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NOVEL POLYPEPTIDE AND PRODUCTION METHOD THEREOF

Foreign code F110002755
File No. S2009-0528-N0
Posted date Apr 13, 2011
Country WIPO
International application number 2008JP066568
International publication number WO 2009035092
Date of international filing Sep 12, 2008
Date of international publication Mar 19, 2009
Priority data
  • P2007-238437 (Sep 13, 2007) JP
Title NOVEL POLYPEPTIDE AND PRODUCTION METHOD THEREOF
Abstract Disclosed are: a novel polypeptide which does not have the risk of causing the infection with any pathogenic bacterium or the transfer of any pathogenic factor, which produces no adverse side effect, and which is useful as a carrier for various physiologically active substances and apatite-type substances; and a method for producing the polypeptide. Specifically disclosed are: a polypeptide which comprises a peptide unit having an amino acid sequence represented by the formula: -Pro-X-Gly- [wherein X represents Pro or Hyp] and a peptide unit having an amino acid sequence represented by the formula: -Pro-Hyp(O-Y-Z)-Gly- [wherein Y represents a carbonyl group, a saturated or unsaturated hydrocarbon group which has or does not have a carbonyl group, or a saturated or unsaturated hydrocarbon group which has an aromatic group and which has or does not have a carbonyl group; and Z represents a carboxyl group]; and a method for producing the polypeptide.
Outline of related art and contending technology BACKGROUND ART
Collagen, fibrous proteins found in any multicellular organism and, as a major component of the skin and bone of the total protein occupies 25% mammals. Is a typical collagen molecules, the three collagen polypeptide chains referred to as a triple-helical structure in the form of a coiled-coil to the bottom surface. Proline (Pro) collagen and glycine (Gly) exist in the, amino acid residues are both important to the formation of stable triple-helical structure is.
The use of collagen as biomaterials is, for example, porcine skin tissue as it is or after freeze-drying, such as a burn the skin by a method for implanting at the site of injury, such as the enzyme-treated cells by a method in which the components are removed, the acidic solution and collagen solubilized by enzyme treatment, to reconstruct the desired form is a method of using. Non-patent document 1 is, methods for their preparation and general collagen described qualitative method.
Various proposals have been for the usage of the collagen has been made. For example, in patent document 1, the cuticles of the skin, and order to smooth the skin, animal tissue containing collagen was modified after is esterified with an alcohol, for producing a modified collagen for collagen, using the cosmetic base is and has been proposed.
In addition, the patent document 2, soluble collagen, methylene chain alkylene imidate ester groups at both ends of a divalent crosslinking by a crosslinking treatment, after heat denaturation in the high rate of the triple-helical structure, a method for producing a water-soluble crosslinked collagen is described.
In addition, the Patent Document 3, first the synthesis of collagen 1 is reacted with a hydrophilic polymer that forms the polymer matrix and collagen synthesis -, collagens - synthetic polymer matrix, the synthetic hydrophilic polymer of the first 2, the biologically active material, glycosaminoglycan and derivatives thereof, chemical cross-linking agent, esterifying agent, amidation agent, acylating agent, amino acid, polypeptide by acid and the like, less immunogenicity, a variety of medical applications to provide a biocompatible implant useful in the preparation of collagen - synthetic polymer matrix is described.
In addition, Patent Document 4 is, in the form of a substantially non-pH7 chemical modifications collagen fibers with a covalently bound hydrophilic synthetic polymer conjugate is described. In this document, the coupling body is, particularly useful in ophthalmic devices, biocompatible optically transparent have been described.
In addition, the patent document 5, collagen matrix and pulverized, and centrifuged at field core is less secure this pulverized matrix, and the precipitate was homogenized into a paste, the paste is casting, 37 °C or less by casting the paste is dried at a temperature of the particulate matter to the collagen membranes described be produced. This collagen film-like material, biocompatible non-inflammatory, and artificial implant may be useful for the repair of tissue as described.
In addition, Patent Document 6 is, as it is or after decalcified fish scales, by pepsin treatment, high-purity soluble fish scale collagen and its manufacturing method is described.
In addition, the patent document 7, 70-90% ethanol in the medium, ejected from a nozzle and a collagen solution, the film-shaped or thread to yield, after drying, cut or by grinding, the dried granular or powdery soluble collagen described a method of producing.
In addition, the patent document 8, an unsintered hydroxyapatite single crystal, with a low antigenic collagen fibers attached to at least a portion of, the biological hard tissue such as bone repair described used as a material.
In addition, the patent document 9, a prion in an animal or human-derived collagen to remove, collagen solution was removed and the cells and tissue fragments, obtained by this method of treatment with an alkali and the collagen is described.
In addition, the non-patent document 2 is, as with the method of chemical synthesis of collagen and the like, of the p- nitrophenyl Pro-Ser-Gly, or Pro-Ala-Gly of p- nitrophenyl esters may be dissolved in dimethylformamide, triethylamine was added by 24 for one hour, soluble polyamide having a molecular weight of 16,000-21,000 have been reported can be obtained. Circular dichroism spectrum of the soluble polyamide having a triple-helical structure is estimated, is no description regarding the nature of the resulting polymer.
In addition, the non-patent document 3, derived from elastin Val-Pro-Gly-Val-Gly sequence 50 - mer peptide was dissolved in dimethyl sulfoxide, 2 equivalents of ethyl 1 - - (3 - - dimethylaminopropyl) carbodiimide -3-1 equivalents of 1 - hydroxybenzotriazole, and 1.6 equivalents of between 14 N- methylmorpholine addition was allowed to stand, 5 million molecular weight cutoff dialysis membrane and dialyzed to report a method for obtaining a polyamide.
In addition, the patent document 10, represented by the following formula (1) - (3) cleaved collagen polypeptide in the tissue to be formed is disclosed.
- (OC - (CH2) m-CO) p-(Pro-Y-Gly) n-a (1) - (OC - (CH2) m-CO) q-(Z) r-b (2) - HN-R-NH-c (3) (in the formula, m is an integer of 1-18, p and q is 0 or 1 are the same or different, represents Y is Pro or Hyp, n is an integer of 1-20. Z is from 1-10 amino acid residues represent a peptide chain, r represents an integer of 1-20 is, R is a linear or branched alkylene group. A and b (molar ratio) the ratio of a/b=100/0-30/70 and, c=a and p=1 when q=0, p=0 and q=1 and when c=b, p=1 and q=1 and when c=a + b, p=0 and when q=0 is c=0.)
On the other hand, the above-mentioned Patent Document 9 as described, sheep tremor disease and bovine spongiform encephalopathy of is referred to as the causative agent of infectious prion protein, human creutzfeldt - this infectious protein is one of the causes of the infection Jakob-as. Non-patent document 4 is, prion is protein, the normal sterilization, sterilization method this inactivation difficult, since the infecting species beyond the pointed out.
In general, the medical device or medicaments, cosmetic collagen derived from bovine and pigs are often used as a raw material. Therefore, the normal sterilization, sterilization method that cannot be removed in a pathogen such as a prion infection (or virulence factors) are at risk of (or transfer) always present in the.
In addition, a variety of cell types in the native collagen is included in the bond sites for, the use of the cell selectivity is not exerted. For example, collagen is used as the material of the nerve and axonal guidance, axonal outgrowth than the surrounding fibroblast cell migration, and the speed increase rate increases and the scar tissue cannot be extended axons. For this reason, the collagen in the material that prevents the migration of fibroblasts by covering the periphery of the power storage device of is required.
On the other hand, in vivo, certain ceramics (for example, bioactive glass as Bioglass (registered trademark) or, crystallized glass A-W(Cerabone (registered trademark) A-W) and the like) is known to bind to the bone. The binding of the ceramic and bone, in vivo (or human body fluids in the aqueous solution having a concentration ions near) in, the hydroxyapatite layer is formed in the ceramic surface due to the. The connection mechanism is, first, the ceramic formed on the surface of the silica ion or a silanol group, or an aqueous solution of calcium and phosphate in vivo react and form hydroxyapatite, the nuclear-based aqueous solution vivo or supersaturated calcium and phosphate are grown takes in the ion considered.
Is Patent Document 11, the plate-like, rod-like, fibrillar, various shapes such as the shape of the metal particle, such as ceramics substrate, coated with a liquid or gel silica hydrosol, silica gel and dried by heating and then attached to the substrate, supersaturated with respect to the hydroxyapatite in an amount of an aqueous solution containing calcium and phosphate ions by immersing (pseudo body fluid) to, to the surface of the substrate is coated with a layer of hydroxyapatite bioactive layer has been proposed that a coating method. In this document, the coating material is apatite, artificial bone, biological embedding material, the biological an implanted medical device, such as surgical instruments to described may also be utilized. However, in such the inorganic biomaterial, biocompatible cell adhesiveness is insufficient.
In addition, the organic-inorganic hybrid material as biomaterials has been studied. For example, in patent document 12, an average fiber length of 60 μm or more of hydroxyapatite and collagen (mammalian, avian, fish derived from collagen or collagen-like protein, such as gene recombinant collagen) and complex of the organic-inorganic composite composed of the biomaterial is disclosed. In addition, in this document, the composite material, and calcium ions in the reaction vessel a phosphate ion, for example, a liquid feeding speed of the starting material to control the density and the like, is maintained at a specific concentration, the resulting composite can be produced by compression-molding is also described. In addition, the non-patent document 5, of 0.1M CaCl2 and 0.1M of NaH2 PO4 presence, acid solubilized rat tail tendon-derived collagen by neutralizing, collagen and hydroxyapatite are combined method described. However, in such complexes, as collagen, natural collagen is selected for use, of the pathogen (or pathogenic factor) infection are at risk of (or transfer).
In addition, the patent document 13, the base having a sericin, calcium ions and phosphate ions contacting the aqueous solution, deposition of apatite to the base as the powder is disclosed.
In addition, the patent document 14, a specific peptide substrate immobilized to a medical dressing material is disclosed, the medical dressing material, physiological activity is high, and a particularly strong cell growth promoting action and/or a cell adhesion has the effect, of the biological tissue healing, adhesive, reinforcement and/or playback is useful as a material or agent described.
In addition, the patent document 15, treatment of fractures, osteoporosis and periodontal disease in bone mass and bone loss diseases, the prevention of fractures in osteoporosis and rheumatoid arthritis and the like useful, a peptide having osteogenic activity as an active ingredient and a bone formation accelerator is disclosed.
In addition, the non-patent document 6, the alginate gel cross-linked with ethylenediamine, a peptide having osteogenic activity, Lys-Ile-Pro-Lys-Ala-Ser-Ser-Val-Pro-Thr-Glu-Leu-Ser-Ala-Ile-Ser-Thr-Leu-Tyr-Leu-NH2 materials obtained by combining the lower legs of the rat and implanted into a muscle, over 7 or more weeks induced calcification of bone-like described.
In addition, the non-patent document 7, the perspective view of the alginate gel, a peptide having the action of a neural stem cell, Tyr-Arg-His-Ala-Trp-Ser-Glu-Asn-Leu-Ala-Gln-Cys-Phe-Asn-NH2 on materials obtained by combining the, culturing rat hippocampus-derived neural stem cells and neuronal cell differentiation into significantly facilitates the is described.
Scope of claims (In Japanese)請求の範囲 [1]
 式:
[化1]

(式中、XはProまたはHypを表す)
で表されるアミノ酸配列を有するペプチドユニットと、式:
[化2]

(式中、Yはカルボニル基、カルボニル基を有するかまたは有しない飽和または不飽和の炭化水素基、または芳香族基を含む、カルボニル基を有するかまたは有しない飽和または不飽和の炭化水素基を表し、Zはカルボキシル基を表す)
で表されるアミノ酸配列を有するペプチドユニットとを含むポリペプチド。

[2]
 Yが、-(C=O)-(CH 2)n- (式中、nは0または1~18の整数を表す);-(C=O)-(CH 2)n-(CH=CH)m-(CH 2)k- (式中、nおよびkは独立して0または1~18の整数を表し、mは1~18の整数を表す);および-(C=O)-(CH 2)n-(C 6H 4)-(CH 2)k- (式中、nおよびkは独立して0または1~18の整数を表し、C 6H 4はフェニレン基を表す)よりなる群から選択される1つ以上の基である請求項1記載のポリペプチド。

[3]
 前記ペプチドユニット(1)と前記ペプチドユニット(2)との割合が、モル比にて(1)/(2)=99.9/0.1~1/99である請求項1記載のポリペプチド。

[4]
 円二色性スペクトルにおいて、波長220~230nmに正のコットン効果を示し、波長195~205nmに負のコットン効果を示す請求項1ないし3のいずれか1項に記載のポリペプチド。

[5]
 該ポリペプチドの少なくとも一部分が三重らせん構造を形成する請求項4記載のポリペプチド。

[6]
 分子量5×10 3~5×10 6の範囲にピークを示す請求項1ないし5のいずれか1項に記載のポリペプチド。

[7]
 コラーゲン組織を形成可能である請求項1ないし6のいずれか1項に記載のポリペプチド。

[8]
 請求項1ないし7のいずれか1項に記載のポリペプチドに、ペプチド、タンパク質、ポリペプチド、核酸、糖、多糖類、脂質、ポリエチレングリコール誘導体、抗菌剤、アパタイト類およびこれらの複合体よりなる群から選択される1以上の物質が結合したポリペプチド誘導体。

[9]
 請求項8記載の物質が、請求項1ないし7のいずれか1項に記載のポリペプチドのジカルボン酸リンカーを介してポリペプチドのHyp残基に結合したポリペプチド誘導体。

[10]
 請求項1ないし9のいずれか1項に記載のポリペプチドまたはポリペプチド誘導体にアパタイト類が担持されているポリペプチド誘導体。

[11]
 式:
[化3]

(式中、oは1以上の整数を表す)       
で表されるアミノ酸配列を有するペプチドユニットと、式:
[化4]

(式中、pは1以上の整数を表す)       
で表されるアミノ酸配列を有するペプチドユニットとを縮合させて得られるポリペプチドに、式:
[化5]

(式中、Yはカルボニル基、カルボニル基を有するかまたは有しない飽和または不飽和の炭化水素基、または芳香族基を含む、カルボニル基を有するかまたは有しない飽和または不飽和の炭化水素基を表し、Zはカルボキシル基を表す)
で表される化合物またはその無水物を反応させることを含む請求項1に記載のポリペプチドの製造方法。

[12]
 請求項11に記載の製造方法において、さらに、該ポリペプチドに、ペプチド、タンパク質、ポリペプチド、核酸、糖、多糖類、脂質、ポリエチレングリコール誘導体、抗菌剤、アパタイト類およびこれらの複合体よりなる群から選択される1つ以上の物質を反応させることを含むポリペプチド誘導体の製造方法。

[13]
 請求項12に記載の製造方法において、1つ以上の該物質を、該ポリペプチドのカルボン酸リンカーを介してポリペプチドのHyp残基に結合させることを含むポリペプチド誘導体の製造方法。

[14]
 請求項1ないし9のいずれか1項に記載のポリペプチドまたはポリペプチド誘導体と、カルシウムイオンおよびリン酸イオンを含む水溶液とを接触させてアパタイト類をポリペプチドまたはポリペプチド誘導体に沈着させることを含むアパタイト類が担持されているポリペプチド誘導体の製造方法。

[15]
 該アパタイト類がヒドロキシアパタイトである請求項14記載の製造方法。

[16]
 請求項14または15記載の製造方法によって得られる、アパタイト類が担持されているポリペプチド誘導体。

[17]
 式:
[化6]

で表されるアミノ酸配列を有するペプチドユニットと、式:
[化7]

(式中、AAはOH、Tyr-Arg-His-Ala-Trp-Ser-Glu-Asn-Leu-Ala-Gln-Cys-Phe-Asn-NH 2
Lys-Ile-Pro-Lys-Ala-Ser-Ser-Val-Pro-Thr-Glu-Leu-Ser-Ala-Ile-Ser-Thr-Leu-Tyr-Leu-NH 2、またはGly-Arg-Gly-Asp-Serを表す)
で表されるアミノ酸配列を有するペプチドユニットとを含むポリペプチド誘導体。






  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • NATIONAL UNIVERSITY CORPORATION NARA INSTITUTE OF SCIENCE AND TECHNOLOGY
  • Inventor
  • TANIHARA, Masao
IPC(International Patent Classification)
Specified countries National States: AE AG AL AM AO AT AU AZ BA BB BG BH BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IS JP KE KG KM KN KP KR KZ LA LC LK LR LS LT LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PG PH PL PT RO RS RU SC SD SE SG SK SL SM ST SV SY TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ MD RU TJ TM
EPO: AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MT NL NO PL PT RO SE SI SK TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG
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