JP2005074079A - Hemostatic material - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a hemostatic material which is excellent in a hemostatic effect, in vivo decomposing and absorbing property, uniformity in quality, and stability, and which reduces the risk of mixing an animal-derived pathogen. <P>SOLUTION: This hemostatic material contains thrombin and a synthetic polypeptide which can form a tri-helix structure. The molecular weight of the polypeptide can indicate a peak in the range of 5×10<SP>4</SP>-100×10<SP>4</SP>. The polypeptide may contain a peptide unit expressed at least by the formula, Pro-X-Gly (X indicates Pro or Hyp (hydroxyproline) in the formula). Thrombin can be a recombinant. The rate of thrombin can be a degree of 0.1-500 unit (U) with respect to the polypeptide in the hemostatic material. A binder component having in vivo decomposing and absorbing property can be contained in the hemostatic material. The material can be formed on a base material. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a novel hemostatic material having excellent hemostatic effect and excellent biocompatibility or biocompatibility.

  Conventionally, as a hemostatic method using a hemostatic material, a method of spraying or sticking a hemostatic material such as oxidized cellulose, collagen, gelatin, calcium alginate, thrombin, or fibrin adhesive to a bleeding site has been adopted. These hemostatic materials are used in the form of powder, liquid, fiber, cloth (nonwoven fabric), film, sponge or the like.

  Japanese Patent Application Laid-Open No. 7-255830 (Patent Document 1) discloses a bioabsorbable surgical hemostatic material composed of a neutralized oxidized cellulose cloth containing 0.5 to 4.0% by weight of calcium. ing. Japanese Patent Application Laid-Open No. 2003-26578 (Patent Document 2) discloses a hemostatic material comprising a maleate of deacetylated chitin. Japanese Unexamined Patent Publication No. 2002-369874 (Patent Document 3) discloses a hemostatic material comprising a water-soluble fiber aggregate such as carboxymethylcellulose. Japanese Patent Laid-Open No. 2002-60341 (Patent Document 4) discloses that a hemostatic material mainly composed of a calcium salt of a nucleic acid can be applied to a site where there is a large amount of bleeding, and there is no problem of antigenicity. Is disclosed. Japanese Patent Application Laid-Open No. 11-322614 (Patent Document 5) discloses a wound hemostatic material having a cell adhesion promoting effect containing carboxymethyl cellulose as a component. Japanese Patent Application Laid-Open No. 9-169653 (Patent Document 6) discloses a chitin hemostatic agent composed of chitin fibers having an orientation degree of 50 to 98%. Japanese Patent Application Laid-Open No. 9-103479 (Patent Document 7) discloses a medical material (bioadhesive, hemostatic material, etc.) obtained by crosslinking gelatin with succinimidated polyglutamic acid. JP-A-7-118157 (Patent Document 8) is obtained by polymerizing D, L-lactide and polyethylene glycol, and the molar ratio of ethylene oxide unit to lactic acid unit is 52:48 to 30:70. There is disclosed a hemostatic material having a molecular weight of 7800-15000.

  Japanese Patent Application Laid-Open No. 9-2971 (Patent Document 9) discloses a stable tissue adhesive containing a prothrombin activator or a prothrombin containing less prothrombin than 5 units / g fibrinogen. In JP-A-8-35193 (Patent Document 10), collagen fibers obtained by discharging an acidic solution of soluble collagen into an aqueous salt solution are cut, and the fibers are dispersed in a solvent and made into paper. A method for producing a collagen fiber nonwoven sheet is disclosed. This document also discloses that the obtained nonwoven sheet is useful as a hemostatic material that can be quickly and effectively applied to a wound part. Japanese Patent Publication No. 61-34830 (Patent Document 11) discloses a wound healing material comprising a collagen carrier partially or wholly coated with a mixture of a fibrinogen component and a thrombin component. This document discloses natural collagen or chemically modified collagen as the collagen, and discloses that the thrombin component can be derived from animals or humans.

  However, with these conventional hemostatic materials, it is difficult to effectively stop bleeding particularly when the bleeding moment is strong or the amount of bleeding is large. In particular, even if a powder or liquid hemostatic material is sprayed directly on the bleeding site, the hemostatic component is easily washed away by the blood flow. In addition, although the conventional hemostatic material has some temporary hemostatic properties, it has a low biodegradable absorbability and contains a large amount of cytotoxic substances, so depending on the type of hemostatic material, May need to be removed and there is a risk of rebleeding.

  On the other hand, in biomaterial applications such as hemostatic materials, raw materials derived from animals (such as cattle and horses) are often used. However, when animal-derived raw materials are used, biodegradability and absorbability can be enhanced, but there is a risk of contamination with pathogens, and the quality is not stable. For example, the causative substance of bovine spongiform encephalopathy and sheep tremor is an infectious protein called prion, and this infectious protein is said to be one of the causes of human Creutzfeldau-Jakob disease. It is pointed out that prion is a protein, is hardly inactivated by normal sterilization and sterilization methods, and is infected across species (Non-patent Document 1).

The present inventors have disclosed a method for producing recombinant thrombin by genetic recombination in International Publication No. 03/004641 (Patent Document 12).
JP-A-7-255830 (Claim 1) JP 2003-26578 A (Claim 1, paragraph number [0044]) JP 2002-369874 A JP 2002-60341 A (Claim 1, paragraph number [0013]) Japanese Patent Laid-Open No. 11-322614 (Claim 1, paragraph number [0026]) JP-A-9-169653 (Claim 1, paragraph number [0004]) JP-A-9-103479 (Claims 1 and 3, paragraph number [0004]) JP 7-118157 A (Claim 1, paragraph number [0030]) Japanese Patent Laid-Open No. 9-2971 (Claim 1, paragraph number [0015]) JP-A-8-35193 (Claim 1, paragraph numbers [0001] and [0008]) Japanese Examined Patent Publication No. 61-34830 (Claim 1, column 4, lines 30-35, column 5, lines 8-14) International Publication No. 03/004641 Pamphlet (Claims) Nature Review, Vol.2, pp.118-126, 2001

  Accordingly, an object of the present invention is to provide a hemostatic material that has excellent hemostasis, high biocompatibility and biocompatibility, uniform quality, and excellent stability.

  Another object of the present invention is that when human plasma-derived or recombinant thrombin (especially recombinant thrombin) is used as thrombin, there is a risk of causing infection of pathogens or transmission of pathogenic factors, and undesirable side effects. It is to provide a hemostatic material with a small amount.

  Still another object of the present invention is to provide a hemostatic material that can be decomposed and absorbed in vivo.

  Another object of the present invention is to provide a hemostatic material that can be molded into various forms and can effectively stop hemostasis depending on the application.

  As a result of intensive studies to achieve the above-mentioned problems, the present inventors have found that combining a chemically synthesized polypeptide having a triple helical structure with thrombin has excellent hemostasis and biocompatibility and biocompatibility. I found it expensive and completed the present invention.

That is, the hemostatic material of the present invention is a hemostatic material containing thrombin and a polypeptide, and the polypeptide is composed of a synthetic polypeptide capable of forming a triple helical structure. The molecular weight of the polypeptide may show a peak in the range of 5 × 10 ^{4 to} 100 × 10 ⁴ . The polypeptide may contain at least a peptide unit represented by the formula -Pro-X-Gly- (wherein X represents Pro or Hyp). The thrombin may be a recombinant. In the hemostatic material, the ratio of thrombin may be about 0.1 to 500 units (U) with respect to 1 mg of the polypeptide. The hemostatic material may further contain a binder component (polysaccharide or derivative thereof, peptides, biodegradable absorbable polyester, etc.) having biodegradability. The ratio (weight ratio) of the binder component may be about 0.01 / 99.99 to 95/5 with respect to the total amount of thrombin and polypeptide = total amount of binder component / thrombin and polypeptide. The hemostatic material may be formed on a substrate.

  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.

  In the present invention, thrombin and a polypeptide having a collagen-like structure are combined, so that hemostasis is excellent, biocompatibility and biocompatibility are high, quality is uniform, and stability is excellent. In addition, when human thrombin or recombinant thrombin (especially recombinant thrombin) is used as thrombin, there is little risk of pathogen infection and transmission of pathogenic factors and undesirable side effects, and safety is improved. high. Moreover, it can be decomposed and absorbed in vivo. Moreover, the hemostatic material of the present invention has high moldability, can be molded into various forms, and can effectively stop hemostasis depending on the application.

  The hemostatic material of the present invention is composed of thrombin and a synthetic polypeptide capable of forming a triple helical structure.

[Thrombin]
The thrombin is not particularly limited as long as it has a blood coagulation effect. For example, thrombin obtained by allowing calcium salt and thromboplastin to act on prothrombin extracted and purified from plasma of a human or non-human animal is used. it can. When human-derived thrombin is used, infection with pathogens derived from non-human animals can be prevented.

  In addition, as thrombin, recombinant thrombin obtained by genetic recombination, for example, thrombin gene derived from human or non-human animal, prothrombin gene, etc. is used to produce in hosts such as E. coli, yeast, insect cells, animal cells, etc. And recombinant thrombin. Such recombinant thrombin has a constant quality and can be supplied stably. When a recombinant thrombin is used, the risk of pathogen infection derived from animal plasma can be greatly reduced.

  For example, a pre-thrombin-2 gene is amplified using a human-derived prothrombin gene as a template, and this prethrombin gene is used as a high-expression vector using, for example, an animal cell (chicken, hamster, mouse, human-derived cell, etc.) as a host. The plasmid is constructed, and the resulting expression plasmid is introduced into animal cells so that the recombinant prethrombin is highly expressed. The obtained recombinant prethrombin is further transformed into a substrate using recombinant ecarin. Thrombin can be obtained by activating as above. The obtained thrombin can be used after purification by chromatography or the like. For details of the method for producing recombinant thrombin, reference can be made to WO 03/004641 pamphlet by the present inventors.

  Thrombin can be used alone or in combination of two or more.

[Polypeptide]
The polypeptide contained in the hemostatic material is not particularly limited as long as it is a synthetic polypeptide capable of forming a triple helical structure. The synthetic polypeptide only needs to have a triple helical structure in at least a part of the polypeptide. Synthetic polypeptides having a triple helix structure form a collagen-like structure.

The molecular weight of the polypeptide may show a peak in the range of, for example, 1 × 10 ^{4 to} 500 × 10 ⁴ , preferably 2 × 10 ^{4 to} 300 × 10 ⁴ , more preferably 5 × 10 ^{4 to} 100 × 10 ^4. Good. If the molecular weight is too small, the solubility is too high and the hemostatic effect is easily impaired, and if it is too large, the solubility is too low and the processability may be lowered.

  Examples of the synthetic polypeptide having a triple helical structure include a polypeptide containing at least a peptide unit represented by the formula -Pro-X-Gly- (wherein X represents Pro or Hyp). A synthetic polypeptide containing the peptide unit exhibits a very stable triple helix structure. Moreover, since it has a collagen-like fibrous form, it is excellent in processability and the strength of the resulting molded product is also excellent.

The synthetic polypeptide may be a polypeptide (Pro-X-Gly) _n (wherein n represents an integer of 1 to 20,000) composed only of the peptide unit -Pro-X-Gly-. It may be a polypeptide composed of the peptide unit -Pro-X-Gly- and other amino acid residues.

  In the above formula, the coefficient n is preferably 2 to 20,000, more preferably 10 to 10,000 (for example, 30 to 10,000), particularly about 100 to 5,000 (for example, 150 to 4,000). It may be an integer.

In addition to (Pro-Pro-Gly) _n and (Pro-Hyp-Gly) _n , the polypeptide composed only of the polypeptide unit includes Unit-Pro-Pro-Gly- and Unit-Pro-Hyp-Gly. And a polypeptide in which the total number of repetitions of both units is n is also included.

  Other amino acid residues include Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Hyp, Ile, Leu, Lys, Met, Phe, Pro, Sar, Ser, Thr, Trp, Tyr, Val etc. are mentioned. These amino acid residues may be used alone or in combination of two or more.

  In addition, the polypeptide has a dicarboxylic acid residue (such as a residue of an aliphatic dicarboxylic acid such as an alkanedicarboxylic acid) or a diamine residue (residual aliphatic diamine residue) as long as it does not inhibit hemostasis, biodegradability and the like. Group) and / or a lactam residue.

  Polypeptides can be used alone or in combination of two or more.

  The polypeptide may be a physiologically or pharmacologically acceptable salt, such as an inorganic acid (hydrochloric acid, sulfuric acid, phosphoric acid, etc.), an 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 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. 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. Therefore, it can be proved that the polypeptide forms a triple helical structure by measuring a circular dichroism spectrum for 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. (JM Biol., Vol. 63 pp. 85-99, 1972).

  These polypeptides can form a collagen tissue (collagenous tissue). Polypeptide chains that form the triple helical structure self-assemble to form fibrils of several nm to several tens of nm, and these fibrils are arranged to form a fiber structure of several nm to several tens of nm. can do. These can be observed with a transmission electron microscope, a scanning electron microscope, or an atomic force microscope.

  Unlike collagen derived from mammals, the polypeptide has no risk of infection or transmission of pathogens or pathogenic factors [eg, proteins converted to pathogenicity (eg, abnormal prions, etc.)] and is safe. high. Further, it can form a collagen-like tissue, and is excellent in cell affinity and biocompatibility.

  The hemostatic material of the present invention is not particularly limited as long as it contains the thrombin and the polypeptide, and is liquid (solution or suspension), non-liquid [for example, granular, fibrous, two-dimensional form (woven fabric) , Non-woven fabrics, films, sheets, etc.) and three-dimensional forms (sponges, etc.).

  Since the polypeptide has high film formability or moldability and can be easily formed into a desired shape, a hemostatic material containing thrombin and polypeptide may be appropriately formed and used. Moreover, you may shape | mold or form into a film using thrombin, polypeptide, and the binder component (or film-forming component) which has a biodegradable absorbability as needed.

  Binder components include polysaccharides or derivatives thereof [locust bean gum, guar gum, tragacanth gum, alginic acid or a salt thereof (such as sodium alginate), propylene glycol alginate, pectin, starch, amylose, amylopectin, agarose, agar, chitin, chitosan, carrageenan , Hyaluronic acid, chondroitins (chondroitin sulfate, chondroitin sulfate sodium, chondroitin heparin, etc.), dextran, cellulose or derivatives thereof (cellulose, methylcellulose, ethylcellulose, carboxymethylcellulose or salts thereof, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, etc. Cellulose such as cellulose ethers and cellulose acetate Esters, etc.), peptides (polylysine, polyglutamine, polyglutamic acid and other polypeptides; gelatin, casein, albumin, etc. proteins), polyester resins (glycolic acid, lactic acid, 3-hydroxybutyric acid, 4- Hydroxybutyric acid, 3-hydroxypropionic acid and other hydroxycarboxylic acid homopolymers or copolymers (lactic acid-glycolic acid copolyester, etc.), hydroxycarboxylic acid and propionic acid, lactone (butyrolactone, caprolactone, etc.) copolyester, etc. Biodegradable absorbable polyester, etc.). These binder components can be used alone or in combination of two or more. When such a binder component is used, the strength and moldability of the hemostatic material can be improved, and water absorption can be adjusted.

  The hemostatic material of the present invention includes other additives such as other hemostatic components (fibrinogen, oxidized cellulose, etc.), cell adhesive proteins (fibronectin, vitronectin, laminin, etc.), antibacterial agents, preservatives, salts (physiologically) Acceptable salts, etc.).

  The hemostatic material may be a hemostatic material in which thrombin is applied or impregnated to a base material formed of a polypeptide. The form of the substrate formed of the polypeptide is not particularly limited, and is granular (for example, about 1 to 300 μm), one-dimensional shape (fibrous or thread-like, linear, rod-like, etc.), It may be a two-dimensional shape (such as a film (or sheet) or plate), a three-dimensional shape (such as a tube), and the like. Furthermore, the polypeptide substrate may be a non-porous body or a porous body. The polypeptide substrate was obtained, for example, by (1) extruding a solution or suspension of a polypeptide from a nozzle and coagulating it in a solution containing a high-concentration salt or a solvent that does not dissolve the polypeptide. Fibrous polypeptide, (2) Non-woven fabric obtained from the fibrous polypeptide using a wet or dry papermaking method, (3) An aqueous solution or suspension of the polypeptide may be left as it is, or a crosslinking agent may be added if necessary. A gel-like product is prepared by adding, placing and cross-linking, and a sponge-like porous material obtained by freeze-drying, and (4) stirring and foaming an aqueous solution or suspension of the polypeptide and drying. Examples thereof include a porous body obtained by the above. In addition, the polypeptide substrate may be crosslinked with a physiologically acceptable crosslinking agent, for example, dialdehydes such as glyoxal, glutaraldehyde, succinaldehyde, dextran dialdehyde, aldehyde starch, etc., if necessary. .

  Further, the hemostatic material may be formed on the substrate by applying a hemostatic agent component containing at least thrombin and a polypeptide to the substrate. As such a base material, in many cases, usually has biocompatibility and biocompatibility, for example, polysaccharides or derivatives thereof (alginate, chitin, chitosan, hyaluronic acid, polygalactosamine, curdlan, Polysaccharides such as pullulan, xanthan and dextran, cellulose or derivatives thereof exemplified in the above-mentioned binder component, protein (gelatin, casein, albumin, etc.), polypeptide (polylysine, polyglutamine, polyglutamic acid, etc.), vinyl alcohol resin (Polyvinyl alcohol resins, ethylene-vinyl alcohol copolymers, etc.), polyvinyl pyrrolidone resins, acrylic resins (poly (meth) acrylic acid, (meth) acrylic acid copolymers such as (meth) acrylic acid copolymers, etc.) ), Halogen-containing resin (polytetrafluoro Fluorine resins such as ethylene, polyvinyl chloride resins such as polyvinyl chloride), polyurethane resins, silicone resins, polyester resins exemplified in the above-mentioned binder component, polyamide resins (nylon 6, nylon 66, etc.) The substrate can be used alone or in combination of two or more.

  The substrate may be non-biodegradable or bioerodible, but advantageously has a biodegradability in vivo. Such a biodegradable substrate can be composed of a biodegradable resin. Examples of the biodegradable resin include various resins such as the polysaccharide or a derivative thereof and the polyester resin. The base material may be a composite base material using two or more kinds of materials.

  The form of the substrate is not particularly limited, and a substrate having the same form as that exemplified in the section of the polypeptide substrate can be used depending on the application. The base material may be a non-porous material (for example, a granular porous material, a cellulose fiber paper, a two-dimensional porous material such as a nonwoven fabric or a woven fabric, a three-dimensional material such as a cylindrical shape). A porous material). If necessary, the substrate may be surface-treated with a surface treatment agent (for example, a physiologically acceptable surface treatment agent).

(Ratio of each component)
The ratio of thrombin in the hemostatic material is not particularly limited as long as a hemostatic effect is obtained, and is, for example, 0.1 to 500 units, preferably 0.2 to 300 units, more preferably 0. It can be selected from a range of about 3 to 200 units. If the ratio of thrombin is too small, the hemostatic effect is insufficient, and if it is too large, the thrombin may not work effectively.

Further, depending on the form of the hemostatic material, for example, in a liquid hemostatic material, the ratio (concentration) of thrombin is, for example, 3 to 200 units / mL, preferably 5 to 150 units / mL, and more preferably 10 to 100 units. / ML may be sufficient. In the non-liquid hemostatic material, the ratio of thrombin is 0.1 to 30 units, preferably 0.3 to 10 units, and more preferably about 0.5 to 5 units with respect to 1 g of the hemostatic material. Good. In the hemostatic material formed on the base material, the ratio of thrombin is, for example, 10 to 500 units / cm ² , preferably 20 to 300 with respect to 1 cm ² of the surface area of the application site of the hemostatic agent component in the base material. It may be about unit / cm ² , more preferably about 30 to ²⁰⁰ unit / cm ² .

  The ratio of thrombin is, for example, 0.1 to 500 units, preferably 0.1 to 300 units (for example, 0.1 to 100 units), more preferably 0.5 to 50 units per 1 mg of polypeptide. It may be about a unit (for example, 1 to 20 units).

  The ratio of the polypeptide is not particularly limited as long as it promotes the hemostatic action of thrombin, enhances adhesion to the tissue, and maintains the strength and flexibility of the molded body. For example, a hemostatic material (hemostatic agent component) It may be about 0.01 to 95% by weight, preferably 0.05 to 90% by weight, more preferably about 0.1 to 85% by weight. Further, depending on the form of the hemostatic material, for example, in the liquid hemostatic material, the ratio of the polypeptide is, for example, 0.01 to 20% by weight, preferably 0.01 to 10% by weight, more preferably the whole hemostatic material. May be in the range of 0.05 to 5% by weight. In the non-liquid hemostatic agent, the ratio of the polypeptide may be, for example, 1 to 95% by weight, preferably 5 to 90% by weight, more preferably about 5 to 80% by weight of the entire hemostatic material (hemostatic agent component). Good.

  The ratio of the binder component may be in a range that does not inhibit the hemostatic action of the hemostatic material and exhibits preferable strength and water absorption. For example, the total amount of binder / thrombin and polypeptide relative to the total amount of thrombin and polypeptide (Weight ratio) = 0.01 / 99.99 to 95/5, preferably 0.05 / 99.95 to 90/10, more preferably about 0.1 / 99.9 to 85/15 it can. Also, depending on the form of the hemostatic material, for example, in the case of a liquid hemostatic material, the ratio (weight ratio) is, for example, 0.01 / 99.99 to 20/80, preferably 0.01 / 99.99 to 10 / 90, more preferably about 0.05 / 99.95 to 5/95. In a non-liquid hemostatic material, the ratio (weight ratio) may be, for example, about 1/99 to 90/10, preferably 2/98 to 70/30, and more preferably about 2/98 to 60/40. .

(Method for producing hemostatic material)
The hemostatic material of the present invention can be produced by a conventional method. For example, a liquid hemostatic material is obtained by dissolving a hemostatic component containing at least thrombin and a polypeptide in water, physiological saline, an organic solvent (such as a hypoallergenic organic solvent such as propanol or glycerin), or a mixed solvent thereof. It can be prepared by dispersing. The granular hemostatic material is, for example, a mixture of a granular polypeptide obtained by pulverizing a polypeptide, spray drying a solution or suspension of the polypeptide, and granular thrombin, It can be prepared by spray drying a solution or suspension containing the peptide. In addition, a solution or suspension containing a hemostatic component and, if necessary, a binder component is cast on a peelable support (for example, a glass plate, a fluororesin (polytetrafluoroethylene) sheet, a fluororesin-coated vat, etc.). And a sheet and a film-like hemostatic agent can be obtained by drying. A sponge-like hemostatic agent can be obtained by adding a cross-linking agent, if necessary, to a solution (or suspension) or gel-like product containing a hemostatic agent component, and allowing it to stand or freeze-drying. For example, a fibrous hemostatic agent is spun by injecting a solution or suspension containing a hemostatic agent component into an aqueous solution containing a high-concentration salt (such as sodium sulfate) or a coagulation bath such as ethanol using a nozzle. Can be obtained. The obtained fibrous hemostatic agent may be molded by a conventional method to prepare a woven or non-woven hemostatic agent.

  The hemostatic material formed on the substrate can be produced by applying a hemostatic component containing at least thrombin and a polypeptide to at least the surface of the substrate. For example, after applying or spraying (or impregnating) the solution or suspension of the hemostatic agent component onto the surface of the base material, the hemostatic material having the surface of the base material coated with the hemostatic agent component is obtained by drying. be able to. Alternatively, a hemostatic material retaining the hemostatic agent component may be prepared by impregnating a porous substrate (nonwoven fabric or the like) with a solution or suspension of the hemostatic agent component. In addition, the hemostatic component may be applied to a part of the base material that is applied to a living body (a part that comes into contact with a body fluid or blood as well as a body tissue). It may be applied to the whole, or it may be applied to at least one surface in the case of a two-dimensional shaped substrate, and it is applied to the application site (full surface, inner surface, outer surface, etc.) for a living body in the case of a three-dimensional shaped substrate. do it.

  A hemostatic material in which thrombin is applied to a polypeptide base material is prepared by the same method as described above, for example, by applying a component containing at least thrombin to the polypeptide base material by application, spraying or impregnation. it can.

  The hemostatic material of the present invention is useful for stopping bleeding associated with damage to the skin and organs of humans and animals. In particular, since the hemostatic material of the present invention is excellent in hemostasis and tissue adhesiveness, it is also effective for hemostasis when there is a large amount of bleeding due to damage to internal organs such as lungs and liver or surgery.

  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
1 g of a peptide represented by the formula: Pro-Hyp-Gly (Peptide Institute, Inc.) was dissolved in 20 mL of 10 mM phosphate buffer (pH 7.4), and 473 mg of 1-hydroxybenzotriazole, 3.35 g of 1-Ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride was added and stirred at 4 ° C. for 2 hours and then stirred at 20 ° C. for 46 hours. The reaction solution was dialyzed against Milli Q water (ultra pure water) for 48 hours.

  The resulting dialyzed solution was diluted 50 times with water, and gel permeation chromatography (Amersham Biosciences, AKTApurifier system, column: Superdex 200 HR 10/30, flow rate: 0.5 mL / min) The eluent: 10 mM phosphate buffer (pH 7.4) containing 150 mM NaCl, a polypeptide peak was observed in the molecular weight range of 100,000 to 600,000.

  Moreover, when the obtained solution after dialysis was diluted 100 times with water and the circular dichroism spectrum was measured, a positive cotton effect was observed at 225 nm and a negative cotton effect was observed at 198 nm. The formation was confirmed.

Chemistry that forms a calibration curve from the absorbance at 215 nm of the peptide represented by the formula: (Pro-Hyp-Gly) ₁₀ (SEQ ID NO: 1) (Peptide Institute, Inc.) and forms the resulting triple helical structure The concentration of the synthetic polypeptide was measured and was about 20 mg / mL.

  Neoveil (Gunze Co., Ltd.), a polyglycolic acid-based non-woven fabric cut in 3 cm square, was impregnated with about 700 U of recombinant thrombin (Chemical and Serological Therapy Research Institute: see WO 03/004641 pamphlet) Thereafter, it was freeze-dried. Further, after impregnating 0.5 mL of a chemically synthesized polypeptide that forms a triple helix structure diluted to about 20 mg / mL, it was freeze-dried to obtain a nonwoven hemostatic material.

Comparative Example 1
In Example 1, a non-woven hemostatic material was obtained in the same manner except that 0.5 mL of porcine-derived Type III collagen (Nitta Gelatin Co., Ltd.) was used instead of the chemically synthesized polypeptide forming a triple helical structure. It was.

Example 2
2.5 mL of a 1% by mass aqueous solution of sodium alginate (manufactured by Kimika Co., Ltd., 99 mPa · s), 2.5 mL of a chemically synthesized polypeptide that forms a triple helical structure of 20 mg / mL obtained in Example 1, 2000 U / mL recombinant thrombin (Chemical and Serum Therapy Laboratory: see WO 03/004641 pamphlet) 0.34 mL is mixed and cast into a Teflon (registered trademark) tray with an internal dimension of 3 cm square Then, it was air-dried at room temperature to obtain a sheet-like hemostatic material.

Example 3
2.5 mL of a 1% by mass aqueous solution of sodium alginate (manufactured by Kimika Co., Ltd., 99 mPa · s), 2.5 mL of a chemically synthesized polypeptide that forms a triple helical structure of 20 mg / mL obtained in Example 1, 2000 U / mL recombinant thrombin (Chemical and Serum Therapy Laboratory: see WO 03/004641 pamphlet) 0.34 mL is mixed and cast into a Teflon (registered trademark) tray with an internal dimension of 3 cm square And then freeze-dried to obtain a sponge-like hemostatic material.

Test Example The liver of a Japanese white rabbit was exposed to produce a round epithelial wound with a diameter of 12 mm. Thereafter, the wound was covered with the hemostatic material obtained in Examples 1 to 3 and Comparative Example 1 and pressed for 1 minute. Thereafter, the blood leaking from the hemostatic material was absorbed with a filter paper until the hemostasis was stopped, and the total amount of bleeding was measured by its weight.

  As a result, the total bleeding amount of the hemostatic material obtained in Example 1 was 0.316 g (n = 3 average value), Example 2 was 0.521 g (n = 3 average value), and Example 3 was 0. .296 g (average value of n = 3). On the other hand, in Comparative Example 1, it was 1.895 g (n = 3 average value).

  As is clear from the test examples, compared with Comparative Example 1, in Examples 1 to 3, the total amount of bleeding was clearly small and the hemostatic effect was excellent.

Claims (9)

  A hemostatic material comprising thrombin and a polypeptide, wherein the polypeptide is composed of a synthetic polypeptide capable of forming a triple helical structure. The hemostatic material according to claim 1, wherein the polypeptide has a molecular weight peak in the range of 5 x 10 ^{4 to} 100 x 10 ⁴ .   The hemostatic material according to claim 1 or 2, wherein the polypeptide contains at least a peptide unit represented by the formula -Pro-X-Gly- (wherein X represents Pro or Hyp).   The hemostatic material according to any one of claims 1 to 3, wherein thrombin is a recombinant.   The hemostatic material according to any one of claims 1 to 4, wherein the ratio of thrombin is 0.1 to 500 units with respect to 1 mg of the polypeptide.   The hemostatic material according to any one of claims 1 to 5, further comprising a binder component having biodegradability.   The hemostatic material according to claim 6, wherein the binder component comprises at least one selected from polysaccharides or derivatives thereof, peptides, and biodegradable absorbable polyester.   The hemostatic material according to claim 6, wherein the ratio (weight ratio) of the binder component is 0.01 / 99.99 to 95/5 with respect to the total amount of thrombin and polypeptide.   The hemostatic material according to any one of claims 1 to 7, which is formed on a substrate.