放性製剤を製造する際に有利である。  The freeze-dried product of metastin or a derivative thereof or a salt thereof obtained by treating with ammonium acetate as described above has a small particle size and has excellent operability, and thus is advantageous in producing a sustained-release preparation. .
こうして得られる本発明の徐放性製剤は、そのままあるいは所望により製剤学的 に許容される添加剤 （例えば、 安定化剤、 保存剤、 無痛化剤等） を用いて種々の剤 形に製造して投与することができる。 このような製剤としては、 例えば非経口剤 （ 例えば注射剤、 埋め込み剤、 坐剤等） 、 経口剤 （例えばカプセル剤、 錠剤、 顆粒剤 、 散剤等の固形製剤、 シロップ剤、 乳剤、 懸濁剤等の液剤等） 等が挙げられる。 製 剤学的に許容される添加剤としての安定剤としては、 例えばヒト血清アルブミン、 ポリエチレングリコール等、 保存剤としては、 例えばべンジルアルコール、 フエノ ール等、 無痛化剤としては、 例えば塩化ベンザルコニゥム、 塩酸プロ力イン等が挙 げられる。本発明の製剤におけるメタスチンもしくはその誘導体またはその塩の含 有量は、 製剤全体に対して通常約 0. 0 1ないし約 1 0 0 % (w/w) の範囲から 適宜選択することができる。 The sustained-release preparation of the present invention thus obtained can be produced in various forms as it is or using pharmaceutically acceptable additives (eg, stabilizers, preservatives, soothing agents, etc.) as desired. Can be administered. Examples of such preparations include parenteral preparations (eg, injections, implants, suppositories, etc.), oral preparations (eg, solid preparations such as capsules, tablets, granules, powders, etc., syrups, emulsions, suspensions) And the like). Made Stabilizers as pharmaceutically acceptable additives include, for example, human serum albumin and polyethylene glycol; preservatives such as benzyl alcohol and phenol; and soothing agents such as benzalkonium chloride. And pro-hydrochloric acid. The content of metastin or a derivative thereof or a salt thereof in the preparation of the present invention can be appropriately selected usually from the range of about 0.01 to about 100% (w / w) based on the whole preparation.
本発明の徐放性製剤は、 メタスチンもしくはその誘導体またはその塩を、水性溶 剤 （例、 蒸留水、 生理的食塩水、 リンゲル液等） または油性溶剤 （例、 ォリーブ油 、 ゴマ油、 綿実油、 コーン油等の植物油；プロピレングリコール等） に溶解、 懸濁 または乳化した後、 市販の徐放性製剤用容器 [例、 デュロス （商品名、 アルザ社製 The sustained-release preparation of the present invention may be prepared by adding metastin or a derivative thereof or a salt thereof to an aqueous solvent (eg, distilled water, physiological saline, Ringer's solution, etc.) or an oily solvent (eg, olive oil, sesame oil, cottonseed oil, corn oil) Or vegetable oils such as propylene glycol, etc.), and then dissolved in a suspension or emulsified, and then sold in a container for a sustained-release preparation [eg, Duros (trade name, manufactured by Alza).
) ] に封入することによつても製造される。 )].
本発明の徐放性製剤は、例えばマイクロカプセルとして、 あるいはマイクロカブ セルを原料物質として種々の剤形に製剤化してなる製剤として、 非経口剤 （例、 筋 肉内、 腹腔内、 皮下、 臓器等への注射剤または埋め込み剤、 鼻腔、 直腸、 子宮等へ の経粘膜剤等） 、 経口剤 （例、 カプセル剤 （例、 硬カプセル剤、 軟カプセル剤等） 、 顆粒剤、 散剤等の固形製剤、 懸濁剤等の液剤等） 等として投与することができる 本発明の製剤は特に注射用であることが好ましい。 例えば、 徐放性製剤がマイク 口カプセルである場合、 マイクロカプセルを分散剤 （例、 Tween80、 HC0- 60等の界 面活性剤、 カルポキシメチルセルロース、 アルギン酸ナトリウム、 ヒアルロン酸等 の多糖類等） 、 保存剤 （例、 メチルパラベン、 プロピルパラベン等） 、 等張化剤 （ 例、 塩化ナトリウム、 マンニトール、 ソルビトール、 ブドウ糖等） 等と共に水性懸 濁剤とすることにより実用的な徐放性注射剤が得られる。 また、 ゴマ油、 コーン油 等の植物油あるいはこれにレシチン等のリン脂質を混合したもの、あるいは中鎖脂 肪酸トリグリセリド （例、 ミグリオ一ル 812) と共に分散して油性懸濁剤として実 際に使用できる徐放性注射剤とする。  The sustained-release preparation of the present invention may be a parenteral preparation (eg, intramuscular, intraperitoneal, subcutaneous, organ, etc.) as a microcapsule or a preparation prepared by using microcapsules as a raw material in various dosage forms. Solids such as injections or implants for intranasal preparations, transmucosal preparations for the nasal cavity, rectum, uterus, etc., oral preparations (eg, capsules (eg, hard capsules, soft capsules, etc.), granules, powders, etc.) Formulations, liquid preparations such as suspensions, etc.) The preparations of the present invention are particularly preferably for injection. For example, when the sustained-release preparation is a microcapsule, the microcapsule may be used as a dispersant (eg, a surfactant such as Tween80, HC0-60, or a polysaccharide such as carboxymethylcellulose, sodium alginate, or hyaluronic acid). A practical sustained-release injection can be obtained by using an aqueous suspension with a preservative (eg, methyl paraben, propyl paraben, etc.), an isotonic agent (eg, sodium chloride, mannitol, sorbitol, glucose, etc.). . Also, vegetable oils such as sesame oil and corn oil, or mixtures of these with phospholipids such as lecithin, or dispersed with medium-chain fatty acid triglycerides (eg, Migliol 812), are used as oily suspending agents. It should be a sustained-release injection.
徐放性製剤が例えばマイク口カプセルである場合、マイク口カプセルの粒子径は 、 懸濁注射剤として使用するためには、 その分散度、 通針性を満足する範囲であれ ばよく、 例えば平均粒子径として約 0. 1ないし約 300 /ζπιの範囲が挙げられる。 平均 粒子径は、 好ましくは約 1ないし約 150 ^m、 特に好ましくは約 2ないし約 100 m の範囲である。 When the sustained-release preparation is, for example, a microphone mouth capsule, the particle size of the microphone mouth capsule is within a range that satisfies the degree of dispersion and needle penetration for use as a suspension injection. The average particle diameter may be, for example, in the range of about 0.1 to about 300 / ζπι. The average particle size is preferably in the range from about 1 to about 150 m, particularly preferably in the range from about 2 to about 100 m.
上記のマイクロカプセルを無菌処理するには、製造全工程を無菌にする方法、 ガ ンマ線で滅菌する方法、 防腐剤を添加する方法などが挙げられるが、特に限定され ない。  Aseptic treatment of the above microcapsules includes, but is not particularly limited to, a method of sterilizing the entire production process, a method of sterilizing with a gamma ray, and a method of adding a preservative.
本発明の製剤は安全で低毒性であるので、 例えば、 ヒトゃ哺乳動物 （例えばサル 、 マントヒヒ、 チンパンジー、 ブタ、 ゥシ、 ヒッジ、 ゥマ、 ィヌ、 ネコ、 マウス、 ラット等） に対して投与することができる。  Since the preparation of the present invention is safe and has low toxicity, it can be used, for example, for human mammals (for example, monkeys, baboons, chimpanzees, pigs, sea lions, sheep, sheep, dogs, dogs, cats, mice, rats, etc.). Can be administered.
本発明の製剤は、メタスチンの生理活性が関与する全ての疾患の治療または予防 に用いることができる。 特に、 本発明の製剤は、 癌転移抑制活性を有するため、 あ らゆる癌 （例えば、 肺癌、 胃癌、 肝癌、 膝癌、 大腸癌、 直腸癌、 結腸癌、 前立腺癌 、 卵巣癌、 子宮頸癌、 乳癌、 腎癌、 膀胱癌、 脳腫瘍等） の治療または予防に有効に 用いることができる。 さらに、 本発明の製剤は、 膝臓機能調節作用を有するため、 種々の膝臓疾患 （例えば、 急性または慢性膝炎、 膝癌等） の治療または予防にも有 効に用いることができる。  The preparation of the present invention can be used for treating or preventing all diseases in which the physiological activity of metastin is involved. In particular, since the preparation of the present invention has a cancer metastasis inhibitory activity, it can be used for any cancer (eg, lung cancer, stomach cancer, liver cancer, knee cancer, colon cancer, rectal cancer, colon cancer, prostate cancer, ovarian cancer, cervical cancer). , Breast cancer, kidney cancer, bladder cancer, brain tumor, etc.). Further, since the preparation of the present invention has a function of regulating knee function, it can be effectively used for treatment or prevention of various knee diseases (for example, acute or chronic knee inflammation, knee cancer, etc.).
また、 本発明の製剤は、 胎盤機能調節作用を有するため、 絨毛癌、 胞状奇胎、 侵 入奇胎、 流産、 胎児の発育不全、 糖代謝異常、 脂質代謝異常または分娩異常の治療 または予防に有効に用いることができる。  In addition, since the preparation of the present invention has a placental function regulating action, it is useful for treating or preventing choriocarcinoma, hydatidiform mole, invasive mole, miscarriage, fetal growth deficiency, abnormal glucose metabolism, abnormal lipid metabolism or parturition. It can be used effectively.
本発明の製剤の投与量は、有効成分であるメタスチンもしくはその誘導体または その塩の種類と含有量、 剤形、 放出の持続期間、 投与対象、 投与ルート、 投与目的 、対象疾患、 症状等に応じて適宜選択することができるが、 所望の持続期間中に当 該有効成分が医薬上有効な濃度で体内に維持される量であればよい。例えば成人の 癌患者（体重約 6 O k g ) の治療において、 例えば約 1ヶ月型徐放性注射剤として 投与する場合、 1回あたり、 メタスチンもしくはその誘導体またはその塩として例 えば約 0 . 1ないし約 1 0 O m g Z k g体重、好ましくは約 1ないし 5 O m g / k g体重の範囲の量を用いる。 投与回数は、 1週間に 1回、 2週間に 1回、 1力月に 1回、 2力月に 1回、 6力月に 1回等、 KiSS- 1ペプチドの種類と含量、 剤形、 放出 の持続期間、 対象疾病、 対象動物等によって適宜選ぶことができる。 好ましくは 1 週間ないし 2力月型徐放性製剤、さらに好ましくは 1週間ないし 1力月型徐放性製 剤が挙げられる。 The dosage of the preparation of the present invention depends on the type and content of the active ingredient metastin or a derivative thereof or a salt thereof, dosage form, duration of release, administration subject, administration route, administration purpose, target disease, symptoms, etc. The amount of the active ingredient may be appropriately maintained in the body at a pharmaceutically effective concentration for a desired duration. For example, in the treatment of an adult cancer patient (body weight of about 6 O kg), for example, when administered as a sustained-release injection for about one month, one dose of metastin or a derivative or salt thereof may be, for example, about 0.1 to 0.1 mg / day. An amount in the range of about 10 O mg Z kg body weight, preferably in the range of about 1 to 5 O mg / kg body weight is used. The administration frequency is once a week, once every two weeks, once every 1 month, once every 2 months, once every 6 months, etc., the type and content of the KiSS-1 peptide, dosage form, release Can be appropriately selected depending on the duration of the disease, the target disease, the target animal, and the like. Preferred is a one-week to two-month sustained-release preparation, more preferably a one-week to one-month sustained-release preparation.
また、 本発明の製剤は、 メタスチンもしくはその誘導体またはその塩が医薬上有 効である種々の疾患のためのその他の医薬、 特に癌治療のための化学療法剤、 ホル モン療法剤、 免疫療法剤等の薬剤 （以下、 併用薬剤と略記する） と組み合わせて用 いることができる。 この際、 本発明の製剤及び併用薬剤の投与時期は限定されず、 これらを投与対象に対し、 同時に投与してもよいし、 時間差をおいて投与してもよ レ^併用薬剤の投与量は、 臨床上用いられている用量を基準として適宜選択するこ とができる。 また、 本発明の製剤と併用薬剤の配合比は、 投与対象、 投与ルート、 対象疾患、 症状、 組み合わせ等に応じて適宜選択することができる。  In addition, the preparation of the present invention may be used as a medicament for various diseases in which metastin or a derivative thereof or a salt thereof is pharmaceutically effective, particularly a chemotherapeutic agent for cancer treatment, a hormonal therapeutic agent, and an immunotherapeutic agent. (Hereinafter abbreviated as concomitant drug). At this time, the timing of administration of the preparation of the present invention and the concomitant drug is not limited, and these may be administered to the subject simultaneously or with a time lag. The dose can be appropriately selected based on the clinically used dose. The mixing ratio of the preparation of the present invention and the concomitant drug can be appropriately selected according to the administration subject, administration route, target disease, symptom, combination, and the like.
化学療法剤としては、 例えばアルキル化剤 （例えばサイクロフォスフアミド、 ィ フォスフアミド、 二ムスチン、 ラエムスチン、 カルポコン等） 、 代謝拮抗剤 （例え ばメソトレキセー卜、 5—フルォロウラシル、 テガフール、 カルモフール、 UFT、 ドキシフルリジン、 シタラビン、 エノシタビン、 メルカプトプリン、 メルカプトプ リンリポシド、 チォグァニン等) 、 抗癌性抗生物質 (例えばマイトマイシン、 アド リアマイシン、 ダウノルビシン、 ェピルビシン、 ピラルビシン、 イダルビシン、 ブ レオマイシン、 ペブロマイシン、 ァクチノマイシン等） 、 植物由来抗癌剤 （例えば ビンクリスチン、 ビンブラスチン、 ビンデシン、 エトポシド、 カンプトテシン、 ィ リノテカン等） 、 シスブラチン、 カルポプラチン、 ネダプラチン、 パクリタキセル Chemotherapeutic agents include, for example, alkylating agents (eg, cyclophosphamide, diphosphamide, dimustine, laemustine, carpocon, etc.), antimetabolites (eg, methotrexate, 5-fluorouracil, tegafur, carmofur, UFT, doxyfluridine, Cytarabine, enocitabine, mercaptopurine, mercaptoprin liposide, thioguanine, etc., anticancer antibiotics (for example, mitomycin, adriamycin, daunorubicin, epirubicin, pyrarubicin, idarubicin, bleomycin, pebromycin, clitincin-derived anticancer agents, etc.) , Vinblastine, vindesine, etoposide, camptothecin, irinotecan, etc.), cisplatin, carpoplatin, neda Platinum, paclitaxel
、 ドセタキセル、 エス卜ラムスチン等が挙げられる。 , Docetaxel, estramustine and the like.
ホルモン療法剤としては、 例えば副腎皮質ホルモン薬 （例えばプレドニゾロン、 プレドニゾン、 デキサメタゾン、 酢酸コルチゾン等） 、 エストロゲン薬 （例えばェ ス卜ラジオ一ル、 ェチニルエストラジオール、 ホスフェストロール、 クロロトリア 二セン等) 、 抗エストロゲン薬 (例えばェピチォス夕ノール、 メピチォスタン、 夕 モキシフェン、 クロミフェン等） 、 黄体ホルモン薬 （例えば力プロン酸ヒドロキシ プロゲステロン、 ジドロゲステロン、 メドロキシプロゲステロン、 ノルェチステロ ン、 ノルェチンドロン等） 、 LHRH誘導体 （例えば酢酸リュープロレリン等） 等が挙 げられる。 Examples of hormonal therapies include corticosteroids (eg, prednisolone, prednisone, dexamethasone, cortisone acetate, etc.), estrogen drugs (eg, estradiol, ethinylestradiol, phosphfestrol, chlorotriacene, etc.), Anti-estrogen drugs (such as epithios-nol, mepithiostan, evening moxifen, clomiphene, etc.), luteinizing hormones (such as hydroxyprogesterone haplonate, dydrogesterone, medroxyprogesterone, norethisterone, norletindrone, etc.), LHRH derivatives (such as leuprorelin acetate) Etc.) I can do it.
免疫療法剤としては、例えば微生物又は細菌成分（例えばムラミルジペプチド誘 導体、 ピシバニ一ル等） 、 免疫増強活性のある多糖類 （例えばレンチナン、 シゾフ イラン、 クレスチン等） 、 遺伝子工学的手法で得られるサイト力イン （例えばイン ターフェロン、 インターロイキン 2 (IL-2) 、 インタ一ロイキン 12 (IL- 12)、 腫 瘍壊死因子 （TNF) 等） 、 コロニー刺激因子 （例えば顆粒球コロニー刺激因子、 ェ リスロポェチン等） 等が挙げられる。  Examples of the immunotherapeutic agent include microorganisms or bacterial components (eg, muramyl dipeptide derivative, picibanil, etc.), polysaccharides having immunopotentiating activity (eg, lentinan, schizophyllan, krestin, etc.), and genetic engineering techniques. Cytokinin (eg, interferon, interleukin 2 (IL-2), interleukin 12 (IL-12), tumor necrosis factor (TNF), etc.), colony stimulating factor (eg, granulocyte colony stimulating factor, erythropoietin Etc.).
更に、 動物モデルや臨床で悪液質改善作用が認められている薬剤、 即ち、 シクロ ォキシゲナーゼ阻害剤 (例えばインドメタシン等) 〔キャンサー ·リサーチ (Can cer Res each) 、 第 49卷、 5935ないし 5939頁、 1989年〕 、 プロゲス テロン誘導体 (例えばメゲステロールァセテ一ト) 〔ジャーナル ·ォブ'クリニ力 ル《オンコロジ一 （Journal of Clinical Oncology) 、 第 12巻、 213ないし 2 25頁、 1994年〕 、 糖質ステロイド （例えばデキサメサゾン等） 、 メトクロブ ラミド系薬剤、 テトラヒドロカンナビノール系薬剤 （文献は何れも上記と同様） 、 脂肪代謝改善剤 （例えばエイコサペンタエン酸等） 〔プリティシュ ·ジャーナル · ォブ 'キャンサー （British Journal of Cancer) 、 第 68巻、 314ないし 31 8頁、 1993年〕 、 成長ホルモン、 IGF—1、 あるいは悪液質を誘導する因子 である TNF— a、 L I F、 IL一 6、 オンコスタチン Mに対する抗体等も本発明 製剤と併用することができる。  Furthermore, drugs that have been shown to improve cachexia in animal models and clinically, such as cyclooxygenase inhibitors (e.g., indomethacin) (Cancer Reseach, Vol. 49, pp. 5935-5939, 1989], progesterone derivatives (eg, megestrol acetate) [Journal of Clinical Oncology, Journal of Clinical Oncology, Vol. 12, 213 to 225, 1994], sugar Steroids (eg, dexamethasone), metoclobramides, tetrahydrocannabinols (all of which are the same as described above), fat metabolism improvers (eg, eicosapentaenoic acid, etc.) [Pretish Journal of Cancer ( British Journal of Cancer), 68, 314-318, 1993], growth hormone, IGF-1, or cachexia Is a factor which induces TNF- a, L I F, IL one 6, antibody against Oncostatin M can also be used in combination with the present invention formulation.
その他、胎盤や膝臓の疾患の治療又は予防に用いられる一般的な薬剤も併用薬剤 として用いられる。 その様な薬剤の例としては、 臨床上通常用いられる抗炎症剤、 解熱鎮痛剤、 抗菌剤、 抗ウィルス剤、 ホルモン剤等が挙げられる。  In addition, general drugs used for treatment or prevention of diseases of the placenta and the knee are also used as concomitant drugs. Examples of such agents include anti-inflammatory agents, antipyretic analgesics, antibacterial agents, antiviral agents, hormonal agents, etc., which are commonly used in clinical practice.
以下に、本発明を実施例及び実験例を示して更に詳細に説明するが、 本発明の範 囲はこれらに限定されるものではない。  Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples, but the scope of the present invention is not limited thereto.
本明細書および図面において、塩基やアミノ酸などを略号で表示する場合、 IU In the present specification and drawings, when abbreviations are used for bases and amino acids, etc.
PAC I UB Commi s i'on on B i ochemi c a l Nomen c 1 a t u r eによる略号あるいは当該分野における慣用略号に基づくものであ り、 その例を下記する。 またアミノ酸に関し光学異性体があり得る場合は、 特に明 示しなければ L体を示すものとする t This is based on the abbreviation by PAC I UB Commision on Biochemi cal Nomenclature or a common abbreviation in the field, and examples are as follows. When amino acids may have optical isomers, If not shown, it means L-body t
DNA デォキシリポ核酸 cDNA 相補的デォキシリポ核酸DNA Deoxylipo nucleic acid cDNA Complementary deoxylipo nucleic acid
A アデニン · A Adenine ·
T チミン  T thymine
G グァニン  G Guanin
C  C
G 1 y グリシン  G 1 y Glycine
A 1 a ァラニン  A 1 a Alanin
Va 1 バリン  Va 1 Valine
Leu ロイシン  Leu Leucine
I 1 e  I 1 e
S e r セリン  S e r serine
Th r スレオニン  Th r threonine
Cy s  Cy s
Me t メチォニン  Me t Methionin
G 1 u グルタミン酸  G 1 u Glutamic acid
As p ァスパラギン酸  As p Aspartic acid
L y s リジン  Lys lysine
A r g アルギニン  A r g Arginine
H i s ヒスチジン  H is histidine
Ph e フエ二ルァラニン Ty r チロシン  Ph e Phenylalanine Ty r Tyrosine
T r p トリプトファン  T r p tryptophan
P r o プロリン  Pro proline
A s n ァスパラギン  A s n asparagine
G 1 n ダル夕ミン 本願明細書の配列表の配列番号は、 以下の配列を示す。 G 1 n Dal Yu Min The sequence numbers in the sequence listing in the present specification indicate the following sequences.
〔配列番号： 1〕  [SEQ ID NO: 1]
ヒト由来 KiSS-1ペプチドのアミノ酸配列を示す。  2 shows the amino acid sequence of human KiSS-1 peptide.
〔配列番号： 2〕  [SEQ ID NO: 2]
配列番号： 1で表されるアミノ酸配列の第 4 0番目から第 5 4番目の部分アミノ 酸配列を示す。  This shows a partial amino acid sequence of the 40th to 54th amino acids of the amino acid sequence represented by SEQ ID NO: 1.
〔配列番号： 3〕  [SEQ ID NO: 3]
配列番号： 1で表されるアミノ酸配列の第 4 5番目から第 5 4番目の部分アミノ 酸配列を示す。  This shows a partial amino acid sequence of the 45th to 54th amino acids of the amino acid sequence represented by SEQ ID NO: 1.
〔配列番号： 4〕  [SEQ ID NO: 4]
配列番号： 1で表されるアミノ酸配列の第 4 6番目から第 5 4番目の部分アミノ 酸配列を示す。  4 shows a partial amino acid sequence of the 46th to 54th amino acids of the amino acid sequence represented by SEQ ID NO: 1.
〔配列番号： 5〕  [SEQ ID NO: 5]
配列番号： 1で表されるアミノ酸配列の第 4 7番目から第 5 4番目の部分アミノ 酸配列を示す。  4 shows a partial amino acid sequence of the 47th to 54th amino acids of the amino acid sequence represented by SEQ ID NO: 1.
〔配列番号 6〕  (SEQ ID NO: 6)
配列番号 1で表されるアミノ酸配列をコードする D NAの塩基配列を示す。 〔配列番号 7〕  1 shows the nucleotide sequence of DNA encoding the amino acid sequence represented by SEQ ID NO: 1. (SEQ ID NO: 7)
配列番号 2で表されるァミノ酸配列をコードする D NAの塩基配列を示す。 〔配列番号 8〕  2 shows the nucleotide sequence of DNA encoding the amino acid sequence represented by SEQ ID NO: 2. (SEQ ID NO: 8)
配列番号 3で表されるアミノ酸配列をコードする D N Aの塩基配列を示す。 〔配列番号 9〕  3 shows the nucleotide sequence of DNA encoding the amino acid sequence represented by SEQ ID NO: 3. (SEQ ID NO: 9)
配列番号 4で表されるアミノ酸配列をコードする D NAの塩基配列を示す。 〔配列番号 1 0〕  2 shows the nucleotide sequence of DNA encoding the amino acid sequence represented by SEQ ID NO: 4. (SEQ ID NO: 10)
配列番号 5で表されるアミノ酸配列をコードする D NAの塩基配列を示す。 実施例 実施例 1 3 shows the nucleotide sequence of DNA encoding the amino acid sequence represented by SEQ ID NO: 5. Example Example 1
乳酸ーグリコール酸共重合体（乳酸ノグリコ一ル =50/50, ポリスチレン換 算平均分子量 =12， 000， 粘度 =0.145dl/g) 1.9 gをジクロロメタン 3.0mlに溶解した。 この有機溶媒液に配列番号： 1で表されるヒト由来 KiSS-Ιぺ プチド （メタスチン （1— 54) ) の凍結乾燥粉末 10 Omgを添加し、 ポリトロン (キネマチ力社） を用いて微粒化する。 この SZO分散液を 0.1%ポリビニルァ ルコール水溶液 800mlに添加し、 ホモミキサーを用いて撹拌 '乳化する。 室温で 3時間撹拌してジクロロメタンを揮散させた後、 遠心分離 （約 2， 000 rpm)する ことによりマイクロカプセルを分取する。次いで蒸留水 400 mlを用いて 2回洗浄 後、 D—マンニトール 0.2 gを添加し凍結乾燥する。 更に残留溶媒除去のため、 46 °Cで 3日間真空乾燥して徐放性メタスチン（1— 54)含有マイクロカプセル を得る。 実施例 2  1.9 g of a lactic acid-glycolic acid copolymer (noglycol lactate = 50/50, polystyrene equivalent average molecular weight = 12,000, viscosity = 0.145 dl / g) was dissolved in 3.0 ml of dichloromethane. Add 10 Omg of lyophilized powder of human KiSS-Ι ぺ peptide (metastin (1-54)) represented by SEQ ID NO: 1 to this organic solvent solution, and atomize using Polytron (Kinemachi Rikisha). . This SZO dispersion is added to 800 ml of a 0.1% aqueous solution of polyvinyl alcohol, and the mixture is stirred and emulsified using a homomixer. Stir at room temperature for 3 hours to evaporate dichloromethane, and centrifuge (about 2,000 rpm) to collect microcapsules. After washing twice with 400 ml of distilled water, add 0.2 g of D-mannitol and freeze-dry. Further, in order to remove the residual solvent, vacuum drying is performed at 46 ° C for 3 days to obtain microcapsules containing sustained-release metastin (1-54). Example 2
乳酸ーグリコール酸共重合体（乳酸/ダリコール =50/50, ポリスチレン換 算平均分子量 =12, 000,粘度 =0.145dl/g) 1.89 gと酸化亜鉛 10m gとをジクロロメタン 3.0 mlに溶解した。 この有機溶媒液に配列番号： 1で表され るヒト由来 KiSS- 1ペプチド （メタスチン （1— 54) ) の凍結乾燥粉末 10 Omg を添加し、 ポリトロン （キネマチ力社） を用いて微粒化した。 この S/〇分散液を 0.1 %ポリビニルアルコール水溶液 80 Offllに添加し、 ホモミキサーを用いて撹 拌-乳化した。 室温で 3時間撹拌してジクロロメタンを揮散させた後、 遠心分離 （ 約 2, 00 Orpm)することによりマイクロカプセルを分取した。次いで蒸留水 40 Oinlを用いて 2回洗净後、 D—マンニトール 0.2 gを添加し凍結乾燥した。 更に 残留溶媒除去のため、 46°Cで 3日間真空乾燥して徐放性メタスチン （1— 54) 含有マイクロカプセルを得た。 実施例 3 乳酸ーグリコール酸共重合体 （乳酸 Zグリコール =65/35, 粘度 =0. 16 Odl/g) 1.69 gと酸化亜鉛 1 Omgとをジクロロメタン 2.7mlに溶解する。 こ の有機溶媒液に配列番号： 1で表されるヒト由来 KiSS- 1ペプチド （メタスチン （1 -54) ) の凍結乾燥粉末 30 Omgを添加し、 ポリトロン (キネマチ力社) を用い て微粒化する。 この S/O分散液を 0. 1 %ポリビニルアルコール水溶液 800ml に添加し、 ホモミキサーを用いて撹拌 ·乳化する。室温で 3時間撹拌してジクロロ メタンを揮散させた後、 遠心分離 （約 2, 00 Orpffl) することによりマイクロカブ セルを分取する。次いで蒸留水 400mlを用いて 2回洗浄後、 D—マンニトール 0 . 2 gを添加し凍結乾燥する。 更に残留溶媒除去のため、 46°Cで 3日間真空乾燥 して徐放性メタスチン （1— 54) 含有マイクロカプセルを得る。 実施例 4 1.89 g of lactic acid-glycolic acid copolymer (lactic acid / daricol = 50/50, polystyrene equivalent average molecular weight = 12,000, viscosity = 0.145 dl / g) and 10 mg of zinc oxide were dissolved in 3.0 ml of dichloromethane. To this organic solvent solution, 10 Omg of a freeze-dried powder of human KiSS-1 peptide (metastin (1-54)) represented by SEQ ID NO: 1 was added, and pulverized using Polytron (Kinemachi Rikisha). This S / II dispersion was added to 80% of a 0.1% aqueous solution of polyvinyl alcohol, and the mixture was stirred and emulsified using a homomixer. After stirring at room temperature for 3 hours to evaporate dichloromethane, microcapsules were collected by centrifugation (about 2,000 rpm). Then, after washing twice with 40 Oinl of distilled water, 0.2 g of D-mannitol was added and freeze-dried. Furthermore, in order to remove the residual solvent, vacuum drying was performed at 46 ° C for 3 days to obtain microcapsules containing sustained-release metastin (1-54). Example 3 1.69 g of lactic acid-glycolic acid copolymer (lactic acid Z glycol = 65/35, viscosity = 0.16 Odl / g) and 1 Omg of zinc oxide are dissolved in 2.7 ml of dichloromethane. Add 30 Omg of lyophilized powder of human KiSS-1 peptide (metastin (1-54)) represented by SEQ ID NO: 1 to this organic solvent solution, and pulverize using Polytron (Kinemachi Rikisha). . This S / O dispersion is added to 800 ml of a 0.1% aqueous solution of polyvinyl alcohol, and the mixture is stirred and emulsified using a homomixer. After stirring at room temperature for 3 hours to evaporate dichloromethane, centrifuge (about 2,000 Orpffl) to collect the microcapsules. Then, after washing twice with 400 ml of distilled water, 0.2 g of D-mannitol is added and lyophilized. Further, in order to remove residual solvent, vacuum drying is performed at 46 ° C for 3 days to obtain microcapsules containing sustained-release metastin (1-54). Example 4
ポリグリセリン脂肪酸エステルの 1つであるへキサグリセ口一ルぺンタステア レート (HexaGlycerol PentaStearate, HGPS) 156 Omgを 70°Cで加熱融解後、 配列番号： 1で表されるヒト由来 KiSS- 1ペプチド （メタスチン （1— 54) ) の凍 結乾燥粉末 4 Omgを添加し、 攪拌混合後、 内径 2. 0匪の移植針に吸引し、 室温に て冷却固化する。 固化したメタスチン （1— 54)含有 HGPSを取り出すため移植針 を 66 で 1ないし 3分間加熱し押し出し成形する。得られた円柱のペレットを長 さ 10蘭に切断し、 長径 2. 0丽、 長さ 10讓の徐放性メタスチン （1一 54) 含 有 HGPS製剤を得る。 実施例 5  HexaGlycerol PentaStearate (HGPS), one of the polyglycerol fatty acid esters, is heated and melted at 70 ° C after heating and melting at 156 Omg. Add 4 Omg of the freeze-dried powder of (1-54)), mix with stirring, aspirate into a 2.0-millimeter implanted needle, and cool and solidify at room temperature. To remove the solidified metastin (1-54) -containing HGPS, heat the implant with a 66 for 1 to 3 minutes and extrude it. The obtained cylindrical pellets are cut into 10 orchids to obtain a sustained-release metastin (1-154) containing 2.0 mm in length and 10 m in length and containing HGPS. Example 5
乳酸ーグリコール酸共重合体 （乳酸/ダリコール =65/35, 粘度 =0. 16 0dl/g) 1. 69 gと酸化亜鉛 1 Omgとをジクロロメタン 2. 7mlに溶解する。 こ の有機溶媒液に配列番号： 3で表されるヒト由来 KiSS-1ペプチド （メタスチン （4 5-54) ) の凍結乾燥粉末 30 Omgを添加し、 ポリトロン （キネマチ力社） を用 いて微粒化する。この SZO分散液を 0. 1 %ポリビニルアルコール水溶液 80 Offl 1に添加し、 ホモミキサーを用いて撹拌.乳化する。 室温で 3時間撹拌してジクロ ロメタンを揮散させた後、 遠心分離 （約 2, 00 Orpm)することによりマイクロ力 プセルを分取する。 次いで蒸留水 400mlを用いて 2回洗浄後、 D—マンニ 1 ^一ル 0. 2 gを添加し凍結乾燥する。 更に残留溶媒除去のため、 46°Cで 3日間真空乾 燥して徐放性メタスチン （45— 54) 含有マイクロカプセルを得る。 実施例 6 1.69 g of lactic acid-glycolic acid copolymer (lactic acid / daricol = 65/35, viscosity = 0.160 dl / g) and 1 Omg of zinc oxide are dissolved in 2.7 ml of dichloromethane. To this organic solvent solution, 30 Omg of a freeze-dried powder of human-derived KiSS-1 peptide (metastin (45-54)) represented by SEQ ID NO: 3 was added and atomized using Polytron (Kinemachi Rikisha). I do. This SZO dispersion is added to a 0.1% aqueous solution of polyvinyl alcohol (80 Offl 1), and the mixture is stirred and emulsified using a homomixer. Stir at room temperature for 3 hours After volatilizing methane, centrifuge (about 2,000 Orpm) to collect micro force cells. Then, after washing twice with 400 ml of distilled water, 0.2 g of D-manni is added and freeze-dried. Further, in order to remove the residual solvent, the microcapsules containing sustained-release metastin (45-54) are obtained by vacuum drying at 46 ° C for 3 days. Example 6
乳酸ーグリコール酸共重合体 （乳酸 Zグリコ一ル =65/35， 粘度 =0. 16 Odl/g) 1. 69 gと酸化亜鉛 1 Oiiigとをジクロロメタン 2. 7ndに溶解する。 こ の有機溶媒液に 25 Omg/ml濃度の配列番号： 3で表されるヒト由来 KiSS- 1ぺプチ ド （メタスチン （45— 54) ) の水溶液を 1· 2 ml添加し、 ポリトロン （キネマ チカ社） を用いて微粒化する。 この WZO乳化液を 0. 1 %ポリビニルアルコール 水溶液 800mlに添加し、 ホモミキサーを用いて撹拌 ·乳化する。 室温で 3時間撹 拌してジクロロメタンを揮散させた後、 遠心分離（約 2, 00 Orpm) することによ りマイクロカプセルを分取する。次いで蒸留水 400 mlを用いて 2回洗浄後、 D— マンニトール 0. 2 gを添加し凍結乾燥する。 更に残留溶媒除去のため、 46 で 3日間真空乾燥して徐放性メタスチン（45— 54)含有マイクロカプセルを得る  Dissolve 1.69 g of lactic acid-glycolic acid copolymer (lactic acid Z-glycol = 65/35, viscosity = 0.16 Odl / g) and zinc oxide 1 Oiiig in dichloromethane 2.7nd. To this organic solvent solution was added 1.2 ml of an aqueous solution of human-derived KiSS-1 peptide (metastin (45-54)) represented by SEQ ID NO: 3 at a concentration of 25 Omg / ml, and Polytron (Kinematica) was added. Atomization using This WZO emulsion is added to 800 ml of a 0.1% aqueous solution of polyvinyl alcohol, and the mixture is stirred and emulsified using a homomixer. After stirring at room temperature for 3 hours to evaporate dichloromethane, the microcapsules are collected by centrifugation (about 2,000 rpm). Then, after washing twice with 400 ml of distilled water, 0.2 g of D-mannitol is added and lyophilized. Furthermore, in order to remove residual solvent, vacuum-dry at 46 for 3 days to obtain microcapsules containing sustained-release metastin (45-54)
実施例 7 Example 7
乳酸ーグリコール酸共重合体 （乳酸 Zグリコール =65/35, 粘度 =0. 1 6 0dl/g) 1. 69 gと酸化亜鉛 1 Omgとをジクロロメタン 2. 7mlに溶解する。 こ の有機溶媒液に配列番号： 2で表されるヒト由来 KiSS- 1ペプチド （メタスチン （4 0- 54) ) の凍結乾燥粉末 30 Omgを添加し、 ポリトロン （キネマチ力社） を用 いて微粒化する。この S/0分散液を 0. 1 %ポリビニルアルコール水溶液 80 On 1に添加し、 ホモミキサーを用いて撹拌 ·乳化する。 室温で 3時間撹拌してジクロ ロメタンを揮散させた後、 遠心分離（約 2， 00 Orpm) することによりマイクロ力 プセルを分取する。次いで蒸留水 400mlを用いて 2回洗浄後、 D—マンニ! ^一ル 0. 2 gを添加し凍結乾燥する。 更に残留溶媒除去のため、 46°Cで 3日間真空乾 燥して徐放性メタスチン （40— 54) 含有マイクロカプセルを得る。 実施例 8 Lactic acid-glycolic acid copolymer (lactic acid Z glycol = 65/35, viscosity = 0.160 dl / g) 1.69 g and 1 Omg of zinc oxide are dissolved in 2.7 ml of dichloromethane. To this organic solvent solution, 30 Omg of a freeze-dried powder of human-derived KiSS-1 peptide (metastin (40-54)) represented by SEQ ID NO: 2 was added and atomized using a Polytron (Kinemachi Rikisha). I do. This S / 0 dispersion is added to a 0.1% aqueous solution of polyvinyl alcohol 80 On 1 and stirred and emulsified using a homomixer. After stirring for 3 hours at room temperature to evaporate dichloromethane, centrifuge (about 2,000 rpm) to collect micro force cells. Then, after washing twice with 400 ml of distilled water, 0.2 g of D-manni! Vacuum dry at 46 ° C for 3 days to remove residual solvent Dry to obtain microcapsules containing sustained-release metastin (40-54). Example 8
乳酸ーグリコール酸共重合体 （乳酸 Zグリコール =65/35, 粘度 =0.16 Odl/g) 1.69 gと酸化亜鉛 1 Omgとをジクロロメタン 2.7mlに溶解した。 こ の有機溶媒液に 25 Omg/ml濃度の配列番号： 2で表されるヒト由来 KiSS- 1ぺプチ ド （メタスチン （40— 54) ) の水溶液を 1. 2 ml添加し、 ポリトロン （キネマ チカ社） を用いて微粒化する。 この W/O乳化液を 0.1 %ポリビニルアルコール 水溶液 800mlに添加し、 ホモミキサーを用いて撹拌 ·乳化する。 室温で 3時間撹 拌してジクロロメタンを揮散させた後、 遠心分離（約 2, 00 Orpm)することによ りマイクロカプセルを分取する。次いで蒸留水 40 Oiiilを用いて 2回洗浄後、 D— マンニ卜ール 0.2 gを添加し凍結乾燥する。 更に残留溶媒除去のため、 46T:で 3日間真空乾燥して徐放性メタスチン（40— 54)含有マイクロカプセルを得る  1.69 g of lactic acid-glycolic acid copolymer (lactic acid Z glycol = 65/35, viscosity = 0.16 Odl / g) and 1 Omg of zinc oxide were dissolved in 2.7 ml of dichloromethane. To this organic solvent solution was added 1.2 ml of an aqueous solution of human-derived KiSS-1 peptide (metastin (40-54)) represented by SEQ ID NO: 2 at a concentration of 25 Omg / ml, and Polytron (Kinematica) was added. Atomization using This W / O emulsion is added to 800 ml of a 0.1% aqueous solution of polyvinyl alcohol, and the mixture is stirred and emulsified using a homomixer. After stirring at room temperature for 3 hours to evaporate dichloromethane, the microcapsules are collected by centrifugation (about 2,000 rpm). Then, after washing twice with 40 Oiiil of distilled water, 0.2 g of D-mannitol is added and lyophilized. In order to remove residual solvent, vacuum-dry with 46T: for 3 days to obtain microcapsules containing sustained-release metastin (40-54)
実施例 9 Example 9
乳酸-グリコール酸共重合体 （乳酸 Zグリコール =50/50, ポリスチレン換 算平均分子量 = 12, 000,粘度 =0.145dl/g) 1.97 gと酸化亜鉛 10m gとをジクロロメタン 2.7mlに溶解した。 この有機溶媒液に配列番号： 1で表され るヒト由来 KiSS-1ペプチド （メタスチン （1-54) ) の凍結乾燥粉末 2 Omgを添 加し、 ポリトロン （キネマチ力社） を用いて微粒化した。 この SZO分散液を 0. 1 %ポリビニルアルコール水溶液 800mlに添加し、 ホモミキサーを用いて撹拌 · 乳化した。 室温で 3時間撹拌してジクロロメタンを揮散させた後、 遠心分離（約 2 , 00 Orpm) することによりマイクロカプセルを分取した。 次いで蒸留水 400m 1を用いて 2回洗浄後、 D-マンニトール 0.2 gを添加し凍結乾燥した。 更に残留 溶媒除去のため、 46°Cで 3日間真空乾燥して徐放性メタスチン （1-54) 含有 マイクロカプセルを得た。 実施例 10 1.97 g of lactic acid-glycolic acid copolymer (lactic acid Z glycol = 50/50, polystyrene equivalent average molecular weight = 12,000, viscosity = 0.145 dl / g) and 10 mg of zinc oxide were dissolved in 2.7 ml of dichloromethane. 2 Omg of a freeze-dried powder of human KiSS-1 peptide (metastin (1-54)) represented by SEQ ID NO: 1 was added to this organic solvent solution, and the mixture was atomized using Polytron (Kinemachi Rikisha). . This SZO dispersion was added to 800 ml of a 0.1% aqueous solution of polyvinyl alcohol, and the mixture was stirred and emulsified using a homomixer. After stirring at room temperature for 3 hours to evaporate dichloromethane, microcapsules were collected by centrifugation (about 2,000 rpm). Then, after washing twice with 400 ml of distilled water, 0.2 g of D-mannitol was added and freeze-dried. Furthermore, in order to remove the residual solvent, vacuum drying was performed at 46 ° C for 3 days to obtain microcapsules containing sustained-release metastin (1-54). Example 10
乳酸-グリコール酸共重合体 （乳酸/ダリコール =50/50， ポリスチレン換 算平均分子量 =12, 000, 粘度 =0. 1 5dl/g) 2.148 gと酸化亜鉛 1 2 mgとをジクロロメタン 3.3 mlに溶解した。 この有機溶媒液に配列番号： 1で表 されるヒト由来 KiSS- 1ペプチド （メタスチン （1ー54) ) の凍結乾燥粉末 240 fflgを添加し、 ポリトロン （キネマチ力社） を用いて微粒化した。 この SZO分散液 を 0.1 %ポリビニルアルコール水溶液 80 Omlに添加し、 ホモミキサーを用いて 撹拌 '乳化した。 室温で 3時間撹拌してジクロロメタンを揮散させた後、 遠心分離 (約 2, 00 Orpm)することによりマイクロカプセルを分取した。次いで蒸留水 4 00mlを用いて 2回洗浄後、 D—マンニトール 0.24 gを添加し凍結乾燥した。 更に残留溶媒除去のため、 46 °Cで 3日間真空乾燥して徐放性メタスチン（1— 5 4)含有マイクロカプセルを得た。 実施例 11  Lactic acid-glycolic acid copolymer (lactic acid / dalicol = 50/50, polystyrene equivalent average molecular weight = 12,000, viscosity = 0.15 dl / g) Dissolve 2.148 g and 12 mg of zinc oxide in 3.3 ml of dichloromethane did. To this organic solvent solution, 240 fflg of a freeze-dried powder of human-derived KiSS-1 peptide (metastin (1-54)) represented by SEQ ID NO: 1 was added, and the mixture was pulverized using Polytron (Kinemachi Rikisha). This SZO dispersion was added to a 0.1% aqueous polyvinyl alcohol solution (80 Oml) and stirred and emulsified using a homomixer. After stirring at room temperature for 3 hours to evaporate dichloromethane, microcapsules were collected by centrifugation (about 2,000 rpm). Then, after washing twice with 400 ml of distilled water, 0.24 g of D-mannitol was added and freeze-dried. Further, in order to remove the residual solvent, the microcapsules containing sustained-release metastin (1-54) were obtained by vacuum drying at 46 ° C for 3 days. Example 11
乳酸-グリコール酸共重合体 （乳酸/ダリコール =50Z50, ポリスチレン換 算平均分子量 =12, 000, 粘度 =0.145dl/g) 2.16 gをジクロロメ夕 ン 3.3mlに溶解した。 この有機溶媒液に配列番号： 1で表されるヒト由来 KiSS- 1 ペプチド （メタスチン （1— 54) ) の凍結乾燥粉末 24 Omgを添加し、 ポリトロ ン （キネマチ力社） を用いて微粒化した。 この S/O分散液を 0.1%ポリビニル アルコール水溶液 800mlに添加し、 ホモミキサーを用いて撹拌 ·乳化した。 室温 で 3時間撹拌してジクロロメタンを揮散させた後、 遠心分離 （約 2， 00 Orpm)す ることによりマイクロカプセルを分取した。次いで蒸留水 40 Omlを用いて 2回洗 浄後、 D—マンニトール 0.24 gを添加し凍結乾燥した。 更に残留溶媒除去のた め、 46 °Cで 3日間真空乾燥して徐放性メタスチン （1— 54)含有マイクロカブ セルを得た。 実験例 1  2.16 g of a lactic acid-glycolic acid copolymer (lactic acid / daricol = 50Z50, polystyrene equivalent average molecular weight = 12,000, viscosity = 0.145 dl / g) was dissolved in 3.3 ml of dichloromethane. To this organic solvent solution, 24 Omg of a freeze-dried powder of human-derived KiSS-1 peptide represented by SEQ ID NO: 1 (metastin (1-54)) was added, and the mixture was atomized using Polytron (Kinemachi Rikisha). . The S / O dispersion was added to 800 ml of a 0.1% aqueous solution of polyvinyl alcohol, and the mixture was stirred and emulsified using a homomixer. After stirring at room temperature for 3 hours to evaporate dichloromethane, microcapsules were collected by centrifugation (about 2,000 rpm). Then, after washing twice with 40 Oml of distilled water, 0.24 g of D-mannitol was added and freeze-dried. Furthermore, in order to remove the residual solvent, vacuum drying was performed at 46 ° C for 3 days to obtain a microcapsule containing sustained-release metastin (1-54). Experimental example 1
実施例 10で調製した徐放性メタスチン（1— 54)含有マイクロカプセルをラ ット皮下にメタスチン （1ー 5 4 ) 量として 6 mg相当量を投与し、 血中メタスチン ( 1 - 5 4 ) 濃度の経時変化を 2週間にわたり測定した。 結果を図 1に示す。 図 1で示されるように血中メタスチン（1一 5 4 )濃度は 2週間にわたり検出さ れた。 これより本発明の徐放性製剤が優れた徐放性を有することは明らかである。 実験例 2 The microcapsules containing sustained release metastin (1-54) prepared in Example 10 were The metastin (1-54) was administered subcutaneously at a dose of 6 mg equivalent, and the time-dependent change in blood metastin (1-54) concentration was measured over 2 weeks. The results are shown in Figure 1. As shown in FIG. 1, blood metastin (1-154) concentration was detected over a two-week period. It is apparent from this that the sustained-release preparation of the present invention has excellent sustained-release properties. Experimental example 2
( 1 ) iiOT7T175発現 B16マウスメラノーマ細胞の作製  (1) Preparation of iiOT7T175 expressing B16 mouse melanoma cells
hOT7T175の cDNA (W0 00/24890に記載の配列番号： 6 ) を、 優性選択マーカ一と して neofを有する pAKKO- neo哺乳動物細胞発現プラスミドに定法を用いて連結し、 ίι 0T7T175発現コンストラクトを得た。 Eiiectene (QIAGEN社） トランスフエクシヨン 試薬を用いて、 該 1ι0Τ7Π75発現コンストラクトを B16細胞に導入した。該 B16細胞を 最終濃度 1. 2 mg/ml の G418含有 RPMI 1640選択培地中で選択培養し、 形成されてき たコロニーをクローニングリングを用いて単離し、 17クローンの G418耐性 β 16/hl7 5細胞株を単離した。 hOT7T175 cDNA (SEQ ID NO: 6 described in WO 00/24890) was ligated to a pAKKO-neo mammalian cell expression plasmid having neo f as a dominant selectable marker using a standard method, and the ίι0T7T175 expression construct was ligated. Obtained. The 1ι0Τ7Π75 expression construct was introduced into B16 cells using Eiiectene (QIAGEN) transfection reagent. The B16 cells were selectively cultured in RPMI 1640 selection medium containing G418 at a final concentration of 1.2 mg / ml, and the formed colonies were isolated using a cloning ring to obtain 17 clones of G418-resistant β16 / hl75 cells. The strain was isolated.
該遺伝子導入 Β16Λ175細胞株 17クローンについて、 最終濃度 1. 2 mg/ml の G418 含有 RPMI 1640選択培地を用いて T25フラスコ中にて培養し、 RNeasy (QIAGEN)を用い て総 RNAを抽出した。 それぞれのクローンから抽出した 1 gの RNAを鐯型として用 いて、 Superscript II (Gibco BRL)によって cDNA合成を実施した。 T7T175に対す る TadManプライマ一、 TaqManプローブセットを用いて TadMan PCRを実施し、 cl. 4 をはじめとする数株の受容体発現株を選別した。  The 17 transgenic {16} -175 cell lines were cultured in T25 flasks using RPMI 1640 selection medium containing G418 at a final concentration of 1.2 mg / ml, and total RNA was extracted using RNeasy (QIAGEN). CDNA synthesis was performed using Superscript II (Gibco BRL) using 1 g of RNA extracted from each clone as type III. TadMan PCR was performed using TadMan primer set and TaqMan probe set for T7T175, and several receptor expression strains including cl. 4 were selected.
選別した 5クローン（cl. 4、 cl. 17、 cl. 19、 cl. 2、 cl. 20)について、 メタスチンお よびメタスチン（4 5— 5 4 ) に対する応答性を、 FLIPRを用いた細胞内 Ca2+濃度変 化の検出により検討した。 その結果、 TaQMan PCRによって高発現量を検出した （c a. 1000-7000 copies/ng total 腿) 細胞株 3クローン （cl. 4、 cl. 17、 cl. 19) に ついて、 それぞれ最終濃度 1(Γ6 Μのメタスチンまたは 10—8 Μのメタスチン （4 5— 5 4 ) に応答することを確認した。 The responsiveness to metastin and metastin (45-54) of the selected 5 clones (cl.4, cl.17, cl.19, cl.2, cl.20) was determined by intracellular Ca using FLIPR. It was examined by detecting changes in 2+ concentration. As a result, for three clones (cl. 4, cl. 17, cl. 19) with high expression levels detected by TaQMan PCR (ca. 1000-7000 copies / ng total thigh), the final concentration was 1 (each). It was confirmed to respond to Γ of 6 Micromax metastin or 10- 8 Micromax of metastin (4 5 5 4).
以降の転移実験では cl. 4 (B16- BL6/1U75)を用いることにした。  In subsequent transfer experiments, cl. 4 (B16-BL6 / 1U75) was used.
( 2 ) h0T7T175発現 B16マウスメラノーマ細胞 (B16-BL6/hl75)を用いた自然転移モ デルでのメタスチンの癌転移抑制作用の検討 (2) Spontaneous metastasis using h0T7T175-expressing B16 mouse melanoma cells (B16-BL6 / hl75) Investigation of the metastatic effect of metastin on cancer in Dell
メタスチンの癌転移抑制作用について、 まず自然転移モデルを用いて検討した。 上記 （1 ) で作製した B16- BL6/11175細胞および空のベクターのみを発現させた B 16 - BL6/mock細胞を、 3 x 105 cel ls/20 χ 1/mouseの割合で、 C57BL/6 (6週齢、 雌、 日本チャールズリパー） マウスの右前足 （forefoot pad) に皮下投与した。 細胞投 与 18日後にケ夕ミン麻酔下で蒸留水 （大塚蒸留水） に溶解した 1 mMのメタスチン、 および vehicleとしての蒸留水を 100 // 1づっ充填した Alzet浸透圧ポンプ（0. 25 \ /hour, 14 days release, Model 1002, Alza社） をマウス背部の皮下に埋め込み、 メタスチンの持続投与を開始した。細胞投与 21日後に、 エーテル麻酔下でマウスに 形成した腫瘍を切除し、傷口をイソジン液で消毒後縫合した。細胞投与 3 5日後に 、 マウスの腹部大動脈より脱血して肺を摘出した。 摘出した肺は、 1. 4 ピクリン酸 1水和物、 4. 8%中性ホルマリンおよび 4. 4%酢酸水溶液 （Bouin液） 中にて 4°C、 16 時間固定後、肺に形成した癌転移結節数を実態顕微鏡下で計数した。結果を表 1に 示す。 First, the metastatic effect of metastin on cancer metastasis was examined using a spontaneous metastasis model. The B16-BL6 / 11175 cells prepared in (1) above and the B16-BL6 / mock cells expressing only the empty vector were transformed into C57BL / 6 at a ratio of 3 × 10 5 cels / 20201 / mouse. (6 weeks old, female, Japanese Charles Ripper) Mice were subcutaneously administered to the right forefoot pad. Eighteen days after cell administration, an Alzet osmotic pump (0.25%) filled with 100 // 1 of 1 mM metastin dissolved in distilled water (Otsuka distilled water) and distilled water under vehicle anesthesia under keummin anesthesia. / hour, 14 days release, Model 1002, Alza) was implanted subcutaneously on the back of the mouse, and continuous administration of metastin was started. Twenty-one days after cell administration, the tumor formed in the mouse was excised under ether anesthesia, and the wound was disinfected with Isodine solution and sutured. 35 days after cell administration, blood was removed from the abdominal aorta of the mouse and the lung was removed. The removed lung was fixed in 4 picric acid monohydrate, 4.8% neutral formalin and 4.4% acetic acid aqueous solution (Bouin solution) at 4 ° C for 16 hours, and the cancer formed in the lung The number of metastatic nodules was counted under a stereoscopic microscope. Table 1 shows the results.
Figure imgf000033_0001
Figure imgf000033_0001
*： 21日目の腫瘍サイズ *: Tumor size on day 21
* *： Vehicle群に比して有意差なし  * *: No significant difference compared to Vehicle group
* * * :危険率1 %以下 （Vehicle群比； Student' s t検定） ， 平均値士標準誤差 メタスチン投与群では、 vehicle投与群に比較して有意な肺への転移結節数の低 下が認められた。 産業上の利用可能性 * * *: Risk factor 1% or less (Vehicle group ratio; Student's test), standard error of the mean value The metastin group showed a significant decrease in the number of metastatic nodules to the lung compared to the vehicle group Was done. Industrial applicability
メタスチンもしくはその誘導体またはその塩は、徐放性製剤化することによって 、 より有効に薬理効果を発揮する。 メタスチンもしくはその誘導体またはその塩を 含有する本発明の徐放性製剤は、メタスチンの生理活性障害を原因とする全ての疾 患の治療または予防に有用である。特に本発明の製剤は、癌転移抑制活性を有する ため、 あらゆる癌の治療または予防に有用である。 また、 本発明の製剤は、 胎盤機 能調節作用を有するため、 絨毛癌、 胞状奇胎、 侵入奇胎、 流産、 胎児の発育不全、 糖代謝異常、 脂質代謝異常または分娩異常の治療または予防に有用である。 更に、 本発明の製剤は、膝臓機能調節作用を有するため、膝臓疾患の治療または予防にも 有用である。  Metastin or a derivative thereof or a salt thereof exhibits a more effective pharmacological effect by forming a sustained-release preparation. The sustained-release preparation of the present invention containing metastin or a derivative thereof or a salt thereof is useful for treating or preventing all diseases caused by impaired metastin bioactivity. In particular, since the preparation of the present invention has a cancer metastasis inhibitory activity, it is useful for treating or preventing any cancer. In addition, since the preparation of the present invention has a placental function regulating action, it is useful for treating or preventing choriocarcinoma, hydatidiform mole, invasive mole, miscarriage, fetal growth deficiency, glucose metabolism disorder, lipid metabolism disorder or delivery abnormality. Useful. Further, since the preparation of the present invention has a knee function-regulating action, it is also useful for treating or preventing knee disease.

Claims

請求の範囲 The scope of the claims
1 . メタスチンもしくはその誘導体またはその塩を含有する徐放性製剤。 1. Sustained-release preparations containing metastin or a derivative or salt thereof.
2 . メタスチンを含有する請求項 1記載の徐放性製剤。  2. The sustained-release preparation according to claim 1, comprising metastin.
3 . メタスチンまたはその誘導体が、 配列番号： 1で表わされるアミノ酸配列の N 末端から 4 7ないし 5 4番目のアミノ酸配列を含有し、かつ 8ないし 5 4個のアミ ノ酸残基からなるぺプチドもしくはその誘導体である請求項 1記載の徐放性製剤。3. Metastin or a derivative thereof comprises a peptide comprising the amino acid sequence at the 47th to 54th position from the N-terminus of the amino acid sequence represented by SEQ ID NO: 1 and consisting of 8 to 54 amino acid residues 2. The sustained-release preparation according to claim 1, which is a derivative thereof.
4. ペプチドが、 配列番号： 1で表わされるアミノ酸配列における N末端から 4 7 ないし 5 4番目のアミノ酸配列を C末端に有するペプチドである請求項 3記載の 徐放性製剤。 4. The sustained-release preparation according to claim 3, wherein the peptide is a peptide having an amino acid sequence at positions 47 to 54 from the N-terminus in the amino acid sequence represented by SEQ ID NO: 1 at the C-terminus.
5 . ペプチドが、 8ないし 1 5個のアミノ酸残基からなるペプチドである請求項 3 または 4記載の徐放性製剤。  5. The sustained-release preparation according to claim 3, wherein the peptide is a peptide consisting of 8 to 15 amino acid residues.
6 . ペプチドが、 配列番号： 2、 配列番号： 3、 配列番号： 4または配列番号： 5 で表わされるァミノ酸配列からなるぺプチドである請求項 3記載の徐放性製剤。  6. The sustained-release preparation according to claim 3, wherein the peptide is a peptide comprising an amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5.
7 . メタスチンもしくはその誘導体またはその塩及びキャリアーを含有する請求項 1記載の徐放性製剤。 7. The sustained-release preparation according to claim 1, comprising metastin or a derivative thereof or a salt thereof and a carrier.
8 . キヤリァ一がポリマーである請求項 7記載の徐放性製剤。  8. The sustained-release preparation according to claim 7, wherein the carrier is a polymer.
9 . ポリマーが生体内分解性ポリマーである請求項 8記載の徐放性製剤。  9. The sustained-release preparation according to claim 8, wherein the polymer is a biodegradable polymer.
1 0 .生体内分解性ポリマーが脂肪族ポリエステルである請求項 9記載の徐放性製 剤。  10. The sustained-release preparation according to claim 9, wherein the biodegradable polymer is an aliphatic polyester.
1 1 .脂肪族ポリエステルが乳酸 Zグリコール酸重合体である請求項 1 0記載の徐 放性製剤。  11. The sustained-release preparation according to claim 10, wherein the aliphatic polyester is a lactic acid-Z glycolic acid polymer.
1 2 .乳酸/ダリコール酸重合体の乳酸/ダリコール酸組成比が約 1 0 0 / 0ない し約 4 0 / 6 0である請求項 1 1記載の徐放性製剤。  12. The sustained release preparation according to claim 11, wherein the lactic acid / daricholic acid polymer has a lactic acid / daricholic acid composition ratio of about 100/0 to about 40/60.
1 3. 乳酸 Zグリコール酸重合体の重量平均分子量が約 3， 0 0 0ないし約 8 0， 0 0 0である請求項 1 1記載の徐放性製剤。 13. The sustained-release preparation according to claim 11, wherein the weight average molecular weight of the lactic acid-Z glycolic acid polymer is about 3,000 to about 800,000.
1 4. ポリマーが生体内非分解性ポリマ一である請求項 8記載の徐放性製剤。 14. The sustained-release preparation according to claim 8, wherein the polymer is a non-degradable polymer in vivo.
1 5 .生体内非分解性ポリマーがポリグリセリン脂肪酸エステルである請求項 1 4 記載の徐放性製剤。 15. The non-degradable polymer in vivo is a polyglycerin fatty acid ester. The sustained-release preparation according to the above.
16. 癌の治療又は予防剤である請求項 1記載の徐放性製剤。  16. The sustained-release preparation according to claim 1, which is an agent for treating or preventing cancer.
17. 胎盤機能改善剤である請求項 1記載の徐放性製剤。  17. The sustained-release preparation according to claim 1, which is a placental function improving agent.
18. 非経口投与剤である請求項 1記載の徐放性製剤。  18. The sustained-release preparation according to claim 1, which is a parenteral administration preparation.
19. 皮下投与剤である請求項 1記載の徐放性製剤。  19. The sustained-release preparation according to claim 1, which is a subcutaneous preparation.
20. 筋肉内投与剤である請求項 1記載の徐放性製剤。  20. The sustained-release preparation according to claim 1, which is an intramuscular preparation.
21. 腹腔内投与剤である請求項 1記載の徐放性製剤。  21. The sustained-release preparation according to claim 1, which is an intraperitoneal preparation.
22.徐放性製剤を製造するためのメタスチンもしくはその誘導体またはその塩の 使用。  22. Use of metastin or a derivative or salt thereof for producing a sustained-release preparation.
23.哺乳動物に対して請求項 1記載の徐放性製剤の有効量を投与することを特徴 とする癌の治療又は予防方法  23. A method for treating or preventing cancer, comprising administering to a mammal an effective amount of the sustained-release preparation according to claim 1.
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