HK1204774B - Sheet molding and hemostatic material - Google Patents

Description

Sheet-formed body and hemostatic material

Technical Field

The present invention relates to a sheet-shaped body and a hemostatic material comprising the same. More specifically, the present invention relates to a sheet molding containing fibrinogen and/or thrombin, which is excellent in elution and supporting properties of these hemostatic proteins and which has excellent hemostatic properties, and a hemostatic material containing the same.

Background

Fibrinogen is a coagulation factor that is present in the final stage of the blood coagulation cascade. When a blood vessel is damaged, activation of the coagulation system occurs, and finally the activated thrombin converts soluble fibrinogen into insoluble fibrin. The fibrin has binding power, and has important functions of stopping bleeding and healing wound.

In medical fields, particularly in surgical operations, tissue adhesion operations such as hemostasis and tissue sealing are important, and fibrin paste adhesive materials to which the present principle is applied are widely used in surgical fields.

As for the method of using the fibrin paste adhesive material, various studies have been made up to now to improve it, and the following preparations exist: a liquid preparation in which a fibrinogen solution and a thrombin solution are applied or sprayed to an affected part (a two-liquid mixed preparation: see Japanese patent publication No. Hei 9-2971 and International publication No. WO 97/33633); a sheet preparation in which a sheet obtained by mixing and immobilizing fibrinogen and thrombin on a support such as collagen is attached to an affected part (see Japanese patent application laid-open No. 2004-521115).

However, since the conventional liquid preparations are used by dissolving the freeze-dried fibrinogen and thrombin separately at the time of use, the freeze-dried preparations require a time of about several minutes for dissolution, and thus the liquid preparations are not satisfactory in terms of handling an emergency operation and simplicity.

In addition, since the higher the fibrinogen concentration, the stronger the binding force can be obtained, the greater the binding force of the above-mentioned fibrin paste binder, and it is necessary to allow a small amount of thrombin with a high concentration to act on the fibrinogen with a high concentration in order to achieve a strong binding force.

However, in the conventional liquid preparation, the maximum efficacy of fibrinogen cannot be exerted because the concentration is halved by mixing the fibrinogen solution and the thrombin solution in equal amounts. Further, since the concentration of fibrinogen is actually limited to about 10% solution, it is difficult to improve the concentration in a system in which two solutions are mixed in equal amounts.

From this point of view, since the sheet preparation can apply the fibrinogen solution to the affected part at a high concentration, it is theoretically expected that the sheet preparation has a stronger adhesive force than the two-liquid mixed preparation. In addition, if the preparation is a tablet preparation, compression hemostasis and compression sealing can be performed on a bleeding part with exudative ejection property, and excellent convenience can be expected.

When a sheet-like tissue adhesive material is used, since a fibrinogen solution can be applied to an affected part at a high concentration, it is necessary to increase the permeability of the sheet preparation into the tissue when applied to a wound site. Further, since the sheet preparation may be rounded or curved in order to be closely adhered to a wound site, it is necessary to increase the flexibility of the sheet and the holding force of the two components so that the sheet is not broken and the fibrinogen component and the thrombin component are not dropped by such forces.

A sheet-like tissue-adhesive material and a sheet-like hemostatic material, each of which comprises an active ingredient fixed to various substrates, have been reported (see Japanese patent publication No. 61-34830, Japanese patent publication No. 2002-513645, International publication No. WO2004/064878 and International publication No. WO 2005/113030). Japanese patent publication No. 61-34830 discloses a sheet preparation in which fibrinogen and thrombin are immobilized on the surface layer of collagen derived from horses, and the sheet preparation has been put into practical use (tachcocomb (registered trademark)). However, since the collagen matrix is thick and hard, the adhesiveness to the wound site is reduced, and it is sometimes difficult to effectively seal the wound site. In addition, the support of the preparation is equine collagen, and when the subject to which the preparation is applied is a human, there is a risk of the expression of antibodies against different proteins and the common infection of humans and animals such as prion diseases, and therefore the preparation is not ideal.

Japanese patent laid-open publication No. 2002-513645 discloses a paper-like composition in which hemostatic compounds are uniformly dispersed. The composition is produced by forming fibrous pulp containing a bioabsorbable polymer and a hemostatic compound (mainly thrombin and fibrinogen) in a nonaqueous solvent and subjecting the fibrous pulp to a papermaking treatment. Compared with the aforementioned TachoComb, the hemostatic time is reduced by 14 times, and the adhesive can be re-adhered in use. However, since it is in a paper form, there is room for improvement in tissue followability.

International publication WO2004/064878 and International publication WO2005/113030 disclose a material in which a sheet in which thrombin is immobilized on a bioabsorbable synthetic nonwoven fabric and a fibrinogen solution are used in combination. These compositions are compounded by immersing a nonwoven fabric in an aqueous solution of an active ingredient and freeze-drying the resultant. The method has the following problems: the productivity in the freeze-drying step is poor, the flexibility of the sheet is low, and the immobilized protein is poor in the supporting property and is peeled off from the sheet.

In japanese patent application laid-open No. 2009-183649, a sheet-like tissue hemostatic material in which an intermediate layer made of a cellulose derivative is provided between a fibrinogen-containing layer and a thrombin-containing layer is shown for the purpose of controlling the coagulation reaction, but there are problems that the solubility of fibrinogen contained in the tissue hemostatic material is insufficient, the handling property is poor because it is a freeze-dried product, and trimming cannot be performed.

Further, as a sheet preparation, japanese patent application laid-open No. 2010-069031 discloses a sheet-like fibrin paste adhesive material comprising a bioabsorbable support to which fibrinogen containing a nonionic surfactant is fixed and a bioabsorbable support to which thrombin is fixed; a hemostatic three-layer bandage (bandage) comprising a fibrinogen layer, a thrombin layer, an absorbent material layer and the like is disclosed in Japanese patent laid-open publication No. 2002-515300; further, japanese patent application laid-open No. 2009-533135 discloses a porous wound care product including a first absorbent nonwoven fabric, one or more second absorbent woven fabrics or gray fabrics, and thrombin and/or fibrinogen. However, since these hemostatic materials are produced by freeze-drying fibrinogen and thrombin, the detachment of fibrinogen and thrombin is likely to occur, and the flexibility is insufficient, and since fibrinogen is adjacent to thrombin, the above-described tissue adhesion effect is insufficient. In addition, in the case of a form in which fibrinogen is in direct contact with thrombin, a slight amount of water advances the coagulation reaction during storage to form fibrin, which has a problem of storage stability. Further, since a freeze-drying step is required, there is a problem that the production thereof takes time and labor.

As described above, there has not yet been established a sheet-like hemostatic material that can be expected to have a practical effect and convenience by combining fibrinogen and thrombin. In addition, a fibrinogen solution having a high concentration is required to exert a strong tissue adhesion effect, but since fibrinogen has poor solubility, fibrinogen hardly dissolves when fibrinogen is fixed to a support in a conventional composition state, and thus it cannot be expected to exert a sufficient drug effect.

Disclosure of Invention

The present invention aims to provide a sheet molded body having good loading properties and elution properties of a hemostatic protein, in other words, fibrinogen and/or thrombin, flexibility (tissue-following properties), and excellent hemostatic properties.

Another object of the present invention is to provide a hemostatic material that is suitable for use in a wound site and that can achieve an excellent hemostatic effect, the hemostatic material comprising the sheet molded body of the present invention.

Other objects and advantages of the present invention will become apparent from the following description.

According to the present invention, the above objects and advantages of the present invention can be achieved by a sheet molded body of a polymer composition comprising: a protein selected from at least one of fibrinogen and thrombin, and at least one polymer selected from an aliphatic polyester and a water-soluble polymer.

According to the present invention, the above object and advantages of the present invention can be achieved by a laminated sheet molded body comprising, as a combination of the sheet molded bodies of the above paragraphs: the adhesive sheet comprises a first sheet molded body layer comprising fibrinogen and a water-soluble polymer, and a second sheet molded body layer comprising thrombin and an aliphatic polyester.

Further, according to the present invention, thirdly, the above object and advantages of the present invention can be achieved by a hemostatic material comprising the above sheet molded body or laminated sheet molded body of the present invention. That is, these molded bodies are applied to a wound site and can be used for treating the wound site as a hemostatic material.

Drawings

FIG. 1 is a diagram showing the thrombin elution properties of a polyglycolic acid-polylactic acid copolymer containing thrombin eluted from a molded fiber.

Detailed Description

The sheet molded body of the present invention is a sheet molded body of a polymer composition containing at least one protein selected from fibrinogen and thrombin and at least one polymer selected from an aliphatic polyester and a water-soluble polymer (hereinafter, also referred to as "base polymer"). Here, "containing a protein" refers to a state in which at least a part of the protein enters the inside of the base polymer composition. Such a structure is different from a freeze-dried complex in which a protein is present on the surface of a composition or in the voids of a composition, and is excellent in the protein-supporting property.

The sheet-shaped body of the present invention is not particularly limited as long as it is in a sheet form, and preferable sheet-shaped bodies include fiber-shaped bodies and film-shaped bodies. The fiber formed body means a three-dimensional formed body in which one or more fibers obtained are layered and formed by weaving, knitting, or other methods. Specific examples of the form of the fiber molded product include nonwoven fabrics. Further, pipes, nets, and the like processed based on this are also included in the fiber formed body. The film molded product is a film molded product produced by an extrusion molding method such as a blow extrusion molding method or a T-die extrusion molding method, or a molding method such as a rolling method or a casting method (casting method).

The sheet molded body of the present invention can exert its effect by itself, and can exert its effect by combining with a second sheet containing a complementary protein in a fibrin paste (fibrinogen in the case of thrombin, or thrombin in the case of fibrinogen). When used alone, the fibrous molded body containing thrombin in the fatty acid polyester is preferable.

The sheet molded body may be used as a sheet molded body constituting a laminated sheet molded body with the second sheet molded body of the present invention, and the laminated sheet molded body with the second sheet molded body of the present invention is a laminated sheet molded body containing: the sheet-like molded article comprises a first sheet-like molded article containing fibrinogen and a water-soluble polymer, and a second sheet-like molded article containing thrombin and an aliphatic polyester.

The hemostatic proteins usable in the present invention, i.e., fibrinogen and thrombin, may be prepared from animals or may be produced by gene recombination techniques. If it is of animal origin, it is preferably of human origin. In addition, proteins with altered amino acid sequences can also be used.

It should be noted that, in the case of the polymer composition described above, particularly, in the case of containing fibrinogen and thrombin, fibrin is sometimes partially produced during storage, and a composition containing such fibrin is also within the scope of the present invention.

Pharmaceutically acceptable additives may be added to the hemostatic protein that can be used in the present invention. Examples of such additives include at least one selected from blood coagulation factor XIII, albumin, isoleucine, glycine, arginine, glutamic acid, phenylalanine, histidine, a surfactant, sodium chloride, sugar alcohol (e.g., glycerol, mannitol), trehalose, sodium citrate, aprotinin, and calcium chloride.

The hemostatic protein or the mixture of the hemostatic protein and the additive, which can be used in the present invention, may be present in the form of molecules dispersed in the base polymer, and preferably particles in which the respective molecules are aggregated (hereinafter, including mixed particles of the hemostatic protein and the additive, which may be referred to as "protein particles"). This may improve the elution of the hemostatic protein, and may improve the flexibility of the sheet when the sheet molded body is a fibrous molded body.

In the present invention, the average particle size of the protein particles contained is 0.1 to 200 μm. It is technically difficult to produce particles having a particle size of less than 0.1. mu.m. On the other hand, if it exceeds 200 μm, the resulting sheet molding tends to be brittle and difficult to handle, which is not preferable. Preferably 0.5 to 150 μm, and more preferably 1 to 100 μm.

When the sheet molded product of the present invention is a fiber molded product, the sheet molded product contains protein-containing particles in an amount of usually 1 to 200% by weight, preferably 10 to 100% by weight, more preferably 20 to 100% by weight, and further preferably 50 to 100% by weight, based on the base polymer. If the amount is less than this range, the elution property of the protein derived from the sheet molded body, and the flexibility or hemostatic property of the sheet molded body may be poor, and if the amount is more than this range, the self-supporting property of the sheet molded body itself may be lowered, which is not preferable. In addition, in the case of the film molded product, the protein-containing particles are contained in an amount of usually 100% by weight or more, preferably 500% by weight or more, and more preferably 800 to 950% by weight based on the base polymer. If the amount is less than this range, the hemostatic properties may be poor, and if the amount is more than this range, the film formability may be poor.

Specific examples of the aliphatic polyester usable in the present invention include polylactic acid, polyglycolic acid, polylactic acid-polyglycolic acid copolymer, polycaprolactone, polyglycerol sebacic acid, polyhydroxyalkanoic acid, polybutylene succinate, and derivatives thereof. Among these, polylactic acid, polyglycolic acid, polycaprolactone, copolymers thereof, and mixtures thereof are preferably selected.

When a copolymer of polylactic acid is used, a monomer component that imparts stretchability may be contained. Examples of the monomer component for imparting stretchability include flexible components such as caprolactone monomers, ethylene glycol, 1, 2-propanediol, 1, 3-propanediol, 1, 2-butanediol, 1, 4-butanediol, polycaprolactone diol, polyalkylene carbonate diol, and polyethylene glycol units. The smaller the amount of these soft components is, the more preferable the amount is, the less than 50mol% based on the polymer unit. When the soft component is more than this range, the self-supporting property is easily lost, and the soft component is too soft to handle.

When polylactic acid or a copolymer thereof is used, the monomers constituting the polymer are L-lactic acid and D-lactic acid, but are not particularly limited, and the optical purity, molecular weight, composition ratio of L-form to D-form, or sequence of the polymer are not particularly limited, and a polymer having a large amount of L-form is preferred, and a stereocomplex of poly-L-lactic acid and poly-D-lactic acid may be used³~5×10⁶Preferably 1 × 10⁴~1×10⁶More preferably 5 × 10⁴~5×10⁵. The terminal structure of the polymer and the catalyst for polymerizing the polymer can be arbitrarily selected.

As the water-soluble polymer usable in the present invention, preferable examples of the polymer include a polymer having an N-vinyl cyclic lactam unit and a water-soluble cellulose derivative.

Examples of the polymer having an N-vinyl cyclic lactam unit include homopolymers and copolymers obtained by polymerizing or copolymerizing N-vinylpyrrolidone and N-vinylcaprolactam. Specific examples of such homopolymers include poly (N-vinyl-2-pyrrolidone), poly (N-vinyl-5-methyl-2-pyrrolidone), poly (N-vinyl-2-piperidone), poly (N-vinyl-6-methyl-2-piperidone), poly (N-vinyl-caprolactam), and poly (N-vinyl-7-methyl-caprolactam).

Specific examples of the copolymer include copolymers obtained by copolymerizing vinyl acetate, (meth) acrylic acid esters, (meth) acrylic acid, maleic acid esters, maleic acid, acrylonitrile, styrene, alkyl vinyl ethers, N-vinyl imidazole, vinyl pyridine, allyl alcohol, olefins, and the like with N-vinyl pyrrolidone, N-vinyl caprolactam, and the like. The ester includes alkyl esters having 1 to 20 carbon atoms, dimethylaminoalkyl esters and quaternary salts thereof, hydroxyalkyl esters, and the like. Such a backbone polymer may be used alone, or two or more kinds may be used in combination. Polyvinylpyrrolidone is most preferable from the viewpoint of ease of production and acquisition.

The average molecular weight of the polymer having an N-vinyl cyclic lactam unit usable in the present invention is not particularly limited, but is usually 1 × 10³~5×10⁶Preferably 1 × 10⁴~1×10⁶More preferably 5 × 10⁴~5×10⁵. The terminal structure of the polymer and the catalyst for polymerizing the polymer can be arbitrarily selected.

The water-soluble cellulose derivative is selected from hydroxypropyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, and a mixture thereof.

Among these, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, and a mixture thereof are preferable, and hydroxypropyl cellulose is most preferable.

The molecular weight of the water-soluble cellulose derivative to be used in the present invention is not particularly limited, and is usually 1 to 10000mPas, preferably 2 to 5000mPas, and more preferably 2 to 4000mPas, when the viscosity is measured at 20 ℃ at a concentration of 2%, for example.

In the sheet molded body of the present invention, other polymers and other compounds may be used in combination within a range not impairing the object. Such as polymer copolymerization, polymer blending, compound mixing. Specific examples of the compound mixture include phospholipids and surfactants.

The polymer usable in the present invention is preferably high in purity, and particularly preferably contains a small amount of residues such as additives, plasticizers, residual catalysts, residual monomers, residual solvents used in molding or subsequent processing. Particularly, when used for medical treatment, suppression to below the safety standard is required.

The average thickness of the sheet molded body of the present invention is usually 10 to 1000 μm, preferably 50 to 200 μm, and more preferably 100 to 150 μm in the case of a fiber molded body. If the content is less than this range, the strength of the sheet molded body cannot be maintained and the sheet molded body cannot be trimmed, and if the content is more than this range, the sheet molded body is undesirably reduced in flexibility and/or hemostatic properties. When the sheet molding is a film molding, the average thickness is usually 5 to 200 μm, preferably 10 to 100 μm.

When the sheet molding of the present invention contains fibrinogen, the amount of fibrinogen is preferably 0.05 to 30mg/cm²The range of (b) includes fibrinogen. The fibrinogen content is less than 0.05mg/cm²When the amount of the protein is larger than 30mg/cm, the effect based on the protein characteristics is not exhibited²In this case, the fiber molded body itself becomes brittle, which is not preferable. The content is preferably 0.1-25 mg/cm²More preferably 0.2 to 25mg/cm². In particular, when the sheet molding is a film molding, the fibrinogen content is 2mg/cm from the viewpoint of hemostatic properties²Below, preferably 1.5mg/cm²The concentration is more preferably 1.4mg/cm or less²The following.

When the sheet molding of the present invention contains thrombin, the content thereof is preferably 0.1 to 100U/cm²The range of (1). The thrombin content is less than 0.1U/cm²The hemostatic effect is no longer than 100U/cm²In this case, the sheet molding itself becomes brittle, which is not preferable. The content is preferably 2-80U/cm²More preferably 5 to 50U/cm²。

In the present invention, the fiber-formed body means a three-dimensional formed body formed by stacking one or more obtained fibers and by weaving, knitting or other methods. Specific examples of the form of the fiber molded product include nonwoven fabrics. Further, pipes, nets, and the like processed based on this are also included in the fiber formed body.

When the sheet molded body of the present invention is a fiber molded body, the average fiber diameter is preferably 0.01 to 50 μm. When the average fiber diameter is less than 0.01. mu.m, the strength of the fiber molded product cannot be maintained, which is not preferable. When the average fiber diameter is larger than 50 μm, the specific surface area of the fiber is small, and the solubility of the hemostatic protein is not preferable. The average fiber diameter is more preferably 0.02 to 30 μm. The fiber diameter represents the diameter of a fiber cross section. The cross-sectional shape of the fiber is not limited to a circular shape, and may be an elliptical shape or a deformed shape. The fiber diameter at this time is calculated as the average of the length of the ellipse in the major axis direction and the length of the ellipse in the minor axis direction. When the cross section of the fiber is neither circular nor elliptical, the fiber diameter is calculated by approximating a circle or an ellipse.

When the sheet molded body of the present invention is a fiber molded body, the basis weight thereof is preferably0.1~50mg/cm². The weight per unit area is less than 0.1mg/cm²In this case, the hemostatic protein cannot be sufficiently supported, which is not preferable. In addition, the weight per unit area is more than 50mg/cm²In this case, the possibility of inflammation is increased, which is not preferable. Further preferably 0.2 to 20mg/cm²。

When the sheet molding of the present invention is a fiber molding, the bulk density is preferably 100 to 200mg/cm³. The bulk density is less than 100mg/cm³In this case, the handling property is not preferable because it is lowered. In addition, the bulk density is more than 200mg/cm³In the case, the fiber molded body has less voids, and the flexibility and the elution property of the hemostatic protein are lowered, which is not preferable.

When the sheet molded body of the present invention is a fiber molded body, the production method thereof may employ any method that can be employed in the production of plastic fibers, and is not particularly limited, and is preferably performed by solution molding in order to prevent the activity of the hemostatic protein from being lowered and to easily disperse the hemostatic protein or particles containing the hemostatic protein. The fibrous formed body is preferably a long fiber. The long fibers mean: specifically, in the step of processing the fiber molded body by spinning, the fiber molded body formed without performing the step of cutting the fiber can be formed by an electrospinning method, a spunbond method, a melt blowing method, or the like, and the electrospinning method is preferably used.

The electrospinning method is a method in which a high voltage is applied to a solution obtained by dissolving a polymer in a solvent to obtain a fiber molded body on an electrode. The steps include: a step of dissolving a polymer in a solvent to prepare a solution; applying a high voltage to the solution; a step of ejecting the solution; forming a fiber molded body by evaporating the solvent from the ejected solution; a step of eliminating the charge of the formed fiber molded body as an optional step; and a step of accumulating the fiber formed body by eliminating the electric charge.

The stage of producing the spinning solution in the electrospinning method will be described. The spinning dope used in the present invention is preferably a suspension comprising a base polymer solution and hemostatic protein particles.

The concentration of the base polymer in the suspension is preferably 1 to 30% by weight. When the concentration of the polymer is less than 1% by weight, it is difficult to form a fibrous molded article, and therefore it is not preferable. When the amount is more than 30% by weight, the fiber diameter of the obtained fiber molded product becomes large, and the viscosity of the suspension becomes high, which is not preferable. The concentration of the polymer in the suspension is more preferably 1.5 to 20% by weight.

The solvent for the water-soluble polymer is not particularly limited as long as it can dissolve the water-soluble polymer, form a suspension with the hemostatic protein particles, and evaporate at the stage of spinning to form a fiber, and one kind of the solvent may be used alone, or a plurality of kinds of the solvents may be combined. Examples thereof include chloroform, 2-propanol, toluene, benzene, benzyl alcohol, methylene chloride, carbon tetrachloride, cyclohexane, cyclohexanone, trichloroethane, methyl ethyl ketone, ethyl acetate, acetone, ethanol, methanol, tetrahydrofuran, 1, 4-dioxane, 1-propanol, phenol, pyridine, acetic acid, formic acid, hexafluoro-2-propanol, hexafluoroacetone, N-dimethylformamide, N-dimethylacetamide, acetonitrile, N-methyl-2-pyrrolidone, N-methylmorpholine-N-oxide, 1, 3-dioxolane, water, and a mixed solvent of these solvents. Among these, dichloromethane, chloroform, 2-propanol, ethanol, and N, N-dimethylformamide are preferably used from the viewpoint of handling properties, physical properties, and the like.

The solvent for dissolving the aliphatic polyester is not particularly limited as long as it can dissolve the aliphatic polyester, form a suspension with the hemostatic protein particles, and evaporate at the stage of spinning to form fibers, and one kind of solvent may be used alone, or a combination of a plurality of kinds of solvents may be used. Examples thereof include chloroform, 2-propanol, toluene, benzene, benzyl alcohol, methylene chloride, carbon tetrachloride, cyclohexane, cyclohexanone, trichloroethane, methyl ethyl ketone, ethyl acetate, and a mixed solvent thereof. In addition, in the range of forming the emulsion, a solvent such as acetone, ethanol, methanol, tetrahydrofuran, 1, 4-dioxane, 1-propanol, phenol, pyridine, acetic acid, formic acid, hexafluoro-2-propanol, hexafluoroacetone, N-dimethylformamide, N-dimethylacetamide, acetonitrile, N-methyl-2-pyrrolidone, N-methylmorpholine-N-oxide, 1, 3-dioxolane, or the like may be included. Among these, dichloromethane and ethanol are preferably used from the viewpoint of handling properties and physical properties.

The method for preparing the suspension is not particularly limited, and ultrasonic waves and various stirring methods can be used. As the stirring method, a high-speed stirring such as a homomixer, or a stirring method such as a super-fine pulverizer or a ball mill may be used. Among them, a dispersion method by ultrasonic treatment is preferable.

Alternatively, the spinning solution can be prepared by forming a suspension of the hemostatic protein particles with a solvent and then adding a water-soluble polymer or an aliphatic polyester.

In addition, the hemostatic protein particles may be subjected to a fine treatment prior to preparation of the suspension. The fine treatment may be dry grinding or wet grinding, and in the present invention, any method may be employed, or both may be combined. Examples of the dry pulverization treatment include a treatment using a ball mill, a treatment using a planetary mill or a vibration mill, a milling treatment using a pestle, an impact mill such as a media-stirring type pulverizer or a hammer mill, and a milling treatment using a jet mill or a stone mill. On the other hand, examples of the wet pulverization treatment include: a treatment of stirring with a high-shear stirring apparatus, a kneading apparatus, or the like in a state where a hemostatic protein is dispersed in an appropriate dispersion medium; ball mill, bead mill, etc. in a state of being dispersed in a medium. Further, hemostatic protein granules produced by a spray dryer may also be used.

Next, a description will be given of a stage of applying a high voltage to the solution, a stage of ejecting the solution, and a stage of forming a fiber molded body by evaporating the solvent from the ejected solution.

In the method for producing a fibrous molded article of the present invention, in order to form a fibrous molded article by spraying a suspension comprising a polymer solution and hemostatic protein particles, a high voltage needs to be applied to the suspension. The method of applying the voltage is not particularly limited as long as the method is a method of forming a fiber compact by discharging the suspension, and there are the following methods: a method of inserting an electrode into the solution to apply a voltage, a method of applying a voltage to a solution ejection nozzle, and the like.

In addition, an auxiliary electrode different from the electrode applied to the solution may be provided. The value of the applied voltage is not particularly limited as long as the fiber molded body can be formed, and is preferably in the range of 5 to 50 kV. When the applied voltage is less than 5kV, the spinning solution is not discharged and a fiber molded body is not formed, which is not preferable, and when the applied voltage is more than 50kV, discharge is generated from the electrode to the ground electrode, which is not preferable. More preferably 10 to 30 kV. The desired potential may be produced by any appropriate method.

In this way, immediately after the suspension of the polymer solution and the hemostatic protein particles is discharged, the solvent used is volatilized, and a fibrous molded article is formed. In general, spinning is performed at room temperature in the air, and when evaporation is insufficient, spinning may be performed at a negative pressure or at a high temperature. The temperature at which spinning is performed depends on the evaporation behavior of the solvent and the viscosity of the spinning solution, and is usually in the range of 0 to 50 ℃.

Next, a description will be given of a stage of accumulating the formed fiber compact by eliminating the electric charge. The method of accumulating the fiber formed body by eliminating the electric charge is not particularly limited, and a method of accumulating the fiber formed body by collecting the fiber formed body to the ground electrode while eliminating the electric charge may be mentioned. Further, a method of eliminating electric charges before accumulation by an ionizer or the like is also exemplified. In this case, the method of accumulating the fiber formed body is not particularly limited, and a general method includes a method of dropping and accumulating the fiber formed body by its own weight by losing electrostatic force by charge disappearance. Further, the following method may be performed as necessary: a method of attracting and accumulating the fiber formed body on the web by eliminating the electrostatic force, a method of causing air in the apparatus to be convected and accumulating the air on the web, and the like. The ionizer is a device that generates ions by a built-in ion generator, and discharges the ions to a charged object to eliminate the charges of the charged object. As a preferred ion generating device constituting the ionizer usable in the method for producing a fiber molded body of the present invention, a device for generating ions by applying a high voltage to a built-in discharge needle can be mentioned.

The electrospinning method is well known, and the apparatus and conditions are not limited as long as the fibrous molded article of the present invention can be produced, and for example, the descriptions of international publication No. WO2004/072336 and international publication No. 2005/087988 can be referred to, except for the examples described later.

When the sheet molded body of the present invention is a film molded body, the production method thereof may be any method conventionally used as a method for producing a film. For example, a casting method (casting method) can be mentioned. The molding in this way may be performed by melt molding or solution molding, and in order to prevent the activity of the hemostatic protein from being lowered and to facilitate the dispersion of the hemostatic protein, solution molding is preferably performed.

Next, the laminated sheet molded body of the present invention will be explained.

The laminated sheet molded body of the present invention comprises: the adhesive comprises a first polymer composition layer containing fibrinogen and a water-soluble polymer, and a second polymer composition layer containing thrombin and an aliphatic polyester.

Polymers of N-vinyl cyclic lactam units, polyethylene oxide, polyvinyl alcohol, hyaluronic acid, dextran, pullulan or starch, or mixtures thereof.

The water-soluble polymer is preferably a cellulose derivative or a polymer having an N-vinyl cyclic lactam unit, or a mixture thereof.

The cellulose derivative is specifically selected from hydroxypropyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, and mixtures thereof.

Among these, preferred is selected from hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropylmethyl cellulose, polyvinylpyrrolidone, and a mixture thereof, and most preferred is hydroxypropyl cellulose or polyvinylpyrrolidone.

The average molecular weight of the water-soluble polymer having an N-vinyl cyclic lactam unit is not particularly limited, but is 1 × 10³~5×10⁶Preferably 1 × 10⁴~1×10⁶More preferably 5 × 10⁴~5×10⁵. The terminal structure of the polymer and the catalyst for polymerizing the polymer can be selected arbitrarily.

When the water-soluble polymer is a cellulose derivative, the concentration is preferably in the range of 0.01 to 10000mPas, more preferably 0.1 to 5000mPas, further preferably 0.1 to 1000mPas, and most preferably 0.1 to 100mPas, when the viscosity is measured at 20 ℃.

In the water-soluble polymer, other polymers and other compounds may be used in combination as long as the purpose thereof is not impaired. Such as polymer copolymerization, polymer blending, compound mixing.

The water-soluble polymer is preferably high in purity, and particularly preferably contains a small amount of residues such as a plasticizer, a residual catalyst, a residual monomer, and a residual solvent used in molding or subsequent processing. Particularly, when used for medical treatment, it is necessary to suppress the level to be lower than a safety standard value.

In addition, the layer formed of the water-soluble polymer and fibrinogen may further contain pharmaceutically allowable additives. Examples of such additives include the same additives as described in the description of the sheet molded body. In particular, when the fibrinogen is in the form of particles having an average particle diameter of 0.01 to 100. mu.m, these additives are preferably added to the particles.

The aliphatic polyester can be the same as described in the description of the sheet-shaped article.

In addition, other polymers and other compounds may be used in combination in the aliphatic polyester within a range not impairing the object. Such as polymer copolymerization, polymer blending, and compound mixing.

The aliphatic polyester is preferably high in purity, and particularly preferably contains a small amount of residues such as additives, plasticizers, residual catalysts, residual monomers, residual solvents used in molding or subsequent processing, and the like. Particularly, when used for medical treatment, it is necessary to suppress the level to be lower than a safety standard value.

In addition, the layer formed of the aliphatic polyester and thrombin may further contain pharmaceutically allowable additives. Examples of such additives include at least one selected from the group consisting of polyhydric alcohols, surfactants, amino acids, oligosaccharides (oligosaccharides), sodium chloride, sodium citrate, and calcium chloride. This may improve stability, solubility, flexibility, and the like of thrombin.

The first polymer composition layer formed of the water-soluble polymer and fibrinogen is preferably formed of a fibrous shaped body or film. The fiber formed body means a three-dimensional formed body formed by stacking one or more obtained fibers and by weaving, knitting, or other methods. Specific examples of the form of the fiber molded product include nonwoven fabrics. Further, pipes, nets, and the like processed based on this are also included in the fiber formed body.

The film can be produced by any method conventionally used. For example, a casting method (casting method) can be mentioned. The molding in this way may be performed by melt molding or solution molding, and in order to prevent the activity of the hemostatic protein from being lowered and to facilitate the dispersion of the hemostatic protein, solution molding is preferably performed.

The average fiber diameter of a fiber molded body formed of a water-soluble polymer and fibrinogen is 0.01 to 50 [ mu ] m. When the average fiber diameter is less than 0.01. mu.m, the strength of the fiber molded product cannot be maintained, which is not preferable. When the average fiber diameter is larger than 50 μm, the specific surface area of the fiber is decreased, and the solubility is not preferable. The average fiber diameter is more preferably 0.02 to 30 μm. The fiber diameter represents the diameter of a fiber cross section. The cross-sectional shape of the fiber is not limited to a circular shape, and may be an elliptical shape or a deformed shape. The fiber diameter at this time is calculated as the average of the length of the ellipse in the major axis direction and the length of the ellipse in the minor axis direction. When the cross section of the fiber is neither circular nor elliptical, the fiber diameter is calculated by approximating a circle or an ellipse.

The average thickness of the laminated sheet molded product of the present invention is preferably 50 to 350 μm, more preferably 100 to 300 μm, and further preferably 100 to 250 μm.

The basis weight of the fibrous formed body formed by the water-soluble polymer and the fibrinogen is preferably 0.1-50 mg/cm². The weight per unit area is less than 0.1mg/cm²In this case, fibrinogen cannot be sufficiently supported, which is not preferable. In addition, the weight per unit area is more than 50mg/cm²In this case, the possibility of inflammation is increased, which is not preferable.

The bulk density of the fiber formed body formed by the water-soluble polymer and the fibrinogen is preferably 100-200 mg/cm³. The bulk density is less than 100mg/cm³In this case, the handling property is not preferable because it is lowered. In addition, the bulk density is more than 200mg/cm³In the case, the fiber molded body has less voids and is undesirably reduced in flexibility and solubility.

The fibrous shaped body formed by water-soluble polymer and fibrinogen is usually 0.05-30 mg/cm²The range of (b) includes fibrinogen. The fibrinogen content is less than 0.05mg/cm²The hemostatic effect can not be shownMore than 30mg/cm²In this case, the fiber molded body itself becomes brittle, which is not preferable. The content is preferably 0.1-25 mg/cm²More preferably 0.2 to 25mg/cm². In particular, when the sheet molding is a film molding, the fibrinogen content is 2mg/cm from the viewpoint of hemostatic properties²Below, preferably 1.5mg/cm²The concentration is preferably 1.4mg/cm or less²The following.

The fibrous formed body formed of the water-soluble polymer and fibrinogen is preferably formed of a long fiber. The long fibers mean: specifically, in the step of processing the fiber molded body by spinning, the fiber molded body formed without performing the step of cutting the fiber can be formed by an electrospinning method, a spunbond method, a melt blowing method, or the like, and the electrospinning method is preferably used.

In the electrospinning method, when the water-soluble polymer described in the description of the sheet-shaped body is mixed with fibrinogen powder to prepare a suspension, the size of the fibrinogen powder is preferably in the range of 0.01 to 100 μm. It is technically difficult to produce a fibrinogen powder having a particle size of less than 0.01. mu.m, but if it exceeds 100. mu.m, the dispersibility is poor and the fiber molded product is brittle, which is not preferable.

The film formed of the water-soluble polymer and fibrinogen may be produced by any conventionally used method. For example, a casting method (casting method) can be mentioned. The molding in this way may be performed by melt molding or solution molding, and in order to prevent the activity of the hemostatic protein from being lowered and to facilitate the dispersion of the hemostatic protein, solution molding is preferably performed.

The content of the water-soluble polymer in the film comprising the water-soluble polymer and fibrinogen varies depending on the type of the polymer, and is preferably 0.1 to 50% by weight, more preferably 0.5 to 20% by weight. The fibrinogen protein particles are usually contained in an amount of 100 wt% or more, preferably 500 wt% or more, and more preferably 800 to 950 wt% based on the water-soluble polymer, depending on the type of the polymer. If the amount is less than this range, the hemostatic properties may be poor, and if the amount is more than this range, the film formability may be poor.

The average thickness of the film formed by the water-soluble polymer and the fibrinogen is preferably 10 to 1000 μm.

The film formed by the water-soluble polymer and the fibrinogen is preferably 0.05-10 mg/cm²The range of (b) includes fibrinogen. Less than 0.05mg/cm²It is difficult to exhibit a hemostatic effect, more than 10mg/cm²In this case, the film itself becomes fragile, which is not preferable. The content is more preferably 0.1-8 mg/cm²More preferably 0.2 to 4mg/cm²。

In the present invention, the second polymer composition layer formed of the aliphatic polyester and thrombin is preferably formed of a fiber-forming body. The fiber formed body is defined as described above.

The average fiber diameter of a fiber molded body formed of an aliphatic polyester and thrombin is 0.01 to 50 [ mu ] m. When the average fiber diameter is less than 0.01. mu.m, the strength of the fiber molded product cannot be maintained, which is not preferable. When the average fiber diameter is larger than 50 μm, the specific surface area of the fiber is small, and thrombin release property is not preferable. The average fiber diameter is more preferably 0.02 to 30 μm.

The average thickness of the fiber molded body formed by the aliphatic polyester and the thrombin is 10-1000 μm. When the average thickness is less than 10 μm, the strength of the fiber molded body cannot be maintained and trimming cannot be performed, which is not preferable. When the average thickness is more than 1000 μm, the flexibility of the fiber molded product is undesirably reduced. The average thickness is more preferably 20 to 500 μm.

The fiber molded body formed by the aliphatic polyester and the thrombin has a unit area weight of 0.1-50 mg/cm². The weight per unit area is less than 0.1mg/cm²In this case, thrombin cannot be sufficiently supported, which is not preferable. In addition, the weight per unit area is more than 50mg/cm²In this case, the possibility of causing inflammation is high, which is not preferable. Further excellenceIs selected to be 0.2-20 mg/cm²。

The fiber molded body formed by the aliphatic polyester and the thrombin has the volume density of 100-200 mg/cm³. The bulk density is less than 100mg/cm³In this case, the handling property is not preferable because it is lowered. In addition, the bulk density is more than 200mg/cm³In the case, the fiber molded body is undesirably low in void content, and is undesirably low in flexibility and thrombin releasing ability.

In the present invention, the fiber molded body formed of the aliphatic polyester and thrombin is preferably 0.1 to 100U/cm²Comprising thrombin. The thrombin content is less than 0.1U/cm²In the case of this, the hemostatic effect is not sufficient, and therefore, this is not preferred. More than 100U/cm²In this case, the fiber molded body itself becomes brittle, which is not preferable. The content is preferably 2-80U/cm²More preferably 5 to 50U/cm². The protein-containing particles of thrombin are usually contained in an amount of 1 to 200% by weight, preferably 10 to 100% by weight, more preferably 20 to 100% by weight, and still more preferably 50 to 100% by weight, based on the aliphatic polyester. If the amount is less than this range, the elution of thrombin, and the flexibility or hemostatic properties of the sheet molded body may be poor, and if the amount is more than this range, the self-supporting properties of the sheet molded body may be lowered, which is not preferable.

The fiber molded body formed of the aliphatic polyester and thrombin is preferably formed of a long fiber. The long fibers and the production method thereof are as described above.

The fiber molded body formed of the aliphatic polyester and thrombin can be produced by an electrospinning method. The electrospinning method has been described in the description of the sheet molding. When the aliphatic polyester is mixed with thrombin powder to prepare a suspension, the size of the thrombin powder is not particularly limited, but is preferably in the range of 0.01 to 100 μm. It is technically difficult to prepare a thrombin powder having a particle size of less than 0.01. mu.m, and when it exceeds 100. mu.m, the dispersibility is poor and the fibrous molded article is brittle, which is not preferable.

The surface of the sheet molded product of the present invention or the surface of each layer of the laminated sheet molded product may be optionally subjected to a process of further laminating a cotton-like fibrous structure or a process of sandwiching a cotton-like structure between the laminated sheet molded products of the present invention to form a sandwich structure, within a range not to impair the object of the present invention.

The fiber of the fiber molded article in the sheet molded article and the laminate sheet molded article of the present invention may optionally contain a chemical. When the molding is carried out by the electrospinning method, the agent to be used is not particularly limited as long as it is soluble in an organic solvent or an aqueous solution and the physiological activity thereof is not impaired by the dissolution.

The laminate sheet molded body of the present invention comprises: the adhesive layer may further comprise 1 or more layers of a first polymer composition layer composed of a water-soluble polymer and fibrinogen and 1 or more layers of a second polymer composition composed of an aliphatic polyester and thrombin, and further layers other than these layers may be provided. The order in which these layers are laminated is not limited, and the layers may have portions in which the same type of layer is adjacent.

The laminate sheet molded body of the present invention may be one in which a first polymer composition layer made of a water-soluble polymer and fibrinogen and a second polymer composition layer made of an aliphatic polyester and thrombin are laminated, or may be laminated on any of the layers formed by a common coating method. The coating method is not particularly limited, and examples thereof include electrospinning, electrospray, casting, dipping, spraying, pressing, and hot pressing. Among these, the electrospinning method is preferable as a method of laminating a layer formed of a fiber-formed body on a layer formed of a fiber-formed body. The fibrous molded article formed of the aliphatic polyester and the thrombin may be laminated on the fibrous molded article formed of the water-soluble polymer and the fibrinogen, or the fibrous molded article formed of the water-soluble polymer and the fibrinogen may be laminated on the fibrous molded article formed of the aliphatic polyester and the thrombin.

When the laminate sheet molded body of the present invention is applied to a wound site as a hemostatic material, it is preferably applied so that a layer formed of a water-soluble polymer and fibrinogen contacts the wound site. By doing so, immediately after the layer formed of the water-soluble polymer and fibrinogen comes into contact with the wound site, the layer formed of the water-soluble polymer and fibrinogen is dissolved, fibrinogen sufficiently permeates the wound site, and thrombin is immediately released from the layer formed of the aliphatic polyester and thrombin, whereby a coagulation reaction proceeds with the generation of fibrin. The aliphatic polyester in the layer formed of the aliphatic polyester and thrombin functions as a reinforcing material necessary for compression hemostasis and then decomposes with time.

The sheet molded body and the laminate sheet molded body of the present invention are thin and excellent in flexibility, and therefore have good adhesion to a wound site. Further, since the sheet molded article and the fiber molded article of the laminated sheet molded article of the present invention contain fibrinogen and/or thrombin as active ingredients, they are excellent in carrying property unlike freeze-dried products. On the other hand, since the solubility of fibrinogen, the releasability of thrombin, and the elution property in the fibrinogen layer are also excellent, a hemostatic effect is exhibited in a short time. In addition, the amount of fibrinogen required for exhibiting a hemostatic effect in a short time is small, and the composition is excellent in terms of cost. Further, the sheet molded body of the present invention is excellent in visibility of a wound site after application to the wound site by selecting a material to be used. This makes it possible to visually confirm the hemostatic state that has been difficult for the conventional products, and to easily identify the site to be sutured when suturing. Further, the sheet molded article and the laminated sheet molded article of the present invention do not require a freeze-drying step in production, and therefore have excellent productivity.

Examples

The embodiments of the present invention will be described below with reference to examples, but the scope of the present invention is not limited thereto.

< measurement methods for examples 1 to 6 and 16 to 29 and comparative examples 1 and 2 >

Protein particle size (average particle size):

the freeze-dried fibrinogen powder was pulverized in a mortar, and then photographed at 1000-fold magnification by a digital microscope (keyencecorportation: trade name "VHX-100"), 10 particles were randomly selected from the obtained photograph, and the diameters thereof were measured, and the average value thereof was defined as the average particle diameter.

Average fiber diameter:

the surface of the obtained fiber molded body was photographed at a magnification of 3000 times by a scanning electron microscope (KEYENCE CORPORATION, trade name "VE 8800"), and 20 points were randomly selected from the obtained photograph, and the fiber diameters were measured, and the average of all the fiber diameters was determined as the average fiber diameter. n = 20.

Average thickness 3 a:

the film thickness of the obtained fiber molded body was measured with a high-precision digital length measuring machine (Mitutoyo Corporation: trade name "LITEMATIC VL-50") at a measuring force of 0.01N and N =15, and the average value was calculated. In the present measurement, the measurement is performed with the minimum measurement force that can be used by the measurement device.

Weight per unit area:

the obtained fiber molded body was cut into 50mm × 100mm, and the weight thereof was measured and converted to calculate the weight per unit area.

Bulk density of 5A

The bulk density was calculated from the values of the weight per unit area and the average thickness measured above.

6A. dissolution test: the obtained fiber molded body was cut into 1cm × 1cm, and 15 μ L of physiological saline was added to confirm the solubility.

ELISA assay 7A

(1) Fibrinogen

In ELISAAnti-human fibrinogen antibody (DAKO A0080) was immobilized at 10. mu.g/mL in plates (NUNC 468667). After washing with PBS containing 0.05% tween 20, Block Ace (DS Pharma Biomedical co., ltd. UK-B80) was added to each well for masking. After washing with PBS containing 0.05% tween 20, the specimen was added. A calibration curve was prepared using human fibrinogen (Enzyme Research Laboratories No. FIB 3) as a standard. After washing with PBS containing 0.05% Tween 20, an anti-human fibrinogen antibody labeled with HRP (CPL 5523) was added, and after the reaction, the reaction mixture was washed with PBS containing 0.05% Tween 20, and then TMB reagent (KPL 50-76-0250-65-02) was added and allowed to stand for 6 minutes to develop color. Addition of 1M H₃PO₄To stop the color development, OD450-650nm was measured with a microplate reader.

(2) Thrombin

Anti-human thrombin antibody (AffinityBiologic, Inc., No. SAHT-AP) was immobilized at 5. mu.g/mL in an ELISA plate (NUNC 468667). After washing with PBS containing 0.05% tween 20, Block Ace (DSPharma Biomedical co., ltd. UK-B80) was added to each well for masking. After washing with PBS containing 0.05% tween 20, the specimen was added. A standard curve was prepared using human thrombin (Haematologic Technologies, Inc.: HCT-0020) as a standard. After washing with PBS containing 0.05% tween 20, 0.1 μ g/mL of HRP-labeled anti-human thrombin antibody (Affinity Biologicals, inc., No. saht-HRP) was added. After the reaction, the reaction mixture was washed with PBS containing 0.05% tween 20, and a TMB reagent (DaKoS 1599) was added thereto and left to stand for 10 minutes to develop color. Addition of 0.5M H₂SO₄To stop the color development, OD450-650nm was measured with a microplate reader.

Thrombin Activity assay

To a 2008 tube manufactured by FALCON Corporation, 20. mu.L of the sample, 60. mu.L of 50mM Tris-HCl (pH 8.5) +50mM NaCl buffer, and 0.1% PLURONIC F-6820. mu.L were added, and the mixture was incubated at 37 ℃ for 3 minutes. As a standard, a product obtained by diluting purified human plasma-derived alpha-thrombin (purchased from Haematologic Technologies, Inc.: HCT-0020) to 5, 2.5, 1.25, 0.625, 0.3125U/mL with the same buffer was used. To the reaction solution, 100. mu.L of Test Team chromogenic substrate S-2238 (1 mM: first chemical industry) was added and mixed with stirring, and after reaction at 37 ℃ for 5 minutes, 800. mu.L of 0.1M citric acid solution was added to stop the reaction. The reaction solution (200. mu.L) was transferred to a 96-well plate, and the OD405/650 was measured.

Example 1

A freeze-dried fibrinogen powder (BOLHEAL (registered trademark, the same shall apply hereinafter)) for tissue adhesion was pulverized in a mortar to prepare a pulverized freeze-dried fibrinogen powder having an average particle size of 14 μm. The pulverized freeze-dried fibrinogen powder was dispersed in ethanol, and then polyvinylpyrrolidone (K90, product of wako pure chemical industries) was dissolved to 10wt%, thereby preparing a dope of freeze-dried fibrinogen powder/polyvinylpyrrolidone =100/100 (w/w). The resultant was spun by an electrospinning method at a temperature of 22 ℃ and a humidity of 26% or less to obtain a sheet-like fiber molded body. The inner diameter of the discharge nozzle was 0.8mm, the voltage was 13.5kV, the flow rate of the spinning solution was 1.2mL/h, and the distance from the discharge nozzle to the flat plate was 15 cm. The resulting fiber molded body had an average fiber diameter of 0.51 μm, an average thickness of 285 μm, and a basis weight of 2.35mg/cm²The bulk density of the powder was 82mg/cm³The resulting fiber molded body was dissolved in 1 second or less, and the obtained sheet was cut into 0.5cm × 0.5.5 cm, and the immobilized protein mass was 0.54mg/cm as a result of ELISA measurement by extracting the protein with 62.5. mu.L physiological saline². The resulting sheet can be trimmed with scissors.

Example 2

The freeze-dried fibrinogen powder pulverized in example 1 was dispersed in ethanol, and then polyvinylpyrrolidone (K90, Wako pure chemical industries, Ltd.) was dissolved to 10wt%, thereby preparing a spun yarn of fibrinogen freeze-dried powder/polyvinylpyrrolidone =100/200 (w/w)And (4) liquid. The resultant was spun by an electrospinning method at a temperature of 22 ℃ and a humidity of 26% or less to obtain a sheet-like fiber molded body. The inner diameter of the discharge nozzle was 0.8mm, the voltage was 17kV, the flow rate of the spinning solution was 1.2mL/h, and the distance from the discharge nozzle to the flat plate was 15 cm. The resulting fiber molded body had an average fiber diameter of 0.33. mu.m, an average thickness of 469 μm, and a basis weight of 5.28mg/cm²The bulk density of the resin composition was 113mg/cm³The resulting fiber molded body was dissolved in 1 second or less, and the obtained sheet was cut into 0.5cm × 0.5.5 cm, and the immobilized protein mass was 1.61mg/cm as a result of ELISA measurement by extracting the protein with 62.5. mu.L of physiological saline². The resulting sheet can be trimmed with scissors.

Example 3

The freeze-dried fibrinogen powder pulverized in example 1 was dispersed in 2-propanol, and then hydroxypropyl cellulose (6-10 mPas, wako pure chemical industries, Ltd.) was dissolved to 16wt% to prepare a dope of freeze-dried fibrinogen powder/hydroxypropyl cellulose =20/100 (w/w). The resultant was spun by an electrospinning method at a temperature of 22 ℃ and a humidity of 26% or less to obtain a sheet-like fiber molded body. The inner diameter of the discharge nozzle was 0.8mm, the voltage was 11kV, the flow rate of the spinning solution was 1.2mL/h, and the distance from the discharge nozzle to the flat plate was 15 cm. The resulting fiber molded body had an average fiber diameter of 0.86. mu.m, an average thickness of 137. mu.m, and a basis weight of 1.59mg/cm²The bulk density of the powder is 116mg/cm³The resulting fiber molded body was dissolved in 1 second or less, and the obtained sheet was cut into 0.5cm × 0.5.5 cm, and the immobilized protein mass was 0.17mg/cm as a result of ELISA measurement by extracting the protein with 62.5. mu.L physiological saline². The resulting sheet can be trimmed with scissors.

Comparative example 1

The pulverized fibrinogen freeze-dried powder in example 1 was dissolved in 1,1,1,3,3, 3-hexafluoro-2-propanol/MINIMUM ESSENTIAL MEDIUM EAGLE (manufactured by Sigma-Aldrich Corporation) 10 × (9/1 = v/v) to 15 w/v%. The resultant was spun by an electrospinning method at a temperature of 22 ℃ and a humidity of 26% or less to obtain a sheet-like fiber molded body. The inner diameter of the discharge nozzle was 0.8mm, the voltage was 23.5kV, the flow rate of the spinning solution was 2.45mL/h, and the distance from the discharge nozzle to the flat plate was 12 cm. When the dissolution test of the obtained fiber molded body was performed, no dissolution occurred.

Comparative example 2

A freeze-dried fibrinogen powder (all contained in the BOLHEAL tissue-binding solution) was dissolved in a fibrinogen-dissolving solution, and then hydroxypropyl cellulose (6-10 mPas, Wako pure chemical industries, Ltd.) was dissolved to 16wt% to prepare a fibrinogen freeze-dried powder/hydroxypropyl cellulose =20/100 (w/w) dope, but the hydroxypropyl cellulose was phase-separated from fibrinogen, and fibrinogen was precipitated, and electrospinning was not possible.

Example 4

The freeze-dried fibrinogen powder pulverized in example 1 was dispersed in 2-propanol, and then hydroxypropyl cellulose (6-10 mPas, wako pure chemical industries, Ltd.) was dissolved to 16wt% to prepare a dope of freeze-dried fibrinogen powder/hydroxypropyl cellulose =40/100 (w/w). The resultant was spun by an electrospinning method at a temperature of 22 ℃ and a humidity of 26% or less to obtain a sheet-like fiber molded body. The inner diameter of the discharge nozzle was 0.8mm, the voltage was 12.5kV, the flow rate of the spinning solution was 1.2mL/h, and the distance from the discharge nozzle to the flat plate was 15 cm. The resulting fiber molded body had an average fiber diameter of 0.43 μm, an average thickness of 152 μm, and a basis weight of 1.86mg/cm²The bulk density of the resin composition was 122mg/cm³The resulting fiber molded body was dissolved in 1 second or less, and the obtained sheet was cut into 0.5cm × 0.5.5 cm, and the immobilized protein mass was 0.30mg/cm as a result of ELISA measurement by extracting the protein with 62.5. mu.L physiological saline². The resulting sheet can be trimmed with scissors.

Example 5

The freeze-dried fibrinogen powder pulverized in example 1 was dispersed in 2-propanol, and then hydroxypropyl cellulose (6-10 mPas, wako pure chemical industries, Ltd.) was dissolved to 16wt% to prepare a dope of freeze-dried fibrinogen powder/hydroxypropyl cellulose =100/100 (w/w). The resultant was spun by an electrospinning method at a temperature of 22 ℃ and a humidity of 26% or less to obtain a sheet-like fiber molded body. The inner diameter of the discharge nozzle was 0.8mm, the voltage was 12.5kV, the flow rate of the spinning solution was 1.2mL/h, and the distance from the discharge nozzle to the flat plate was 15 cm. The resulting fiber molded body had an average fiber diameter of 0.35. mu.m, an average thickness of 191. mu.m, and a weight per unit area of 2.74mg/cm²The bulk density of the resin composition was 143mg/cm³The resulting fiber molded body was dissolved in 1 second or less, and the obtained sheet was cut into 0.5cm × 0.5.5 cm, and the immobilized protein mass was 0.51mg/cm as a result of ELISA measurement by extracting the protein with 62.5. mu.L physiological saline². The resulting sheet can be trimmed with scissors.

Example 6

< preparation of layer comprising aliphatic polyester and Thrombin >

A thrombin lyophilized powder (VIAL 3 for tissue adhesion) pulverized in a mortar in the same manner as in example 1 was dispersed in ethanol, and then methylene chloride was added to dissolve 10wt% of polylactic acid (PL 18 Purac biological material company), thereby preparing a dope of thrombin lyophilized powder/polylactic acid =100/100 (w/w). The sheet-like fiber compact was obtained by electrospinning at a temperature of 22 ℃ and a humidity of 26% or less, wherein the sheet was cut into 2cm × 2cm with an inner diameter of an ejection nozzle of 0.8mm, a voltage of 15kV, a flow rate of the dope of 3.0mL/h, and a distance from the ejection nozzle to a plate of 25 cm., and the sheet was extracted with 1mL of physiological saline to carry out the activityAnd (4) performing ELISA measurement. As a result, the activity was measured at 23U/cm²ELISA measurement of 16. mu.g/cm²。

< evaluation test of tissue adhesion Effect >

In order to confirm the activity of fibrinogen, an adhesion test was performed using a combination of the layer formed of the water-soluble polymer and fibrinogen prepared in example 5 and the layer formed of the aliphatic polyester and thrombin prepared in example 6. The adhesive force was determined by adhering the skin of the rabbit to the sheet (2 cm. times.2 cm) to confirm whether fibrin gel was formed or not and whether the sheet was adhered. At this time, 200. mu.L of water was added to the layer formed of the water-soluble polymer and fibrinogen in advance for the overlapped sheet, and the layer formed of the water-soluble polymer and fibrinogen was attached to the skin of the rabbit after 40 seconds. Thereafter, the sheet was left to stand at 37 ℃ for 3 minutes, and then the adhesiveness between the sheet and the skin was confirmed. As a control, a collagen sheet preparation (product name: Tachocomb/CSL ベーリング (strain)) containing a fibrin adhesive material immobilized thereon was used: a preparation in which a sheet of spongy equine collagen is used as a support and is fixed to one surface of the sheet by vacuum drying: 2 cm. times.2 cm). As a result, the sheet to be evaluated showed an adhesive force higher than that of the collagen sheet preparation of the comparative control.

< examination >

In comparative example 1,1,1,3,3, 3-hexafluoro-2-propanol/MINIMUM ESSENTIAL MEDIUM EAGLE10 × (9/1 = v/v) was used in order to produce a fibrous molded body from a fibrinogen freeze-dried powder by an electrospinning method. Fibrinogen is poorly soluble in aqueous solvents, and therefore additives for increasing the solubility of fibrinogen are included in the fibrinogen freeze-dried powder. In comparative example 1, even though the freeze-dried fibrinogen powder was used as it was, no elution of fibrinogen therefrom was observed when the freeze-dried fibrinogen powder was formed into a fibrous molded body.

On the other hand, as in examples 1 to 5, the fibrinogen was dissolved within 1 second by preparing particles having an average particle diameter of 0.01 to 100 μm and then dissolving the particles in water and ethanol via a dispersion thereof to obtain a form containing a soluble polymer. In addition, from example 6, it is clear that: the physiological activity of the hemostatic protein in the sheet molded body of the present invention is maintained.

On the other hand, in comparative example 2, a method of dissolving a freeze-dried fibrinogen powder of BOLHEAL in a fibrinogen solution and mixing the same with a water-soluble cellulose derivative solution was attempted in accordance with international publication No. WO2009/031620, but a uniform composition could not be obtained.

< measurement methods for examples 7 to 13 >

Dispersibility of fibrinogen, thrombin, and fibrin in the dope for spinning:

the dispersibility of these proteins was confirmed by visually observing the dispersion of fibrinogen, thrombin, and fibrin immediately before the addition of the aliphatic polyester.

Thickness of fiber formed body: the measurement was carried out by the same method as in 1A.

Fiber diameter (average fiber diameter): measured by the same method as in 2A.

Handling of the sheet:

whether the obtained fiber formed body could be easily handled was qualitatively evaluated.

Example 7

The fibrinogen freeze-dried powder (for BOLHEAL tissue binding: VIAL 1) was micronized using an air jet mill (product of セイシン, Inc.: ultra small amount lab jet mill). This was added to ethanol (manufactured by Wako pure chemical industries, Ltd.) and treated for 5 minutes by an ultrasonic bath to prepare a fibrinogen dispersion liquid having excellent dispersibility. Methylene chloride (manufactured by Wako pure chemical industries, Ltd.) and L-form 100% polylactic acid (Purasorb PL18, manufactured by Purac) were added to the obtained dispersion, and the polylactic acid was dissolved to prepare a uniform solution. The polylactic acid solution for spinning was prepared so that the polylactic acid concentration was 10wt%, the fibrinogen freeze-dried powder concentration was 4 wt% (1.8 wt% based on fibrinogen), and the ratio of ethanol to methylene chloride was 1:8 by weight. When the protein/organic solvent dispersion before polylactic acid addition was visually observed, it was found that: without precipitation, in a uniformly dispersed state. The obtained polylactic acid solution was spun at a humidity of 30% or less by an electrospinning method to obtain a sheet-like fiber molded body. The inner diameter of the discharge nozzle was 0.8mm, the voltage was 12kV, and the distance from the discharge nozzle to the flat plate was 25 cm. The plate was used as the cathode during spinning. The resulting fiber molded body had an average fiber diameter of 3.3 μm and a thickness of 161 μm, and was flexible and handleable. In the case where methylene chloride was used instead of ethanol (example 14), the handleability of the resulting fiber molded article was lowered, and ethanol was considered to be more preferable from this viewpoint.

Example 8

A thrombin dispersion having excellent dispersibility was prepared by adding thrombin freeze-dried powder (for tissue adhesion: VIAL 3) to ethanol (manufactured by Wako pure chemical industries, Ltd.) and subjecting the mixture to a treatment for 5 minutes by an ultrasonic bath. Methylene chloride (manufactured by Wako pure chemical industries, Ltd.) and L-form 100% polylactic acid (Purasorb PL18, manufactured by Purac) were added to the obtained dispersion, and the polylactic acid was dissolved to prepare a uniform solution. The obtained polylactic acid solution for spinning was prepared so that the polylactic acid concentration was 10% by weight, the thrombin freeze-dried powder concentration was 4% by weight (0.045% by weight in terms of thrombin), and the ratio of ethanol to dichloromethane was 1:8 by weight. When the protein/organic solvent dispersion before polylactic acid addition was visually observed, it was found that: without precipitation, in a uniformly dispersed state. The obtained polylactic acid solution was spun at a humidity of 30% or less by an electrospinning method to obtain a sheet-like fiber molded body. The inner diameter of the discharge nozzle was 0.8mm, the voltage was 12kV, and the distance from the discharge nozzle to the flat plate was 25 cm. The plate was used as the cathode during spinning. The resulting fiber molded body had an average fiber diameter of 6.2 μm and a thickness of 170 μm, and was flexible and handleable. In addition, when dichloromethane was used instead of the above ethanol (example 15), the handleability of the obtained fiber molded body was lowered, and from this viewpoint, ethanol was considered to be more preferable.

Example 9

A thrombin dispersion having excellent dispersibility was prepared by adding thrombin freeze-dried powder (for tissue adhesion: VIAL 3) to ethanol (manufactured by Wako pure chemical industries, Ltd.) and subjecting the mixture to a treatment for 5 minutes by an ultrasonic bath. Methylene chloride (manufactured by Wako pure chemical industries, Ltd.) and L-form 100% polylactic acid (Purasorb PL18, manufactured by Purac) were added to the obtained dispersion, and the polylactic acid was dissolved to prepare a uniform solution. The obtained polylactic acid solution for spinning was prepared so that the polylactic acid concentration was 10% by weight, the thrombin freeze-dried powder concentration was 7% by weight (0.078% by weight in terms of thrombin), and the ratio of ethanol to dichloromethane was 1:8 by weight. When the protein/organic solvent dispersion before polylactic acid addition was visually observed, it was found that: without precipitation, in a uniformly dispersed state. The obtained polylactic acid solution was spun at a humidity of 30% or less by an electrospinning method to obtain a sheet-like fiber molded body. The inner diameter of the discharge nozzle was 0.8mm, the voltage was 12kV, and the distance from the discharge nozzle to the flat plate was 25 cm. The plate was used as the cathode during spinning. The resulting fiber molded body had an average fiber diameter of 8.1 μm and a thickness of 175 μm, and was flexible and handleable.

Example 10

A thrombin dispersion having excellent dispersibility was prepared by adding thrombin freeze-dried powder (for tissue adhesion: VIAL 3) to ethanol (manufactured by Wako pure chemical industries, Ltd.) and subjecting the mixture to a treatment for 5 minutes by an ultrasonic bath. Methylene chloride (manufactured by Wako pure chemical industries, Ltd.) and L-form 100% polylactic acid (Purasorb PL18, manufactured by Purac) were added to the obtained dispersion, and the polylactic acid was dissolved to prepare a uniform solution. The obtained polylactic acid solution for spinning was prepared so that the polylactic acid concentration was 10% by weight, the thrombin lyophilized powder concentration was 10% by weight (0.11% by weight in terms of thrombin), and the ratio of ethanol to dichloromethane was 1:8 by weight. When the protein/organic solvent dispersion before polylactic acid addition was visually observed, it was found that: without precipitation, in a uniformly dispersed state. The obtained polylactic acid solution was spun at a humidity of 30% or less by an electrospinning method to obtain a sheet-like fiber molded body. The inner diameter of the discharge nozzle was 0.8mm, the voltage was 12kV, and the distance from the discharge nozzle to the flat plate was 25 cm. The plate was used as the cathode during spinning. The resulting fiber molded body had an average fiber diameter of 9.4 μm and a thickness of 210 μm, and was flexible and handleable.

Example 11

A thrombin dispersion having excellent dispersibility was prepared by adding thrombin freeze-dried powder (for tissue adhesion: VIAL 3) to ethanol (manufactured by Wako pure chemical industries, Ltd.) and subjecting the mixture to a treatment for 5 minutes by an ultrasonic bath. To the obtained dispersion, methylene chloride (manufactured by Wako pure chemical industries, Ltd.) and a polyglycolic acid-polylactic acid copolymer (Purasorb PDLG5010, manufactured by Purac) were added, and the polyglycolic acid-polylactic acid copolymer was dissolved to prepare a uniform solution. The polyglycolic acid-polylactic acid copolymer solution for spinning obtained was prepared so that the polymer concentration was 10% by weight, the thrombin lyophilized powder concentration was 5% by weight (0.06% by weight in terms of thrombin), and the ratio of ethanol to dichloromethane was 1:8 by weight. When the protein/organic solvent dispersion before the polyglycolic acid-polylactic acid copolymer was added was visually observed, it was found that: without precipitation, in a uniformly dispersed state. The obtained polyglycolic acid-polylactic acid copolymer solution was spun at a humidity of 30% or less by an electrospinning method to obtain a sheet-like fiber molded body. The inner diameter of the discharge nozzle was 0.8mm, the voltage was 15kV, and the distance from the discharge nozzle to the flat plate was 25 cm. The plate was used as the cathode during spinning. The resulting fiber molded body had an average fiber diameter of 4.8 μm and a thickness of 330 μm, and was flexible and handleable.

Example 12

A thrombin dispersion having excellent dispersibility was prepared by adding thrombin freeze-dried powder (for tissue adhesion: VIAL 3) to 2-propanol (manufactured by Wako pure chemical industries, Ltd.) and subjecting the mixture to a treatment for 5 minutes by an ultrasonic bath. To the obtained dispersion, methylene chloride (manufactured by Wako pure chemical industries, Ltd.) and a polyglycolic acid-polylactic acid copolymer (Purasorb PDLG5010, manufactured by Purac) were added, and the polyglycolic acid-polylactic acid copolymer was dissolved to prepare a uniform solution. The polyglycolic acid-polylactic acid copolymer solution for spinning obtained was prepared so that the polymer concentration was 10% by weight, the thrombin lyophilized powder concentration was 5% by weight (0.06% by weight in terms of thrombin), and the ratio of 2-propanol to methylene chloride was 1:8 by weight. When the protein/organic solvent dispersion before the polyglycolic acid-polylactic acid copolymer was added was visually observed, it was found that: without precipitation, in a uniformly dispersed state. The obtained polyglycolic acid-polylactic acid copolymer solution was spun at a humidity of 30% or less by an electrospinning method to obtain a sheet-like fiber molded body. The inner diameter of the discharge nozzle was 0.8mm, the voltage was 15kV, and the distance from the discharge nozzle to the flat plate was 25 cm. The plate was used as the cathode during spinning. The resulting fiber molded body had an average fiber diameter of 6.4 μm and a thickness of 320 μm, and was flexible and handleable.

Example 13

A thrombin lyophilized powder (prepared by lyophilizing recombinant thrombin 1mg/mL, 3.4% sodium chloride, 1.2% sodium citrate, 0.29% calcium chloride, and 1% mannitol pH 7) was dispersed in ethanol, and then dichloromethane was added to dissolve a polyglycolic acid-polylactic acid copolymer (Purasorb PDLG5010, Purac corporation) to 10% by weight, thereby preparing a spinning dope of thrombin lyophilized powder/polyglycolic acid-polylactic acid copolymer =100 (1.67 in terms of thrombin)/100 (w/w). The resultant was spun by an electrospinning method at a temperature of 26 ℃ and a humidity of 29% or less to obtain a sheet-like fiber molded body. The inner diameter of the discharge nozzle was 0.8mm, the voltage was 20V, the flow rate of the spinning solution was 4.0mL/h, and the distance from the discharge nozzle to the grounded plate was 35 cm. The resulting fiber shaped body had a thickness of 136 μm, was flexible and was handleable. The thrombin elution property of the obtained sheet was confirmed by an elution test. The test method is as follows.

< dissolution test >

(1) The sample was punched out to a size of 6mm in diameter, and the mass was measured.

(2) The sample was placed in a microtube, and a dissolution test was performed using a physiological saline solution or a physiological saline solution containing HPC.

(3) The sampling time was 10, 30, 60, 120 seconds.

(4) The collected sample was measured by a liquid chromatograph, and the thrombin content was determined from the peak area.

(5) The dissolution rate was determined by the following equation.

Dissolution rate (%) = resulting thrombin content/theoretical immobilized thrombin content × 100.

The theoretical immobilized thrombin content was calculated from the weight% of thrombin to be charged and the weight per unit area of the fiber molded body.

The results of the dissolution test are shown in fig. 1. The elution rate of the physiological saline containing HPC is improved compared with that of the physiological saline. Thus, it can be seen that: the laminated sheet molded body of the present invention contains HPC in the sheet, which contributes to an increase in the elution rate of thrombin.

< measurement methods for examples 14 to 15 and comparative examples 3 to 4 >

Hemostatic protein particle size (average particle size):

the spinning solution was photographed at a magnification of 1000 times by a digital microscope (KEYENCE CORPORATION: trade name "VHX-100"), 10 particles were randomly selected from the obtained photograph, and the diameter was measured, and the average value thereof was defined as the average particle diameter.

Thickness of fiber formed body: the measurement was carried out by the same method as in 1A.

Fiber diameter (average fiber diameter): measured by the same method as in 2A.

Dissolution test of hemostatic protein:

the obtained fiber compact was cut into 2cm × 2cm, and immersed in 1mL of physiological saline for 3 minutes or 3 hours, whereby the water-soluble component was dissolved out. The weight change before and after measurement was measured at n =3 to 6, and the average value of the extraction rate calculated by the following equation was obtained. The theoretical weight of the water-soluble component is calculated from the weight% of the hemostatic protein to be charged and the basis weight of the fibrous molded body.

Extraction rate [% ] = (weight reduction [ mg ]/theoretical weight of water-soluble component [ mg ]) × 100.

Test for carrying hemostatic protein:

the obtained fiber-formed product was cut into pieces of 1cm × 1cm, and the pieces were cut into approximately 4 equal parts with scissors. The weight was measured before and after the measurement, and the weight change was calculated.

Weight change [% ] = (weight after division [ mg ]/weight before division [ mg ]) × 100.

Flexibility test of fiber molded body:

the flexibility was measured by the following procedure, using the glass slide method (JIS-L-19068.19.2B method) as a reference, with the dimensions of the test piece set at 0.5 cm. times.3.5 cm. The test piece was set in a state where the test machine main body was aligned with the upper surface of the movable stage and a portion having a width of 0.5cm was sandwiched between the cover glass and the test machine main body. The movable stage was lowered, and the lowering length value of the free end of the test piece when it left the movable stage was calculated from the scale (the larger the value, the higher the flexibility).

Example 14

The fibrinogen freeze-dried powder (for BOLHEAL tissue binding: VIAL 1) was micronized using an air jet mill (product of セイシン, Inc.: ultra small amount lab jet mill). This was added to methylene chloride (manufactured by Wako pure chemical industries, Ltd.), and the mixture was treated with an ultrasonic bath for 5 minutes to prepare a fibrinogen dispersion. 100% L-form polylactic acid (Purasorb PL18, Purac) was added to the solution to dissolve the polymer. The polymer solution for spinning thus obtained was prepared so that the polymer concentration was 10% by weight and the fibrinogen freeze-dried powder concentration was 4% by weight (1.8% by weight based on fibrinogen). The particle size of the fibrinogen dispersed in the spinning solution was 12 μm. The obtained polymer solution was spun at a humidity of 30% or less by an electrospinning method to obtain a sheet-like fiber molded body. The inner diameter of the discharge nozzle was 0.8mm, the voltage was 12kV, and the distance from the discharge nozzle to the flat plate was 25 cm. The plate was used as the cathode during spinning. The fiber compact obtained had an average fiber diameter of 14.9 μm and a thickness of 325 μm. The amount of fibrinogen contained in the tablet calculated from the weight of the tablet and the feed ratio was 0.43mg/cm². The extraction rate after 3 hours of immersion was 40%. No weight change occurred in the loading test (maintained at 100%). The value obtained by the flexibility test was 2.7 cm.

Example 15

Adding thrombin lyophilized powder (BOLHEAL VIAL 3) (containing 1.12% thrombin (750 unit) in 40mg lyophilized powder) into the mixtureA thrombin dispersion was prepared by treating methylene chloride (manufactured by Wako pure chemical industries, Ltd.) for 5 minutes with an ultrasonic bath. 100% L-form polylactic acid (Purasorb PL18, Purac) was added to the solution to dissolve the polymer. The polymer solution for spinning was prepared so that the polymer concentration was 10% by weight and the thrombin freeze-dried powder concentration was 4% by weight (0.045% by weight or 750 units (U)/g in terms of thrombin). The particle size of thrombin dispersed in the spinning solution was 9 μm. The obtained polymer solution was spun at a humidity of 30% or less by an electrospinning method to obtain a sheet-like fiber molded body. The inner diameter of the discharge nozzle was 0.8mm, the voltage was 12kV, and the distance from the discharge nozzle to the flat plate was 25 cm. The plate was used as the cathode during spinning. The resulting fiber compact had an average fiber diameter of 16.6 μm and a thickness of 291. mu.m. The amount of thrombin contained in the tablet calculated from the weight of the tablet and the charge ratio was 31.39U/cm²。

Comparative example 3

A thrombin dispersion was prepared by adding thrombin freeze-dried powder (for tissue adhesion: VIAL 3) to ethanol (manufactured by Wako pure chemical industries, Ltd.) and subjecting the mixture to a treatment for 5 minutes by an ultrasonic bath. Methylene chloride (manufactured by Wako pure chemical industries, Ltd.) and L-form 100% polylactic acid (Purasorb PL18, manufactured by Purac) were added to the obtained dispersion, and the polymer was dissolved to prepare a uniform solution. The polymer solution for spinning was prepared so that the polymer concentration was 10% by weight, the thrombin lyophilized powder concentration was 4% by weight (0.045% by weight or 750U/g in terms of thrombin), and the ratio of ethanol to methylene chloride was 1:8 by weight. The particle size of thrombin dispersed in the spinning solution was 12 μm. The polymer solution was air-dried to prepare a solid state. The dissolution test was carried out in the same manner as for the fibrous molded article, and as a result, about 3% of the water-soluble component was extracted after 3 minutes of immersion.

Comparative example 4

ネオベール (polyglycolic acid nonwoven fabric) (i.e., (see)Registered trademark, manufactured by GUNZE ltd.), fibrinogen-immobilized sheets were prepared by the following procedure (freeze-drying method), and 1.25mL of fibrinogen solution contained in a commercially available kit for biological tissue adhesive material (product name: boleal: manufactured by general institute of treasury, chemist and serotherapy) was dyed on the above-mentioned molded fiber (5 × 5cm, manufactured by waybill)²) The above. The subject was frozen and then lyophilized for 24 hours to obtain a fibrinogen-immobilized sheet. In the supporting test, the supported fibrinogen collapsed and was lost as a powder (weight was 89%). The value obtained by the flexibility test was 0.7 cm.

< measuring methods in examples 16 to 29 >

Protein powder particle size (average particle size)

The fibrinogen freeze-dried powder was pulverized in a mortar, and the particle size distribution was measured with a laser diffraction particle size distribution measuring apparatus (Malvern: trade name "Master sizer 2000"), and the D50 value (median particle size) was defined as the average particle size.

2D. measurement of fibrinogen content

The resulting sheet was cut into 0.5cm phi, and fibrinogen was extracted with a 0.1% TFA solution and quantified by high performance liquid chromatography.

< test conditions >

A detector: ultraviolet absorption photometer (measuring wavelength: 214 nm)

Column: agilent Bio SEC-3 (3 μm, 30nm, 4.6X 300mm, Agilent Technologies)

Column temperature: 35 deg.C

Temperature of the sampler: 5 deg.C

Mobile phase: water with 0.1% TFA/acetonitrile with 0.1% TFA =50/50

Flow rate: 0.5mL/min

Analysis time: for 10 min.

Example 16

< production of sheet molded article comprising Water-soluble Polymer and fibrinogen >

After dispersing fibrinogen freeze-dried powder (boleal tissue adhesion: VIAL 1) in 2-propanol, hydroxypropyl cellulose (6-10 mPas, manufactured by wako pure chemical industries, ltd.) was dissolved to 16wt%, and a spinning dope of fibrinogen freeze-dried powder/hydroxypropyl cellulose =100 (46 in terms of fibrinogen)/100 (w/w) was prepared. The resultant was spun by an electrospinning method at a temperature of 22 ℃ and a humidity of 26% or less to obtain a sheet-like fiber molded body. The inner diameter of the discharge nozzle was 0.8mm, the voltage was 12.5kV, the flow rate of the spinning solution was 1.2mL/h, and the distance from the discharge nozzle to the grounded plate was 15 cm. The resulting fiber molded body had an average fiber diameter of 0.35. mu.m, an average thickness of 191. mu.m, and a weight per unit area of 2.74mg/cm²The bulk density of the resin composition was 143mg/cm³The resulting sheet was cut into 0.5cm × 0.5.5 cm, and the protein was extracted with 62.5. mu.L of physiological saline, and subjected to ELISA measurement ("method described in ELISA measurement (1) fibrinogen" 7A.) and, as a result, the immobilized protein mass was 0.51mg/cm²。

Example 17

< production of sheet molded article comprising aliphatic polyester and Thrombin >

After dispersing a thrombin lyophilized powder (for tissue adhesion: VIAL3 in ethanol), dichloromethane was added to dissolve 10wt% of polylactic acid (PL 18 pure biological company), and a spinning solution of thrombin lyophilized powder/polylactic acid =100 (1.1/100 (w/w in terms of thrombin) was prepared. The resultant was spun by an electrospinning method at a temperature of 22 ℃ and a humidity of 26% or less to obtain a sheet-like fiber molded body. Ejection nozzleThe inner diameter of the nozzle (D) was 0.8mm, the voltage was 15kV, the flow rate of the spinning solution was 3.0mL/h, and the distance from the discharge nozzle to the grounded plate was 25 cm. The resulting fiber molded body had an average fiber diameter of 9.37 μm, an average thickness of 210 μm, and a weight per unit area of 3.15mg/cm²The bulk density of the powder was 150mg/cm³The resulting sheet was cut to 2cm × 2cm, and the protein was extracted with 1mL of physiological saline, and the activity ("method described in" assay for thrombin activity 8A ") ELISA assay (" method described in "assay for thrombin by ELISA (2) 7A") was performed, and as a result, the activity was measured at 23.0U/cm²ELISA measurement of 16. mu.g/cm²。

Example 18

< evaluation test of Structure adhesion Effect of laminated sheet molded body >

In order to confirm the effect of using the sheet molded body composed of the water-soluble polymer and fibrinogen prepared in example 16 in combination with the sheet molded body composed of the aliphatic polyester and thrombin prepared in example 17, the adhesive force was compared. The adhesive force was determined by adhering the skin of the rabbit to the sheet (2 cm. times.2 cm) to confirm whether fibrin gel was formed or not and whether the sheet was adhered. At this time, for the overlapped sheets, 200 μ L of water was previously added to the sheet formed body formed of the water-soluble polymer and fibrinogen, and after 40 seconds, the sheet formed body formed of the water-soluble polymer and fibrinogen was attached to the skin of a rabbit. Thereafter, the sheet was left to stand at 37 ℃ for 3 minutes, and then the adhesiveness between the skin and the sheet molded body was confirmed. As a control, a collagen sheet preparation (product name: Tachocomb/CSL ベーリング (strain)) containing a fibrin adhesive material immobilized thereon was used: a preparation in which a sheet of spongy equine collagen is used as a support and is fixed to one surface of the sheet by vacuum drying: 2 cm. times.2 cm). As a result, the laminate sheet molded product of the present invention exhibited an adhesive force higher than that of the comparative collagen sheet preparation.

Example 19

< production of sheet molded article comprising Water-soluble Polymer and fibrinogen >

After dispersing fibrinogen freeze-dried powder (boleal tissue adhesion: VIAL 1) in 2-propanol, hydroxypropyl cellulose (6-10 mPas, manufactured by wako pure chemical industries, ltd.) was dissolved to 16wt%, and a spinning dope of fibrinogen freeze-dried powder/hydroxypropyl cellulose =100 (46 in terms of fibrinogen)/100 (w/w) was prepared. The resultant was spun by an electrospinning method at a temperature of 22 ℃ and a humidity of 26% or less to obtain a sheet-like fiber molded body. The inner diameter of the discharge nozzle was 0.8mm, the voltage was 12.5kV, the flow rate of the spinning solution was 1.2mL/h, and the distance from the discharge nozzle to the grounded plate was 15 cm. The resulting fiber molded body had an average fiber diameter of 0.35. mu.m, an average thickness of 191. mu.m, and a weight per unit area of 2.74mg/cm²The bulk density of the resin composition was 143mg/cm³The obtained tablet was cut into 0.5cm × 0.5.5 cm, and protein was extracted with 62.5. mu.L of physiological saline, and then subjected to ELISA measurement (the method described in "7A. ELISA measurement (1) fibrinogen"), and as a result, the immobilized protein mass was 0.78mg/cm²。

Example 20

< production of sheet molded article comprising aliphatic polyester and Thrombin >

After dispersing a thrombin lyophilized powder (for tissue adhesion: VIAL3 in ethanol), dichloromethane was added to dissolve 10wt% of polylactic acid (PL 18 pure biological company), and a spinning solution of thrombin lyophilized powder/polylactic acid =100 (1.1/100 (w/w in terms of thrombin) was prepared. The resultant was spun by an electrospinning method at a temperature of 22 ℃ and a humidity of 26% or less to obtain a sheet-like fiber molded body. The inner diameter of the ejection nozzle is 0.8mm, and the voltage is15kV, the flow rate of the spinning solution was 3.0mL/h, and the distance from the discharge nozzle to the grounded plate was 25 cm. The resulting fiber molded body had an average fiber diameter of 9.37 μm, an average thickness of 210 μm, and a weight per unit area of 3.15mg/cm²The bulk density of the powder was 150mg/cm³The resulting sheet molded body was cut into 2cm × 2cm, and the protein was extracted with 1mL of physiological saline, and the activity ("method described in 8A thrombin activity measurement") ELISA measurement ("method described in 7A ELISA measurement (2) thrombin") was performed, and as a result, the activity measurement value was 14.7U/cm²The ELISA measurement value was 11.4. mu.g/cm²。

Example 21

< hemostatic Effect on exudative liver hemorrhage in rabbits >

The hemostatic effect obtained when the sheet molded body composed of the water-soluble polymer and fibrinogen prepared in example 19 and the sheet molded body composed of the aliphatic polyester and thrombin prepared in example 20 were used in combination was compared with the hemostatic effect obtained when tachcocomb was used.

As a model of hemostasis for animals, rabbits were used. The rabbit was subjected to laparotomy, a part of the liver was cut out, and a sheet molded body composed of a water-soluble polymer and fibrinogen and a sheet molded body composed of an aliphatic polyester and thrombin were applied to the bleeding site in a superposed manner to observe the hemostatic effect (presence or absence of hemostasis, amount of bleeding). The test method is as follows.

(1) Selactar10mg/kg (about 1.0 mL) and Ketalar50mg/kg (about 3.0 mL) were administered intramuscularly.

(2) Body weight was measured and the abdomen was shaved and kept in a supine position.

(3) Continuous anesthesia was performed via the auricular vein (2% Ketalar, physiological saline supplemented with 20U/mL heparin).

(4) A median incision was made from just below the xiphoid sternal process to the lower abdomen.

(5) 300U/kg of heparin sodium injection was administered by auricular vein.

(6) Liver lobes (lateral left lobe, medial left lobe, right lobe) having a sufficient thickness for wound preparation were drawn out using forceps for intestine, gauze, and the like.

(7) A wound of 10mm in diameter and 4mm in depth was made on the liver lobe with a skin punch (leather 12509 ンチ) and the portion was cut with a scalpel.

(8) The bleeding from the cut wound was absorbed by BENSHEET for 10 seconds, and the weight thereof was measured. Wounds with bleeding on the wound part of 0.5g or more were used for the test.

(9) A layer of a water-soluble polymer and fibrinogen cut to 2.5cm square and a layer of an aliphatic polyester and thrombin were superimposed on the wound site, and the layer of a water-soluble polymer and fibrinogen was applied to the bleeding site and pressed for 1 minute. When Tachocomb was used as a control, the cut pieces were cut into 2.5cm square, and 312.5. mu.L of physiological saline was added dropwise to apply the pressure to the bleeding sites for 1 minute.

(10) After the compression, whether or not bleeding occurred was confirmed, and the bleeding from the wound site was absorbed by benset, and the weight thereof was measured.

As a result, when the laminate sheet molded product of the present invention was used, blood was stopped (n = 1), and the amount of bleeding 1 minute after application was very small, 0.003 g. On the other hand, when tachcocomb was used as a control (n = 5), the amount of bleeding 1 minute after application was 1.57g, the hemostatic effect was insufficient, and the amount of bleeding was large.

Example 22

< production of sheet molded article comprising aliphatic polyester and Thrombin >

Freeze drying thrombin to obtain powderRecombinant thrombin 1mg/mL, 3.4% sodium chloride, 1.2% sodium citrate, 0.29% calcium chloride, and 1% mannitol pH 7) was dispersed in ethanol, and then dichloromethane was added to dissolve polyglycolic acid-polylactic acid copolymer (Purasorb PDLG5010, Purac) to 10% by weight, thereby preparing a thrombin lyophilized powder/polyglycolic acid-polylactic acid copolymer =100 (1.67/100 (w/w) in terms of thrombin). The resultant was spun by an electrospinning method at a temperature of 22 ℃ and a humidity of 26% or less to obtain a sheet-like fiber molded body. The inner diameter of the discharge nozzle was 0.8mm, the voltage was 20kV, the flow rate of the spinning solution was 4.0mL/h, and the distance from the discharge nozzle to the grounded plate was 35 cm. The resulting fiber molded body had an average fiber diameter of 3.8 μm, an average thickness of 127 μm, and a weight per unit area of 1.38mg/cm²The bulk density of the powder was 109mg/cm³. The resulting sheet was cut into 1cm and proteins were extracted with 200. mu.L of physiological saline, followed by thrombin activity measurement (the method described in "8A thrombin activity measurement"). As a result, the activity was measured to be 18.3U/cm²。

Example 23

< production of a molded article of a laminate sheet comprising a layer comprising a water-soluble polymer and fibrinogen and a layer comprising an aliphatic polyester and thrombin >

A freeze-dried fibrinogen powder (prepared by freeze-drying recombinant fibrinogen 10mg/mL, 10mM arginine, 130mM sodium chloride, and 0.5% mannitol at pH 8.5) was pulverized in a mortar to prepare a pulverized freeze-dried fibrinogen powder having an average particle size of 30 μm. The pulverized fibrinogen lyophilized powder was dispersed in 2-propanol, and then hydroxypropylcellulose (2.0 to 2.9mPas, manufactured by Nippon Caoka corporation) and polyethylene glycol (molecular weight: 400, manufactured by Kasei Kogyo Co., Ltd.) were dissolved in such a manner that 15% by weight was obtained, thereby preparing fibrinogen lyophilized powder/hydroxypropylcellulose/polyethylene glycol =51 (25.92/34/15 (w/w/w) as fibrinogenThe coating liquid of (1). Using the obtained coating liquid, a film was produced by a casting method. The coating interval was 127 μm, and the coating speed was 30.1 mm/sec. After the film was produced, the layer comprising the aliphatic polyester and thrombin prepared in example 22 was stacked from the top within 1 minute to obtain a laminate sheet molded body comprising a layer comprising a water-soluble polymer and fibrinogen and a layer comprising the aliphatic polyester and thrombin. The average film thickness of the resulting laminate sheet was 157 μm. The obtained laminate sheet molded body was subjected to electron beam sterilization at 20 kGy. The resulting laminate sheet was cut into 1cm and protein was extracted with 200. mu.L of physiological saline, and then subjected to ELISA measurement of fibrinogen ("method described in" ELISA measurement of fibrinogen (7A.) "(1)"). As a result, the immobilized protein mass was 0.58mg/cm²。

Example 24

< hemostatic Effect on exudative liver hemorrhage in rabbits >

The hemostatic effect of the laminate sheet molded body comprising the layer composed of the water-soluble polymer and fibrinogen and the layer composed of the aliphatic polyester and thrombin prepared in example 23 was compared with that of the case of using TachoSil.

As a model of hemostasis for animals, rabbits were used. The rabbit was subjected to laparotomy, a part of the liver was cut out, and a laminate sheet molded body comprising a layer formed of a water-soluble polymer and fibrinogen and a layer formed of an aliphatic polyester and thrombin was applied to the bleeding site to observe the hemostatic effect (presence or absence of hemostasis, amount of bleeding). The test method was the same as that described in example 21.

As a result, when the laminate sheet molded product of the present invention was used (n = 4), the amount of bleeding 1 minute after application was very small, and was 0.003 g. On the other hand, when TachoSil was used as a control (n = 4), the amount of bleeding 1 minute after application was 0.65g, and the hemostatic effect was insufficient, and the amount of bleeding was large.

Example 25

< production of sheet molded article comprising aliphatic polyester and Thrombin >

A thrombin lyophilized powder (prepared by lyophilizing recombinant thrombin 1mg/mL, 3.4% sodium chloride, 1.2% sodium citrate, 0.29% calcium chloride, and 1% mannitol pH 7) was dispersed in ethanol, and then dichloromethane was added to dissolve a polyglycolic acid-polylactic acid copolymer (Purasorb PDLG5010, Purac corporation) to 10% by weight, thereby preparing a spinning dope of thrombin lyophilized powder/polyglycolic acid-polylactic acid copolymer =100 (1.67 in terms of thrombin)/100 (w/w). The resultant was spun by an electrospinning method at a temperature of 22 ℃ and a humidity of 26% or less to obtain a sheet-like fiber molded body. The inner diameter of the discharge nozzle was 0.8mm, the voltage was 20kV, the flow rate of the spinning solution was 4.0mL/h, and the distance from the discharge nozzle to the grounded plate was 35 cm. The resulting fiber molded body had an average fiber diameter of 2.97 μm, an average thickness of 137 μm, and a basis weight of 1.49mg/cm²The bulk density of the powder is 108mg/cm³。

Example 26

< production of a molded article of a laminate sheet comprising a layer comprising a water-soluble polymer and fibrinogen and a layer comprising an aliphatic polyester and thrombin >

A freeze-dried fibrinogen powder (prepared by freeze-drying recombinant fibrinogen 10mg/mL, 10mM arginine, 130mM sodium chloride, and 0.5% mannitol at pH 8.5) was pulverized in a mortar to prepare a pulverized freeze-dried fibrinogen powder having an average particle size of 30 μm. The pulverized freeze-dried fibrinogen powder was dispersed in 2-propanol, and then hydroxypropyl cellulose (2.0-2.9 mPas, Nippon Cao Kao Co., Ltd.) and polyethylene glycol (molecular weight: 400 Sanyo chemical Co., Ltd.) were dissolved to 2.9 wt% to prepare fibrinogenDry powder/hydroxypropylcellulose/polyethylene glycol =90 (36.98 as fibrinogen)/7/3 (w/w/w) of the coating liquid. Using the obtained coating liquid, a film was produced by a casting method. The coating interval was 50.8 μm and the coating speed was 30.1 mm/sec. After the film was produced, the layer comprising the aliphatic polyester and thrombin prepared in example 25 was stacked from the top within 1 minute to obtain a laminate sheet molded body comprising a layer comprising a water-soluble polymer and fibrinogen and a layer comprising the aliphatic polyester and thrombin. The average film thickness of the resulting laminate sheet was 169. mu.m. The obtained laminate sheet was cut into 0.5cm, fibrinogen was extracted with a 0.1% TFA solution, and the amount was determined by high performance liquid chromatography ("method described in" 2d. measurement of fibrinogen content "). As a result, the immobilized protein mass was 0.54mg/cm²。

Example 27

< hemostatic Effect on exudative liver hemorrhage in rabbits >

The hemostatic effect of the laminate sheet molded body comprising the layer composed of the water-soluble polymer and fibrinogen and the layer composed of the aliphatic polyester and thrombin prepared in example 26 was compared with that of the case of using TachoSil.

As a model of hemostasis for animals, rabbits were used. The rabbit was subjected to laparotomy, a part of the liver was cut out, and a laminate sheet molded body comprising a layer formed of a water-soluble polymer and fibrinogen and a layer formed of an aliphatic polyester and thrombin was applied to the bleeding site to observe the hemostatic effect (presence or absence of hemostasis, amount of bleeding). The test method was the same as that described in example 21.

As a result, when the laminate sheet molded product of the present invention was used (n = 6), the amount of bleeding 1 minute after application was very small, and was 0.003 g. On the other hand, when TachoSil was used as a control (n = 4) as in example 24, the bleeding amount 1 minute after application was 0.65g, and the hemostatic effect was insufficient and the bleeding amount was large.

Example 28

The freeze-dried fibrinogen powder (prepared by freeze-drying recombinant fibrinogen 10mg/mL, 10mM arginine, 110mM sodium chloride, 1% glycine, 0.2% mannitol, 0.25% phenylalanine, 0.4% histidine, and 0.1% 3 sodium citrate, pH 8.5) was pulverized in a mortar to prepare a pulverized fibrinogen freeze-dried powder having an average particle size of 22 μm. After dispersing the pulverized freeze-dried fibrinogen powder in 2-propanol, hydroxypropylcellulose (2.0 to 2.9mPas, japan koha) was dissolved in an amount of 4.2 wt% to prepare a coating liquid of freeze-dried fibrinogen powder/hydroxypropylcellulose/= 90 (26.55 in terms of fibrinogen)/10 (w/w). Using the obtained coating liquid, a film was produced by a casting method. The coating interval was 101.6 μm, and the coating speed was 30.1 mm/sec. After the film was produced, a sheet-like fibrous molded body having a ratio of thrombin lyophilized powder/polyglycolic acid-polylactic acid copolymer =20/100, 40/100, 60/100, 80/100, and 100/100 prepared by the method described in example 25 was stacked from the top within 3 minutes, thereby obtaining a laminated sheet molded body including a layer formed of a water-soluble polymer and fibrinogen and a layer formed of an aliphatic polyester and thrombin. The hemostatic effect of these laminate sheet molded bodies was confirmed by evaluating the efficacy of the drug using a rat encapsulation model (a test was performed using a bleeding model with a wound on the rat liver). the test sheet molded bodies were pressed on the wound site for a fixed period of time (5 minutes in this example), and then the bleeding was evaluated by visual observation for 1 minute thereafter. The test was performed with n =6, and whether bleeding occurred or not was confirmed.

As a result, as shown in table 1, the ratio of the thrombin lyophilized powder/polyglycolic acid-polylactic acid copolymer evaluated was in the range of 20/100 to 100/100, and hemostatic effects exceeding TachoSil were confirmed.

Example 29

A sheet-like molded fiber having different thrombin contents (thrombin content 0.23U/cm) was obtained by the method described in example 25²、2.8U/cm²、11.4U/cm²、28.5U/cm²). Next, a laminate sheet molded body containing a layer formed of a water-soluble polymer and fibrinogen and a layer formed of an aliphatic polyester and thrombin, with a constant fibrinogen content, was obtained by the method described in example 26. The obtained laminate sheet molded bodies having a constant fibrinogen content and different thrombin contents were used to evaluate the influence of the thrombin content on the adhesive force. The test method is as follows.

(1) The laminate sheet molded body and a positive control preparation (Tachosil) (1 cm. times.1 cm) were adhered to the bottom (1 cm. times.1 cm) of a plastic rectangular column with a double-sided tape.

(2) The laminate sheet molded body and the positive control preparation (TachoSil) were immersed in 1mL of physiological saline for 10 seconds to adhere to the eucalyptus globulus plate.

(3) A load of 100g was applied for 5 minutes from above the square column.

(4) The four-cornered column was pulled at a speed of 30mm/min, and the tension was measured with a digital dynamometer.

The test was performed with n =5, and the average value of the tensile force was evaluated as the adhesive force (g). As a result, as shown in table 2, any of the laminate sheets showed a higher adhesive force than TachoSil.

Example 30

A sheet molding comprising an aliphatic polyester and thrombin was obtained in the same manner as in example 25 (the thrombin content was 24.2U/cm)²). The hemostatic effect of the sheet molded body was confirmed by the method described in example 28 (compression time: 3 minutes). The experiment was performed with n = 6. As a result, 6/6 confirmed hemostasis.

Example 31

A sheet molding comprising an aliphatic polyester and thrombin was obtained in the same manner as in example 25 (thrombin content: 19-26U/cm)²) Subsequently, a laminated sheet molded body including layers made of a water-soluble polymer and fibrinogen having different contents and a layer made of an aliphatic polyester and thrombin was obtained by the method described in example 26. The hemostatic effect of the resulting laminate sheet molded body having a thrombin content within a certain range and different fibrinogen contents was confirmed by the method described in example 28. As a result, as shown in Table 3, although a high hemostatic effect was observed in any fibrinogen content, the fibrinogen content was 1.47mg/cm²The effect is slightly reduced.

Industrial applicability

The sheet molding of the present invention is used as a hemostatic material and is useful in the medical product manufacturing industry.

Claims (22)

1. A sheet-formed article of a polymer composition comprising: at least one protein selected from fibrinogen and thrombin, and at least one polymer selected from an aliphatic polyester and a water-soluble polymer, at least a part of the protein entering the interior of the polymer.

2. The sheet molding product according to claim 1, wherein the at least one polymer selected from the group consisting of aliphatic polyesters and water-soluble polymers is a polymer selected from the group consisting of cellulose derivatives, polymers having N-vinyl cyclic lactam units, polyethylene oxide, polyvinyl alcohol, hyaluronic acid, dextran, pullulan, starch, and mixtures thereof.

3. The sheet-formed body of claim 1, wherein the at least one polymer selected from the group consisting of aliphatic polyesters and water-soluble polymers is a polymer selected from the group consisting of hydroxypropyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, and a mixture thereof.

4. The sheet-shaped body of claim 1, wherein the at least one polymer selected from the group consisting of aliphatic polyesters and water-soluble polymers is a polymer selected from the group consisting of polyglycolic acid, polylactic acid, polycaprolactone, copolymers thereof, and mixtures thereof.

5. The sheet molding as claimed in any one of claims 1 to 4, wherein the sheet molding is a film molding or a fiber molding.

6. A laminated sheet molded body comprising: the adhesive comprises a first polymer composition layer containing fibrinogen and a water-soluble polymer, wherein at least a part of the fibrinogen enters the water-soluble polymer, and a second polymer composition layer containing thrombin and an aliphatic polyester, wherein at least a part of the thrombin enters the aliphatic polyester.

7. The laminate sheet forming body as claimed in claim 6, wherein the water-soluble polymer is at least one selected from the group consisting of cellulose derivatives, polymers having N-vinyl cyclic lactam units, polyethylene oxide, polyvinyl alcohol, hyaluronic acid, dextran, pullulan, starch, and mixtures thereof.

8. The laminate sheet-shaped article of claim 6, wherein the water-soluble polymer is at least one selected from the group consisting of hydroxypropyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, and a mixture thereof.

9. The laminate sheet-shaped article of any one of claims 6 to 8, wherein the aliphatic polyester is at least one selected from the group consisting of polyglycolic acid, polylactic acid, polycaprolactone, copolymers thereof, and mixtures thereof.

10. The laminate shaped body as claimed in any one of claims 6 to 8, wherein the first polymer composition layer comprises at least one additive selected from blood coagulation factor XIII, albumin, isoleucine, glycine, arginine, glutamic acid, phenylalanine, histidine, a surfactant, sodium chloride, a sugar alcohol, trehalose, sodium citrate, aprotinin and calcium chloride.

11. The laminate sheet-shaped article of any one of claims 6 to 8, wherein the first polymer composition layer is formed of a film-shaped article or a fiber-shaped article.

12. The laminate sheet-shaped article as claimed in any one of claims 6 to 8, wherein the first polymer composition layer is formed of a film-shaped article.

13. The laminate sheet molding of claim 12, wherein the water-soluble polymer is contained in the film molding in an amount of 0.1 to 50 wt%.

14. The laminate sheet molding of claim 12, wherein the film molding comprises 0.05 to 10mg/cm²Fibrinogen of (4).

15. The laminate sheet-shaped article of claim 12, wherein the film-shaped article is produced from a suspension solution formed from a water-soluble polymer solution and fibrinogen powder.

16. The laminate sheet-shaped article as claimed in claim 6 to 8, wherein the second polymer composition layer is formed of a fiber-shaped article.

17. The laminate sheet-shaped article as claimed in any one of claims 6 to 8, wherein the fibrinogen or the mixture of the fibrinogen and the additive and/or the thrombin or the mixture of the thrombin and the additive are dispersed in the form of protein particles in at least one polymer selected from the group consisting of aliphatic polyesters and water-soluble polymers.

18. The laminate shaped body as claimed in claim 17, wherein the fibrinogen or the mixture of fibrinogen and an additive and/or the thrombin or the mixture of thrombin and an additive is thrombin or the mixture of thrombin and an additive.

19. The laminate sheet-shaped article of claim 18, wherein the weight ratio of the protein particles to the aliphatic polyester is 10:10 to 100.

20. The sheet molding according to claim 1, which is a fiber molding produced from a suspension solution comprising an aliphatic polyester solution and thrombin powder.

21. A hemostatic material comprising the sheet molded article according to any one of claims 1 to 5 or the laminate sheet molded article according to any one of claims 6 to 8.

22. A tissue-adhesive material or tissue-sealing material comprising the sheet molded body according to any one of claims 1 to 5 or the laminated sheet molded body according to any one of claims 6 to 8.