Hemostatic material composition

Abstract

To provide a hemostatic material composition having excellent storage stability.SOLUTION: A hemostatic material composition contains a polysaccharide having a reactive group that reacts with a protein to form a crosslinking structure, a hydrophobic solvent, and a crosslinked particle containing one or more compounds selected from the group consisting of a crosslinked protein and a crosslinked polysaccharide.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to hemostatic compositions. [Background technology]

[0002] Uncontrolled bleeding remains a leading cause of death in traumatic and surgical injuries. Therefore, the development of effective therapeutic approaches to control bleeding is of great clinical and societal importance. To date, hemostatic materials used in surgery include fibrin glue, oxidized cellulose nonwoven fabric, collagen sponge, starch microparticles, and thrombin-containing gelatin paste, and these are used in a variety of forms. Among these, FloSeal (registered trademark, manufactured by Baxter) and Surgeflow (manufactured by Echicon) are widely known as thrombin-containing gelatin pastes. Thrombin activates fibrinogen contained in blood to form a fibrin network, which, together with blood cells, forms a clot to seal the bleeding site. Thrombin is purified from appropriately selected human blood through virus inactivation and removal processes, but there is a problem that the risk of infection transmission cannot be completely eliminated. Therefore, there is a need for the development of a hemostatic agent with excellent safety.

[0003] Patent Document 1 discloses a hemostatic composition containing polyethylene glycol having an N-hydroxysuccinimide ester group, polyethylene glycol as a hydrophilic solvent, and crosslinked gelatin particles. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 6195568 Summary of the Invention [Problem to be solved by the invention]

[0005] The present inventors have now discovered a new problem with the hemostatic composition disclosed in Patent Document 1: the N-hydroxysuccinimide ester group of polyethylene glycol reacts with trace amounts of water contained in the hydrophilic solvent and with hydroxyl groups of the hydrophilic solvent, resulting in decomposition, and the hemostatic properties decrease over time, leaving room for improvement in the storage stability. An object of one embodiment of the present disclosure is to provide a hemostatic composition that has excellent storage stability. [Means for solving the problem]

[0006] The specific means for solving the problems are as follows: <1> A hemostatic composition comprising a polysaccharide having a reactive group that reacts with a protein to form a crosslinked structure, a hydrophobic solvent, and crosslinked particles containing one or more compounds selected from the group consisting of crosslinked proteins and crosslinked polysaccharides. <2> the crosslinked particles comprise the crosslinked protein; and The cross-linked protein is cross-linked gelatin. <1> The hemostatic composition according to claim 1. <3> The average primary particle size of the crosslinked particles is 20 μm to 700 μm. <1> or <2> The hemostatic composition according to claim 1. <4> the reactive group is one or more groups selected from the group consisting of -CON(COCH2)2, an N-hydroxysuccinimide ester group, an imide ester group, an aldehyde group, a carboxy group, an isocyanate group, a maleimide group, and a haloacetyl group; <1> ~ <3> The hemostatic composition according to any one of the above. <5> The reactive group is an N-hydroxysuccinimide ester group. <1> ~ <4> The hemostatic composition according to any one of the above. <6> The polysaccharide contains a structure derived from one or more polysaccharides selected from the group consisting of cellulose, dextran, dextrin, pullulan, hyaluronic acid, alginic acid, xanthan gum, chitin, chitosan, and modified forms thereof. <1> ~ <5> The hemostatic composition according to any one of the above. <7> the polysaccharide comprises a constitutional unit derived from the monosaccharide having the reactive group; and the content of the structural unit derived from the monosaccharide having the reactive group relative to 100 mol % of the polysaccharide is 1 mol % to 40 mol %; <1> ~ <6> The hemostatic composition according to any one of the above. <8> The structural unit derived from the monosaccharide having the reactive group is represented by the following general formula (1): <7> The hemostatic composition according to claim 1.

[0007] [ka] In the general formula (1), L is a group represented by the following formula:

[0008] [ka]

[0009] In the above formula, n and m represent integers of 1 to 3, * represents the site linking to the oxygen atom on the tetrahydrofuran side, and ** represents the site linking to the oxygen atom on the N-hydroxysuccinimide ester group side.

[0010] <9> The weight-average molecular weight of the polysaccharide is 2,000 to 2,500,000. <1> ~ <8> The hemostatic composition according to any one of the above. <10> The hydrophobic solvent is at least one selected from the group consisting of vegetable oil, paraffin-based solvent, olefin-based solvent, silicone oil-based solvent, fatty acid ester-based solvent, alkyl halide-based solvent, and terpene-based solvent. <1> ~ <9> The hemostatic composition according to any one of the above. <11> The kinematic viscosity of the above hydrophobic solvent at 25°C is 1 mm 2 / sec~1000mm 2 / sec above <1> ~ <10> The hemostatic composition according to any one of the above. <12> The content of the hydrophobic solvent relative to the total mass of the hemostatic material composition is 30% by mass to 80% by mass. <1> ~ <11> The hemostatic composition according to any one of the above. <13> The above composition does not contain water, or contains water and the water content relative to the total mass of the hemostatic material composition is 0.5 mass% or less. <1> ~ <12> The hemostatic composition according to any one of the above. [Effects of the Invention]

[0011] According to the present disclosure, a hemostatic composition having excellent storage stability can be provided. DETAILED DESCRIPTION OF THE INVENTION

[0012] In the present disclosure, numerical ranges indicated using "to" include the numerical values ​​before and after "to" as the minimum and maximum values, respectively. In the numerical ranges described in stages in this disclosure, the upper or lower limit value described in one numerical range may be replaced with the upper or lower limit value of another numerical range described in stages. Furthermore, in the numerical ranges described in this disclosure, the upper or lower limit value of that numerical range may be replaced with a value shown in the examples.

[0013] In this disclosure, the term "hydrophobic solvent" refers to a solvent that has low miscibility with water, i.e., a solvent that does not dissolve 20 g or more in 1 L of ion-exchanged water (pH 6-8) at 20°C to form a homogeneous aqueous solution, i.e., a solvent with a solubility in water of less than 20 g / L. In addition, in this disclosure, a solvent with a solubility of 20 g / L or more is referred to as a hydrophilic solvent. Here, the pH of ion-exchanged water is a value measured at 20°C within 3 minutes after the ion-exchanged water comes into contact with air.

[0014] [Hemostatic material composition] The hemostatic composition of the present disclosure contains a polysaccharide (hereinafter also referred to as a "specific polysaccharide") having a reactive group (hereinafter also referred to as a "specific reactive group") that reacts with a protein to form a crosslinked structure, a hydrophobic solvent, and crosslinked particles containing one or more compounds selected from the group consisting of crosslinked proteins and crosslinked polysaccharides.

[0015] The hemostatic composition of the present disclosure has excellent storage stability. The reason for this effect is presumed to be as follows, but is not limited to this. The hemostatic composition of the present disclosure contains a hydrophobic solvent, and the specific polysaccharide is present in a dispersed state in the hydrophobic solvent. The specific reactive group of the specific polysaccharide is unlikely to be decomposed in the hydrophobic solvent, and is presumed to exhibit excellent storage stability. Furthermore, it is believed that the inclusion of crosslinked particles in the hemostatic composition of the present disclosure allows the crosslinked particles to absorb trace amounts of water in the composition, inhibiting the decomposition of the specific polysaccharides and thereby exhibiting excellent storage stability. Furthermore, among crosslinked particles, crosslinked gelatin has a protein structure and therefore has a high affinity for water, which is believed to more significantly enhance the storage stability effect.

[0016] The state of the hemostatic composition of the present disclosure is not particularly limited, but from the viewpoint of ease of application to living bodies, it is preferably in a paste state.

[0017] [Specified polysaccharide] The specific polysaccharide has a specific reactive group that reacts with a protein to form a crosslinked structure. The specific reactive group is not particularly limited as long as it is a group that can react with an amino group or the like possessed by a protein to form a crosslinked structure. From the viewpoint of improving hemostatic properties, however, it is preferably one or more groups selected from the group consisting of -CON(COCH2)2, an N-hydroxysuccinimide ester group (hereinafter also referred to as "NHS-ester group"), an imide ester group, an aldehyde group, a carboxy group, an isocyanate group, a maleimide group, and a haloacetyl group, and an NHS-ester group is more preferred. The specific polysaccharide may have two or more types of specific reactive groups, and the hemostatic composition of the present disclosure may contain two or more types of specific polysaccharides. In the present disclosure, polysaccharides that do not have a specific reactive group are also referred to as non-specific polysaccharides.

[0018] Certain polysaccharides contain constitutional units derived from two or more monosaccharides. From the viewpoint of improving storage stability and hemostatic properties, as well as biocompatibility, the specific polysaccharide preferably contains a structure derived from one or more polysaccharides selected from the group consisting of cellulose, dextran, dextrin, pullulan, hyaluronic acid, alginic acid, xanthan gum, chitin, chitosan, and modified forms thereof, and more preferably contains a structure derived from dextran. The structure of the polysaccharide is preferably modified with a specific reactive group, and may be carboxy-modified. In the present disclosure, the modified product refers to a compound into which a functional group such as a carboxy group has been introduced by reacting cellulose or the like with the functional group.

[0019] From the viewpoint of improving storage stability and hemostatic properties, the specific polysaccharide contains a structural unit derived from a monosaccharide having a specific reactive group (hereinafter also referred to as a "specific structural unit"), and the content of the specific structural unit relative to 100 mol% of the specific polysaccharide is preferably 1 mol% to 40 mol%, more preferably 3 mol% to 30 mol%, and even more preferably 5 mol% to 25 mol%.

[0020] From the viewpoints of improving storage stability and hemostatic properties, and biocompatibility, the specific constitutional unit is preferably a constitutional unit represented by the following general formula (1).

[0021] [ka]

[0022] In the general formula (1), L is a group represented by the following formula: In the following formula, n and m represent integers of 1 to 3, * represents the site linking to the oxygen atom on the tetrahydrofuran side, and ** represents the site linking to the oxygen atom on the N-hydroxysuccinimide ester group side.

[0023] [ka]

[0024] Among the above groups, L is preferably a group represented by the following formula, from the viewpoint of achieving the highest reaction rate of the reactive group and improving hemostatic properties.

[0025] [ka]

[0026] From the viewpoint of biocompatibility, the specific polysaccharide preferably contains a constitutional unit represented by the following formula (2) in addition to the constitutional unit represented by the above formula (1).

[0027] [ka]

[0028] When the specific polysaccharide contains a constitutional unit represented by formula (2), the content of the constitutional unit represented by formula (2) relative to 100 mol % of the specific polysaccharide is preferably 40 mol % to 90 mol %, and more preferably 45 mol % to 80 mol %.

[0029] The specific polysaccharide can contain a constitutional unit represented by the following formula (3), which can lower the pH when dissolved in water. The specific reactive group such as an N-hydroxysuccinimide ester group is inhibited from decomposing in a low pH solution, and therefore can inhibit decomposition reactions due to slight moisture in the atmosphere or in a composition.

[0030] [ka]

[0031] When the specific polysaccharide contains a constitutional unit represented by formula (3), the content of the constitutional unit represented by formula (3) relative to 100 mol % of the specific polysaccharide is preferably 5 mol % to 40 mol %, and more preferably 8 mol % to 30 mol %.

[0032] The weight average molecular weight (Mw) of the specific polysaccharide is preferably 2,000 to 2,500,000, more preferably 5,000 to 2,000,000, further preferably 10,000 to 750,000, and particularly preferably 20,000 to 200,000. When the Mw of the specific polysaccharide is 2000 or more, the specific reactive group of the specific polysaccharide reacts with protein to form a crosslinked structure, resulting in a good coating and improved hemostatic properties.When the Mw of the specific polysaccharide is 2,500,000 or less, the specific polysaccharide can be easily synthesized, and the hemostatic composition containing the specific polysaccharide has excellent handleability. Furthermore, since the Mw of the specific polysaccharide is 2000 or more, the number of specific reactive groups in the specific polysaccharide can be easily adjusted, thereby improving hemostatic properties and storage stability. The number of specific polysaccharides can be adjusted by adjusting the proportion of monosaccharides having reactive groups used in the synthesis of the specific polysaccharide. After the hemostatic composition has been applied to the area where bleeding has been stopped, the excess portion is preferably removed, typically with physiological saline, etc. By ensuring that the Mw of the specific polysaccharide is 2,500,000 or less, the removability can be improved.

[0033] In the present disclosure, Mw is determined by dissolving a sample in ultrapure water to a concentration of 0.1% by mass, filtering the solution using a filter with a pore size of 0.45 μm, and then subjecting the resulting filtrate to gel permeation chromatography (GPC) measurement under the following measurement conditions: (Measurement conditions) Column: TSK gel G6000PW XL +G4000PWXL +G2500PW XL ·Flow rate: 0.7mL / min Column temperature: 40℃ ·Injection volume: 100μL ·Eluent: 100mM NaNO3aq. Sample concentration: 1000 ppm Detection: RI (Refractive Index Detector) Analysis time: 60 minutes Standard sample: Pullulan 5.8k, 12.2k, 23.7k, 48.0k, 100k, 186k, 380k, 853k (Shodex Standard P-82)

[0034] From the viewpoint of improving storage stability and hemostatic properties, the content of the specific polysaccharide relative to the total mass of the hemostatic composition is preferably 1% by mass to 60% by mass, more preferably 5% by mass to 50% by mass, and even more preferably 15% by mass to 30% by mass.

[0035] From the viewpoint of improving storage stability, hemostatic properties, and removability, the solubility of the specific polysaccharide in 100 g of a hydrophobic solvent at 20° C. is preferably 1% by mass or less, more preferably 0.5% by mass or less, and even more preferably 0.1% by mass or less. The lower limit of the solubility is not particularly limited and may be 0% by mass. For example, a solubility of 1% by mass in 100 g of a hydrophobic solvent at 20° C. means that the amount of a particular polysaccharide that dissolves in 100 g of the hydrophobic solvent is 1 g.

[0036] [Hydrophobic solvent] From the viewpoint of improving storage stability, hemostatic properties, and removability, the hydrophobic solvent preferably has a solubility of less than 20 g / L, more preferably less than 5 g / L, and even more preferably less than 1 g / L in 1 L of ion-exchanged water at 20° C. The lower limit of the solubility is not particularly limited and may be 0 g / L.

[0037] The type of hydrophobic solvent is not particularly limited, but from the viewpoint of improving storage stability and hemostatic properties, as well as ease of application to living organisms, the hydrophobic solvent is preferably one or more selected from the group consisting of vegetable oil, paraffin-based solvent, olefin-based solvent, silicone oil-based solvent, fatty acid ester-based solvent, halogenated alkyl solvent, and terpene-based solvent, more preferably one or more selected from the group consisting of vegetable oil and fatty acid ester-based solvent, and even more preferably vegetable oil. In the present disclosure, the term "vegetable oil" is not particularly limited as long as it is an oil made from a plant, and may be a derivative thereof, with vegetable oils containing fatty acid glycerides being preferred. Examples of vegetable oils include linseed oil, perilla oil, olive oil, grapeseed oil, corn oil, coconut oil, sesame oil, rice bran oil, soybean oil, rapeseed oil, palm oil, sunflower oil, safflower oil, cottonseed oil, peanut oil, avocado oil, almond oil, argan oil, cocoa oil, shea butter, camellia oil, peanut oil, and castor oil. Examples of the paraffinic solvent include octane, decane, dodecane, and tridecane. Examples of the olefin solvent include decene and dodecene. Examples of silicone oil solvents include dimethyl silicone oil, methylphenyl silicone oil, and cyclic dimethyl silicone oil. Examples of fatty acid ester solvents include methyl laurate, methyl palmitate, methyl stearate, methyl oleate, methyl palmoleate, 2-ethylhexyl caprylate, glycerin monostearate, glycerin monobehenate, glycerin mono-12-hydroxystearate, glycerin monooleate, glycerin monocaprylate, glycerin monocaprate, glycerin monolaurate, alkyl oleates (ethyl oleate, methyl oleate, etc.), oleic acid triglyceride, alkyl linoleates (methyl linoleate, ethyl linoleate, etc.), linoleic acid triglyceride, and dioleic acid glyceride. Examples of the alkyl halide solvent include methylene chloride and 1,1,1-trichloroethane. Terpene solvents include D-limonene. The hemostatic composition of the present disclosure may contain two or more hydrophobic solvents.

[0038] The molecular weight of the hydrophobic solvent is preferably 300 to 1,500. For medical applications, high-frequency treatments and other procedures can cause instantaneous high temperatures. For this reason, from the perspective of safety in actual surgery, it is preferable that the hydrophobic solvent be nonvolatile. A nonvolatile solvent refers to a solvent with a boiling point of 50°C or higher, preferably 100°C or higher, and more preferably 200°C or higher. In this disclosure, the measurement of boiling point means the boiling point under 1 atmosphere (101,325 Pa). The boiling point is measured using a boiling point meter, for example, a boiling point measuring device manufactured by Titan Technologies (product name "DosaTherm300")

[0039] In order to improve ease of application to living bodies, ease of removal by washing with water from living bodies, and to prevent liquid separation over time, the kinematic viscosity of the hydrophobic solvent at 25°C is 1 mm 2 / sec~1000mm 2 / sec is preferred, and 5 mm 2 / sec~200mm 2 / sec is more preferable, and 20 mm 2 / sec~100mm 2 / sec is even more preferable. In the present disclosure, the kinematic viscosity is measured in accordance with JIS K 2283 (2000).

[0040] From the viewpoint of improving storage stability and hemostatic properties, the content of the hydrophobic solvent relative to the total mass of the hemostatic composition is preferably 25% by mass to 75% by mass, more preferably 30% by mass to 70% by mass, and even more preferably 40% by mass to 60% by mass.

[0041] [Crosslinked particles] The hemostatic composition of the present disclosure contains crosslinked particles comprising one or more compounds selected from the group consisting of crosslinked proteins and crosslinked polysaccharides. When the hemostatic composition containing the crosslinked particles is mixed with blood, the water in the blood is absorbed by the crosslinked particles, increasing the protein concentration in the blood and improving reactivity with the specific polysaccharide, thereby further improving hemostatic properties. Furthermore, in order to improve the hemostatic effect, the hemostatic composition applied to the bleeding site may be compressed with gauze or the like impregnated with saline or the like. By incorporating crosslinked particles into the hemostatic composition, it is possible to prevent the hemostatic composition from being transferred to the gauze or the like, which peels off after compression. This is thought to be because the crosslinked particles take up the physiological saline or the like impregnated into the gauze or the like, and swell depending on the degree of crosslinking, thereby reducing the contact area between the gauze and the hemostatic composition. In the present disclosure, a crosslinked particle refers to a particle in which two or more proteins are crosslinked. Also, in the present disclosure, a crosslinked protein refers to a particle in which two or more proteins are crosslinked, and a crosslinked polysaccharide refers to a particle in which two or more polysaccharides are crosslinked.

[0042] Examples of the cross-linked protein include cross-linked gelatin, cross-linked collagen, cross-linked albumin, cross-linked hemoglobin, cross-linked fibrinogen, cross-linked fibrin, cross-linked casein, cross-linked elastin, and cross-linked keratin. Examples of polysaccharides that constitute the above-mentioned cross-linked polysaccharides include cross-linked cellulose, cross-linked dextran, cross-linked dextrin, cross-linked pullulan, cross-linked hyaluronic acid, cross-linked alginic acid, cross-linked xanthan gum, cross-linked chitin, cross-linked chitosan, and modified forms thereof. The hemostatic composition of the present disclosure may contain two or more types of crosslinked particles. From the viewpoint of improving storage stability, hemostatic properties, and transcriptional inhibition properties, it is preferred that the crosslinked particles contain a crosslinked protein, and that the crosslinked protein is crosslinked gelatin.

[0043] From the viewpoint of improving water uptake and hemostatic properties, the crosslinked protein and crosslinked polysaccharide are preferably hydrophilic polymers.

[0044] From the viewpoint of improving storage stability and hemostatic properties, and of suppressing the transfer of the hemostatic composition to the gauze, the sum of the content of crosslinked proteins and crosslinked polysaccharides relative to the total mass of the crosslinked particles is preferably 50% by mass or more, more preferably 65% ​​by mass or more, even more preferably 75% by mass or more, particularly preferably 90% by mass or more, and may be 100% by mass.

[0045] From the viewpoints of improving water absorption, improving storage stability and hemostatic properties, inhibiting transfer of the hemostatic composition to gauze, and inhibiting penetration into blood vessels, etc., the average primary particle size of the crosslinked particles is preferably 20 μm to 700 μm, more preferably 100 μm to 700 μm, and even more preferably 200 μm to 700 μm.

[0046] In the present disclosure, the average primary particle size is measured by arithmetically averaging the sizes of 50 particles randomly selected from a scanning electron microscope (SEM) image. Note that the particle size of the particles is measured as the primary particle size.

[0047] From the viewpoints of improving storage stability, improving water absorption and hemostatic properties, inhibiting transfer of the hemostatic composition to gauze, and inhibiting penetration into blood vessels, etc., the content of crosslinked particles relative to the total mass of the hemostatic composition is preferably 1% by mass to 50% by mass, more preferably 8% by mass to 40% by mass, and even more preferably 20% by mass to 40% by mass.

[0048] [Ingredients other than specific polysaccharides, hydrophobic solvents, and crosslinked particles] The hemostatic composition of the present disclosure may contain components other than the specific polysaccharide, hydrophobic solvent, and crosslinked particles, including stabilizers such as ascorbic acid, antibiotics, vasoconstrictors, pigments, growth factors, bone morphogenetic proteins, analgesics, anti-inflammatory agents, and antifibrinolytic agents such as tranexamic acid. The hemostatic composition of the present disclosure may contain water, but from the viewpoint of storage stability, the water content relative to the total mass of the hemostatic composition is preferably 5% by mass or less, more preferably 3% by mass or less, even more preferably 1% by mass or less, particularly preferably 0.5% by mass or less, most preferably 0.1% by mass or less, and most preferably zero. [Example]

[0049] The above-described embodiment will be specifically described below using examples, but the above-described embodiment is not limited to these examples. Note that the units of values ​​in Tables 1 to 3 are % by mass unless otherwise specified.

[0050] (Synthesis Example 1) 10 g of carboxymethyl dextran sodium A (manufactured by Meito Sangyo Co., Ltd., CMD-D40, Mw 40000) was dissolved in 100 g of distilled water, and 13 g of 10% by mass aqueous hydrochloric acid solution was added. The resulting solution was reprecipitated in 1600 mL of methanol, and the residue was washed with methanol and then vacuum-dried at room temperature (25°C) for 24 hours to obtain 10.44 g of carboxymethyl dextran X. Next, 10 g of carboxymethyl dextran X was dissolved in 100 g of dimethylacetamide (DMAc), and 3.78 g of N-hydrosuccinimide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and 6.30 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC hydrochloride, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were added, followed by stirring at 40°C for 4 hours. The resulting solution was reprecipitated in 350 mL of methanol, and the residue was washed with methanol and acetone and then vacuum-dried at room temperature (25°C) for 24 hours to obtain 10.6 g of carboxymethyldextran A having N-hydrosuccinimide ester groups (hereinafter also referred to as "NHS-carboxymethyldextran"). Note that NHS-carboxymethyldextran A was composed of the structural units represented by the above-mentioned general formula (1), the structural units represented by formula (2), and the structural units represented by formula (3).

[0051] 10 mg of NHS-carboxymethyldextran A was dissolved in 1 g of heavy water, heated to 40°C, and hydrolysis was carried out for 24 hours. A nuclear magnetic resonance spectrometer (BRUKER, 400 MHz NMR, 16 accumulations) was used to obtain a hydrolyzate of NHS-carboxymethyldextran A. From the spectrum obtained, the ratio of the protons of the free N-hydroxysuccinimide (2.67 ppm to 2.74 ppm) to the protons at position 1 of the glucopyranose ring (4.82 ppm to 5.47 ppm) was determined. The content of the structural unit derived from a monosaccharide having an N-hydroxysuccinimide ester group (the structural unit represented by general formula (1)) relative to 100 mol% of NHS-carboxymethyldextran A was calculated to be 14.8 mol%. Similarly, the contents of the constitutional unit represented by formula (2) and the constitutional unit represented by formula (3) were calculated to be 70 mol % and 15.2 mol %, respectively. The Mw of NHS-carboxymethyldextran A was 45,000.

[0052] (Synthesis Example 2) 19.6 g of sodium hydroxide was dissolved in 106 g of distilled water, and 10 g of pullulan (manufactured by Tokyo Chemical Industry Co., Ltd.) was added little by little to the solution and dissolved, followed by stirring for 90 minutes. After stirring, an aqueous solution of chloroacetic acid (7 g of chloroacetic acid dissolved in 63 g of distilled water) was added, and the mixture was reacted at 60° C. for 6 hours. After cooling to room temperature (25°C), 65 g of 20% by mass aqueous hydrochloric acid solution was added and stirred for 2 hours. The resulting solution was reprecipitated in 2500 mL of methanol, and the residue was washed with methanol and then vacuum-dried at room temperature (25°C) for 24 hours to obtain 10.85 g of carboxymethyl pullulan. NHS-carboxymethylpullulan was obtained in the same manner as in Synthesis Example 1, except that carboxymethyldextran X was changed to carboxymethylpullulan and the amounts of N-hydroxysuccinimide and EDC hydrochloride added were changed to 1.69 g and 2.81 g, respectively. The NHS-carboxymethylpullulan was composed of the structural units represented by the above-mentioned general formula (1), the structural units represented by formula (2), and the structural units represented by formula (3). Furthermore, the content of the structural unit derived from a monosaccharide having an N-hydrosuccinimide ester group (structural unit represented by general formula (1)) relative to 100 mol % of NHS-carboxymethylpullulan was calculated to be 6.3 mol %. Similarly, the contents of the constitutional unit represented by formula (2) and the constitutional unit represented by formula (3) were calculated to be 70 mol % and 23.7 mol %, respectively. The Mw of the NHS-carboxymethylpullulan was 12,000.

[0053] (Synthesis Example 3) 19.6 g of sodium hydroxide was dissolved in 106 g of distilled water, and 10 g of dextrin (Sandec (registered trademark) #100, manufactured by Sanwa Starch Industrial Co., Ltd.) was gradually added thereto, dissolved, and stirred for 90 minutes. After stirring, an aqueous solution of chloroacetic acid (7 g of chloroacetic acid dissolved in 63 g of distilled water) was added, and the mixture was reacted at 60° C. for 6 hours. After cooling to room temperature (25°C), 65 g of 20% by mass aqueous hydrochloric acid solution was added and stirred for 2 hours. The resulting solution was reprecipitated in 2500 mL of methanol, and the residue was washed with methanol and then vacuum-dried at room temperature (25°C) for 24 hours to obtain 10.25 g of carboxymethyl dextrin. NHS-carboxymethyldextrin was obtained in the same manner as in Synthesis Example 1, except that carboxymethyldextran X was replaced with carboxymethyldextrin and the amounts of N-hydroxysuccinimide and EDC hydrochloride added were changed to 3.31 g and 5.51 g, respectively. The NHS-carboxymethyldextrin was composed of the structural unit represented by the above-mentioned general formula (1), the structural unit represented by formula (2), and the structural unit represented by formula (3). Furthermore, the content of the structural unit derived from a monosaccharide having an N-hydrosuccinimide ester group (structural unit represented by general formula (1)) relative to 100 mol % of NHS-carboxymethyldextrin was calculated to be 10 mol %. Similarly, the contents of the constitutional unit represented by formula (2) and the constitutional unit represented by formula (3) were calculated to be 70 mol % and 20 mol %, respectively. The Mw of the NHS-carboxymethyldextrin was 18,000.

[0054] (Synthesis Example 4) 10 g of carboxymethyl dextran X synthesized in Synthesis Example 1 was dissolved in 100 g of DMAc, and 5.67 g of N-hydrosuccinimide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and 9.45 g of EDC hydrochloride (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were added, followed by stirring at 40°C for 4 hours. The resulting solution was reprecipitated in 350 mL of methanol, and the residue was washed with methanol and acetone and then vacuum dried at room temperature (25°C) for 24 hours to obtain 11.2 g of NHS-carboxymethyl dextran B. The NHS-carboxymethyl dextran B was composed of the structural unit represented by the above-mentioned general formula (1), the structural unit represented by formula (2), and the structural unit represented by formula (3). Furthermore, the content of the structural unit derived from a monosaccharide having an N-hydrosuccinimide ester group (structural unit represented by general formula (1)) relative to 100 mol % of NHS-carboxymethyldextran B was calculated to be 21 mol %. Similarly, the contents of the constitutional unit represented by formula (2) and the constitutional unit represented by formula (3) were calculated to be 70 mol % and 9 mol %, respectively. The Mw of NHS-carboxymethyl dextran B was 47,000.

[0055] (Synthesis Example 5) 10 g of carboxymethyl dextran X synthesized in Synthesis Example 1 was dissolved in 100 g of DMAc, and 1.32 g of N-hydrosuccinimide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and 2.20 g of EDC hydrochloride (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were added, followed by stirring at 40°C for 4 hours. The resulting solution was reprecipitated in 350 mL of methanol, and the residue was washed with methanol and acetone and then vacuum dried at room temperature (25°C) for 24 hours to obtain 10.1 g of NHS-carboxymethyl dextran C. The NHS-carboxymethyl dextran C was composed of the structural unit represented by the above-mentioned general formula (1), the structural unit represented by formula (2), and the structural unit represented by formula (3). Furthermore, the content of the structural unit derived from a monosaccharide having an N-hydrosuccinimide ester group (structural unit represented by general formula (1)) relative to 100 mol % of NHS-carboxymethyldextran C was calculated to be 2.2 mol %. Similarly, the contents of the constitutional unit represented by formula (2) and the constitutional unit represented by formula (3) were calculated to be 70 mol % and 27.8 mol %, respectively. The Mw of NHS-carboxymethyldextran C was 44,000.

[0056] (Synthesis Example 6) NHS-carboxymethyldextran D was obtained in the same manner as in Synthesis Example 1, except that carboxymethyldextran sodium A was changed to carboxymethyldextran sodium B (manufactured by Meito Sangyo Co., Ltd., CMD-500, Mw 500000). Note that NHS-carboxymethyldextran D was composed of the constitutional unit represented by the above-mentioned general formula (1), the constitutional unit represented by formula (2), and the constitutional unit represented by formula (3). Furthermore, the content of the structural unit derived from a monosaccharide having an N-hydrosuccinimide ester group (structural unit represented by general formula (1)) relative to 100 mol % of NHS-carboxymethyldextran D was calculated to be 15 mol %. Similarly, the contents of the constitutional unit represented by formula (2) and the constitutional unit represented by formula (3) were calculated to be 70 mol % and 15 mol %, respectively. The Mw of NHS-carboxymethyl dextran D was 530,000.

[0057] (Synthesis Example 7) NHS-carboxymethyldextran E was obtained in the same manner as in Synthesis Example 1, except that carboxymethyldextran sodium A was replaced with carboxymethyldextran sodium C (manufactured by Meito Sangyo Co., Ltd., CMD-L, Mw 10000). NHS-carboxymethyldextran G was composed of the constitutional unit represented by the above-mentioned general formula (1), the constitutional unit represented by formula (2), and the constitutional unit represented by formula (3). Furthermore, the content of the structural unit derived from a monosaccharide having an N-hydrosuccinimide ester group (structural unit represented by general formula (1)) relative to 100 mol % of NHS-carboxymethyldextran E was calculated to be 15 mol %. Similarly, the contents of the constitutional unit represented by formula (2) and the constitutional unit represented by formula (3) were calculated to be 70 mol % and 15 mol %, respectively. The Mw of NHS-carboxymethyldextran E was 11,000.

[0058] (Synthesis Example 8) 5 g of diphenylmethane diisocyanate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in a mixed solution of 90 g of toluene (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and 5 g of diphenylmethane diisocyanate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and 5 g of chitosan (manufactured by General Science Corporation, Mw 15000, powder form, deacetylation degree 84%) was dispersed therein. The resulting dispersion was stirred at 100°C for 24 hours, cooled to room temperature (25°C), filtered, and the resulting powder was washed with toluene and acetone and air-dried to obtain an isocyanated chitosan powder. The Mw of the isocyanated chitosan was 15,000.

[0059] (Synthesis example A) High-grade gelatin (Mw 60,000) manufactured by Nippi Corporation was irradiated with gamma rays to an absorbed dose of 50 kGy to obtain cross-linked gelatin. Crosslinked gelatin was crushed in a mortar and pestle, and then sieved using a mesh sieve to obtain crosslinked gelatin particles A having an average primary particle diameter of 20 μm or more and less than 100 μm, crosslinked gelatin particles B having an average primary particle diameter of 100 μm or more and less than 200 μm, and crosslinked gelatin particles C having an average primary particle diameter of 200 μm or more and less than 700 μm.

[0060] <Examples 1 to 20 and Comparative Examples 1 to 6> The components shown in Tables 1 to 3 were mixed to produce a paste-like hemostatic composition.

[0061] Details of the components shown in Tables 1 to 3 are as follows. (hydrophobic solvent) Vegetable oil A: Japanese Pharmacopoeia olive oil, manufactured by Kenei Pharmaceutical Co., Ltd., kinematic viscosity 72 mm at 25°C 2 / sec, non-volatile Vegetable oil B: Kaneda Co., Ltd., Japanese Pharmacopoeia soybean oil, kinematic viscosity at 25°C 60mm 2 / sec, non-volatile paraffin solvent: liquid paraffin NikP manufactured by Nichi-Iko Pharmaceutical Co., Ltd., kinematic viscosity at 25°C 37mm 2 / sec, non-volatile Silicone oil solvent A: Shin-Etsu Chemical Co., Ltd., KF-96-10cs, kinematic viscosity 10 mm at 25°C 2 / sec, non-volatile Silicone oil solvent B: Shin-Etsu Chemical Co., Ltd., KF-96-500cs, kinematic viscosity at 25°C 500mm 2 / sec, non-volatile Fatty acid ester solvent: methyl oleate, manufactured by Kao Corporation, Exepar (registered trademark) M-OL, kinematic viscosity at 25°C 5mm 2 / sec, non-volatile When 20 g of the above hydrophobic solvent was added to 1 L of ion-exchanged water (pH 7) at 20°C, it separated into two layers and did not become a homogeneous aqueous solution, confirming that its solubility in water was less than 20 g / L.

[0062] (hydrophilic solvent) Polyethylene glycol: Fujifilm Wako Pure Chemical Industries, Ltd., PEG200, kinematic viscosity at 25°C 62mm 2 / sec, non-volatile Polyethylene glycol / polypropylene glycol copolymer: BASF Pluronic L61, kinematic viscosity 300mm at 25°C 2 / sec, non-volatile Dimethyl sulfoxide (DMSO): Fujifilm Wako Pure Chemical Industries, Ltd., non-volatile When 20 g of the hydrophilic solvent was added to 1 L of ion-exchanged water (pH 7) at 20° C., a homogeneous aqueous solution was obtained, and it was confirmed that the solubility in water was 20 g / L or more.

[0063] (non-specific polysaccharide) Dextran 40000: Fujifilm Wako Pure Chemical Industries, Ltd. Pentaerythritol poly(ethylene glycol) ether tetrasuccinimidyl glutarate: Yuka Sangyo Co., Ltd., SUNBRIGHT PTE-100GS, Mw 10000

[0064] (crosslinked particles) Cross-linked polysaccharide particles: DFE Pharma, Primellose, cross-linked sodium carboxymethylcellulose, average primary particle size 50 μm

[0065] <<Storage stability evaluation>> The hemostatic compositions produced in Examples 1 to 20 and Comparative Examples 1 to 6 were stored in an environment at 50°C for 10 days. The pigs were subjected to abdominal sectioning, and a bleeding wound measuring 8 mm in diameter and 2 mm deep was created on the surface of the liver using a biopsy trephine. After storage, 0.5 g of each hemostatic composition was applied to the bleeding wound to cover it, forming a film, and the wound was then compressed with gauze moistened with physiological saline. The condition of the bleeding wound was visually observed 1 minute, 2 minutes, 5 minutes, and 10 minutes after application of the hemostatic composition, and evaluated based on the following evaluation criteria, with the results summarized in Tables 1 to 3. A rating of 3 to 5 was considered to indicate good storage stability. (Evaluation criteria) 5: Observation of the bleeding wound after 1 minute confirmed that hemostasis had been achieved. 4: When the bleeding wound was observed after 1 minute, it was found that bleeding had not stopped. However, when the bleeding wound was observed after 2 minutes, it was confirmed that bleeding had stopped. 3: When the condition of the bleeding wound was observed after 2 minutes, it was found that hemostasis had not been achieved, but when the condition of the bleeding wound was observed after 5 minutes, it was confirmed that hemostasis had been achieved. 2: Observation of the bleeding wound after 5 minutes showed that hemostasis had not been achieved, but observation of the bleeding wound after 10 minutes confirmed that hemostasis had been achieved. 1: Even after 10 minutes of observation of the bleeding wound, bleeding had not stopped.

[0066] <<Hemostatic evaluation>> The pigs were subjected to abdominal sectioning, and a bleeding wound measuring 8 mm in diameter and 2 mm deep was created in the liver using a biopsy trephine. 0.5 g of each of the hemostatic compositions produced in Examples 1 to 20 and Comparative Examples 1 to 6 was applied to the bleeding wound to cover it, forming a film, and then the wound was compressed with gauze moistened with saline. The condition of the bleeding wound was visually observed 1 minute, 2 minutes, 5 minutes, and 10 minutes after application of the hemostatic composition, and evaluated based on the following evaluation criteria, as summarized in Tables 1 to 3. A rating of 3 to 5 was considered to indicate good hemostatic properties. (Evaluation criteria) 5: Observation of the bleeding wound after 1 minute confirmed that hemostasis had been achieved. 4: When the bleeding wound was observed after 1 minute, it was found that bleeding had not stopped. However, when the bleeding wound was observed after 2 minutes, it was confirmed that bleeding had stopped. 3: When the condition of the bleeding wound was observed after 2 minutes, it was found that hemostasis had not been achieved, but when the condition of the bleeding wound was observed after 5 minutes, it was confirmed that hemostasis had been achieved. 2: Observation of the bleeding wound after 5 minutes showed that hemostasis had not been achieved, but observation of the bleeding wound after 10 minutes confirmed that hemostasis had been achieved. 1: Even after 10 minutes of observation of the bleeding wound, bleeding had not stopped.

[0067] <<Removability evaluation>> The hemostatic compositions produced in Examples 1 to 20 and Comparative Examples 1 to 6 were applied to a slide glass plate (2.5 cm×7.5 cm) to a thickness of 2 mm. After application, the slide glass plate was gently immersed in 50 cc of physiological saline, removed after 10 seconds and 1 minute, and visually observed. The results were evaluated based on the following evaluation criteria, and the results are summarized in Tables 1 and 2. A rating of 3 to 5 was considered to indicate good removability. (Evaluation criteria) 5: When the glass slide plate was removed 10 seconds after the start of immersion, no residual hemostatic composition was found. 4: A small amount of hemostatic composition was found remaining on the glass slide plate when it was removed 10 seconds after the start of immersion, but no hemostatic composition was found remaining on the glass slide plate when it was removed 1 minute after the start of immersion. 3: A small amount of residual hemostatic composition was confirmed on both the glass slide plates removed 10 seconds and 1 minute after the start of immersion. 2: A large amount of the hemostatic composition remained on both the glass slide plates removed 10 seconds and 1 minute after the start of immersion. 1: When the glass slide plates were removed 10 seconds and 1 minute after the start of immersion, there was no sign of the hemostatic composition being removed, and it was confirmed that almost the entire amount of the applied hemostatic composition remained.

[0068] <<Evaluation of transcriptional repression>> 0.5 g of each of the hemostatic compositions produced in Examples 1 to 20 and Comparative Examples 1 to 6 was applied to the surface of an excised pig liver to form a coating, which was then pressed with gauze moistened with saline for 2 minutes. The gauze was then slowly peeled off, and the surface of the gauze and the surface of the excised liver were visually observed and evaluated based on the following evaluation criteria, and the results are summarized in Tables 1 to 3. A rating of 3 to 5 was considered to indicate good transcriptional suppression. (Evaluation criteria) 5: No transfer of the hemostatic composition to the gauze was observed, and no peeling of the capsule on the surface of the excised liver was observed. 4: A small amount of the hemostatic composition was transferred to the gauze, but no peeling of the capsule on the surface of the excised liver was observed. 3: Slight transfer of the hemostatic composition to the gauze was observed, and slight peeling of the capsule on the surface of the excised liver was observed. 2: There was a lot of transfer of the hemostatic composition to the gauze, and there was a lot of peeling of the capsule on the surface of the excised liver. 1: A large amount of the hemostatic composition was transferred to the gauze, and most of the capsule on the surface of the excised liver was peeled off.

[0069] [Table 1]

[0070] [Table 2]

[0071] [Table 3]

[0072] Tables 1 and 2 show that the hemostatic composition of the present disclosure, which contains a specific polysaccharide, a hydrophobic solvent, and crosslinked particles, has excellent storage stability. Table 3 shows that there is room for improvement in the storage stability of hemostatic compositions containing hydrophilic solvents. Furthermore, Table 3 shows that there is room for improvement in hemostatic properties and storage stability of hemostatic material compositions that do not contain specific polysaccharides.

Claims

1. The present invention comprises a polysaccharide having a reactive group that reacts with a protein to form a crosslinked structure, a hydrophobic solvent, and crosslinked particles containing one or more compounds selected from the group consisting of crosslinked proteins and crosslinked polysaccharides, The hydrophobic solvent is a vegetable oil. Hemostat composition.

2. the cross-linked particles comprise the cross-linked protein; and The hemostatic composition of claim 1, wherein the cross-linked protein is cross-linked gelatin.

3. 3. The hemostatic composition according to claim 1, wherein the crosslinked particles have an average primary particle size of 20 μm to 700 μm.

4. The reactive group is —CON(COCH ２ ) ２ 4. The hemostatic composition according to claim 1, wherein the hydroxyl group is one or more groups selected from the group consisting of an N-hydroxysuccinimide ester group, an imide ester group, an aldehyde group, a carboxy group, an isocyanate group, a maleimide group, and a haloacetyl group.

5. The hemostatic composition according to any one of claims 1 to 4, wherein the reactive group is an N-hydroxysuccinimide ester group.

6. The hemostatic composition according to any one of claims 1 to 5, wherein the polysaccharide comprises a structure derived from one or more polysaccharides selected from the group consisting of cellulose, dextran, dextrin, pullulan, hyaluronic acid, alginic acid, xanthan gum, chitin, chitosan, and modified forms thereof.

7. the polysaccharide comprises a constitutional unit derived from the monosaccharide having the reactive group; and The hemostatic composition according to any one of claims 1 to 6, wherein the content of structural units derived from monosaccharides having the reactive group relative to 100 mol% of the polysaccharide is 1 mol% to 40 mol%.

8. The hemostatic composition according to claim 7, wherein the structural unit derived from a monosaccharide having a reactive group is represented by the following general formula (1): 【Chemistry 1】 In the general formula (1), L is a group represented by the following formula: 【Chemistry 2】 In the above formula, n and m represent integers of 1 to 3, * represents the site of linkage to the oxygen atom on the tetrahydropyran ring side, and ** represents the site of linkage to the oxygen atom on the N-hydroxysuccinimide ester group side.

9. The hemostatic composition according to any one of claims 1 to 8, wherein the polysaccharide has a weight average molecular weight of 2,000 to 2,500,000.

10. The kinematic viscosity of the hydrophobic solvent at 25°C is 1 mm ２ / sec ~ 1000mm ２ The hemostatic composition according to any one of claims 1 to 9, wherein the viscosity is 1 / sec.

11. The hemostatic composition according to any one of claims 1 to 10, wherein the content of the hydrophobic solvent relative to the total mass of the hemostatic composition is 30 mass % to 80 mass %.

12. The hemostatic composition according to any one of claims 1 to 11, which does not contain water, or which contains water and the water content relative to the total mass of the hemostatic composition is 0.5 mass % or less.

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