Hemostatic composition and container containing the composition

Abstract

The present invention relates to a hemostatic composition comprising collagen, a stabilizer and thrombin and a container comprising the composition. The hemostatic composition and the container comprising the composition can be used for patients with urgent bleeding through a simple method, are non-toxic, do not cause blood infection, have a fast biodegradation rate, and have a good hemostatic effect, so that they can be effectively used as a hemostatic agent.

Description

Technical Field

[0001] This application claims the benefit of priority based on Korean Patent Application No. 10-2019-0062613, filed on May 28, 2019, and all contents disclosed in the Korean Patent Application document are incorporated herein as a part of this specification.

[0002] The present invention relates to a hemostatic composition and a container containing the composition. Background Art

[0003] Research and development in the field of tissue adhesives, including sealants and hemostats, has grown rapidly. Since the U.S. FDA approved fibrin sealants in 1998, new tissue adhesives have been continuously developed every year. The above-mentioned tissue adhesives have attracted much attention as materials that can replace suturing, clipping, moxibustion and other techniques used in surgical or medical operations in the prior art.

[0004] Although existing surgical techniques such as suturing have strong tensile strength, they cause pain to patients and require removal after surgery. On the contrary, although tissue adhesives have many advantages such as rapid bonding, ease of use, and no need for removal after surgery, they have limitations such as low adhesiveness and tensile strength and significant reduction in adhesiveness in the presence of moisture. In order to overcome the above limitations of tissue adhesives, continuous research has been conducted.

[0005] Since tissue adhesives are in direct contact with tissues, biocompatibility is required. In addition, in the case of medical adhesives, since they are usually used in the human body, after the adhesive essentially flows into body fluids and blood, the human body directly intervenes, so more stringent conditions are required to eliminate toxicity and harmfulness, requiring more stringent biocompatibility and biodegradable materials.

[0006] Currently, commercialized and / or practical tissue adhesives include cyanoacrylate instant adhesives, fibrin glue, gelatin, and polyurethane. Recently, cyanoacrylate instant adhesives have attracted much attention in the research of instant adhesives with high functionality and high performance. In particular, medical instant adhesives for tissue sutures that are biocompatible, flexible, and low in toxicity not only have hemostatic and antibacterial effects, but can also replace sutures, so research is actively carried out, mainly in developed countries.

[0007] The above-mentioned cyanoacrylate instant adhesives have been commercialized in the form of products such as Dermabond (Johnson & Johnson) and Indermil (US Surgical). Although the above-mentioned cyanoacrylate instant adhesives have the advantages of being a single substance that can be cured by moisture in a short time at room temperature without an initiator, transparent in appearance, and adhesive cavity, they have the disadvantages of weak impact resistance and low heat resistance. In addition, due to its high toxicity, it is rarely used now. Although it is partially used in clinical practice in other countries except the United States, its use is limited due to its toxicity and fragility to some tissues.

[0008] In addition, fibrin glue adhesive was first approved by the FDA for use in cardiac surgery in 1998. After that, the research and development of fibrin tissue adhesives was very active, and products such as Tisseel VH (Baxer) and Evicel TM (Johnson & Johnson) have been commercialized. Fibrin-based tissue adhesives, together with cyanoacrylates, occupy most of the tissue adhesives. Fibrin tissue adhesives use the cross-linking reaction of fibrin to use fibrinogen, thrombin, calcium chloride and enzyme inhibitors as tissue adhesives, and are applied to the suturing of peripheral nerves, the suturing of microvessels, etc., to implement clinical applications of replacing or strengthening sutures. The above-mentioned fibrin glue adhesive has the advantages of not being affected by the moisture of the bonding site, rapid bonding, no coagulation barriers with platelets, and good biocompatibility. However, it has the limitations of weak bonding, fast biodegradation, and the risk of blood infection.

[0009] In addition, gelatin has been developed as a tissue adhesive derived from the human body, and a tissue adhesive formed by cross-linking gelatin-resorcinol-formalin (GRF). In addition, although tissue adhesives such as gelatin-glutaraldehyde have been developed, which have high tissue adhesion, there is a disadvantage that formalin or glutaraldehyde used as a cross-linking agent also undergoes a cross-linking reaction with proteins in the human body, thereby generating tissue toxicity.

[0010] In addition, polyurethane adhesives have been developed as elastic adhesives that can maintain the flexibility of the joint after curing. The characteristics of this adhesive are that it improves the adhesion to the tissue by absorbing water on the surface of human tissue, and cures within a few minutes after reacting with water to make the cured product flexible. It has the advantage that the cured adhesive is suitable for biodegradation. However, it has the disadvantage that the aromatic diisocyanate used as the synthetic raw material is toxic to the human body.

[0011] In addition, hemostatic agents containing only collagen components, such as "Avitene" (Alcon) and "Helitene" (Duhamed), are non-blood preparations but are very expensive. As a single component, they have only a tissue adhesion effect and are therefore used only as hemostatic agents.

[0012] In addition, as another prior art, there are "Fibrin monomer based tissue adhesive, US Patent 5464471, 1995", "Hemostatic and tissue adhesive, US Patent 5883078, 1999", "Fibrinogen / chitosan hemostatic agents, US Patent 5773033, 1998", "Therapeutic fibrinogen compositions, US Patent 5605887, 1997" and the like. However, these adhesive hemostatic products use blood products such as fibrinogen, thrombin, blood coagulants, and peptidase, as well as proteins derived from cows, and thus there is a risk of infection with specific diseases caused thereby and the problem of high product prices due to excessive costs required for raw material procurement, storage, and manufacturing processes.

[0013] In addition, FloSeal (Baxter) containing gelatin matrix components is the most used product, which is a product that adds thrombin mixed in calcium chloride solution to the gelatin matrix to have a hemostatic effect. This is particularly useful for bleeding situations that cannot be effectively regulated or cannot be regulated by ligature or general measures during surgical operations on various areas other than the eyes. However, this is roughly divided into three parts: thrombin, gelatin, and calcium chloride solution. Each solution cannot be mixed at once, but needs to be mixed in two steps, so it takes some time. Therefore, when performing a surgical operation, in an emergency situation where serious ligation and bleeding occur, there is no ample time for mixing according to the complicated product instructions, and the process carried out in sequence may threaten the life of the bleeding patient who cannot wait, which is very cumbersome for the user.

[0014] In addition, since the above-mentioned hemostatic products are used on the human body, the highest safety standards for the quality of their ingredients, storage safety, and simple usage methods must be provided. In particular, when two or more ingredients are mixed, they must be prepared in a "ready-to-use" form and provided in an easy-to-mix state.

[0015] Therefore, it is necessary to develop a hemostatic agent that is very low in toxicity, easy to store and use, simple in method of use, can be used for patients in urgent need of treatment, and has good biodegradability.

[0016] Prior art literature

[0017] [Patent Literature]

[0018] (Patent Document 1) U.S. Patent Registration No. 5464471

[0019] (Patent Document 2) U.S. Patent Registration No. 5773033

[0020] (Patent Document 3) U.S. Patent Registration No. 5605887 Summary of the invention

[0021] Problem that the invention aims to solve

[0022] In order to solve the above problems, the inventors have developed a hemostatic composition comprising collagen, a stabilizer, and thrombin, and a container comprising the composition, which can be used for patients with urgent bleeding through a simple method, is non-toxic, does not cause blood infection, has a fast biodegradation rate, and has a good hemostatic effect, thereby completing the present invention.

[0023] Therefore, an object of the present invention is to provide a hemostatic composition comprising collagen, a stabilizer, and thrombin, and a container comprising the composition.

[0024] Another object of the present invention is to provide a kit comprising a first container filled with the hemostatic composition and a second container filled with a diluent.

[0025] Another object of the present invention is to provide a hydrogel produced using the above hemostatic kit.

[0026] Solutions to the problem

[0027] To achieve the above object, according to one aspect of the present invention, there is provided a hemostatic composition in the form of a powder comprising collagen, a stabilizer and thrombin, wherein the collagen and thrombin are disposed adjacent to each other.

[0028] More specifically, the present invention provides a hemostatic composition comprising a first layer comprising collagen powder, a second layer comprising stabilizer powder and a third layer comprising thrombin powder, which are layered and filled in a container, wherein the first layer comprising collagen powder and the third layer comprising thrombin powder are adjacent to each other and / or the second layer comprising stabilizer powder is arranged at a position to be first mixed with a diluent.

[0029] The above-mentioned hemostatic composition can be arranged in the order of stabilizer, thrombin, collagen or stabilizer, collagen, thrombin; or can be arranged in the order of thrombin, collagen, stabilizer or collagen, thrombin, stabilizer.

[0030] More specifically, the above-mentioned hemostatic composition can be arranged in the order of a second layer comprising stabilizer powder, a third layer comprising thrombin powder, a first layer comprising collagen powder, or a second layer comprising stabilizer powder, a first layer comprising collagen powder, and a third layer comprising thrombin powder, or can be arranged in the order of a third layer comprising thrombin powder, a first layer comprising collagen powder, a second layer comprising stabilizer powder, or a first layer comprising collagen powder, a third layer comprising thrombin powder, and a second layer comprising stabilizer powder.

[0031] In the above-mentioned hemostatic composition, the above-mentioned collagen is cross-linked collagen.

[0032] In the above-mentioned hemostatic composition, the above-mentioned cross-linked collagen is prepared by the following manufacturing method, comprising:

[0033] S1) a step of treating collagen with ethanol or methanol;

[0034] S2) adding acid to the collagen treated in step S1) to produce a collagen solution with a pH of 2 to 4;

[0035] S3) neutralizing the collagen solution obtained in step S2) and centrifuging to produce esterified collagen;

[0036] S4) a step of adding a cross-linking agent to the esterified collagen obtained in the above step S3) to produce cross-linked collagen; and

[0037] S5) dispersing the cross-linked collagen obtained in step S4) in purified water, followed by freeze-drying.

[0038] In the above hemostatic composition, the molecular weight of the above cross-linked collagen is 100,000 to 1,000,000 Dalton.

[0039] The hemostatic composition contains the collagen in an amount of 40 to 97 wt % based on the total weight of the hemostatic composition.

[0040] In the above-mentioned hemostatic composition, the above-mentioned stabilizer is one or more selected from the group consisting of albumin (human serum albumin), mannitol, sodium acetate (C2H3NaO2), sucrose, trehalose, sorbitol and glycine.

[0041] In the above hemostatic composition, the above stabilizer is mannitol.

[0042] The hemostatic composition contains the stabilizer in an amount of 1 to 30 wt % based on the total weight of the hemostatic composition.

[0043] The hemostatic composition contains 2 to 50 wt % of the thrombin based on the total weight of the hemostatic composition.

[0044] According to another aspect of the present invention, a container comprising the above-mentioned hemostatic composition is provided.

[0045] According to another aspect of the present invention, there is provided a hemostatic kit, comprising: a first container filled with a hemostatic composition in powder form containing collagen, a stabilizer and thrombin; and a second container filled with a diluent; in the first container, the collagen and thrombin are adjacently arranged.

[0046] More specifically, the present invention provides a hemostatic kit, comprising: a first container filled with a hemostatic composition that is layered and comprises a first layer comprising collagen powder, a second layer comprising stabilizer powder, and a third layer comprising thrombin powder; and a second container filled with a diluent; in the first container, the first layer comprising collagen powder and the third layer comprising thrombin powder are arranged adjacent to each other, and the second layer comprising stabilizer powder is arranged at a position to be mixed with the diluent first.

[0047] In the above-mentioned hemostatic kit, the above-mentioned diluent is one or more selected from the group consisting of calcium chloride (CaCl2) solution, sodium chloride (NaCl) solution, human serum albumin (human serum albumin) and sodium acetate (C2H3NaO2) solution.

[0048] In the above-mentioned hemostatic kit, the above-mentioned diluent is a calcium chloride solution; relative to the total weight of the above-mentioned diluent, the above-mentioned calcium chloride is contained in an amount of 0.001 to 30 weight %.

[0049] In the above hemostatic kit, the first container and the second container are combined with each other so that the hemostatic composition and the diluent are mixed to form a hydrogel; the average pore size of the hydrogel is 50 μm to 200 μm.

[0050] In the hemostatic kit, the pH of the hydrogel is 6 to 8.

[0051] In the above-mentioned hemostatic kit, the above-mentioned first container is filled in the order of stabilizer, thrombin, collagen or stabilizer, collagen, thrombin; or filled in the order of thrombin, collagen, stabilizer or collagen, thrombin, stabilizer.

[0052] More specifically, the above-mentioned first container can be filled in the order of a second layer containing stabilizer powder, a third layer containing thrombin powder, and a first layer containing collagen powder, or a second layer containing stabilizer powder, a first layer containing collagen powder, and a third layer containing thrombin powder, or can be filled in the order of a third layer containing thrombin powder, a first layer containing collagen powder, and a second layer containing stabilizer powder, or a first layer containing collagen powder, a third layer containing thrombin powder, and a second layer containing stabilizer powder.

[0053] In the above hemostatic kit, the collagen is cross-linked collagen.

[0054] In the above-mentioned hemostatic kit, the above-mentioned cross-linked collagen is prepared by the following manufacturing method, comprising:

[0055] S1) a step of treating collagen with ethanol or methanol;

[0056] S2) adding acid to the collagen treated in step S1) to produce a collagen solution with a pH of 2 to 4;

[0057] S3) neutralizing the collagen solution obtained in step S2) and centrifuging to produce esterified collagen;

[0058] S4) a step of adding a cross-linking agent to the esterified collagen obtained in the above step S3) to produce cross-linked collagen; and

[0059] S5) dispersing the cross-linked collagen obtained in step S4) in purified water, followed by freeze-drying.

[0060] In the above hemostatic kit, the molecular weight of the above cross-linked collagen is 100,000 to 1,000,000 Dalton.

[0061] In the hemostatic kit, the collagen is contained in an amount of 40 to 97 wt % based on the total weight of the hemostatic composition.

[0062] In the hemostatic kit, the stabilizer is one or more selected from the group consisting of albumin (human serum albumin), mannitol, sodium acetate (C2H3NaO2), sucrose, trehalose, sorbitol and glycine.

[0063] In the above hemostatic kit, the stabilizer is mannitol.

[0064] In the hemostatic kit, the stabilizer is contained in an amount of 1 to 30 wt % based on the total weight of the hemostatic composition.

[0065] The hemostatic kit contains 2 to 50 wt % of the thrombin relative to the total weight of the hemostatic composition.

[0066] Effects of the Invention

[0067] The hemostatic composition comprising collagen, a stabilizer and thrombin of the present invention has a good hemostatic effect.

[0068] In addition, the hemostatic composition of the present invention comprising collagen, a stabilizer and thrombin can be stored easily because the activity of thrombin is stabilized, and can be used conveniently and quickly because the composition can be mixed at a low weight.

[0069] In addition, the container containing the hemostatic composition containing collagen, stabilizer and thrombin of the present invention can be used conveniently and quickly, and because it is non-toxic, there is no problem of blood infection, the biodegradation rate is fast, and it expands when in contact with blood, and the expansion rate is large, so the hemostatic effect is good.

[0070] Therefore, the hemostatic composition of the present invention and the container containing the composition can be used as a hemostatic kit.

[0071] BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 This is a curve obtained by measuring the force required for mixing with the number of times when Example 1 and Comparative Example 1 are mixed with a calcium chloride solution;

[0073] Figure 2 The SEM images of the hydrogels prepared by mixing Example 1 and Comparative Example 1 with a calcium chloride solution;

[0074] Figure 3 The graph is a graph obtained by measuring the expansion ratios of Example 1 and Comparative Example 1;

[0075] Figure 4 A graph showing the area changes of Example 1 and Comparative Example 1;

[0076] Figure 5 This is a curve graph obtained by applying Example 1 and Comparative Example 1 to a rat nephrectomy model and measuring the time required for hemostasis;

[0077] Figure 6 The graphs show the hemostatic ability of Example 1 and Comparative Example 1 after each application to a rat nephrectomy model.

[0078] Best Mode for Carrying Out the Invention

[0079] Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not limited to the examples and is not limited by the examples, but is defined only by the scope of the claims to be described later.

[0080] In the process of describing the present invention in detail, if the detailed description of the related disclosed function or structure is considered to hinder the understanding of the present invention, its detailed description will be omitted.

[0081] When describing the components of the present invention, terms such as first, second, A, B, (a), (b), etc. may be used. The above terms are only used to distinguish one component from another, and the nature or order of the corresponding components is not limited by the above terms.

[0082] In the process of describing the present invention, the terms "approximately", "substantially", "extent" and the like which express degree are used to disclose the inherent manufacturing and material tolerances in the meaning mentioned, and the meaning is close to its numerical value. The purpose is to prevent unscrupulous infringers from improperly using the content that mentions correct or absolute numerical values ​​to help understand.

[0083] The present invention provides a hemostatic composition in the form of a powder comprising collagen, a stabilizer and thrombin, wherein the collagen and thrombin are disposed adjacent to each other.

[0084] Specifically, the hemostatic composition can be filled in the order of stabilizer, thrombin, collagen or stabilizer, collagen, thrombin; or can be filled in the order of thrombin, collagen, stabilizer or collagen, thrombin, stabilizer. The hemostatic composition can be mixed with a diluent when used. In this case, preferably, the stabilizer is arranged at the position where the hemostatic composition is first mixed with the diluent. Therefore, preferably, the stabilizer, thrombin, collagen or stabilizer, collagen, thrombin are arranged in the order of stabilizer, thrombin, collagen or stabilizer, collagen, thrombin.

[0085] The hemostatic composition of the present invention may be in the form of layers of each component in the form of a powder. Specifically, it is a hemostatic composition in which a first layer comprising collagen powder, a second layer comprising stabilizer powder, and a third layer comprising thrombin powder are layered and filled in a container, wherein the first layer comprising collagen powder and the third layer comprising thrombin powder may be arranged adjacent to each other, or further, the second layer comprising stabilizer powder may be arranged at a position first mixed with the diluent. Therefore, the hemostatic composition may be arranged in the order of the second layer comprising stabilizer powder - the third layer comprising thrombin powder - the first layer comprising collagen powder or the second layer comprising stabilizer powder - the first layer comprising collagen powder - the third layer comprising thrombin powder, or the third layer comprising thrombin powder - the first layer comprising thrombin powder - the second layer comprising stabilizer powder, or the first layer comprising collagen powder - the third layer comprising thrombin powder - the second layer comprising stabilizer powder.

[0086] The collagen is contained in the hemostatic composition, and absorbs blood from the bleeding site, pulling platelets to achieve hemostasis. In addition, when the collagen adheres to the platelets, it does not bind in a state of nothingness, but binds to vWF (von Willebrand's factor), which is a substance that acts like an adhesive, to promote the binding of platelets to the GPIb / IX complex, which is a membrane glycoprotein present on the cell surface, thereby achieving primary hemostasis.

[0087] The collagen may be a protein extracted by subjecting tissues of various animals to acid or alkali treatment or to enzyme treatment such as pepsin.

[0088] The collagen may be contained in an amount of 40 to 97% by weight relative to the total weight of the hemostatic composition. If the collagen is contained in an amount of less than 40% by weight, the hemostatic effect may be reduced, and if the collagen is contained in an amount of more than 97% by weight, the chemical stability of the entire hemostatic composition may be reduced or the hemostatic effect may be reduced due to the relatively small amount of thrombin and stabilizer.

[0089] In addition, the collagen may be cross-linked collagen, and in this case, preferably, the molecular weight of the cross-linked collagen is 100,000 to 1,000,000 Dalton, but this is not a limitation. If the molecular weight of the cross-linked collagen is less than 100,000 Dalton, the blood coagulation is not sufficient although the blood absorption capacity is high, which may reduce the hemostatic effect, and if it exceeds 1,000,000 Dalton, the blood absorption capacity may be significantly reduced.

[0090] The cross-linked collagen can be prepared by chemical cross-linking, physical cross-linking or a combination thereof. In this case, the cross-linked collagen prepared by chemical cross-linking is collagen that is self-crosslinked by a cross-linking agent. In addition, the cross-linked collagen prepared by physical cross-linking is collagen that is cross-linked by dry heat treatment, ultraviolet irradiation or gamma ray irradiation.

[0091] Here, the crosslinking agent may be one or more crosslinking agents selected from the group consisting of formaldehyde, glutaraldehyde, carbodiimides (EDC), and polyepoxy compounds, preferably carbodiimides (EDC), but not limited thereto.

[0092] The above cross-linked adhesive has good mechanical strength and can achieve physical compression hemostasis effect, thereby stopping heavy bleeding.

[0093] The cross-linked collagen may be collagen obtained by cross-linking esterified collagen.

[0094] In one embodiment of the present invention, the cross-linked collagen is prepared by the following manufacturing method, comprising:

[0095] S1) a step of treating collagen with ethanol or methanol;

[0096] S2) adding acid to the collagen treated in step S1) to produce a collagen solution with a pH of 2 to 4;

[0097] S3) neutralizing the collagen solution obtained in step S2) and centrifuging to produce esterified collagen;

[0098] S4) a step of adding a cross-linking agent to the esterified collagen obtained in the above step S3) to produce cross-linked collagen; and

[0099] S5) dispersing the cross-linked collagen obtained in step S4) in purified water, followed by freeze-drying.

[0100] The above-mentioned step S1) is a step of treating collagen with ethanol or methanol, a step of dissolving collagen in ethanol or methanol to facilitate subsequent mixing with a cross-linking agent, and a step of preparing a collagen solution.

[0101] Before the above step S1), the method may further include treating the animal skin tissue with an acidic solution to extract atelocollagen.

[0102] The step S2) is a step of adding an acid to the collagen treated in the step S1) to prepare a collagen solution with a pH of 2 to 4. The step S2) is a step of adding an acid to the collagen solution so that the collagen is completely dissolved in ethanol or methanol.

[0103] The above step S3) is a step of making the collagen solution obtained in the above step S2) neutral and then centrifuging to produce esterified collagen, and is a step of removing the telopeptide of the flying helical structure consisting of about 12 to 27 amino acids located at both ends of the collagen, and removing the telopeptide by esterification. Here, the telopeptide is considered to be the main cause of immune response when collagen is added to the human body. Therefore, it is preferred to remove the telopeptide and use telopeptide-deficient collagen or esterified collagen when used as a raw material for pharmaceuticals, etc., to avoid immune response.

[0104] The above-mentioned step S3) may be a step of treating the neutralized collagen solution with pepsin to remove the telopeptide, producing the esterified collagen having an ester functional group for the first time, and then separating the high-purity esterified collagen using a centrifugal separator.

[0105] At this time, preferably, the above centrifugal separation is performed at 1,000 rpm to 30,000 rpm for 5 minutes to 3 hours so that only the esterified collagen is cut, but it is not limited.

[0106] Here, the method for separating high-purity esterified collagen comprises:

[0107] (a) preparing a sample containing atelocollagen from which telopeptide has been removed in a container;

[0108] (b) performing an ultrafiltration process in which the sample containing atelocollagen is flowed from the container into a filtration module having a filtration membrane, and pressure is applied to the filtration module to filter the sample through the filtration membrane;

[0109] (c) collecting the esterified collagen solution that has passed through the ultrafiltration process, been filtered through the filtration membrane, and flowed out from the filtration module;

[0110] (d) measuring the flow rate of the esterified collagen solution filtered through the filter membrane to determine the ultrafiltration rate;

[0111] (e) when the ultrafiltration rate decreases to below a certain rate, stopping the ultrafiltration process;

[0112] (f) adding water to the residue of the sample that fails to pass through the filter membrane and returns to the container, and then allowing the residue to flow into the filter module having the filter membrane to perform a diafiltration process;

[0113] (g) collecting the esterified collagen solution that has passed through the diafiltration process, been filtered through the filtration membrane, and flowed out from the filtration module;

[0114] (h) Repeat the steps of step (f) and step (g).

[0115] In the step (b), the sample containing atelocollagen is flowed from the container into the filtration module having the filtration membrane by the suction action of a pump, and a pressure of about 10 to 30 psi may be applied to the filtration module.

[0116] In the above step (e), when the ultrafiltration rate decreases to below about 1 g / min, the ultrafiltration process is stopped.

[0117] In the above step (f), the same amount of purified water as the solution filtered through the ultrafiltration process may be added to the residue returned to the above container.

[0118] Preferably, the filtration process in the above steps (f) and (g) is performed at least 5 times.

[0119] Therefore, through the above steps (a) to (h), high-purity esterified collagen can be obtained while esterifying the atelocollagen.

[0120] In addition, the above-mentioned step S4) is a step of adding a crosslinking agent to the esterified collagen obtained from the above-mentioned step S3) to produce crosslinked collagen, which is a step of crosslinking the esterified collagen to provide crosslinked collagen that has a physical compression hemostatic effect and does not cause an immune response.

[0121] In addition, as mentioned above, the cross-linking agent in the above step S4) can be one or more cross-linking agents selected from the group consisting of formaldehyde, glutaraldehyde, carbodiimides (EDC), and polyepoxy compounds, which will not be described in detail here.

[0122] The above step S5) is a step of dispersing the cross-linked collagen obtained in the above step S4) in purified water and then freeze-drying it, and is a step of removing the collagen from which the cross-linking agent and telomerase are not removed. Therefore, the above step S5) is to disperse the cross-linked collagen obtained in the above step S4) in purified water, dissolve the collagen from which the trace amount of cross-linking agent and telomerase are not removed in purified water, and freeze-dry it in a state of being dissolved in purified water and remove it. In addition, the freezing conditions at this time can be a common method used in this technical field.

[0123] The stabilizer is contained in the hemostatic composition to stabilize the activity of the thrombin and to rapidly mix the composition at a low weight when mixed with the diluent. Therefore, by containing the stabilizer in the hemostatic composition, when the collagen and thrombin having a strong hemostatic effect are mixed with the diluent, they are rapidly mixed at a low weight, and at the same time, the chemical stability and biological activity of the hemostatic composition can be maintained by the inactive substance.

[0124] The stabilizer is one or more selected from the group consisting of albumin (human serum albumin), mannitol, sodium acetate (C2H3NaO2), sucrose, trehalose, sorbitol and glycine.

[0125] In this case, the stabilizer includes one or more selected from the group consisting of albumin, mannitol, and sodium acetate, so that the chemical stability and biological activity of thrombin can be maintained.

[0126] Furthermore, the stabilizer contains one or more selected from the group consisting of mannitol, sodium acetate, sucrose, trehalose, sorbitol and glycine, thereby suppressing the extension of hemostasis time and maintaining blood coagulation activity.

[0127] In addition, the stabilizer includes one or more selected from the group consisting of mannitol, sodium acetate, sucrose, trehalose, sorbitol and glycine, so that the composition can be mixed quickly at a low weight.

[0128] Preferably, the stabilizer is mannitol, but not limited thereto.

[0129] The above-mentioned mannitol is a white needle-shaped or columnar crystal widely found in plants such as mushrooms and pomegranate roots. It is easily soluble in water and is also used as a laxative and as a substitute for glycerol.

[0130] In particular, the mannitol mentioned above significantly increases the reconstitution speed of the dry powder, maintains the inertia of the reconstituted powder, reduces the hygroscopicity of the dry powder to reduce the cohesive strength between the powders, and thus increases the fluidity of the powder.

[0131] The stabilizer may be included in an amount of 1 to 30% by weight relative to the total weight of the hemostatic composition. If the stabilizer is included in an amount of less than 1% by weight, it is difficult to maintain the chemical stability and biological activity of the collagen and thrombin, and a high weight is required when mixing with a diluent, which reduces the mixing convenience. If the stabilizer is included in an amount of 30% by weight or more, the hemostatic effect of the overall hemostatic composition is reduced due to the relatively small amount of collagen and thrombin included.

[0132] The thrombin contained in the hemostatic composition participates in blood coagulation as a powerful hemostatic factor and acts as a catalyst in the reaction of hydrolyzing soluble fibrinogen in the blood to convert it into insoluble fibrin. In addition, the thrombin can induce platelet activity in the process of forming cellulose using the activity of blood coagulation factors as the secondary hemostatic process.

[0133] The thrombin may be included in an amount of 2 to 50% by weight relative to the total weight of the hemostatic composition. If the amount is less than 2% by weight, the hemostatic effect may be reduced, while if the amount is more than 50% by weight, the chemical stability of the overall hemostatic composition may be reduced or the hemostatic effect may be reduced due to the relatively small amount of collagen and stabilizer.

[0134] The hemostatic composition may be in the form of a powder. Preferably, the hemostatic composition in the form of a dry powder is manufactured to be a powder that is not easily deteriorated into a liquid state by the external environment and moisture. The hemostatic composition is in the form of a powder, and each component has chemical stability, and is easy to maintain a state of not mixing with each other. In addition, it has the advantage of being easy to use and store.

[0135] According to another embodiment of the present invention, a container including the hemostatic composition may be provided.

[0136] In this case, the container containing the hemostatic composition is filled with collagen, stabilizer and thrombin, and the use of the damper is very simple, so it has the advantage of being able to be used for bleeding patients who cannot wait. In particular, during surgical operations, in the urgent state of severe ligation and bleeding, and in a time-sensitive state, it only needs to be mixed with a diluent and immediately applied to the bleeding site, thereby not only improving the convenience of the user, but also having a very simple method of use, so that even non-professional technicians can easily use it.

[0137] According to another aspect of the present invention, there is provided a hemostatic kit, comprising: a first container filled with a hemostatic composition comprising collagen, a stabilizer and thrombin; and a second container filled with a diluent; in the first container, the collagen and thrombin are adjacently arranged.

[0138] Since the above-mentioned hemostatic kit utilizes the above-mentioned hemostatic composition, the collagen, stabilizer and thrombin are also the same, and therefore, they are not described in detail here.

[0139] The first container is filled with a hemostatic composition, and collagen and thrombin are arranged adjacently, and the arrangement order will not be changed by external forces. In addition, according to the arrangement order of collagen, stabilizer, and thrombin filled in the first container, not only the flowability formed by subsequent mixing with a diluent is changed, but also the expansion rate and expansion speed are changed. Therefore, the first container can be filled in the order of stabilizer, thrombin, collagen or stabilizer, collagen, and thrombin; or it can be filled in the order of thrombin, collagen, stabilizer or collagen, thrombin, stabilizer. When the hemostatic combination is used, it can be mixed with a diluent. At this time, in the hemostatic composition, the hemostatic combination and the diluent are mixed by combining the first container filled with the hemostatic composition and the second container filled with the diluent. At this time, preferably, the hemostatic composition arranges the stabilizer at the position where it is first mixed with the diluent. Therefore, preferably, in the first container, considering the position of mixing with the diluent, the hemostatic composition is arranged in the order of stabilizer, thrombin, collagen or stabilizer, collagen, thrombin starting from the nozzle of the first container combined with the diluent.

[0140] The second container is filled with a diluent, which is used to dissolve or disperse the hemostatic composition in the first container and can be used as a solvent or dispersion medium. For example, it is one or more selected from the group consisting of calcium chloride (CaCl2) solution, sodium chloride (NaCl) solution, human serum albumin (human serum albumin) and sodium acetate (C2H3NaO2) solution. Preferably, it is a calcium chloride solution or a sodium chloride solution, but it is not limited.

[0141] When the diluent is a calcium chloride solution, the calcium chloride may be contained in an amount of 0.001 to 30% by weight relative to the total weight of the diluent, and the weight % is not particularly limited as long as it is within a range that allows easy mixing with the hemostatic composition.

[0142] The shape and material of the first container or the second container are not particularly limited, and may be those commonly used in the fields of medicine and biotechnology. For example, the first container or the second container is in the shape of a syringe, and the material of the first container or the second container is a material that does not chemically react with one selected from collagen, stabilizer, and thrombin.

[0143] In addition, in the above-mentioned hemostatic kit, the first container and the second container may be combined with each other so that the hemostatic composition and the diluent are mixed to form a hydrogel.

[0144] In order to combine the first container and the second container with each other, the first container has a coupling protrusion at the front end, and the second container has a coupling groove to be coupled with the coupling protrusion provided at the front end of the first container.

[0145] In addition, the hydrogel is porous and has a high blood absorption rate when used as a hemostatic agent. After absorbing blood, the hydrogel can maintain its shape without affecting the hemostatic effect, thereby being able to exhibit an excellent hemostatic effect.

[0146] In addition, the average pore size of the hydrogel may be 50 μm to 200 μm. If the average pore size is less than 50 μm, the blood absorption rate is reduced, and if it exceeds 200 μm, the hydrogel cannot maintain its shape after absorbing blood, thereby possibly reducing the hemostatic effect.

[0147] In addition, the hydrogel takes 5 seconds to 5 minutes to form, so it can stop severe ligation and bleeding in a time-sensitive state. In addition, since the hydrogel can be formed in a short time, it is easy for users to use.

[0148] The hydrogel has a neutral pH by mixing the hemostatic composition and the diluent, thereby ensuring biocompatibility and biosafety when used in the human body. At the same time, it has low toxicity and can have infection safety. Therefore, the pH of the hydrogel can be 6 to 8.

[0149] As another aspect of the present invention, there is provided use of the above-mentioned hemostatic composition for hemostasis of a bleeding site or treatment of a bleeding wound.

[0150] As another form of the present invention, a method for hemostasis is provided, comprising the step of treating or curing an individual in need of hemostasis with the above-mentioned hemostatic composition. In the present invention, "individual" includes all animals including humans whose part of the body is bleeding and thus in need of hemostasis, and the above-mentioned individual can be treated, treated, applied or administered with the hemostatic composition of the present invention to achieve effective hemostasis. DETAILED DESCRIPTION

[0151] Below, the preferred embodiments and experimental examples of the present invention are described to help better understand the present invention. However, the following embodiments are only used to illustrate the present invention and are not intended to limit the present invention. It should be understood by those skilled in the art that the present invention can be modified, deformed or replaced by equivalents without departing from the spirit and scope of the present invention, which should all be included in the scope of the claims of the present invention.

[0152] Production Example 1: Production of esterified collagen powder

[0153] The esterified collagen powder is produced by the following process:

[0154] ① After adding collagen to ethanol, stir it under refrigeration;

[0155] ② Use 0.5M HCl to adjust the pH of the sample to 6.7±0.3;

[0156] ③Put the above solution into dialysis tubing and perform dialysis using purified water;

[0157] ④ Freeze and freeze-dry the above solution or prepare collagen powder.

[0158] Production Example 2: Production of cross-linked collagen powder

[0159] The cross-linked collagen powder is produced by the following process:

[0160] ① The esterified collagen prepared in the above-mentioned Preparation Example 1 was added into purified water and stirred;

[0161] ② Add EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) to the well-stirred esterified collagen solution;

[0162] ③ Stir thoroughly to allow EDC to fully react with esterified collagen;

[0163] ④ Leave in the refrigerator for 2 to 3 days;

[0164] ⑤ Put the cross-linked collagen solution into purified water for dispersion;

[0165] ⑥ Let the cross-linked collagen solution stand, and when layer separation occurs, only take the cross-linked collagen and discard the solution;

[0166] ⑦ Repeat the above process ⑥ 3 to 5 times to obtain cross-linked collagen;

[0167] ⑧ Add cross-linked collagen to the buffer (sodium phosphate / sodium chloride) and let it stand in the refrigerator for 1 to 2 days to neutralize the buffer.

[0168] ⑨After neutralizing the buffer, wash with purified water, freeze and freeze-dry the collagen;

[0169] ⑩ Put the freeze-dried sample into a blender and grind it;

[0170] The powder was filtered through a sieve to screen out particles with a size of 200 to 355 μm.

[0171] Experimental Example 1: Confirming Flowability

[0172] In order to confirm the difference in fluidity when mannitol, thrombin and collagen as stabilizers are arranged in the hemostatic composition and mixed with a calcium chloride solution as a diluent, examples were prepared as shown in Table 1. At this time, mannitol and thrombin were in powder form and commercially available products were used, and the cross-linked collagen used was obtained in the above-mentioned Production Example 2.

[0173]

Table 1

[0174] distinguish Arrange order Example 1 Mannitol-thrombin-cross-linked collagen Example 2 Mannitol-cross-linked collagen-thrombin Example 3 Thrombin-cross-linked collagen-mannitol Example 4 Cross-linked collagen-thrombin-mannitol Comparative Example 1 Thrombin-mannitol-cross-linked collagen

[0175] The forces required for mixing of Examples 1 to 4 and Comparative Example 1 shown in Table 1 above were measured by the following method, and their structures are shown in Table 2.

[0176] 1) Prepare two syringes, and fill one syringe with the components from the nozzle to the inlet side of the syringe in the same arrangement order as in Examples 1 to 4 and Comparative Example 1, with 90% by weight of cross-linked collagen, 5% by weight of mannitol, and 5% by weight of thrombin relative to the weight of the components filled in the syringe. In addition, fill the remaining syringe with 10% calcium chloride solution.

[0177] 2) Connect the two syringes (rotate the barrel ends towards each other to join).

[0178] 3) The syringe (male type) filled with Example 1 or Comparative Example 1 was fixed (clamped) by a machine.

[0179] 4) Use a machine to push the plunger of a syringe (female type) filled with calcium chloride solution to push the calcium chloride solution into the syringe filled with Example or Comparative Example 1. Measure the force required at this time and mark it as N1.

[0180] 5) Change the position of the syringe and fix (clamp) the syringe on the machine.

[0181] 6) Push the plunger of the syringe (male type) filled with the mixed contents to push the mixed contents into the empty syringe. Measure the force required at this time and mark it as N2.

[0182] 7) Repeat 5) to 6) to measure the force required for mixing. Here, measure the force (N3, N4, ..., N20) required each time step 6) is performed.

[0183]

Table 2

[0184]

[0185] As shown in Table 2 above, the force required for mixing of Comparative Example 1 and Example 1, that is, the magnitude of the pressure generated during mixing, is similar. That is, the maximum weight of Comparative Example 1 and Example 1 decreased sharply in N4 in both groups, and was substantially reduced to N20, which indicates that the overall mixing of the contents was completed after N4, and the pressure generated during mixing gradually decreased and became the same. The experimental results show that not only in the case of Comparative Example 1 filled in the order of thrombin-mannitol-cross-linked collagen, but also in the case of Example 1 arranged in the order of mannitol-thrombin-cross-linked collagen, the pressure required for mixing can be reduced to ensure uniform mixing of the raw materials.

[0186] In addition, not only in Example 1, but also in the case of Example 2 arranged in the order of mannitol-cross-linked collagen-thrombin, similar results to those of Example 1 were obtained. In the cases of Example 3 arranged in the order of thrombin-cross-linked collagen-mannitol and Example 4 arranged in the order of cross-linked collagen-thrombin-mannitol, the pressure generated during mixing was slightly larger than that of Examples 1 and 2.

[0187] Experimental Example 2: Confirmation of microstructure using SEM (Scanning Electron Microscope)

[0188] After executing Example 1, the microstructure images of Comparative Example 1 and Example 1 mixed with calcium chloride solution were obtained using SEM. Figure 2 shown.

[0189] As above Figure 2As shown, a fine and dense porous membrane structure can be confirmed not only in Comparative Example 1 filled in the order of thrombin-mannitol-cross-linked collagen, but also in Example 1 arranged in the order of mannitol-thrombin-cross-linked collagen. Therefore, not only in the case of filling in the order of thrombin-mannitol-cross-linked collagen, but also in the case of arranging in the order of mannitol-thrombin-cross-linked collagen, after mixing with calcium chloride solution, the porous membrane structure has a porous membrane structure that helps to absorb blood and coagulate and stop bleeding through the porous structure.

[0190] Experimental Example 3: Confirming the expansion rate and area change

[0191] In order to confirm the expansion rate and area change of the hemostatic composition according to the present invention after mixing with a diluent, first, Comparative Example 1 and Example 1 were prepared in the manner described in Table 1 of Experimental Example 1. The particle areas of the prepared Comparative Example 1 and Example 1 were measured. After that, after each sample was mixed with a calcium chloride solution, the particle areas were measured at intervals of 1, 2, 3, 5, 10, 20, and 30 minutes, and the area change and expansion rate were calculated. The results are shown in FIG. Figure 3 and Figure 4 shown.

[0192] Expansion rate (%) = ((area of ​​diluted powder per unit time - area of ​​dry powder) / area of ​​dry powder) × 100

[0193] like Figure 3 and Figure 4 As shown, Comparative Example 1 and Example 1 expand rapidly in the first minute, and continue to expand for 2, 3, 5, and 10 minutes, and the area change reaches saturation after 10 minutes.

[0194] Experimental Example 4: Decomposition Test

[0195] In order to measure the biodegradability and decomposition degree of the collagen contained in the hemostatic composition according to the present invention, in order to confirm whether it has biocompatibility and whether it has an infection risk due to a low decomposition degree, a decomposition degree test is implemented. First, the cross-linked collagen of the above-mentioned Example 1 and Comparative Example 1 is prepared, and the weight of each sample is measured. After that, each sample is put into a micro-centrifuge tube, and 1 to 2 mL of 1XPBS buffer containing 25 units / mL of collagenase is adjusted until it is filled. The tube containing the sample is placed in a 37°C water bath and allowed to stand for 24 to 72 hours. After 24, 48, and 72 hours, the sample is taken out, freeze-dried, and then weighed. The remaining weight ratio is calculated using the weight of the sample measured before and after the decomposition degree test using the following calculation formula. The results are shown in Table 3.

[0196] Residual weight ratio (%) = (dry weight of sample after decomposition test / dry weight of sample before decomposition test) × 100

[0197]

Table 3

[0198]

[0199] It was confirmed that the cross-linked collagen of Comparative Example 1 and Example 1 was almost completely degraded by collagenase within 72 hours. Therefore, even if thrombin and collagen are placed adjacent to each other as in the present invention, the cross-linked collagen has a rapid degradability under the same conditions, so it was confirmed that the biodegradability is high and the risk of infection is low.

[0200] Experimental Example 5: Comparison of hemostatic properties in a rat nephrectomy model

[0201] In order to confirm the hemostatic ability of the hemostatic composition according to the present invention, Example 1 and Comparative Example 1 were prepared in the same manner as in Experimental Example 1. The comparison of hemostatic ability was performed using a rat nephrectomy model. After the kidney was cut to cause bleeding, hemostatic compression was applied for 30 seconds using Example 1 or Comparative Example 1, and then the presence or absence of bleeding was confirmed. If bleeding occurred, hemostatic compression was repeated for an additional 30 seconds. The time required for hemostasis was measured and the results are shown in FIG. Figure 5 , and the hemostasis process is Figure 6 shown.

[0202] As mentioned above Figure 5 As shown, the time required for hemostasis was about 68 seconds for Example 1, while it was about 45 seconds for Comparative Example 1. Therefore, even if thrombin and collagen are placed adjacent to each other as in the present invention, excellent hemostasis ability is achieved.

[0203] The above description is only used to illustrate the present invention by way of example, and those skilled in the art to which the present invention belongs should understand that the present invention can be implemented in a deformed form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments and experimental examples should be considered from the perspective of explanation rather than from the perspective of limitation. The scope of the present invention is shown in the above description or claims, and all differences within the same scope thereof should be understood to be within the scope of the present invention.

Claims

1. A hemostatic composition comprising: A first layer comprising cross-linked esterified collagen powder, wherein the size of the cross-linked esterified collagen powder is 200 to 355 μm, and the molecular weight of the cross-linked esterified collagen is 100,000 to 1,000,000 Dalton, a second layer comprising a stabilizer powder, and The third layer contains thrombin powder, Features: These three layers are filled in a layered manner within the container. The invention is characterized in that: the first layer comprising the cross-linked esterified collagen powder and the third layer comprising the thrombin powder are arranged adjacent to each other; and The second layer containing the stabilizer powder is arranged at the position where it is first mixed with the diluent, and The layers of the hemostatic composition are arranged in the order of the second layer comprising stabilizer powder - the third layer comprising thrombin powder - the first layer comprising cross-linked esterified collagen powder, or in the order of the second layer comprising stabilizer powder - the first layer comprising cross-linked esterified collagen powder - the third layer comprising thrombin powder; or The layers of the hemostatic composition are arranged in the order of the third layer comprising thrombin powder - the first layer comprising cross-linked esterified collagen powder - the second layer comprising stabilizer powder, or in the order of the first layer comprising cross-linked esterified collagen powder - the third layer comprising thrombin powder - the second layer comprising stabilizer powder; and The hemostatic composition is in the form of a powder. Keep the ingredients from mixing with each other. The hemostatic composition comprises 40 to 97 wt % of the cross-linked esterified collagen, 1 to 30 wt % of the stabilizer, and 2 to 50 wt % of the thrombin, relative to the total weight of the hemostatic composition, and The stabilizer is one or more selected from the group consisting of albumin, mannitol, sodium acetate, sucrose, trehalose, sorbitol and glycine.

2. The hemostatic composition according to claim 1, characterized in that: The above stabilizer is human serum albumin.

3. A container comprising the hemostatic composition of claim 1.

4. A hemostatic kit, comprising: A first container is filled with a hemostatic composition in a powder form, the hemostatic composition comprising: a first layer comprising cross-linked esterified collagen powder, a second layer comprising stabilizer powder, and a third layer comprising thrombin powder, the cross-linked esterified collagen powder has a size of 200 to 355 μm and a molecular weight of 100,000 to 1,000,000 Dalton; and a second container filled with a diluent; It is characterized in that: in the first container, the first layer containing the cross-linked esterified collagen powder and the third layer containing the thrombin powder are arranged adjacent to each other, and The second layer containing the stabilizer powder is arranged at the position where it is first mixed with the diluent, and In the above-mentioned first container, the second layer containing the stabilizer powder, the third layer containing the thrombin powder and the first layer containing the cross-linked esterified collagen powder are filled in this order, or the second layer containing the stabilizer powder, the first layer containing the cross-linked esterified collagen powder and the third layer containing the thrombin powder are filled in this order; or In the first container, the third layer containing thrombin powder, the first layer containing cross-linked esterified collagen powder, and the second layer containing stabilizer powder are filled in this order, or the first layer containing cross-linked esterified collagen powder, the third layer containing thrombin powder, and the second layer containing stabilizer powder are filled in this order; and The hemostatic composition is in the form of a powder. Keep the ingredients from mixing with each other. The hemostatic composition comprises 40 to 97 wt % of the cross-linked esterified collagen, 1 to 30 wt % of the stabilizer, and 2 to 50 wt % of the thrombin, relative to the total weight of the hemostatic composition, and The stabilizer is one or more selected from the group consisting of albumin, mannitol, sodium acetate, sucrose, trehalose, sorbitol and glycine.

5. The hemostatic kit according to claim 4, characterized in that: The diluent is at least one selected from the group consisting of a calcium chloride solution, a sodium chloride solution, human serum albumin, and a sodium acetate solution.

6. The hemostatic kit according to claim 4, characterized in that: In the above-mentioned hemostatic kit, the first container and the second container are combined with each other so that the hemostatic composition and the diluent are mixed to form a hydrogel; The average pore size of the hydrogel is 50 μm to 200 μm.

7. The hemostatic kit according to claim 4, characterized in that: The above stabilizer is human serum albumin.

Images