JP2005126360A - Collagenase inhibitor composed of new polypeptide - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a collagenase inhibitor that has high inhibitory action on collagenase, no fear of danger of causing infection with a pathogen and transmission of a pathogenic factor and unfavorable adverse effect and is decomposable in vivo. <P>SOLUTION: The new polypeptide comprises a peptide unit represented by formula (1): -Pro-X-Gly- (1) (X is Pro or Hyp; Y is Gln, Asn, Leu, Ile, Val or Ala) and a peptide unit represented by formula (2): -Pro-Y-Gly-Z-Ala-Gly- (2) (Z is Ile or Leu) and is used for inhibiting collagenase activity. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a collagenase inhibitor composed of a novel polypeptide having biodegradability. More specifically, the present invention relates to a collagenase useful as a highly safe biomaterial or medical material, an active ingredient of a pharmaceutical, a cosmetic material or an edible composition additive without the risk of pathogen infection or undesirable side effects. Relates to inhibitors.

  Collagen is a fibrous protein found in all multicellular animals and accounts for 25% of the total protein in mammals as the main component of skin and bone. A typical collagen molecule has a rope-like superhelical structure in which three collagen polypeptide chains are called a triple helical structure. Collagen contains a particularly large amount of proline (Pro) and glycine (Gly), and both amino acid residues are important for the formation of a stable triple helical structure.

  Collagenase (Matrix metalloproteinase-1), an enzyme that specifically degrades collagen, is known to increase its activity and degrade collagen due to inflammatory reaction, inflammatory disease, aging, and the like. Therefore, inhibition of collagenase activity is considered to be effective in suppressing the inflammatory reaction, alleviating various symptoms caused by the inflammatory reaction, preventing aging due to aging of the skin, and preventing osteoporosis.

  Various proposals have been made on the use of substances having a collagenase inhibitory action. For example, Japanese Patent Application Laid-Open No. 2003-192565 (Patent Document 1) discloses an anti-aging cosmetic containing a plant extract having a collagenase inhibitory action and collagen derived from fish.

  JP 2003-183173 A (Patent Document 2) discloses collagenase inhibition using an extract from a plant such as Niktantis Arbolistis, Piper Chaba, Antocephalans indicans, Crotalaria cytisoides as an active ingredient. An external preparation for skin and a food or drink containing the agent are disclosed.

  JP-A No. 2003-176219 (Patent Document 3) discloses an antiseptic / antifungal agent and a degradation product of collagen or gelatin, wherein (Gly-XY) n (where Gly represents a glycine residue, X , Y represents an amino acid residue, and X and Y may be the same or different, and n represents a positive integer) and a tripeptide having an amino acid sequence represented by Is disclosed.

  Japanese Patent Application Laid-Open No. 2002-104986 (Patent Document 4) is an anti-periodontal disease agent containing a ginger extract as an active ingredient, which inhibits collagenase produced by the periodontal disease pathogen Polyphyromonas gingivalis. An anti-periodontal agent having the following has been disclosed.

  JP-A-2001-247469 (Patent Document 5) produces α- and / or γ-mangostin extracted from hypericum mangosteen and its pericarp as an active ingredient and is produced by polyphyromonas gingivalis. An anti-caries and periodontal disease agent having an action of inhibiting collagenase, and oral compositions and foods and drinks containing them are disclosed.

  Japanese Patent Application Laid-Open No. 09-241287 (Patent Document 6) discloses a collagenase inhibitor collected from a culture of microorganisms belonging to the genus Aspergillus such as Aspergillus niger, which comprises an antirheumatic agent, an anti-inflammatory agent, and an anticancer agent. And collagenase inhibitor FO-5904 useful as a therapeutic agent for anti-influenza virus infection is disclosed.

  Japanese Patent Application Laid-Open No. 09-235293 (Patent Document 7) discloses novel triterpenes that are extracted from fruit bodies of mushrooms such as bamboo shoots and have a collagenase inhibitory action. This document describes that the triterpenes are useful as therapeutic agents for diseases such as joint diseases, bone resorption diseases, periodontal diseases, corneal ulcers, and epidermolysis bullosa.

  Japanese Patent Application Laid-Open No. 07-304770 (Patent Document 8) discloses a novel benzoazepinone derivative that has a strong collagenase inhibitory action and is useful as a therapeutic agent for various diseases caused by enhanced collagenase activity.

  Japanese Patent Application Laid-Open No. 07-291873 (Patent Document 9) contains at least one solvent extract selected from the group consisting of guava leaves, Nishikawayanagi and Rishimi, and is a collagenase activity inhibitor useful for food or medicine. Is disclosed.

Japanese Patent Laid-Open No. 05-163287 (Patent Document 10) discloses a peptide derivative used as an inhibitor of bacterial collagenase belonging to the type of zinc metal binding protein, which strongly interacts with the zinc atom in the active site of collagenase. Novel peptide derivatives having a phosphine chelating group PO ₂ —CH ₂ capable of acting on phosphine are disclosed.

  In addition, structural features of a heterotrimer for collagen that mimics the collagenase degradation site of type I collagen have been reported ("J. Mol. Biol., Vol. 319" (Non-patent Document 1), pp. .1235-1242, 2002).

  In addition, regarding the method of chemical synthesis of collagen analogues, p-nitrophenyl ester of a peptide represented by the formula Pro-Ser-Gly or p-nitrophenyl ester of a peptide represented by the formula Pro-Ala-Gly is converted to dimethylformamide. It is reported that a soluble polyamide having a molecular weight of 16,000 to 21,000 can be obtained by dissolving, adding triethylamine and allowing to stand for 24 hours (“J. Mol. Biol., Vol. 63” (Non-patent Document 2). ), Pp.85-99, 1972). These soluble polyamides are estimated to have a triple helical structure from the circular dichroism spectrum, but there is no description regarding the properties of the obtained polymer.

  A 50-mer peptide containing the elastin-derived Val-Pro-Gly-Val-Gly sequence was dissolved in dimethyl sulfoxide, and 2 equivalents of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide and 1 equivalent of 1 It has also been reported that after adding -hydroxybenzotriazole and 1.6 equivalents of N-methylmorpholine and allowing to stand for 14 days, a dialysis membrane having a molecular weight cutoff of 50,000 is dialyzed to obtain a polyamide ("" Int. J. Peptide Protein Res., Vol. 46 "(Non-Patent Document 3), pp. 453-463, 1995).

  On the other hand, the causative substance of sheep tremor and bovine spongiform encephalopathy is an infectious protein called prion, and this infectious protein is said to be one of the causes of human Creutzfelder-Jakob disease. It is pointed out that prion is a protein, is hardly inactivated by ordinary sterilization and sterilization methods, and is infected across species ("Nature Review, Vol. 2" (Non-patent Document 4), pp. 118-126, 2001).

In general, natural products and extracts from these are often used as raw materials in pharmaceuticals, cosmetics, and foods and drinks. Therefore, there is always a risk of infection (or transmission) of unknown pathogens (or pathogenic factors) such as prions that cannot be removed by ordinary sterilization and sterilization methods. On the other hand, the synthetic chemical substances described in Patent Document 8 are not sufficiently decomposed or absorbed in vivo and have a risk of side effects.
JP 2003-192565 A (Claims 1 and 2) JP 2003-183173 A (Claims 3, 6 and 7) JP 2003-176219 A (Claim 1) JP 2002-104986 A (Claim 1) JP 2001-247469 A (Claims 1 to 5, paragraph number [0047]) JP 09-241287 (Claims 1 to 5, paragraph number [0001]) JP 09-235293 A (paragraph number [0008]) JP 07-304770 A (Claim 1, paragraph number [0026]) JP 07-291873 A (Claim 1, paragraph number [0046]) JP 05-163287 A (paragraph number [0001]) "J. Mol. Biol., Vol.319", pp.1235-1242, 2002 "J. Mol. Biol., Vol.63", pp.85-99, 1972 "Int. J. Peptide Protein Res., Vol.46", pp.453-463, 1995 "Nature Review, Vol.2", pp.118-126, 2001

  Accordingly, an object of the present invention is to have a high collagenase inhibitory action, and there is no risk of causing infection of pathogens or transmission of pathogenic factors and there is no risk of undesirable side effects, and a collagenase inhibitor (or novel) that can be degraded in vivo. Polypeptide).

  Another object of the present invention is to provide a collagenase inhibitor that is highly safe and useful as a component of biomaterials or medical materials, pharmaceutical compositions, cosmetics, and edible compositions.

  As a result of intensive studies to solve the above problems, the present inventors have found that when a specific peptide component is condensed, a collagen-like polypeptide is produced without cyclization, can be decomposed in vivo, and has a collagenase inhibitory action. The headline and the present invention were completed.

  That is, the novel polypeptide of the present invention includes a peptide unit having an amino acid sequence represented by the following formula (1) and a peptide unit having an amino acid sequence represented by the following formula (2), and has an activity of collagenase. Used to inhibit.

-Pro-X-Gly- (1)
-Pro-Y-Gly-Z-Ala-Gly- (2)
(In the formula, X represents Pro or Hyp, Y represents Gln, Asn, Leu, Ile, Val or Ala, and Z represents Ile or Leu.)
The ratio (molar ratio) between the peptide unit (1) and the peptide unit (2) may be about (1) / (2) = 99/1 to 1/99.

The polypeptide or collagenase inhibitor of the present invention contains an amino acid sequence recognized by collagenase, which is a collagenolytic enzyme. Therefore, the polypeptide binds to or is degraded by collagenase, or binds to and is degraded by collagenase. Since the polypeptide has such properties, it can inhibit the action of collagenase. In addition, since the polypeptide is a collagen-like polypeptide having an amide bond, it can be decomposed in the living body of a mammal. That is, the polypeptide can be decomposed and absorbed in vivo. In the circular dichroism spectrum, the polypeptide exhibits a positive cotton effect at wavelengths of 220 to 230 nm and a negative cotton effect at wavelengths of 195 to 205 nm. This indicates that at least a part (part or all) of the polypeptide forms a triple helical structure. The polypeptide may exhibit a peak in the molecular weight range of 5 × 10 ^{2 to} 500 × 10 ⁴ . The collagenase inhibitor may be used, for example, as a component of biomaterials or medical materials, pharmaceutical compositions, cosmetics, edible compositions, and the like.

  The present invention also includes cosmetics and edible compositions that contain the polypeptide and are used to inhibit collagenase activity. The present invention also includes a method of inhibiting collagenase activity using the polypeptide.

  The polypeptide or collagenase inhibitor of the present invention has a high collagenase inhibitory action and is capable of degrading in vivo without risk of pathogen infection or side effects. Therefore, since it is excellent in biocompatibility, it is highly safe and useful as a component of biomaterials or medical materials, pharmaceutical compositions, cosmetics, edible compositions, and the like. Furthermore, this polypeptide can be produced by a simple operation called a condensation reaction while suppressing dimerization or cyclization reaction.

  In the present invention, various amino acid residues are described by the following abbreviations.

Ala: L-alanine residue
Arg: L-arginine residue
Asn: L-asparagine residue
Asp: L-aspartic acid residue
Cys: L-cysteine residue
Gln: L-glutamine residue
Glu: L-glutamic acid residue
Gly: Glycine residue
His: L-histidine residue
Hyp: L-hydroxyproline residue
Ile: L-isoleucine residue
Leu: L-leucine residue
Lys: L-lysine residue
Met: L-methionine residue
Phe: L-phenylalanine residue
Pro: L-proline residue
Sar: Sarcosine residue
Ser: L-serine residue
Thr: L-threonine residue
Trp: L-tryptophan residue
Tyr: L-tyrosine residue
Val: L-valine residue In this specification, according to a conventional method, the amino acid sequence of the peptide chain is determined by positioning the N-terminal amino acid residue on the left side and the C-terminal amino acid residue on the right side. Describe.

The novel polypeptide in the present invention needs to contain a peptide unit having an amino acid sequence represented by -Pro-X-Gly-. Since the sequence represented by -Pro-X-Gly- contributes to the stability of the triple helix structure, the stability of the triple helix structure decreases when the ratio of this sequence is small. Furthermore, this unit forms a repeating structure (oligo or polypeptide unit structure) represented by-(Pro-X-Gly) _n -in the polypeptide from the viewpoint of the stability of the triple helical structure. May be. The repeating number n of this arrangement is, for example, about 1 to 5000, preferably about 2 to 3000. X may be either Pro or Hyp, but is more preferably Hyp in view of the stability of the triple helical structure. Hyp is usually a 4Hyp (eg, trans-4-hydroxy-L-proline) residue.

  Further, this polypeptide needs to contain a peptide unit having an amino acid sequence represented by -Pro-Y-Gly-Z-Ala-Gly-. When this sequence is not included or too little, the inhibitory action of collagenase decreases. On the other hand, when the number of the sequences is too large, the stability of the triple helical structure is lowered. Y may be any of Gln, Asn, Leu, Ile, Val or Ala, but Gln, Asn, Leu, Val, Ala, particularly Gln and Leu are more preferable. Z may be Ile or Leu, but Ile is more preferable. The combination of Y and Z is, for example, a peptide in which Y is selected from Gln, Asn, Leu, Ile, Val and Ala (for example, Gln or Leu), and a peptide in which Z is Ile, or Y is Gln, Examples thereof include a peptide selected from Asn, Leu, Ile, Val and Ala (for example, Gln or Leu), and Z is Leu.

  A combination of X, Y and Z is a peptide in which X is Hyp, Y is selected from Gln, Asn, Leu, Ile, Val and Ala (for example, Gln or Leu), Z is Ile or Leu, Examples thereof include a peptide wherein X is Pro, Y is Gln, Asn, Leu, Ile, Val and Ala (for example, Gln or Leu), and Z is Ile or Leu.

  Furthermore, the polypeptide in the present invention may contain other amino acid residues and peptide residues (units) as long as the physical and biological properties of the resulting polypeptide are not impaired. In order for the polypeptides in the present invention to exhibit useful physical and biological properties, for example, Gly, Sar, Ser, Glu, Asp, Lys, His, Ala, Val, Leu, Arg, Pro, Tyr, At least one amino acid residue or peptide residue selected from Ile, in particular at least one amino acid residue or peptide residue selected from Gly, Sar, Ser, Glu, Asp, Lys, Arg, Pro, Val Many have. Specifically, for example, Gly, Sar, Ser, Glu, Asp, Lys, Arg-Gly-Asp, Tyr-Ile-Gly-Ser-Arg, Ile-Lys-Val-Ala-Val, Val-Pro-Gly -Val-Gly, Asp-Gly-Glu-Ala, Gly-Ile-Ala-Gly, His-Ala-Val, Glu-Arg-Leu-Glu, Lys-Asp-Pro-Lys-Arg-Leu, Arg-Ser It preferably contains an amino acid residue or peptide residue represented by -Arg-Lys.

  The polypeptide in the present invention may be a physiologically or pharmacologically acceptable salt. For example, inorganic acid (hydrochloric acid, sulfuric acid, phosphoric acid, etc.), organic acid (acetic acid, trifluoroacetic acid, lactic acid, tartaric acid) , Maleic acid, fumaric acid, oxalic acid, malic acid, citric acid, oleic acid, palmitic acid, etc., metals (alkali metals such as sodium and potassium, alkaline earth metals such as calcium, aluminum, etc.), organic bases (trimethylamine) , Triethylamine, t-butylamine, benzylamine, diethanolamine, dicyclohexylamine, arginine and the like. These salt-forming compounds can be used alone or in combination of two or more. These salts can be obtained by ordinary salt formation reactions.

  In the polypeptide of the present invention, the ratio (molar ratio) between the peptide unit (1) and the peptide unit (2) is (1) / (2) = 99/1 to 1/99, preferably 98/2. ˜2 / 98, more preferably about 95/5 to 5/95.

  The ratio (molar ratio) between the total amount of the peptide unit (1) and the peptide unit (2) and other peptide units is the former / the latter = 100/0 to 50/50, preferably 100/0 to 60. / 40, more preferably about 100/0 to 70/30.

Such a polypeptide forms a chain polypeptide without forming a 6-membered ring by cyclization, and a solvent (water, sulfoxides such as dimethyl sulfoxide, dimethylformamide, dimethylacetamide, N-methyl). It is soluble in a hydrophilic solvent such as pyrrolidone or a mixed solvent thereof. The polypeptide of the present invention has, for example, a molecular weight of 5 × 10 ^{2 to} 500 × 10 ⁴ , preferably a molecular weight of 1 × 10 ^{3 to} 300 × 10 ⁴ , preferably in terms of globular protein, in aqueous gel permeation chromatography (GPC). Shows a peak in the molecular weight range of about 3 × 10 ³ to 200 × 10 ⁴ , more preferably about 5 × 10 ^{3 to} 100 × 10 ⁴ .

  Furthermore, the polypeptide in the present invention exhibits a positive cotton effect at a wavelength of 220 to 230 nm and a negative cotton effect at a wavelength of 195 to 205 nm in a circular dichroism spectrum. Therefore, at least a part (that is, part or all) of the polypeptide can form a triple helical structure, forming a collagen-like polypeptide. The cotton effect refers to a phenomenon that occurs because the optical rotation material has different absorption coefficients for left and right circularly polarized light at a specific wavelength.

  The novel polypeptide in the present invention can be obtained by a conventional method in which an amino acid or a peptide fragment (or segment) is subjected to a condensation reaction. Finally, the peptide unit (1) and the peptide unit (2) are contained in the polypeptide. Is not particularly limited as long as it is contained in, for example, may be obtained by using a condensation reaction of constituent amino acids or a condensation reaction of a peptide fragment and an amino acid, but may be obtained in advance by the formula (1) and / or (2) It is preferable to obtain by the method of preparing the peptide which has an amino acid sequence represented by these, and condensing the peptide component containing this peptide.

  In the method of condensing a peptide component prepared in advance, the peptide chain of the peptide component can be synthesized according to an ordinary peptide synthesis method. The peptide can be prepared, for example, by a solid phase synthesis method or a liquid phase synthesis method. However, the solid phase synthesis method is simple in operation [for example, “Sequence Chemistry Laboratory Lecture 2 Protein Chemistry (Part 2)” (See pages 641-694 of May 20, 1987, issued by Tokyo Chemical Co., Ltd.). For peptide synthesis, conventional methods such as coupling methods using condensing agents, active ester methods (phenyl esters such as p-nitrophenyl ester (ONp) and pentafluorophenyl ester (Opfp), N-hydroxysuccinimide ester ( N-hydroxydicarboxylic acid imide ester such as ONSu), 1-hydroxybenzotriazole ester (Obt), etc.), mixed acid anhydride method, azide method and the like can be used. In a preferred method, at least a condensing agent (preferably a condensing agent described later, particularly a combination of a condensing agent and a condensing aid described later) is often used.

  Furthermore, in peptide synthesis, depending on the type of amino acid or peptide segment, depending on the protecting group of amino group, carboxyl group, and other functional groups (guanidino group, imidazolyl group, mercapto group, hydroxyl group, ω-carboxyl group, etc.) Protection and elimination / removal of the protective group by catalytic reduction or strong acid treatment (anhydrous hydrogen fluoride, trifluoromethanesulfonic acid, trifluoroacetic acid, etc.) are repeated. For example, amino-protecting groups include benzyloxycarbonyl group (Z), p-methoxybenzyloxycarbonyl group (Z (OMe)), 9-fluorenylmethoxycarbonyl group (Fmoc), t-butoxycarbonyl group ( Boc), 3-nitro-2-pyridinesulfenyl group (Npys) and the like, and the protective group for the carboxyl group includes benzyloxy group (OBzl), phenacyloxy group (OPac), t-butoxy group (OBu ), Methoxy group (OMe), ethoxy group (OEt), and the like. An automatic synthesizer may be used for peptide synthesis.

  More specifically, the peptide chain can be prepared by a solid phase synthesis method by a conventional method. As the solid phase resin (or carrier), a polymer insoluble in the reaction solvent, for example, styrene-divinylbenzene copolymer, for example, chloromethylated resin, hydroxymethyl resin, hydroxymethylphenylacetamidomethyl resin, 4-methylbenz A hydrylamine resin can be used.

  In the solid-phase synthesis method, usually (i) the polymer (resin) has a free α-COOH group and a functional group (at least at the C-terminal to N-terminal direction of the target peptide). (Ii) an α-amino group protecting group that forms a peptide bond among the bound amino acids or peptide fragments; (Iii) a step of extending the peptide chain to form a peptide chain corresponding to the target peptide by sequentially repeating the above binding operation and the removing operation; and (iv) a peptide chain that is a polymer (resin ) And removing the protecting group from the protected functional group to produce the desired peptide, and purify the produced peptide to produce the peptide. In the operation (i) for binding the amino acid or peptide fragment, an amino acid (for example, Fmoc-) corresponding to the C-terminus of the peptide chain and having a free α-COOH group and at least the N-terminus protected with a protecting group. Amino acids, Boc-amino acids, etc.) are used. The elimination of the peptide chain from the polymer and the removal of the protecting group are preferably performed simultaneously using trifluoroacetic acid from the viewpoint of suppressing side reactions. Further, the produced peptide can be purified using a separation and purification means such as reverse phase liquid chromatography or gel permeation chromatography.

  A method for condensing peptide components containing peptides synthesized in this manner includes (a) a method for condensing peptide components containing at least peptides having both amino acid sequences represented by formulas (1) and (2). And (b) a method of condensing a peptide component comprising at least a peptide having the amino acid sequence represented by formula (1) and a peptide having the amino acid sequence represented by formula (2).

  In the former method (a), a peptide having both amino acid sequences represented by formulas (1) and (2) (that is, a peptide unit having an amino acid sequence represented by formula (1) and formula (2) Peptides containing both units with the peptide unit having the amino acid sequence represented can be used alone or in combination of two or more. In this method, as the peptide component, in addition to the peptide containing both of the above units, other peptides may be used depending on the target polypeptide. Examples of other peptides include the above-mentioned other amino acid residues and peptides containing peptide residues. These other peptides can also be used alone or in combination of two or more. In this method, the ratio between the unit (1) and the unit (2) can be easily adjusted by co-condensing the peptide having the amino acid sequence represented by the formula (1) or (2). .

  Also in the latter method (b), the peptide (oligo or polypeptide unit) having the amino acid sequence represented by the formula (1) and the peptide having the amino acid sequence represented by the formula (2) are each independently or Two or more types can be used in combination. Also in this method, as the peptide component, in addition to these peptides (1) and (2), other peptides such as the other amino acid residues and peptide residues described above according to the target polypeptide Peptides containing etc. may be used. These other peptides can also be used alone or in combination of two or more.

  The condensation reaction of these peptides is usually performed in a solvent. The solvent may be any solvent as long as it can dissolve or suspend (partially or completely dissolve) the peptide component and the compound, and water and / or an organic solvent can be usually used. Examples of the solvent include water, amides (dimethylformamide, dimethylacetamide, hexamethylphosphoramide, etc.), sulfoxides (dimethylsulfoxide, etc.), nitrogen-containing cyclic compounds (N-methylpyrrolidone, pyridine, etc.), nitriles ( Examples include acetonitrile (such as acetonitrile), ethers (such as dioxane and tetrahydrofuran), alcohols (such as methyl alcohol, ethyl alcohol, and propyl alcohol), and mixed solvents thereof. Of these solvents, water, dimethylformamide, and dimethyl sulfoxide are frequently used.

  The reaction of these peptides can usually be carried out in the presence of at least a dehydrating agent (dehydrating condensing agent) or a condensing agent. When reacted in the presence of a dehydrating condensing agent and a condensing aid, dimerization or cyclization occurs. A polypeptide can be produced smoothly while suppressing the above.

  The dehydrating condensing agent is not particularly limited as long as dehydrating condensation can be efficiently performed in the solvent. For example, a carbodiimide condensing agent [diisopropylcarbodiimide (DIPC), 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide ( EDC = WSCI), 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (WSCI · HCl), dicyclohexylcarbodiimide (DCC), etc.], fluorophosphate condensing agent [O- (7-azabenzotriazole -1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate, O-benzotriazol-1-yl-N, N, N ′, N′-tetramethyluronium hexafluorophosphate, benzo Triazol-1-yl-oxy-tris-pyrrolidinophosphonium hexafluorophosphate Benzotriazol-1-yl - tris (dimethylamino) phosphonium hexafluorophosphate product salt (BOP)], such as diphenylphosphoryl azide (DPPA) can be exemplified. These dehydration condensing agents can be used alone or in combination of two or more. A preferred dehydrating condensing agent is a carbodiimide condensing agent [for example, 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide, 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride].

  The condensation aid is not particularly limited as long as it promotes the reaction of the condensation agent. For example, N-hydroxypolycarboxylic imides [for example, N-hydroxysuccinimide (HONSu), N-hydroxy-5-norbornene N-hydroxydicarboxylic imides such as -2,3-dicarboxylic acid imide (HONB)], N-hydroxytriazoles [for example, N-hydroxybenzotriazoles such as 1-hydroxybenzotriazole (HOBt)], 3- Examples thereof include triazines such as hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine (HOObt), and 2-hydroxyimino-2-cyanoacetic acid ethyl ester. These condensation aids can also be used alone or in combination. Preferred condensation aids are N-hydroxydicarboxylic imides [such as HONSu], N-hydroxybenzotriazole or N-hydroxybenzotriazines [such as HOBt].

  The dehydration condensation agent and the condensation aid can be used in appropriate combination. Examples of the combination of the dehydrating condensing agent and the condensing aid include DCC-HONSu (HOBt or HOObt), WSCI-HONSu (HOBt or HOObt), and the like.

  The amount of the dehydrating condensing agent used is usually 0.7 to 5 mol, preferably 0.8 to 2.5 mol, more preferably, when a non-aqueous solvent not containing water is used per 1 mol of the total amount of the peptide. Is about 0.9-2.3 mol (for example, 1-2 mol). In a solvent containing water (aqueous solvent), since the dehydration condensing agent is deactivated by water, the amount of the dehydrating condensing agent used is generally 2 to 500 mol (for example, 2 to 50 mol), preferably 5 to 250 mol (for example, 5 to 25 mol), more preferably about 10 to 125 mol (for example, 10 to 20 mol). The amount of the condensation aid used is, for example, 0.5 to 5 moles, preferably 0.7 to 2 moles, and more preferably 0.8 to 2 moles per mole of the peptide regardless of the type of solvent. About 1.5 moles.

  In the condensation reaction of the present invention, the pH of the reaction system may be adjusted, or a base that does not participate in the reaction may be added. The pH can be adjusted usually using an inorganic base [sodium hydroxide, potassium hydroxide, sodium carbonate, sodium hydrogen carbonate, etc.], an organic base, an inorganic acid [hydrochloric acid, etc.] or an organic acid. The pH of the solution is adjusted to near neutral (pH = about 6 to 8). Examples of the base not involved in the reaction include tertiary amines such as trialkylamines such as trimethylamine, triethylamine and diisopropylethylamine, and heterocyclic tertiary amines such as N-methylmorpholine and pyridine. . The amount of such base used can usually be selected from a range of about 1 to 2 times the total number of moles of the peptide.

  The formation of a triple helical structure by the polypeptide of the present invention can be usually proved by measuring a circular dichroism spectrum of a solution of the polypeptide. In the circular dichroism spectrum, natural collagen and peptide chains forming a triple helical structure characteristically show a positive cotton effect at wavelengths of 220 nm to 230 nm and a negative cotton effect at wavelengths of 195 nm to 205 nm. Has been reported (J. Mol. Biol., Vol. 63 pp. 85-99, 1972).

  Such a polypeptide contains an amino acid sequence recognized by collagenase, which is a collagenolytic enzyme, binds to collagenase, and is degraded by collagenase. Therefore, since this polypeptide has a high inhibitory action on collagenase activity, it can be used as a collagenase inhibitor.

  Further, since the polypeptide (collagenase inhibitor) of the present invention is a collagen-like polypeptide, infection and transmission of pathogens and pathogenic factors [for example, proteins converted to pathogenicity (for example, abnormal prions, etc.)] There is no danger of. Further, since it can be decomposed and absorbed in the living body, there is no possibility of causing undesirable side effects. Thus, the collagenase inhibitor of the present invention has high safety and excellent biocompatibility. Therefore, the collagenase inhibitor of the present invention is a subject that needs to inhibit collagenase activity (in addition to humans, non-human animals such as monkeys, sheep, cows, horses, dogs, cats, rabbits, rats, mice, etc.) For example, it can be used as components (base materials, active ingredients, additives, etc.) contained in biomaterials, medical materials, pharmaceutical compositions, cosmetics, edible compositions, and the like. In particular, the collagenase inhibitor of the present invention is useful because it can be safely applied to affected areas where collagenase activity is increased (for example, inflammatory sites, joints, caries sites, periodontal sites, bones, corneas, etc.). It is.

  In biomaterials or medical materials, the collagenase inhibitor may be used as an additive. In addition, since the collagenase inhibitor has high film formability or moldability and can be easily formed into a desired shape, a biomaterial or a medical material may be composed of the collagenase inhibitor alone. For example, the biomaterial is used in various forms composed of the collagenase inhibitor, for example, liquid (solution or suspension), powder, two-dimensional form (film or sheet) or three-dimensional form. it can. Examples of the biomaterial or medical material include a covering material or a coating agent, an embedding material, a hemostatic material, an adhesion preventing material, an adhesive material, a lumen material, and a membrane material.

  When a collagenase inhibitor is used as an additive in a biomaterial or a medical material, the ratio of the collagenase inhibitor is, for example, 0.1 to 500 parts by weight, preferably 1 to 300 parts by weight with respect to 100 parts by weight of the base material. It may be about 5 parts by weight, more preferably about 5 to 200 parts by weight.

  The pharmaceutical composition only needs to contain at least a collagenase inhibitor, and may be any of a solid preparation, a semi-solid preparation, and a liquid preparation. The pharmaceutical composition may be a pharmaceutical or a quasi-drug and can be used as a preparation in various dosage forms. Examples of solid preparations include powders, granules, tablets, troches, gummi, pills, capsules and the like, and examples of semisolid preparations include ointments (including creams and eye ointments), Patches, patches, suppositories and the like can be exemplified, and examples of liquid preparations include aerosols, suspensions, emulsions, injections, eye drops, lotions, and liniments. The ratio of the collagenase inhibitor to the whole preparation may be about 0.001 to 99% by weight, preferably 0.01 to 95% by weight, and more preferably about 0.1 to 90% by weight.

  The cosmetic only needs to contain at least the collagenase inhibitor, and includes a powdery cosmetic including a powdery base, a solid or semi-solid base (an aqueous base, a gel base, or an oily base). Any of solid or semi-solid cosmetics and liquid cosmetics containing a liquid base (aqueous or oily base) may be used. In addition, cosmetics usually contain a base (or carrier), an active ingredient (such as a humectant), and an additive, and the collagenase inhibitor is contained as at least one of these ingredients. Just do it.

  The ratio of the collagenase inhibitor can be selected from a wide range, for example, a range of about 0.001 to 99% by weight, depending on the type and dosage form of the cosmetic. When used as a base, the ratio of the collagenase inhibitor may be, for example, about 10 to 99% by weight, preferably about 20 to 99% by weight, based on the entire cosmetic. When used as an active ingredient, the proportion may be, for example, about 0.001 to 95% by weight, preferably about 0.01 to 90% by weight. Moreover, when using as an additive, the said ratio may be 0.001 to 40 weight%, Preferably about 0.01 to 30 weight%.

  The edible composition only needs to contain at least the collagenase inhibitor, and includes a powdery composition containing a powdery base, a solid or semisolid composition containing a solid or semisolid base, and a liquid containing a liquid base. Any of a composition or a mixture thereof may be used. Edible compositions usually contain a base (or carrier), an active ingredient, and additives (food additives, seasonings, etc.). The collagenase inhibitor may be contained as at least one of these components. The edible composition is useful for various foods as well as functional foods such as health foods, health supplement foods, functional foods, special-purpose foods and health functional foods. The edible composition of the present invention includes livestock (cattle, pig, sheep, etc.), pets (mammals such as dogs and cats, birds, reptiles, etc.), fish (cultured fish such as salmon, eel, etc .; goldfish, etc. And feed for laboratory animals (rats, etc.).

  The ratio of the collagenase inhibitor can be selected from a wide range according to the type and form of the edible composition, for example, from about 0.001 to 99% by weight. When used as a base, the ratio of the collagenase inhibitor may be, for example, about 10 to 90% by weight, preferably about 20 to 80% by weight, based on the entire edible composition. When used as an additive, the proportion may be about 0.001 to 40% by weight, preferably about 0.01 to 30% by weight. Moreover, when using as an active ingredient, the said ratio may be about 0.001-90 weight%, for example, Preferably about 0.01-80 weight%.

  The polypeptide or collagenase inhibitor of the present invention is preferably used after sterilization or sterilization. As sterilization and sterilization methods, various sterilization and sterilization methods such as wet heat steam sterilization, gamma ray sterilization, ethylene oxide gas sterilization, drug sterilization, and ultraviolet sterilization are used. Among these methods, gamma ray sterilization and ethylene oxide gas sterilization are preferable because they have little effect on sterilization efficiency and materials.

  The polypeptide or collagenase inhibitor of the present invention has no risk of pathogen infection or transmission of pathogenic factors, and there is no risk of undesirable side effects, and can be degraded in vivo. It can be used for products, cosmetics, edible compositions and the like.

  Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited to these examples.

Example 1
A peptide chain represented by the formula: H- (Pro-Hyp-Gly) ₄ -Pro-Gln-Gly-Ile-Ala-Gly- (Pro-Hyp-Gly) ₄ -OH (SEQ ID NO: 1) It was synthesized by a solid phase synthesis method using a synthesizer. That is, a styrene-divinylbenzene copolymer containing 4- (Nα-9- (fluorenylmethoxycarbonyl) -glycine) -oxymethyl-phenoxy-methyl group at a rate of 0.65 mmol / g (resin) [styrene and divinyl Using 0.1 mmol of a granular resin (HMP glycine, manufactured by Applied Biosystems, USA) consisting of 99 to 1 molar ratio with benzene, the corresponding amino acids are sequentially added from the carboxyl terminus to the amino terminus of the target peptide. Combined. In the binding reaction, Nα-9- (fluorenylmethoxycarbonyl) -L-proline [Fmoc proline], Nα-9- (fluorenylmethoxycarbonyl) -glycine [Fmoc] manufactured by Applied Biosystems, Inc. Glycine], Nα-9- (fluorenylmethoxycarbonyl) -Nγ-trityl-L-glutamine [Fmoc glutamine], Nα-9- (fluorenylmethoxycarbonyl) -L-isoleucine [Fmoc isoleucine], Nα-9 -(Fluorenylmethoxycarbonyl) -L-alanine [Fmoc alanine], Nα-9- (fluorenylmethoxycarbonyl) -Ot-butyl-L-hydroxyproline (Fmoc hydroxyproline) manufactured by Bacchem, 1 mmol each was used for each binding step.

  The obtained peptide resin was treated with 10 mL of trifluoroacetic acid containing 5% water for 3 hours. The resulting solution was added to diethyl ether, and the resulting precipitate was further washed several times with diethyl ether to deprotect the polypeptide and desorb it from the resin. The crude product was purified by a column (Amersham Biosciences, Inc., PD10 column) to obtain a polypeptide. The obtained polypeptide was subjected to column chromatography [Amersham Biosciences, “AKTA explorer10XT”, column: Millipore, “Novapack C18”, 3.9 mmφ × 150 mm, mobile phase: 0.05% trifluoroacetic acid. When used for a mixed solvent of acetonitrile and water containing volume% (acetonitrile concentration was linearly changed from 5 volume% to 50 volume% over 30 minutes), flow rate 1.0 mL / min), retention time was 12.4 minutes A single peak was shown. The molecular weight of the polypeptide determined by FAB mass spectrum was 2681.3 (theoretical value: 2679.9). The ratio of peptide unit (1) to peptide unit (2) ((1) / (2)) is 8/1 (88.9 / 11.1) (molar ratio).

  When the circular dichroism spectrum of the obtained polypeptide was measured, a positive cotton effect was observed at 224 nm and a negative cotton effect was observed at 198 nm, confirming the formation of a triple helical structure.

Example 2
2.5 mL (0.0009 mmol) of H- (Pro-Hyp-Gly) ₄ -Pro-Gln-Gly-Ile-Ala-Gly- (Pro-Hyp-Gly) ₄ -OH obtained in Example 1 was added to 1 mL. Suspended in dimethyl sulfoxide and stirred at room temperature. To this mixture, 0.12 mg (0.0009 mmol) diisopropylethylamine, 0.12 mg (0.0009 mmol) 1-hydroxybenzotriazole, 0.34 mg (0.0018 mmol) 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide Hydrochloric acid was added and stirring was continued at 20 ° C. for 2 days. The resulting reaction solution was diluted 3 times with water and dialyzed against water for 3 days to remove reagents such as a condensing agent and unreacted monomers to obtain a polypeptide. The ratio of peptide unit (1) to peptide unit (2) ((1) / (2)) is 8/1 (88.9 / 11.1) (molar ratio).

  The obtained polypeptide was subjected to gel permeation chromatography (manufactured by Amersham Biosciences, AKTApurifier system, column: Superose 6 HR GL, flow rate: 0.5 mL / min, eluent: 10 mM phosphate buffer containing 150 mM NaCl ( When subjected to pH 7.4)), a polypeptide peak was observed in the molecular weight range of 70,000 to 1,000,000. The molecular weight was calculated using a Gel Filtration HMW Calibration Kit manufactured by Amersham Biosciences as a standard substance.

  When the circular dichroism spectrum of the obtained polypeptide was measured, a positive cotton effect was observed at 223 nm and a negative cotton effect was observed at 198 nm, confirming the formation of a triple helical structure.

Example 3
1.2 mg (0.00045 mmol) of formula: peptide represented by H- (Pro-Hyp-Gly) ₁₀ -OH (SEQ ID NO: 2) (Peptide Institute, Inc.) and 1.2 mg (0.00045 mmol) of Examples Peptide obtained by formula 1: H- (Pro-Hyp-Gly) ₄ -Pro-Gln-Gly-Ile-Ala-Gly- (Pro-Hyp-Gly) ₄ -OH (SEQ ID NO: 1) Is dissolved in 0.25 mL of 10 mM phosphate buffer (pH = 7.4), 0.12 mg (0.0009 mmol) 1-hydroxybenzotriazole, 15.7 mg (0.082 mmol) 1-ethyl-3- (3-dimethylamino) Propyl) -carbodiimide hydrochloride was added and stirring was continued at 20 ° C. for 2 days. The resulting reaction solution was diluted 10-fold with water and dialyzed against water for 3 days to remove reagents such as condensing agents and unreacted monomers to obtain a polypeptide. The ratio of peptide unit (1) to peptide unit (2) ((1) / (2)) is 18/1 (94.7 / 5.3) (molar ratio).

  The obtained polypeptide was subjected to gel permeation chromatography (manufactured by Amersham Biosciences, AKTApurifier system, column: Superose 6 HR GL, flow rate: 0.5 mL / min, eluent: 10 mM phosphate buffer containing 150 mM NaCl ( When subjected to pH 7.4)), a polypeptide peak was observed in the molecular weight range of 140,000 to 1,000,000. The molecular weight was calculated using a Gel Filtration HMW Calibration Kit manufactured by Amersham Biosciences as a standard substance.

  When the circular dichroism spectrum of the obtained polypeptide was measured, a positive cotton effect was observed at 224 nm and a negative cotton effect was observed at 196 nm, confirming the formation of a triple helical structure.

Example 4
2.2 mg (0.00081 mmol) of formula: peptide represented by H- (Pro-Hyp-Gly) ₁₀ -OH (SEQ ID NO: 2) (Peptide Institute, Inc.) and 0.24 mg (0.00009 mmol) of Example Peptide obtained by formula 1: H- (Pro-Hyp-Gly) ₄ -Pro-Gln-Gly-Ile-Ala-Gly- (Pro-Hyp-Gly) ₄ -OH (SEQ ID NO: 1) Is dissolved in 0.25 mL of 10 mM phosphate buffer (pH = 7.4), 0.12 mg (0.0009 mmol) 1-hydroxybenzotriazole, 15.7 mg (0.082 mmol) 1-ethyl-3- (3-dimethylamino) Propyl) -carbodiimide hydrochloride was added and stirring was continued at 20 ° C. for 2 days. The obtained reaction solution was diluted 10-fold with water and dialyzed against water for 3 days to remove reagents such as a condensing agent and unreacted monomers to obtain the polypeptide of the present invention. The ratio of peptide unit (1) to peptide unit (2) ((1) / (2)) is 98/1 (≈99 / 1) (molar ratio).

  The obtained polypeptide was subjected to gel permeation chromatography (manufactured by Amersham Biosciences, AKTApurifier system, column: Superose 6 HR GL, flow rate: 0.5 mL / min, eluent: 10 mM phosphate buffer containing 150 mM NaCl ( When subjected to pH 7.4)), a polypeptide peak was observed in the molecular weight range of 140,000 to 400,000. The molecular weight was calculated using a Gel Filtration HMW Calibration Kit manufactured by Amersham Biosciences as a standard substance.

  When the circular dichroism spectrum of the obtained polypeptide was measured, a positive cotton effect was observed at 224 nm and a negative cotton effect was observed at 197 nm, confirming the formation of a triple helical structure.

Comparative Example 1
A peptide represented by the formula: H- (Pro-Hyp-Gly) ₁₀ -OH (SEQ ID NO: 2) (Peptide Institute, Inc.) was used as Comparative Example 1. The ratio of peptide unit (1) to peptide unit (2) ((1) / (2)) is 1/0 (= 100/0) (molar ratio). When the circular dichroism spectrum of this polypeptide was measured, a positive cotton effect was observed at 225 nm and a negative cotton effect was observed at 196 nm, confirming the formation of a triple helical structure.

Comparative Example 2
A peptide represented by 2.4 mg (0.0009 mmol) of formula: H- (Pro-Hyp-Gly) ₁₀ -OH (SEQ ID NO: 2) (Peptide Institute, Inc.) was suspended in 1 mL of dimethyl sulfoxide, and at room temperature. Stir. To this mixture, 0.12 mg (0.0009 mmol) diisopropylethylamine, 0.12 mg (0.0009 mmol) 1-hydroxybenzotriazole, 0.34 mg (0.0018 mmol) 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide Hydrochloric acid was added and stirring was continued at 20 ° C. for 2 days. The resulting reaction solution was diluted 3 times with water and dialyzed against water for 3 days to remove reagents such as a condensing agent and unreacted monomers to obtain a polypeptide. The ratio of peptide unit (1) to peptide unit (2) ((1) / (2)) is 100/0 (molar ratio).

  The obtained polypeptide was subjected to gel permeation chromatography (manufactured by Amersham Biosciences, AKTApurifier system, column: Superose 6 HR GL, flow rate: 0.5 mL / min, eluent: 10 mM phosphate buffer containing 150 mM NaCl ( When subjected to pH 7.4)), a polypeptide peak was observed in the molecular weight range of 140,000 to 600,000. The molecular weight was calculated using a Gel Filtration HMW Calibration Kit manufactured by Amersham Biosciences as a standard substance.

  When the circular dichroism spectrum of the obtained polypeptide was measured, a positive cotton effect was observed at 224 nm and a negative cotton effect was observed at 196 nm, confirming the formation of a triple helical structure.

Example 5
The peptide chain represented by the formula: H- (Pro-Hyp-Gly) ₄ -Pro-Leu-Gly-Ile-Ala-Gly- (Pro-Hyp-Gly) ₄ -OH (SEQ ID NO: 3) It was synthesized by a solid phase synthesis method using a synthesizer. That is, a styrene-divinylbenzene copolymer containing 4- (Nα-9- (fluorenylmethoxycarbonyl) -glycine) -oxymethyl-phenoxy-methyl group at a rate of 0.65 mmol / g (resin) [styrene and divinyl Using 0.1 mmol of a granular resin (HMP glycine, manufactured by Applied Biosystems, USA) consisting of 99 to 1 molar ratio with benzene, the corresponding amino acids are sequentially added from the carboxyl terminus to the amino terminus of the target peptide. Combined. In the binding reaction, Nα-9- (fluorenylmethoxycarbonyl) -L-proline [Fmoc proline], Nα-9- (fluorenylmethoxycarbonyl) -glycine [Fmoc] manufactured by Applied Biosystems, Inc. Glycine], Nα-9- (fluorenylmethoxycarbonyl) -L-leucine [Fmoc leucine], Nα-9- (fluorenylmethoxycarbonyl) -L-isoleucine [Fmoc isoleucine], Nα-9- (fluorescein) Nylmethoxycarbonyl) -L-alanine [Fmoc alanine], Nα-9- (fluorenylmethoxycarbonyl) -Ot-butyl-L-hydroxyproline [Fmoc hydroxyproline] manufactured by Bacchem, for each binding step 1 mmol each was used.

  The obtained peptide resin was treated with 10 mL of trifluoroacetic acid containing 5% by weight of water for 3 hours. The resulting solution was added to diethyl ether, and the resulting precipitate was further washed several times with diethyl ether to deprotect the polypeptide and desorb it from the resin. The crude product was purified by a column (Amersham Biosciences, PD10 column) to obtain a peptide. The obtained polypeptide was subjected to column chromatography [Amersham Biosciences, “AKTA explorer10XT”, column: Millipore, “Novapack C18”, 3.9 mmφ × 150 mm, mobile phase: 0.05% trifluoroacetic acid. A mixed solvent of acetonitrile and water containing volume% (acetonitrile concentration was linearly changed from 5 volume% to 50 volume% over 30 minutes), flow rate 1.0 mL / min), the retention time was 15 minutes. One peak was shown. The molecular weight of the purified peptide determined by FAB method mass spectrum was 2666.3 (theoretical value: 2664.9). The ratio of peptide unit (1) to peptide unit (2) ((1) / (2)) is 8/1 (88.9 / 11.1) (molar ratio).

  When the circular dichroism spectrum of the obtained polypeptide was measured, a positive cotton effect was observed at 225 nm and a negative cotton effect was observed at 199 nm, confirming the formation of a triple helical structure.

Example 6
1.2 mg (0.00045 mmol) of the polypeptide H- (Pro-Hyp-Gly) ₄ -Pro-Leu-Gly-Ile-Ala-Gly- (Pro-Hyp-Gly) ₄ -OH obtained in Example 5 0.12 mg (0.0009 mmol) 1-hydroxybenzotriazole, 15.7 mg (0.082 mmol) 1-ethyl-3- (3-dimethylaminopropyl) dissolved in 0.25 mL 10 mM phosphate buffer (pH = 7.4) ) -Carbodiimide hydrochloride was added and stirring was continued at 20 ° C. for 2 days. The resulting reaction solution was diluted 10-fold with water and dialyzed against water for 3 days to remove reagents such as condensing agents and unreacted monomers to obtain a polypeptide. The ratio of peptide unit (1) to peptide unit (2) ((1) / (2)) is 8/1 (88.9 / 11.1) (molar ratio).

  The obtained polypeptide was subjected to gel permeation chromatography (manufactured by Amersham Biosciences, AKTApurifier system, column: Superose 6 HR GL, flow rate: 0.5 mL / min, eluent: 10 mM phosphate buffer containing 150 mM NaCl ( When subjected to pH 7.4)), a polypeptide peak was observed in the molecular weight range of 80,000 to 1,000,000. The molecular weight was calculated using a Gel Filtration HMW Calibration Kit manufactured by Amersham Biosciences as a standard substance.

  When the circular dichroism spectrum of the obtained polypeptide was measured, a positive cotton effect was observed at 224 nm and a negative cotton effect was observed at 197 nm, confirming the formation of a triple helical structure.

Test example 1
0.025 mg of each of the polypeptides obtained in Examples 1 to 6 and the polypeptides obtained in Comparative Examples 1 and 2 was added to a 50 mM Tris / HCl buffer solution containing 0.05 mL of 50 mM NaCl and 10 mM CaCl ₂ ( dissolved in pH = 7.5). In addition, 200 ng collagenase (MMP-1, human rheumatoid synovial fibroblast) dissolved in 0.05 mL of the same buffer and 0.01 mL of 1 mM Dnp-Pro-Leu-Gly-Ile-Ala-Gly-Arg-NH ₂ (strain) Peptide Institute) DMSO solution was added and allowed to stand at 37 ° C. for 1 hour. Thereafter, 0.2 mL of acetonitrile was added and centrifuged at 10,000 rpm for 10 minutes. The obtained supernatant was subjected to column chromatography [Amersham Biosciences, “AKTA explorer10XT”, Column: Millipore, “Novapack C18”, 3.9 mmφ × 150 mm, mobile phase: trifluoroacetic acid. Dnp-Pro-Leu- mixed solvent of acetonitrile and water containing 0.05 vol% (acetonitrile concentration was changed linearly from 5 vol% to 50 vol% over 30 minutes), flow rate 1.0 mL / min) The peak area of Dnp-Pro-Leu-Gly produced by degradation of Gly-Ile-Ala-Gly-Arg-NH ₂ by collagenase was detected.

  As a control, the Dnp-Pro-Leu-Gly peak area was determined without adding the polypeptide. With this peak area as “100”, the peak area detected by adding the polypeptides obtained in Examples and Comparative Examples was calculated as a relative value to the control. As a result, when the polypeptides of Examples 1 to 6 were added, the relative values of the detected peak areas were 5, 3, 12, 22, 4, and 2, and the activity of collagenase was inhibited. In contrast, when the polypeptides of Comparative Examples 1 and 2 were added, the relative values of the detected peak areas were 96 and 97, respectively, and the collagenase activity inhibitory action was not observed.

Claims (8)

A polypeptide comprising a peptide unit having an amino acid sequence represented by the following formula (1) and a peptide unit having an amino acid sequence represented by the following formula (2), and used for inhibiting collagenase activity.
-Pro-X-Gly- (1)
-Pro-Y-Gly-Z-Ala-Gly- (2)
(In the formula, X represents Pro or Hyp, Y represents Gln, Asn, Leu, Ile, Val or Ala, and Z represents Ile or Leu.)   The polypeptide according to claim 1, wherein the ratio (molar ratio) between the peptide unit (1) and the peptide unit (2) is (1) / (2) = 99/1 to 1/99.   The polypeptide according to claim 1 or 2, wherein the polypeptide exhibits a positive cotton effect at a wavelength of 220 to 230 nm and a negative cotton effect at a wavelength of 195 to 205 nm in a circular dichroism spectrum.   The polypeptide according to any one of claims 1 to 3, wherein at least a part of the polypeptide is capable of forming a triple helical structure. The polypeptide according to any one of claims 1 to 4, wherein the polypeptide exhibits a peak in a molecular weight range of 5 x 10 ^{2 to} 500 x 10 ⁴ .   A cosmetic comprising the polypeptide according to claim 1 and used for inhibiting the activity of collagenase.   An edible composition comprising the polypeptide according to claim 1 and used for inhibiting collagenase activity. Method for inhibiting collagenase activity using a novel polypeptide comprising a peptide unit having an amino acid sequence represented by the following formula (1) and a peptide unit having an amino acid sequence represented by the following formula (2) .
-Pro-X-Gly- (1)
-Pro-Y-Gly-Z-Ala-Gly- (2)
(Wherein X represents Pro or Hyp, Y represents Gln, Asn, Leu, Ile, Val or Ala, and Z represents Ile or Leu)