Absorbable internal hemostasis kit and absorbable internal hemostatic agent

Abstract

The present invention relates to an absorbable internal hemostasis kit and an absorbable internal hemostatic agent, the kit comprising: a first syringe containing a powder mixture including gelatin, thrombin, and calcium chloride; a second syringe containing a mixed solution including a thermo-responsive gel, purified water, polyvinylpyrrolidone, polyethylene glycol, sodium chloride, and a protein extract obtained through a multi-stage decellularization process; and a connector for connecting the first syringe and the second syringe so that the powder mixture and the mixed solution can be combined, wherein the absorbable internal hemostatic agent according to the present invention promotes and activates primary physiological hemostasis through thrombin and provides a secondary physical hemostatic effect by gelling in a temperature range close to body temperature, while also offering additionally wound-healing effects, so as to achieve faster and more effective internal hemostasis.

Description

Absorbent internal hemostatic kit and absorbent internal hemostatic agent

[0001] The present invention relates to an absorbable hemostatic kit and an absorbable hemostatic agent for internal use, and more specifically, to an absorbable hemostatic kit or an absorbable hemostatic agent for internal use that can perform hemostatic within the body more quickly and effectively by primarily promoting and activating physiological hemostasis by thrombin, gelling in a temperature range similar to body temperature to provide a secondary physical hemostatic effect, and providing an additional wound healing effect.

[0002] Generally, in healthy individuals, when bleeding occurs, natural hemostasis takes place through the complex interaction of platelets and plasma factors, which can prevent loss of bodily function, shock, or disability caused by excessive bleeding. However, when excessive internal bleeding occurs due to unexpected accidents, surgical procedures, diseases, or disorders, natural hemostasis driven by physiological factors does not occur. This leads to secondary side effects caused by blood loss and can obstruct the medical team's view during surgery, causing difficulties in treatment. Consequently, additional measures such as blood transfusions may be required, and in extreme cases, it can lead to death. Therefore, it is crucial to take rapid early action to prevent blood loss in cases of bleeding caused by serious factors such as accidents, surgeries, or diseases.

[0003] Hemostasis methods primarily involve chemical methods, such as the use of hemostatic agents, or mechanical methods that physically block the site of bleeding to prevent blood loss. Chemical hemostasis involves supplying hemostatic factors, such as thrombin and fibrinogen, from an external source to achieve hemostasis. Mechanical hemostasis methods include attaching hemostatic agents in the form of patches—such as sponges, films, fabrics, or non-woven materials—to the bleeding site, or using syringes containing biocompatible materials to inject the biocompatible fluid onto the site. Since these methods utilize biocompatible materials such as cellulose, polysaccharides, collagen, and gelatin, they can be absorbed into the body after a certain period and excreted through metabolism.

[0004] Recently, minimally invasive surgical methods are frequently applied, so the application of hemostatic agents via syringe injection is essential to stop bleeding at the surgical site through the invasive area, and hydrogel formulations are mainly used as such internal hemostatic agents.

[0005] However, syringe-type internal hemostatic agents currently commercialized in the market have problems such as not adhering sufficiently to the site of injury, making it difficult to stop bleeding easily and quickly, and causing secondary infections or insufficient hemostatic effect due to a structure prone to contamination.

[0006] The present invention aims to provide an absorbable hemostatic kit or absorbable hemostatic agent that can perform hemostasis in the body more quickly and effectively by primarily promoting and activating physiological hemostasis by thrombin, gelling in a temperature range similar to body temperature to provide a secondary physical hemostatic effect, and providing an additional wound healing effect.

[0007] One embodiment of the present invention for achieving the above-described purpose relates to an absorbable hemostatic kit comprising: a first syringe containing a powder mixture comprising gelatin, thrombin, and calcium chloride; a second syringe containing a mixture comprising a temperature-sensitive gel, purified water, polyvinylpyrrolidone, polyethylene glycol, sodium chloride, and a multi-stage decellularized protein extract; and a connector connecting the first syringe and the second syringe so that the powder mixture and the mixture can be mixed.

[0008] The above particle mixture may further include at least one of a multi-stage decellularized protein extract, albumin, and mannitol.

[0009] The above mixture may further contain mannitol.

[0010] The above temperature-sensitive gel may be a poloxamer, which is a copolymer of polyethylene oxide (PEO) - polypropylene oxide (PPO) - polyethylene oxide (PEO), having a sol-gel phase transition behavior depending on the temperature change.

[0011] The above poloxamer is in a sol phase at room temperature and may be in a gel phase at body temperature.

[0012] The above multi-stage decellularized protein extract can be prepared by undergoing the following steps: a pretreatment step of preparing and pretreating mammalian tissue excluding humans; a first inactivation step of inactivating viruses contained in the pretreated tissue using alcohol; a first decellularization step of removing cells from the virus-inactivated tissue using an aqueous base solution; a second decellularization step of removing cells by enzymatically treating the first decellularized tissue; and a second inactivation step of inactivating viruses contained in the decellularized tissue using acid.

[0013] The DNA content of the tissue that has undergone the second inactivation step may be 50 ng / mg or less, and the reduction rate (L) of the elastin content of the tissue that has undergone the pretreatment step to the second inactivation step may be 20% or less.

[0014] The above first decellularization step may include a first decellularization step performed through a mixture of n-PrOH and NaOH; and a second decellularization step performed with an aqueous sodium hydroxide solution of greater than 0.05 M and less than 0.2 M.

[0015] It may further include a catheter capable of applying an absorbable hemostatic agent mixed with the above particle mixture and liquid to the affected area.

[0016] Another embodiment of the present invention relates to an absorbable hemostatic agent for internal use, wherein a powder mixture comprising gelatin, thrombin, and calcium chloride is mixed with a mixture comprising a temperature-sensitive gel, purified water, polyvinylpyrrolidone, polyethylene glycol, sodium chloride, and a multi-stage decellularized protein extract, and applied to a wound.

[0017] The above particle mixture may further include at least one of a multi-stage decellularized protein extract, albumin, and mannitol.

[0018] The above mixture may further contain mannitol.

[0019] The above temperature-sensitive gel may be a poloxamer, which is a copolymer of polyethylene oxide (PEO) - polypropylene oxide (PPO) - polyethylene oxide (PEO), having a sol-gel phase transition behavior depending on the temperature change.

[0020] The above poloxamer is in a sol phase at room temperature and can undergo a phase transition to a gel phase at body temperature.

[0021] The above multi-stage decellularized protein extract can be prepared by undergoing the following steps: a pretreatment step of preparing and pretreating mammalian tissue excluding humans; a first inactivation step of inactivating viruses contained in the pretreated tissue using alcohol; a first decellularization step of removing cells from the virus-inactivated tissue using an aqueous base solution; a second decellularization step of removing cells by enzymatically treating the first decellularized tissue; and a second inactivation step of inactivating viruses contained in the decellularized tissue using acid.

[0022] The DNA content of the tissue that has undergone the second inactivation step above may be 50 ng / mg or less.

[0023] The reduction rate (L) of the elastin content of the tissue that has undergone the above pretreatment step to the second inactivation step may be 20% or less.

[0024] The above first decellularization step may include a first decellularization step performed through a mixture of n-PrOH and NaOH; and a second decellularization step performed with an aqueous sodium hydroxide solution of greater than 0.05 M and less than 0.2 M.

[0025] The mixing ratio of the above particle mixture and the mixture may be 1:3 to 20 based on weight.

[0026] The absorbable hemostatic agent of the present invention primarily promotes and activates physiological hemostasis by thrombin, and gels within a temperature range similar to body temperature to provide a secondary physical hemostatic effect, thereby providing a rapid and effective hemostatic function within the body.

[0027] In addition, hemostasis and regeneration effects can be promoted by providing additional wound healing effects through multi-stage decellularized protein extracts.

[0028] FIG. 1 is a photograph of an absorbable internal hemostatic kit according to one embodiment of the present invention.

[0029] Figure 2 is a photograph of the packaging of an absorbent internal hemostatic kit.

[0030] Figure 3(A) is a photograph showing the first syringe and the second syringe connected, and Figure 3(B) is a photograph showing a catheter connected to a syringe containing an absorbable hemostatic agent.

[0031] Figures 4(A) and 4(B) are graphs showing the experimental results of Experimental Example 1.

[0032] Figure 5 is a graph showing the experimental results of Experimental Example 2.

[0033] Figures 6(A) and 6(B) are graphs showing the experimental results of Experimental Example 3.

[0034] Figures 7(A) and 7(B) are graphs showing the experimental results of Experimental Example 4.

[0035] Figure 8 is a table showing the experimental results of Experimental Example 5.

[0036] Figure 9 is a graph showing the experimental results of Experimental Example 7.

[0037] Figure 10 is a graph showing the experimental results of Experimental Example 8, and Figure 11 is a photograph of the experimental results.

[0038] Figure 12 is a graph showing the experimental results of Experimental Example 9.

[0039] Before describing the preferred embodiments of the present invention in detail below, it should be noted that the terms and words used in this specification and claims should not be interpreted as being limited to their ordinary or dictionary meanings, but should be interpreted in a meaning and concept consistent with the technical spirit of the present invention.

[0040] Throughout this specification, when a part is described as "comprising" a certain component, it means that, unless specifically stated otherwise, it does not exclude other components but may include additional components.

[0041] Throughout this specification, "%" used to indicate the concentration of a specific substance means (weight / weight)% for solid / solid, (weight / volume)% for solid / liquid, and (volume / volume)% for liquid / liquid, unless otherwise noted.

[0042] In each step, identification codes are used for convenience of explanation and do not describe the order of the steps; unless the context clearly indicates a specific order, the steps may be performed differently from the specified order. That is, the steps may be performed in the same order as specified, substantially simultaneously, or in the reverse order.

[0043] Hereinafter, embodiments of the present invention are examined. However, the scope of the present invention is not limited to the following preferred embodiments, and those skilled in the art may implement various modified forms of the contents described herein within the scope of the present invention.

[0044] The present invention relates to an absorbable internal hemostatic agent capable of providing a hemostatic effect when injected into the body through a syringe, a method for manufacturing the same, and an absorbable internal hemostatic kit prepared in a form for applying the same to a wound.

[0045] FIG. 1 is a photograph of an absorbable internal hemostatic kit according to one embodiment of the present invention, and FIG. 2 is a photograph of a packaged product in which such a kit is packaged.

[0046] An absorbable hemostatic kit for internal use according to one embodiment of the present invention comprises: a first syringe (100) containing a powder mixture comprising gelatin, thrombin, and calcium chloride; a second syringe (200) containing a mixture comprising a temperature-sensitive gel, purified water, polyvinylpyrrolidone, polyethylene glycol, sodium chloride, and a multi-stage decellularized protein extract; and a connector (300) connecting the first syringe (100) and the second syringe (200) so that the powder mixture and the mixture can be mixed.

[0047] Additionally, it may further include a catheter (400) capable of applying an absorbable hemostatic agent to a wound, the mixture of particles contained in the first syringe (100) and the mixture contained in the second syringe (200).

[0048] The above-described absorbable internal hemostatic kit is configured to connect the discharge ports of a first syringe (100) and a second syringe (200) (see FIG. 3(A)) and to inject the mixture within the second syringe (200) into the first syringe (100) to mix the particle mixture and the mixture, thereby forming an absorbable internal hemostatic agent through this process. Subsequently, the first syringe (100) containing the absorbable internal hemostatic agent is separated from the connector (300) and connected to a catheter (400) as shown in FIG. 3(B), so that the absorbable internal hemostatic agent can be prepared in a form that can be applied to the affected area. In addition, it is also possible to form an absorbable internal hemostatic agent by injecting the particle mixture within the first syringe (100) into the second syringe (200) in a manner different from the example presented here.

[0049] The absorbable hemostatic agent formed by mixing a particle mixture and a liquid through the process described above forms an adhesive hydrogel, so when applied to a wound, it adheres to the wound and enables rapid hemostasis. In addition, since it is formed solely from biocompatible materials, it can naturally decompose after a predetermined period following application to the wound.

[0050] The first syringe (100) contains a particle mixture comprising gelatin, thrombin, and calcium chloride.

[0051] The above particle mixture may contain 50 to 99 wt% gelatin, 0.01 to 25 wt% thrombin, and 0.01 to 25 wt% calcium chloride.

[0052] Gelatin is a hydrogel-forming material that, when reacting with moisture in a mixture, transforms the composition into a gel state, contributing to shape retention and providing adhesiveness, thereby enabling the absorbent internal hemostatic agent applied to the affected area to adhere well to the area. If the gelatin content is insufficient, there is a problem of reduced physical hemostatic performance due to insufficient adhesiveness and weakened gel strength; conversely, if an excessive amount of gelatin is included, the content of factors involved in physiological hemostasis, such as thrombin or calcium chloride, becomes relatively insufficient, leading to a problem of reduced physiological hemostatic efficiency; therefore, it is desirable to include it within the weight range described above.

[0053] Thrombin is added to induce a blood coagulation reaction by hydrolyzing fibrinogen in the blood and converting it into insoluble fibrin, thereby providing a hemostatic effect.

[0054] Calcium chloride performs the function of improving hemostatic performance by promoting the physiological blood coagulation mechanism through its involvement in the production of thrombin and fibrin.

[0055] In addition to the components described above, the particle mixture may further include at least one of a multi-stage decellularized protein extract, albumin, and mannitol. When such additional components are included, the multi-stage decellularized protein extract may be included in an amount of 0.1 to 5 wt%, albumin in an amount of 0.1 to 3 wt%, and mannitol in an amount of 0.1 to 5 wt%.

[0056] Multistage decellularized protein extracts are biocompatible materials produced by multistage decellularization of mammalian-derived tissues. Containing collagen and elastin, they exhibit excellent effects in wound healing, re-epithelialization, promotion of damaged tissue regeneration, and angiogenesis. In particular, elastin facilitates the formation of elastic fiber tissues in damaged tissues, thereby facilitating recovery similar to the original tissue. Furthermore, during the process where damaged blood vessels constrict and hemostasis occurs, elastin promotes the migration of inflammatory cells to the site of injury and the release of related factors. Additionally, during the stage of removing necrotic tissue from the injury site, elastin promotes the migration, adhesion, proliferation, and differentiation of epithelial cells at the site of injury, as well as the proliferation of cells and the extracellular matrix at the site of injury. It also participates in the synthesis of collagen and elastin, which form the basic framework of wound healing, promotes the process of angiogenesis, and plays a role in restoring elasticity similar to that of existing tissue through the promotion of elastin expression. These decellularized protein extracts will be described further below.

[0057] Albumin or mannitol is added to reduce the load applied during mixing when the particle mixture and the mixture are mixed, thereby enabling the two compositions to be mixed quickly and uniformly, and additionally performs the function of stabilizing the activity of thrombin.

[0058] The second syringe (200) contains a mixture comprising a temperature-sensitive gel, purified water, polyvinylpyrrolidone, polyethylene glycol, sodium chloride, and a multi-stage decellularized protein extract.

[0059] The above mixture may comprise 1 to 35 wt% of a temperature-sensitive gel, 45 to 90 wt% of purified water, 0.1 to 20 wt% of polyvinylpyrrolidone, 0.1 to 20 wt% of polyethylene glycol, 0.01 to 5 wt% of sodium chloride, and 0.1 to 10 wt% of a multi-stage decellularized protein extract.

[0060] The temperature-sensitive gel described above may be a poloxamer, which is a copolymer of polyethylene oxide (PEO) - polypropylene oxide (PPO) - polyethylene oxide (PEO), exhibiting a sol-gel phase transition behavior depending on temperature changes. The poloxamer is characterized by being in a sol phase at room temperature and changing into a gel phase at body temperature (approximately 36–37°C). Consequently, it maintains a sol phase within a syringe, existing as an applicable fluid, but rapidly changes into a gel phase due to body temperature upon application to the body. Therefore, the poloxamer significantly contributes to maintaining the initial form by forming a rigid gel phase immediately after the absorbable hemostatic agent is applied to the affected area, thereby preventing the agent from flowing out of the affected area and allowing it to maintain its injected form. Additionally, as an amphiphilic block copolymer, the poloxamer also functions as a carrier or medium for drug delivery.

[0061] The above polyvinylpyrrolidone and polyethylene glycol are water-soluble polymers with characteristics such as low toxicity, biocompatibility, and excellent adhesion, and are added to improve the framework formation and strength of the absorbable hemostatic agent, and sodium chloride is added to improve strength and secure a stable structure when the gel is formed.

[0062] The above-mentioned multi-stage decellularized protein extract is a biocompatible material prepared by multi-stage decellularizing mammalian-derived tissue, and this will be described later.

[0063] The above mixture may further contain mannitol, in which case the mannitol may be included in an amount of 0.1 to 5 wt%.

[0064] The connector (300) may be provided to connect the discharge port of the first syringe (100) and the discharge port of the second syringe (200) so that the particle mixture in the first syringe (100) and the mixture in the second syringe (200) can be mixed.

[0065] Below, we will specifically describe the additional components of the particle mixture contained in the first syringe (100) and the multi-stage decellularized protein extracts that are components contained in the mixture contained in the second syringe (200).

[0066] A multi-stage decellularized protein extract is prepared through the following steps: a pretreatment step of preparing and pretreating mammalian tissues other than human tissues; a first inactivation step of inactivating viruses contained in the pretreated tissues using alcohol; a first decellularization step of removing cells from virus-inactivated tissues using an aqueous base solution; a second decellularization step of removing cells by enzymatically treating the first decellularized tissues; and a second inactivation step of inactivating viruses contained in the decellularized tissues using acid.

[0067] At this time, the DNA content of the tissue that has undergone the second inactivation step is 50 ng / mg or less, preferably 20 ng / mg or less, or 10 ng / mg or less, and more preferably 5 ng / mg or less. In addition, the reduction rate (L) of the elastin content of the multi-stage decellularized protein extract that has undergone the pretreatment step to the second inactivation step is 20% or less.

[0068] Here, the reduction rate (L) of elastin content is defined by the following formula (1), where L0 represents the elastin content of the tissue at the preparation stage, and L f represents the elastin content of a multi-stage decellularized protein extract that has undergone a second inactivation step.

[0069]

[0070] First, the above preparation step is a step of preparing tissues of mammals other than humans. Here, mammals are mammals other than humans, such as pigs, horses, cattle, sheep, etc., and the tissues may be one or more of blood vessels, ligaments, and tendons derived from these mammals.

[0071] To manufacture a multi-stage decellularized protein extract, mammalian tissue is first harvested. In the preparation stage, the harvested mammalian tissue is prepared by removing useless tissue and blood attached to it, and then washing, drying, and cutting it. The prepared mammalian tissue can be stored in a frozen state and thawed for use when required. When using frozen tissue raw materials, the preparation stage may involve removing the frozen tissue raw materials and preparing them.

[0072] Next, the mammalian tissue prepared in the preparation stage is pretreated through the pretreatment stage.

[0073] This step is for washing mammalian tissue; if the mammalian tissue is frozen, it can be thawed, washed, and cut through this step. In this step, distilled water, purified water, saline solution, etc., may be used as the washing water, and preferably, distilled water may be used. Physical stirring may be performed from this step to the second inactivation step.

[0074] Mammalian tissues prepared through this pretreatment step undergo a first inactivation step, a first decellularization step, a second decellularization step, and a second inactivation step, thereby removing cells, crude fats, viruses, and other foreign substances that may induce immune and foreign body reactions. By decellularizing through such a multi-stage process, the decellularization efficiency and foreign substance removal efficiency at each stage are significantly improved, which increases the amount of tissue processed at once and shortens the processing time. Consequently, the productivity and yield of the multi-stage decellularized protein extract can be improved.

[0075] First, the first inactivation step is a step of inactivating viruses contained in pre-treated tissue using alcohol.

[0076] This step is performed to more effectively remove cells, crude fat, and foreign substances during the primary and secondary decellularization steps by first inactivating viruses within the tissue before decellularizing the tissue.

[0077] The alcohol used in this step may be n-propanol, and to obtain a virus inactivation effect, it is preferable to use an aqueous solution of n-propanol at a concentration of 50 to 90 percent, and more preferably, an aqueous solution of n-propanol at a concentration of 65 to 80 percent may be used.

[0078] In the present invention, n-propanol is used as the alcohol for virus inactivation in the first inactivation step. When n-propanol is used for virus inactivation in this manner, the tissue becomes softer; since soft tissue allows the treatment solution to penetrate the tissue more effectively, the effect of performing virus inactivation and decellularization uniformly throughout is obtained. Consequently, the efficiency of inactivation and decellularization treatment is improved, a more uniform tissue can be obtained, and the quality and reliability of the finally obtained multi-stage decellularized protein extract can be enhanced.

[0079] When ethanol, which is conventionally used for virus inactivation, is used, it has the characteristic of fixing and hardening mammalian tissue to be decellularized; however, if ethanol is used instead of n-propanol in this step of the present invention, the tissue becomes hardened, which is undesirable because it reduces the efficiency of the process that loosens the tissue in subsequent steps to improve the penetration power of the treatment material.

[0080] In addition, since iso-propanol has a low viral inactivation effect, if iso-propanol is used in the first inactivation step, a sufficient viral inactivation effect is not obtained, and the decellularization efficiency decreases in the subsequent first and second decellularization steps, so it is preferable to use n-propanol in the first inactivation step.

[0081] At this stage, the pretreated tissue can be treated in an amount of 10 to 25 parts by weight, preferably 20 to 25 parts by weight, per 100 parts by weight of the aqueous propanol solution.

[0082] The above first decellularization step is a step of removing cells by treating the virus-inactivated tissue, which has undergone the first inactivation step, with an aqueous base solution.

[0083] This step comprises a first decellularization step of treating tissue using a mixture of an aqueous base solution and an alcohol; and a second decellularization step of treating tissue using an aqueous base solution. Here, the aqueous base solution is an aqueous sodium hydroxide solution, and it is preferable to use n-propanol as the alcohol; in this case, the first decellularization step is a step performed by treating the tissue with a mixture of n-propanol and sodium hydroxide, and the second decellularization step is a step performed by treating the tissue with an aqueous sodium hydroxide solution.

[0084] Specifically, the first decellularization step may include a first decellularization step performed through a mixture of n-PrOH and NaOH; and a second decellularization step performed with an aqueous sodium hydroxide solution of greater than 0.05 M and less than 0.2 M.

[0085] When the decellularization step is performed in two stages as described above, the first decellularization stage removes not only cells but also foreign substances such as crude fat, allowing for a more effective decellularization process in the second stage.

[0086] In each of the first and second decellularization steps, the tissue can be treated in an amount of 10 to 25 parts by weight, preferably 20 to 25 parts by weight, per 100 parts by weight of the treatment solution. This is close to approximately twice the conventional treatment capacity, and since effective decellularization treatment is possible through the multi-stage method of the present invention, it has the effect of sufficiently treating twice the amount of tissue compared to the conventional method.

[0087] Specifically, the first decellularization step involves treating virus-inactivated tissue with a mixture of n-propanol and sodium hydroxide. In this step, crude fat within the tissue undergoes a saponification reaction with sodium hydroxide, thereby separating and removing the crude fat from the tissue. During this process, n-propanol promotes this saponification reaction and acts to aggregate the products of the reaction, allowing the crude fat to be separated and removed from the tissue more quickly and efficiently.

[0088] At this time, the treatment time for treating the tissue with a mixture of n-propanol and sodium hydroxide may be 10 to 48 hours, preferably 15 to 40 hours, more preferably 18 to 28 hours, and the treatment temperature range may be 0 to 30°C, preferably 10 to 28°C, more preferably 18 to 25°C. When treated for the above time at this temperature range, sufficient crude fat removal efficiency can be obtained without significant damage to the tissue structure or loss of elastin.

[0089] The above mixture of n-propanol and sodium hydroxide may have an n-propanol concentration of 50 to 95%, preferably 60 to 90%, more preferably 65 to 85%, and may be an aqueous solution with a sodium hydroxide concentration greater than 0.05 M and less than 0.2 M. These concentration ranges are ranges in which tissue damage can be minimized in the first decellularization step and foreign substances such as crude fat can be effectively removed at the same time. In particular, if the concentration of sodium hydroxide is below the above range, the processing efficiency of the first decellularization step decreases, and if it exceeds the above range, there is a problem of the tissue structure itself collapsing and dissolving; therefore, it is preferable to use an aqueous solution of n-propanol and sodium hydroxide within the above concentration ranges.

[0090] The aforementioned second decellularization step involves treating the tissue that has undergone the first decellularization step with an aqueous alkaline solution to soften the tissue, thereby loosening its structure while simultaneously allowing the decellularization reaction to occur. Crude fat and various foreign substances contained in the tissue act as physical and chemical barriers during the decellularization process; however, since the tissue has already undergone the first decellularization step to remove crude fat and various foreign substances, the decellularization reaction can be carried out more effectively in the second decellularization step.

[0091] At this stage, the treatment time for treating the tissue in an aqueous alkaline solution may be 10 to 48 hours, preferably 15 to 40 hours, more preferably 18 to 28 hours, and the treatment temperature may be 0 to 30°C, preferably 10 to 28°C, more preferably 18 to 25°C, and when treated for the above time at this temperature range, the tissue structure is sufficiently loosely deformed without collapse of the tissue structure or loss of elastin, and an appropriate decellularization reaction can be achieved.

[0092] The aqueous base solution used as the treatment solution at this time may be an aqueous sodium hydroxide solution, and the concentration of sodium hydroxide contained in the aqueous sodium hydroxide solution may be greater than 0.05 M and less than 0.2 M. If the concentration of sodium hydroxide is 0.05 M or less, the tissue structure is not sufficiently loosened, which causes a problem of reduced decellularization efficiency in the secondary decellularization step described later; and if it is 0.2 M or more, some tissues may disintegrate or dissolve beyond the level of loosening in this step, resulting in a significant decrease in the final yield and loss of elastin, so it is desirable to use an aqueous sodium hydroxide solution of the aforementioned concentration.

[0093] After the first decellularization step, a second decellularization step using enzymes is performed, and between these two steps, a neutralization step and a washing step may be performed.

[0094] The neutralization step is a step of treating the sodium hydroxide used in the first decellularization step with an acidic aqueous solution to neutralize it. The type of acidic aqueous solution used at this time may include, for example, an acidic aqueous solution containing at least one of hydrochloric acid, sulfuric acid, acetic acid, and peracetic acid, but is not limited thereto, and the concentrations thereof may also be appropriately adjusted to suit the working environment or conditions.

[0095] The washing step is a step performed to prevent a decrease in enzyme reactivity due to residues when enzymatically treating in the subsequent secondary decellularization step. A buffer solution may be used as the washing solution in the washing step, for example, a PBS (Phosphate buffered saline) solution may be used, but is not limited thereto.

[0096] Meanwhile, the above-mentioned second decellularization step is a step of removing cells by enzymatically treating the first decellularized tissue, and a step of removing DNA within the first decellularized tissue by treating the first decellularized tissue with a DNA degrading enzyme.

[0097] At this stage, the tissue can be treated in an amount of 10 to 25 parts by weight, preferably 20 to 25 parts by weight, per 100 parts by weight of a treatment solution containing DNA degrading enzymes. This is a treatment capacity nearly double that of the conventional method, as previously explained, and is achievable because the treatment efficiency is increased by the multi-stage decellularization process of the present invention.

[0098] In this step, to obtain enzyme activity and sufficient DNA degradation efficiency, the treatment time for treating the tissue with a DNA degrading enzyme may be 10 to 35 hours, preferably 18 to 30 hours, and the treatment temperature may be 30 to 45°C, preferably 35 to 42°C.

[0099] In the present invention, the concentration of DNA degrading enzyme included in the treatment solution is 0.0001 to 0.005 wt%, which is several to hundreds of times lower than the concentration of DNA degrading enzyme typically used in conventional similar technologies. In the primary decellularized tissue of the present invention, foreign substances including crude fat within the tissue are removed and the tissue structure becomes loose as the primary decellularization step is performed. When DNA degrading enzyme is introduced in this state, the DNA degrading enzyme can penetrate very easily into the tissue structure. Accordingly, since DNA removal efficiency that is at least the same or superior can be secured even when using a low concentration of DNA degrading enzyme, the present invention allows for the use of such a low concentration of DNA degrading enzyme.

[0100] However, if the concentration of the DNA degrading enzyme is below the above range, the DNA degradation efficiency decreases, so it is desirable to use it at a concentration of 0.0001% or higher. If it exceeds the above range, the improvement in DNA degradation efficiency is extremely minimal compared to the amount of additional DNA degrading enzyme, making it uneconomical. Therefore, it is desirable to use the DNA degrading enzyme within the concentration range described above.

[0101] The tissue decellularized through the above second decellularization step undergoes a second inactivation step to further remove viruses, and between these two steps, a decolorization step and / or a degreasing step may be additionally performed. The decolorization step is a step to remove the color of the tissue, which can be performed, for example, by treating the tissue with hydrogen peroxide, and the degreasing step is a step to further remove lipids contained in the tissue, which can be performed, for example, using a ketone solution, preferably an acetone solution.

[0102] Next, a second inactivation step is performed to further inactivate the viruses contained within the tissue that has undergone the second decellularization step. This step involves inactivating the viruses within the tissue using acid, specifically treating the tissue with a mixture of organic acid and alcohol.

[0103] The organic acid used at this stage may be peracetic acid, which acts as an oxidizing agent and can inactivate viruses by causing non-specific free radical damage. The peracetic acid used in this step can be used at a concentration of 0.05 to 1%.

[0104] In addition, the alcohol used in this step may be a lower alcohol having 1 to 4 carbon atoms, such as methanol, ethanol, or propanol, and preferably ethanol. It is desirable to use ethanol because it performs the function of inactivating viruses and fixing tissue structures to make the tissues rigid.

[0105] Through these steps, the virus is finally inactivated, crude fat and DNA are removed, and a multi-stage decellularized protein extract containing various extracellular matrix components, including elastin, can be obtained.

[0106] The DNA content contained in the multi-stage decellularized protein extract obtained in this way is 50 ng / mg or less, preferably 20 ng / mg or less, 10 ng / mg or less, and more preferably 5 ng / mg or less, so that the induction of immunity or various foreign body reactions can be minimized.

[0107] In addition, the multi-stage decellularized protein extract has the advantage of having a very low elastin content reduced through the pretreatment step to the second inactivation step, thereby providing effects such as wound healing and scar suppression by elastin. Specifically, the reduction rate (L) of the elastin content of the tissue that has undergone the pretreatment step to the second inactivation step is 20% or less. This numerical value is very low compared to the case where the reduction rate (L) of the elastin content is very high, at 50% or more, when a conventional decellularization method is applied.

[0108] Meanwhile, after the second inactivation step, additional steps such as washing, packaging, sterilization, and processing may be performed. Washing may be performed using a buffer solution, distilled water, etc., and packaging is performed to prevent contamination and facilitate handling when the multi-stage decellularized protein extract is stored or transported.

[0109] Another embodiment of the present invention relates to an absorbable hemostatic agent for internal use. The absorbable hemostatic agent for internal use according to the present embodiment comprises a particle mixture and a mixture, wherein the particle mixture comprises gelatin, gelatin, thrombin, and calcium chloride, and the mixture comprises a temperature-sensitive gel, purified water, polyvinylpyrrolidone, polyethylene glycol, sodium chloride, and a multi-stage decellularized protein extract.

[0110] Since the particle mixture and mixture of the absorbable internal hemostatic agent according to this embodiment may be the particle mixture and mixture included in the absorbable internal hemostatic kit according to one embodiment of the present invention described above, some overlapping descriptions are omitted.

[0111] The particle mixture and the mixture included in the above-mentioned absorbable hemostatic agent may each be packaged in different containers, such as syringes, and then mixed together for use. The mixing ratio of the particle mixture and the mixture at use may be 1:3 to 20 by weight, and preferably 1:4 to 10. If the mixing ratio falls outside the above range, the viscosity of the absorbable hemostatic agent, which is a mixture of the particle mixture and the mixture, becomes excessively high or low, resulting in poor spreadability; therefore, it is preferable to mix within the weight range described above.

[0112] The above particle mixture may contain 50 to 99 wt% gelatin, 0.01 to 25 wt% thrombin, and 0.01 to 25 wt% calcium chloride. In addition to this, the mixture may further contain at least one of a multi-stage decellularized protein extract, albumin, and mannitol as needed; in this case, the multi-stage decellularized protein extract may be included in an amount of 0.1 to 5 wt%, albumin in an amount of 0.1 to 3 wt%, and mannitol in an amount of 0.1 to 5 wt%. The multi-stage decellularized protein extract mentioned in this embodiment is a biocompatible material prepared by multi-stage decellularization of mammalian-derived tissue, and since it is identical to that mentioned in one embodiment of the present invention, a redundant description is omitted.

[0113] The above mixture may comprise 1 to 35 wt% of a temperature-sensitive gel, 45 to 90 wt% of purified water, 0.1 to 20 wt% of polyvinylpyrrolidone, 0.1 to 20 wt% of polyethylene glycol, 0.01 to 5 wt% of sodium chloride, and 0.1 to 10 wt% of a multi-stage decellularized protein extract.

[0114] The temperature-sensitive gel may be a poloxamer, which is a copolymer of polyethylene oxide (PEO) - polypropylene oxide (PPO) - polyethylene oxide (PEO), exhibiting a sol-gel phase transition behavior depending on temperature changes. Preferably, to improve in vivo stability and ease of use, it is desirable to use a poloxamer with an HLB value of 18 to 23.

[0115] The above mixture may further contain mannitol, in which case the mannitol may be included in an amount of 0.1 to 5 wt%.

[0116] Another embodiment of the present invention relates to a method for preparing an absorbable hemostatic agent for internal use according to another embodiment of the present invention. Since the particle mixture, the mixture, and the components included therein mentioned below are the same as those described in the previously examined embodiments, redundant descriptions are omitted.

[0117] The method for manufacturing an absorbable hemostatic agent for internal use according to the present embodiment comprises: a particle mixture preparation step of preparing a particle (powder) mixture comprising gelatin, thrombin, and calcium chloride; a mixture preparation step of preparing a mixture comprising a temperature-sensitive gel, purified water, polyvinylpyrrolidone, polyethylene glycol, sodium chloride, and a multi-stage decellularized protein extract; and a mixing step of mixing the particle mixture and the mixture.

[0118] The particle mixture preparation step comprises: Step 1A, which involves crushing and sieving gelatin to collect particles that pass through a 30- to 70 mesh sieve; and Step 1B, which involves mixing the gelatin obtained through Step 1A with thrombin and calcium chloride to prepare a particle mixture. Additionally, Step 1C, which involves filling the particle mixture into a first syringe (100) after Step 1B, may be further included.

[0119] Step 1A is a step of selecting gelatin particles having a predetermined particle size by crushing and sieving gelatin and collecting particles that pass through a 30 to 70 mesh sieve.

[0120] Step 1B is a step of preparing a particle mixture by mixing the gelatin obtained through Step 1A, thrombin, and calcium chloride, and the particle mixture can be prepared by mixing in a weight ratio of 50~99 wt% gelatin, 0.01~25 wt% thrombin, and 0.01~25 wt% calcium chloride.

[0121] At this stage, if necessary, at least one of a multi-stage decellularized protein extract, albumin, and mannitol may be further mixed into the particle mixture, in which case the multi-stage decellularized protein extract may be included in an amount of 0.1 to 5 wt%, albumin in an amount of 0.1 to 3 wt%, and mannitol in an amount of 0.1 to 5 wt%.

[0122] Step 1C is the step of preparing the first syringe (100) filled with the particle mixture by filling the particle mixture into the first syringe (100).

[0123] The above-mentioned mixture preparation step comprises: Step 2A, which involves mixing and mechanically stirring a temperature-sensitive gel, purified water, polyvinylpyrrolidone, polyethylene glycol, sodium chloride, and a multi-stage decellularized protein extract; and Step 2B, which involves removing air bubbles from the mixture obtained through Step 2A to prepare the mixture. Additionally, Step 2C, which involves filling the mixture into a second syringe (200) after Step 2B, may be further included.

[0124] Step 2A is the step of mixing and mechanically stirring a temperature-sensitive gel, purified water, polyvinylpyrrolidone, polyethylene glycol, sodium chloride, and a multi-stage decellularized protein extract.

[0125] The temperature-sensitive gel included in the mixture for the second syringe (200) is a poloxamer that exhibits a sol-gel phase transition behavior depending on the temperature change. Since it is water-soluble and does not dissolve easily due to its agglomerating properties, it is desirable to undergo a process of mechanical stirring to ensure it is uniformly dissolved in purified water. Preferably, it is effective to use a blender to stir so that grinding and mixing occur simultaneously.

[0126] At this stage, the mixture may be composed of 1~35 wt% temperature-sensitive gel, 45~90 wt% purified water, 0.1~20 wt% polyvinylpyrrolidone, 0.1~20 wt% polyethylene glycol, 0.01~5 wt% sodium chloride, and 0.1~10 wt% multi-stage decellularized protein extract.

[0127] In addition, the mixture may further include mannitol as needed. In this case, mannitol may be mixed with other components in step 2A, and in this case, mannitol may be mixed in a weight ratio of 0.1 to 5 wt%.

[0128] Step 2B is a step of preparing a mixture by removing air bubbles from the mixture obtained through Step 2A. One or more of the following methods may be applied for removing air bubbles: refrigeration and the use of a vacuum degassing machine.

[0129] The refrigeration method can be performed by storing the mixture obtained through step 2A at a low temperature, for example, at a temperature of 3 to 10°C for 30 minutes to 24 hours. The use of a vacuum degasser can be performed using a standard vacuum degasser used for removing bubbles.

[0130] Step 2C is the step of preparing the second syringe (200) filled with the mixture by filling the mixture into the second syringe (200).

[0131] The above mixing step is a step of mixing the particle mixture and the mixture. In this step, mixing can be performed by connecting the discharge port of the first syringe (100) containing the particle mixture and the discharge port of the second syringe (200) containing the mixture to a connector (300). The mixing method is not particularly limited and, for example, can be mixed by injecting the particle mixture in the first syringe (100) into the second syringe (200), or vice versa, or by repeating this injection operation multiple times. The particle mixture and the mixture can be mixed in a weight ratio of 1:3 to 20, and preferably in a weight ratio of 1:4 to 10.

[0132] Hereinafter, the specific operation and effects of the present invention will be explained through an embodiment of the present invention. However, this is presented as a preferred example of the present invention, and the scope of the rights of the present invention is not limited according to the embodiment.

[0133] [Preparation Example 1]

[0134] 1. Preparation and Preprocessing Steps

[0135] Porcine heart aorta that had been washed, dried, cut, and frozen was prepared, and after thawing, washed with distilled water and cut into 50×50 mm pieces to prepare raw tissue specimens.

[0136] 2. First inactivation step

[0137] Next, 250g of raw tissue specimens were added to 1L of 70% n-propanol and stirred at a temperature of 20℃ and a stirring speed of 150RPM for 24 hours to perform the first inactivation step.

[0138] 3-1. Primary Decellularization Stage (First Decellularization Stage)

[0139] Next, a mixed solution containing 70% n-propanol and 0.1M sodium hydroxide was prepared in distilled water, and the raw tissue specimen was mixed in such a way that 23 parts by weight were included in 100 parts by weight of the mixed solution, and then the first decellularization step was performed by stirring at a temperature of 20℃ and a stirring speed of 150 RPM for 24 hours.

[0140] 3-2. Primary Decellularization Stage (Second Decellularization Stage)

[0141] Next, 24 parts by weight of the tissue specimen that had undergone the first decellularization step were mixed with 100 parts by weight of a pre-prepared 0.1M aqueous sodium hydroxide solution, and then the second decellularization step was performed by stirring for 24 hours under the same conditions as the previous step.

[0142] The tissue specimen that had undergone the second decellularization step was subsequently immersed in an aqueous acetic acid solution and stirred to neutralize it, after which it was washed with a PBS solution.

[0143] 4. Secondary decellularization stage

[0144] Next, the tissue specimen was placed in a treatment solution containing DNase at a concentration of 0.00011 wt%, and a second decellularization step was performed by stirring at a stirring speed of 150 RPM for 23 hours at a temperature of 37°C. The tissue specimen, after the second decellularization step was completed, was decolorized by treating it in a 3% hydrogen peroxide solution at 20°C for 1 hour, and degreased by treating it in a 20% aqueous acetone solution at 20°C for 1 hour.

[0145] 5. Second inactivation step

[0146] Next, the tissue specimen was immersed in a mixed solution containing 70% n-propanol and 0.2% peracetic acid in distilled water, and treated at 20°C for 4 and a half hours to perform a second inactivation step.

[0147] Finally, the tissue specimen that had undergone all of the above processing steps was washed with PBS solution and distilled water to prepare a multi-stage decellularized protein extract according to one embodiment of the present invention.

[0148] [Experimental Example 1]

[0149] A multi-stage decellularized protein extract was prepared using the same method as in Preparation Example 1, wherein the first, second, and second decellularization steps were performed sequentially, and a tissue specimen was collected immediately after each step was performed to measure the elastin content per 1 mg of tissue specimen, and the results are shown in Fig. 4(A). In addition, the elastin content of the original tissue was measured during the preparation step, and the elastin content at the time each step was performed was expressed as a percentage relative to the elastin content of the original tissue and is shown in Fig. 4(B).

[0150] In FIGS. 4(A) and FIGS. 4(B), Process 1 refers to the first decellularization step, Process 2 refers to the second decellularization step, and Process 3 refers to the second decellularization step (as described below with the same meaning).

[0151] Elastin content was determined by testing the Fastin Elastin Assay kit F2000 (Manufacturer: Biocolor) according to the kit's protocol, followed by analysis with a Multimode plate reader Victor Nivo TM Elastin was analyzed at an absorbance of 513 mm using (Manufacturer: Perkin Elmer), and the elastin content was measured by comparing it with a standard material.

[0152] Referring to the results in Figures 4(A) and 4(B), it can be seen that the elastin content is slightly lost as each process proceeds, but at the time the final process is completed, more than 80% of the elastin remains.

[0153] [Experimental Example 2]

[0154] A multi-stage decellularized protein extract was prepared using the same method as in Preparation Example 1, but with the first, second, and second decellularization steps each omitted by only one step, and a specimen of the multi-stage decellularized protein extract that had undergone the final process was collected and the elastin content per 1 mg of the specimen was measured. Then, the elastin content of the original tissue was measured in the same way during the preparation stage, and the L value according to Formula (1) was calculated and the results were listed in Table 1.

[0155] In addition, regarding the elastin content of the original tissue in the preparation stage, the elastin content contained in the multi-stage decellularized protein extract obtained by omitting each step was calculated as a percentage, and the results are shown in Fig. 5.

[0156] The elastin content was measured in the same manner as in Experimental Example 1, and in Table 1 and Figure 5, Process 1 refers to the first decellularization step, Process 2 refers to the second decellularization step, and Process 3 refers to the second decellularization step.

[0157]

[0158] First, looking at Table 1, it can be seen that the L value is 20% or less in both cases where all processes are performed and when one process is omitted, and referring to Fig. 5, it can be seen that the elastin content does not change significantly even if a specific process is omitted. Therefore, from the above experimental results, it was confirmed that the elastin content is not significantly affected even if any one of the first decellularization step, the second decellularization step, and the second decellularization step is omitted in the process according to one embodiment of the invention.

[0159] [Experimental Example 3]

[0160] The experiment was conducted in the same manner as in Experimental Example 1, but DNA content was measured instead of elastin content after each step. DNA content was measured using the analysis kit Quant-iT™ PicoGreen™ dsDNA Assay kits and dsDNA Reagents (Manufacturer: Invitrogen) according to the protocol of the analysis kit, and then the DNA content was measured at an absorbance of 480–520 mm using a Multimode plate reader Victor Nivo™ (Manufacturer: Perkin Elmer).

[0161] The DNA content per 1 mg of tissue after each step is shown in Fig. 6(A), and the DNA content of the tissue after each step relative to the DNA content of the original tissue is shown as a percentage value in Fig. 6(B). In Figs. 6(A) and 6(B), Process 1 refers to the first decellularization step, Process 2 refers to the second decellularization step, and Process 3 refers to the second decellularization step.

[0162] Referring to Figures 6(A) and 6(B), it can be seen that most of the DNA is removed after undergoing the first decellularization step, the second decellularization step, and the second decellularization step.

[0163] [Experimental Example 4]

[0164] The experiment was conducted in the same manner as in Experimental Example 2, but with one step omitted for each, and the DNA content of the tissue matrix prepared was measured in the same manner as in Experimental Example 3.

[0165] The DNA content per 1 mg of tissue matrix obtained by omitting each step is shown in Fig. 7(A), and the DNA content of the tissue obtained by omitting each step relative to the DNA content of the original tissue is shown as a percentage value in Fig. 7(B). In this case, in Figs. 7(A) and 7(B), Process 1 refers to the first decellularization step, Process 2 refers to the second decellularization step, and Process 3 refers to the second decellularization step.

[0166] Referring to Figures 7(A) and 7(B), it can be confirmed that DNA is reliably removed only after undergoing the first decellularization step, the second decellularization step, and the second decellularization step. In particular, it can be seen that the efficiency of DNA removal drops significantly when the second decellularization step or the second decellularization step is omitted; therefore, through the above experiment, it was confirmed that sequentially undergoing each step is more effective for DNA removal.

[0167] [Experimental Example 5]

[0168] A multi-stage decellularized protein extract was prepared using the same method as in Preparation Example 1, but the tissue was treated by varying the concentration of sodium hydroxide included in the treatment solution to 0.005M, 0.1M, 0.2M, and 0.3M during the first decellularization step.

[0169] During the experiment, the condition of the sample during and after the second decellularization step (second process) was photographed and is shown in Fig. 8. In addition, the DNA content contained in 1 mg of the finally obtained multi-stage decellularized protein extract was measured using the same method as in Experimental Example 3, and the yield of the multi-stage decellularized protein extract was calculated by expressing the content of the finally obtained multi-stage decellularized protein extract as a percentage value relative to the content of the initially introduced tissue, and the H&E stained photograph is shown in Fig. 8.

[0170] Referring to Figure 8, when the concentration of NaOH is 0.05M, the yield is good, but the DNA content is high and it is confirmed that there are cell nuclei that were not removed in the tissue, indicating that the decellularization efficiency is low.

[0171] On the other hand, when treated with 0.1M NaOH, it can be seen that the tissue structure is well maintained in the second decellularization step, the DNA content after final treatment is very low at 0.6 ng / mg, and the yield is high at 20.8%.

[0172] When treated with 0.2M NaOH, the DNA content was low, but some of the tissue was disintegrated during the first decellularization step, resulting in a final yield that was less than half of that of the case treated with 0.1M NaOH, and when treated with 0.3M NaOH, the tissue was almost dissolved, making it impossible to obtain tissue.

[0173] Therefore, from the above experimental results, it was confirmed that in order to obtain a multi-stage decellularized protein extract in which DNA within the tissue is sufficiently removed and the tissue does not disintegrate or dissolve during the first decellularization step, the concentration of NaOH included in the treatment solution during the first decellularization step, particularly during the second decellularization step, is greater than 0.05M and less than 0.2M.

[0174] [Experimental Example 6]

[0175] Multistage decellularized protein extracts were prepared using the same method as in Preparation Example 1, but in the first decellularization step, when the concentration of sodium hydroxide included in the treatment solution was 0.005M and 0.1M, the tissues were treated while varying the concentration of Dnase from 0.0001 to 0.003 wt%, and then the DNA content ratio of each tissue matrix and the yield of the tissue matrix obtained were calculated and the results are listed in Table 6.

[0176]

[0177] Referring to the results in Table 2 above, it can be seen that when the concentration of NaOH is constant, the DNA concentration in the final product, the multi-stage decellularized protein extract, decreases as the concentration of DNase increases. However, when the NaOH concentration was 0.1M, the DNA concentration in the final multi-stage decellularized protein extract was actually lower despite treatment with a DNase concentration several to tens of times lower than when it was 0.05M, and it was confirmed that the difference in yield was not significant. Experimental results showed that when the NaOH concentration was 0.05M, the DNA removal efficiency was significantly lower compared to when it was 0.1M, indicating that it is desirable for the concentration of NaOH in the treatment solution used in the first decellularization step to exceed 0.05M.

[0178] [Preparation Example 2]

[0179] Gelatin was crushed to obtain particles that passed through a 30-mesh sieve, and 92.5g of gelatin, 3.5g of thrombin, and 4g of calcium chloride were mixed to prepare a particle mixture.

[0180] 15.6 g of poloxamer, 76.7 g of water for injection, 3.5 g of polyvinylpyrrolidone, 2.0 g of polyethylene glycol, 0.7 g of sodium chloride, and 1.5 g of multi-stage decellularized protein extract were mixed and ground and stirred with a blender to prepare a mixture. The prepared mixture was stored in a refrigerator at 4°C for 1 hour to degas the mixture.

[0181] Next, the absorbable hemostatic agent of the example was prepared by mixing the particle mixture and the mixture in a weight ratio of 1:6.

[0182] [Experimental Example 7]

[0183] The viscosity of the absorbable hemostatic agent prepared in the example according to temperature was measured using a viscometer (DV2THBTJ0, AMETEK BROOKFIELD) and the results are shown in Fig. 9.

[0184] As can be seen from the experimental results in Fig. 9, the absorbable hemostatic agent according to one embodiment of the present invention maintains low viscosity at room temperature, but its viscosity increases rapidly at 37°C, which is similar to body temperature. From these viscosity characteristics according to temperature, it can be anticipated that the absorbable hemostatic agent according to the present invention has excellent application characteristics at room temperature, but when applied to a wound, its viscosity increases due to body temperature, so the applied form is well maintained without collapsing, and thus it can function efficiently and effectively as a hemostatic agent.

[0185] [Experimental Example 8]

[0186] A commercially available absorbable hemostatic agent product C was prepared as Comparative Example 1 and a commercially available absorbable hemostatic agent product P was prepared as Comparative Example 2. The hemostatic performance of the example, Comparative Example 1, and Comparative Example 2 was evaluated using the Lee-White method described below, and the results are shown in FIG. 10.

[0187] The experiment was specifically performed as follows. First, 100 mg of the absorbable hemostatic agent sample was placed in a test tube, and a blood sample was prepared by pipetting 900 μL of blood and a 40 mM CaCl₂ solution into an Ep tube for 15 seconds. Next, the test tube containing the absorbable hemostatic agent sample was laid flat, 1 mL of the blood sample was dispensed, and the tube was placed in a 37°C water bath. The tube was then tilted 30 degrees every 15 seconds to observe whether coagulation occurred. The time until the blood stopped flowing even when tilted up to 180 degrees was measured and recorded.

[0188] In addition, the experimental results obtained by performing the above experiment without using a separate absorbable hemostatic agent—namely, the hemostatic time when blood alone was used as the hemostatic time of the control group—were used to calculate the percentage of the hemostatic time of the hemostatic agent relative to the control group, and the results are shown in Fig. 10. Additionally, photographs of the final results of each experiment, i.e., samples with completed coagulation, are attached in Fig. 11. Three experiments were performed for each hemostatic agent, photographs of all samples are attached in Fig. 11, and a graph based on the average coagulation time is plotted and shown in Fig. 10.

[0189] As a result of the experiment, it took an average of 8 minutes and 5 seconds for the blood to coagulate in the control group, but in the example, comparative example 1 and comparative example 2, the blood coagulated immediately after the sample and blood were mixed.

[0190] Therefore, the results of this experiment confirmed that the absorbable hemostatic agent according to the embodiment of the present invention can achieve a rapid hemostatic function at a level similar to commercial products.

[0191] [Experimental Example 9]

[0192] The hemostatic performance of the Example, Comparative Example 1, and Comparative Example 2 was evaluated using the BCI (blood clotting index) values ​​described below, and the results are shown in FIG. 12.

[0193] First, a sample was prepared by dispensing 100 mg of an absorbable hemostatic agent sample into a 6-well plate, and a blood sample in which a coagulation reaction was initiated was prepared by injecting 500 μL of 40 mM calcium chloride into a 3.2% sodium acetate tube into which 5 mL of blood had been injected.

[0194] 200 μL of a blood sample in which the coagulation reaction was initiated was dispensed into a 6-well plate containing an absorbable hemostatic agent sample and reacted for 1, 10, and 20 minutes, followed by the addition of 10 mL of distilled water and reaction for 5 minutes. After the reaction was completed, 100 μL of distilled water was taken and dispensed into a 96-well plate, and the absorbance at a wavelength of 545 nm was measured.

[0195] In addition, absorbance was measured using the same method with a blood sample alone without using a separate hemostatic agent sample, and the BCI value was calculated using this as a control, and the results are shown in Fig. 12. The BCI value was obtained using (Equation 2).

[0196]

[0197] OD in Formula 2 c represents the absorbance of the control sample, which is blood alone without using a separate hemostatic agent sample, and OD t represents the absorbance of a test sample of blood treated with a hemostatic agent.

[0198] The experimental method for deriving the BCI value evaluates hemostatic performance based on the amount of red blood cells adsorbed to the hemostatic agent, and a lower BCI value indicates superior hemostatic performance.

[0199] In Fig. 12, the bar graph represents the reaction time between the blood sample and the absorbent hemostatic agent sample before dispensing distilled water, where white indicates the BCI value for samples reacted for 5 minutes, light gray for 10 minutes, and dark gray for 20 minutes. Referring to Fig. 12, it was confirmed that the hemostatic performance of the absorbent hemostatic agent of the example is similar to that of Comparative Example 1, a commercial product, and superior to that of Comparative Example 2.

[0200] The absorbable hemostatic agent according to the present invention comprises a powder mixture containing gelatin, thrombin, and calcium chloride; and a mixture containing a temperature-sensitive gel, purified water, polyvinylpyrrolidone, polyethylene glycol, sodium chloride, and a multi-stage decellularized protein extract; which is then mixed and applied to a wound. This primarily promotes and activates physiological hemostasis by thrombin, gels within a temperature range similar to body temperature to provide a secondary physical hemostatic effect, and provides an additional wound healing effect, thereby enabling faster and more effective hemostasis within the body, thus having industrial applicability.

Claims

1. A first syringe containing a powder mixture comprising gelatin, thrombin, and calcium chloride; A second syringe containing a mixture of a temperature-sensitive gel, purified water, polyvinylpyrrolidone, polyethylene glycol, sodium chloride, and a multi-stage decellularized protein extract; and An absorbable hemostatic kit for internal use, comprising: a connector connecting the first syringe and the second syringe so that the particle mixture and the mixture can be mixed.

2. In Paragraph 1, An absorbable hemostatic kit for internal use, characterized in that the above particle mixture further comprises at least one of a multi-stage decellularized protein extract, albumin, and mannitol.

3. In Paragraph 1, The above mixture is characterized by further containing mannitol, an absorbable hemostatic kit for internal use.

4. In accordance with Paragraph 1, The above temperature-sensitive gel is, An absorbable hemostatic kit for internal use characterized by being a poloxamer, which is a copolymer of polyethylene oxide (PEO) - polypropylene oxide (PPO) - polyethylene oxide (PEO), having a sol-gel phase transition behavior depending on temperature changes.

5. In Paragraph 4, The above-mentioned poloxamer is characterized by being in a sol phase at room temperature and in a gel phase at body temperature, an absorbable internal hemostatic kit.

6. In Paragraph 1 or 2, The above-mentioned multi-stage decellularized protein extract is, A pretreatment step for preparing and pretreating mammalian tissues excluding humans; A first inactivation step of inactivating viruses contained in pre-treated tissue using alcohol; Primary decellularization step of removing cells from virus-inactivated tissue using an aqueous base solution; A secondary decellularization step of enzymatically treating the primary decellularized tissue to remove cells; and It is manufactured through a second inactivation step in which viruses contained in decellularized tissue are inactivated using acid; and The DNA content of the tissue that has undergone the second inactivation step is 50 ng / mg or less, and An absorbable hemostatic kit for internal use, characterized in that the reduction rate (L) of the elastin content of the tissue that has undergone the above-mentioned pretreatment step to the second inactivation step is 20% or less.

7. In Paragraph 6, The above first decellularization step is, A first decellularization step performed through a mixture of n-PrOH and NaOH; and An absorbable hemostatic kit for internal use, characterized by comprising a second decellularization step performed with an aqueous sodium hydroxide solution of greater than 0.05 M and less than 0.2 M.

8. In Paragraph 1, An absorbable internal hemostatic kit comprising a catheter capable of applying an absorbable internal hemostatic agent, mixed with the above-mentioned particle mixture and liquid, to a wound.

9. A mixture of particles (powder) comprising gelatin, thrombin, and calcium chloride; and, An absorbable hemostatic agent for internal use, comprising a temperature-sensitive gel, purified water, polyvinylpyrrolidone, polyethylene glycol, sodium chloride, and a multi-stage decellularized protein extract; the mixture of which is applied to the affected area.

10. In Paragraph 9, The above particle mixture is characterized by further comprising at least one of a multi-stage decellularized protein extract, albumin, and mannitol, an absorbable hemostatic agent for internal use.

11. In Paragraph 9, The above mixture is an absorbable hemostatic agent for internal use, characterized by further containing mannitol.

12. In Paragraph 9, The above temperature-sensitive gel is, An absorbable hemostatic agent for internal use, characterized by being a poloxamer, which is a copolymer of polyethylene oxide (PEO) - polypropylene oxide (PPO) - polyethylene oxide (PEO), having a sol-gel phase transition behavior depending on temperature changes.

13. In Paragraph 12, The above-mentioned poloxamer is an absorbable hemostatic agent for internal use, characterized by being in a sol phase at room temperature and undergoing a phase transition to a gel phase at body temperature.

14. In Paragraph 9 or 10, The above-mentioned multi-stage decellularized protein extract is, A pretreatment step for preparing and pretreating mammalian tissues excluding humans; A first inactivation step of inactivating viruses contained in pre-treated tissue using alcohol; Primary decellularization step of removing cells from virus-inactivated tissue using an aqueous base solution; A secondary decellularization step of enzymatically treating the primary decellularized tissue to remove cells; and It is manufactured through a second inactivation step in which viruses contained in decellularized tissue are inactivated using acid; and The DNA content of the tissue that has undergone the second inactivation step is 50 ng / mg or less, and An absorbable hemostatic agent for the body, characterized in that the reduction rate (L) of the elastin content of the tissue that has undergone the above-mentioned pretreatment step to the second inactivation step is 20% or less.

15. In Paragraph 14, The above first decellularization step is, A first decellularization step performed through a mixture of n-PrOH and NaOH; and An absorbable hemostatic agent for the body, characterized by comprising a second decellularization step performed with an aqueous sodium hydroxide solution of greater than 0.05 M and less than 0.2 M.

16. In Paragraph 9, An absorbable hemostatic agent for internal use, characterized in that the mixing ratio of the particle mixture and the mixture is 1:3 to 20 based on weight.

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