Pharmaceutical composition for gastrointestinal hemostasis and wound treatment

Abstract

A pharmaceutical composition for hemostasis in the digestive tract and a pharmaceutical composition for wound treatment are provided. In one embodiment, the pharmaceutical composition may include a first component consisting of a water-soluble copolymer that adheres to gastrointestinal mucosal tissue, a second component consisting of a water-absorbent material, and thrombin. In the pharmaceutical composition for hemostasis in the digestive tract and the pharmaceutical composition for wound treatment according to one embodiment of the present invention, the first component and the second component generate a synergistic effect with thrombin, and the pharmaceutical composition for hemostasis in the digestive tract and the pharmaceutical composition for wound treatment have a superior hemostatic and wound treatment effect to conventionally commercially available hemostatic agents, a superior hemostatic and wound treatment effect to a mixture of thrombin with other conventionally commercially available hemostatic agents, and a superior hemostatic and wound treatment effect to a single use of thrombin.

Description

[Technical field]

[0001] The present invention relates to a pharmaceutical composition for hemostasis in the gastrointestinal tract and wound treatment, and to a pharmaceutical composition for hemostasis and wound treatment in bleeding gastrointestinal ulcers by being effectively administered into the gastrointestinal tract and a pharmaceutical preparation containing the same. Meanwhile, this patent application claims priority to Korean Patent Application No. 10-2022-0019745 filed with the Korean Intellectual Property Office on February 15, 2022 and Korean Patent Application No. 10-2022-0035054 filed with the Korean Intellectual Property Office on March 22, 2022, the disclosures of which are incorporated herein by reference. [Background technology]

[0002] Gastrointestinal bleeding is a common clinical condition, and approximately 50% of gastric ulcer patients are diagnosed with bleeding gastric ulcers. More than 80% of gastrointestinal bleeding occurs in the upper gastrointestinal tract, and upper gastrointestinal bleeding refers to a condition in which bleeding occurs from lesions in the esophagus, stomach, or duodenum, resulting in hematemesis and bloody stools. More than 90% of bleeding lesions in upper gastrointestinal bleeding can be identified by endoscopic examination. Recently, a variety of procedures have been performed, including polypectomy for the stomach and large intestine, mucosal resection for the treatment of early gastric cancer and colon cancer, and therapeutic endoscopic procedures. In some cases, bleeding during or after these procedures can lead to emergency surgery or even death.

[0003] Recently, endoscope-based hemostasis has been used as a treatment for gastrointestinal bleeding. Examples of such endoscopic hemostasis include a method of directly injecting saline solution, epinephrine, or alcohol into the ulcer, a method of using coagulation therapy using electric heat, argon, or laser, and a method of hemostasis using clips. However, the above-mentioned conventional treatment methods reduce bleeding by injecting a liquid medicine around the blood vessels exposed by the ulcer to compress the blood vessels or by ligating the blood vessels themselves, and the ulcers at the wound site are left in a state where the mucosa has fallen off. Therefore, there are frequent cases where hemostasis is not achieved despite endoscopic hemostasis, and research has shown that hemostasis is achieved in only about 70 to 80% of cases when using conventional endoscopic hemostasis. In addition, even after hemostasis is achieved once, rebleeding occurs 3 to 4 days later in 20 to 25% of cases.

[0004] Rebleeding is a condition where blood vessels bleed before the tissue at the ulcer site regenerates and the ulcer is healed. This is a problem that cannot be solved by conventional endoscopic treatments, which stop bleeding while maintaining the exposed state of the ulcer mucosa by sloughing off. That is, conventional endoscopic treatments have the disadvantages of slow healing speed of the wound site and the possibility of rebleeding. In order to solve this problem, Korean Patent Publication No. 10-2006-0040329 has disclosed an internal hemostatic agent and its application method that induces hemostasis by dispersing and coating a polymer solution-type coating agent at the ulcer site inside the body through an endoscope, thereby minimizing the possibility of rebleeding.

[0005] On the other hand, in order to increase the healing rate of the wound site and minimize the possibility of rebleeding, a multifunctional formulation is required that can not only prevent bleeding (i.e., hemostasis), but also effectively protect the wound site and further prevent adhesion. That is, a formulation that can be administered (injected) to the wound site of the digestive tract via an endoscopic catheter and play the role of wound protection, bleeding prevention, and / or adhesion prevention is required. For topical application in the digestive tract, it should have appropriate viscosity conditions, appropriate mucoadhesiveness, and it is necessary to form a physical protective film at the target site to provide physical bleeding prevention.

[0006] To prevent adhesion at the treatment site, gel and sol-gel formulations have been developed. Low-viscosity gel formulations have a large loss when applied to the upper digestive tract and have limited ability to form a physical barrier film, while high-viscosity gel formulations have excellent mucosal adhesion but require a high-pressure drug injection device, which also has the disadvantage of causing significant loss. Sol-gel formulations are formulations that gel at a certain temperature (body temperature) in a sol state before injection into the body, but the sol-gel transition period is small, so there is a problem of gelling in the catheter before application to the body, which also causes significant loss.

[0007] To solve these problems, various hemostatic agents and wound treatment agents have been developed, but there is still a need in clinical practice to develop pharmaceutical compositions and pharmaceutical preparations that can achieve more rapid hemostatic action and prevent rebleeding. Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention aims to provide a pharmaceutical preparation that can be effectively administered into the digestive tract for hemostasis and wound treatment in bleeding digestive tract ulcers, and provides a pharmaceutical composition that can shorten the hemostatic effect and wound treatment time and significantly improve the effects compared to conventional commercially available wound treatment agents such as hemostatic agents and wound dressings.

[0009] In addition, the pharmaceutical formulation prepared from the composition according to one embodiment of the present invention is formulated in a powder form, and thus has suitable flowability, making it easy to pass through a catheter and apply, and provides a pharmaceutical formulation that can minimize loss of the pharmaceutical formulation by forming a barrier through instantaneous gelation at the application site. [Means for solving the problem]

[0010] In order to solve the above problems, the present invention provides a pharmaceutical composition for hemostasis in the gastrointestinal tract and a pharmaceutical composition for wound treatment. In one embodiment, the pharmaceutical composition can include a first component consisting of a water-soluble copolymer that adheres to gastrointestinal mucosal tissue, a second component consisting of a water-absorbent material, and thrombin. Effect of the Invention

[0011] According to one embodiment of the present invention, the hemostatic effect and wound treatment time can be shortened and the effects can be significantly improved compared to conventional wound treatment agents such as commercially available hemostatic agents and wound dressings.

[0012] In addition, according to one embodiment of the present invention, by applying a pharmaceutical composition containing the first component, the second component, and thrombin, a significant synergistic effect in hemostatic effect and wound healing effect can be achieved compared to the use of the first component and the second component separately or the use of thrombin alone.

[0013] In addition, the pharmaceutical formulation prepared from the composition according to one embodiment of the present invention is formulated in a powder form, which has suitable flowability, making it easy to pass through a catheter and apply, and can form a barrier at the application site through instantaneous gelation, minimizing loss of the pharmaceutical formulation. [Brief description of the drawings]

[0014] [Figure 1-7]The blood coagulation time measurement experimental results performed according to Example 1-1 of the present invention are shown, with FIG. 1 showing the experimental results performed on sample S1, FIG. 2 showing the experimental results performed on sample S1-1, FIG. 3 showing the experimental results performed on sample S2, FIG. 4 showing the experimental results performed on sample S2-1, FIG. 5 showing the experimental results performed on sample S3, FIG. 6 showing the experimental results performed on sample S3'-1, and FIG. 7 showing the experimental results performed on sample S4. [Figure 8] FIG. 13 is a diagram showing the measurement results of red blood cell hemolysis carried out according to Experimental Example 1-2-1 of the present invention. [Figure 9-12] The results of thrombus weight measurements performed according to Experimental Example 1-2-2 of the present invention are shown in Fig. 9 for sample S1-1, Fig. 10 for sample S2-1, Fig. 11 for sample S3'-1, and Fig. 12 for sample S4. [Figure 13-19] The results of blood clotting time measurements performed according to experimental examples 1-3 of the present invention are shown in Fig. 14 for sample S5, Fig. 15 for sample S6, Fig. 16 for sample S7, Fig. 17 for sample S8, Fig. 18 for sample S9, and Fig. 19 for sample S10. [Figure 20-23] The figures show endoscopic findings for the success rate of early hemostasis performed according to experimental example 3 of the present invention, with FIG. 20 showing the experimental results for the no treatment group, FIG. 21 showing the experimental results for the Endoclot treatment group, FIG. 22 showing the experimental results for the CGGEL treatment group, and FIG. 23 showing the experimental results for the CGGEL_Thrombin treatment group. [Figure 24] FIG. 13 is a diagram showing the results of an experiment on mucosal regeneration area carried out according to Experimental Example 3 of the present invention. [Figure 25-30] The results of blood clotting time measurements performed according to Experimental Example 2 of the present invention are shown in Fig. 25 for sample S11-1, Fig. 26 for sample S12-1, Fig. 27 for sample S13-1, Fig. 28 for sample S14, Fig. 29 for sample S15, and Fig. 30 for sample S16. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The present invention provides a pharmaceutical composition which achieves hemostatic and wound healing effects more rapidly in gastrointestinal ulcers and blocks the possibility of rebleeding, as compared with previously developed and commercially available hemostatic and wound healing compositions.

[0016] That is, the present invention provides a pharmaceutical composition for hemostasis that comprises a first component consisting of a water-soluble copolymer that adheres to gastrointestinal mucosal tissue, a second component consisting of a water-absorbent material, and thrombin.

[0017] That is, the present invention provides a pharmaceutical composition for wound treatment that comprises a first component consisting of a water-soluble copolymer that adheres to gastrointestinal mucosal tissue, a second component consisting of a water-absorbent material, and thrombin.

[0018] The first component adheres to gastrointestinal mucosal tissue to exert a physical hemostatic effect, and may include at least one of hydroxyethyl cellulose (HEC), sodium alginate, chitosan, hydroxypropyl cellulose (HPC), and polyethylene oxide (PEO).

[0019] The second component can include at least one of croscarmellose sodium (Ac-di-sol), sodium starch glycolate, calcium silicate, and crospovidone.

[0020] For example, the first component and the second component may be included in an amount of 70-95 parts by weight: 5-30 parts by weight based on 100 parts by weight of the pre-composition before the addition of the thrombin. Within the above numerical ranges, a physical hemostatic effect can be exhibited by a protective wall formed by instantaneous Kelling in the gastric mucosa, while at the same time, a sufficient Kell strength, for example, a Kell strength of 0.001 MPa or more, can be exhibited. For example, the first component and the second component may be included in an amount of 70-90 parts by weight: 10-30 parts by weight based on 100 parts by weight of the pre-composition before the addition of the thrombin.

[0021] The thrombin used may be extracted from bovine or porcine plasma.

[0022] The thrombin contained in the pharmaceutical composition of the present invention can promote wound healing by regulating gene expression that performs inflammation and wound healing functions in mononuclear cells, and can express tissue factor (TF), which plays various roles in the wound healing process, by activating protease-activated receptor (PAR), and can accelerate angiogenesis by upregulating VEGF receptor expression in vascular endothelial cells.

[0023] The inventors of the present invention have investigated various substances having physiological hemostatic effects to rapidly achieve hemostasis and wound healing effects in gastrointestinal ulcers and to prevent rebleeding, and as a result of repeated related experiments, have found that thrombin can exert hemostatic and wound healing effects in gastrointestinal ulcers.

[0024] In addition, the inventors of the present invention have conducted repeated experiments and through trial and error on various hemostatic and / or wound healing compositions that can exert a synergistic effect with thrombin, and have confirmed that when thrombin is combined with the first component and the second component, the hemostatic and / or wound healing effect is improved.

[0025] In addition, the inventors of the present invention have confirmed that when the first component, the second component, and thrombin are used in a specific composition range, a synergistic effect of the pharmaceutical composition due to thrombin can be achieved. Although the specific pharmacological mechanism has not yet been elucidated, it has been confirmed through experiments that the physical hemostatic effect of the first component and the second component and the physiological hemostatic effect of thrombin have a synergistic effect within the composition range of the pharmaceutical composition according to the present invention.

[0026] To confirm this, the inventors of the present invention conducted experiments in which thrombin was added to various types of commercially available pharmaceutical compositions for hemostasis and / or wound treatment. As a result, it was confirmed through experiments that when a powder-form pharmaceutical composition having a composition according to one embodiment of the present invention is prepared, a synergistic effect with thrombin is generated, which results in, first, better hemostatic and wound treatment effects than conventionally available hemostatic agents, second, better hemostatic and wound treatment effects than when thrombin is mixed with other conventionally available hemostatic agents, and third, better hemostatic and wound treatment effects than when thrombin is used alone.

[0027] For example, the thrombin may be contained in the pharmaceutical composition at 20 IU / g to 10,000 IU / g. Within this range, the pharmaceutical composition may have additional hemostatic and wound healing effects. As an example, the thrombin may be contained in the pharmaceutical composition at 100 IU / g to 5000 IU / g. Within this range, a synergistic effect of the pharmaceutical composition due to the thrombin may be achieved.

[0028] For example, the thrombin may be included in an amount of 0.003 to 1.603 parts by weight per 100 parts by weight of the pharmaceutical composition. Within this range, the pharmaceutical composition may have additional hemostatic and wound healing effects. The higher the thrombin content, the better the short-term hemostatic effect, but at 1.603 parts by weight or more, the effect may converge.

[0029] For example, the thrombin may be included in an amount of 0.016 to 0.802 parts by weight per 100 parts by weight of the pharmaceutical composition. Within this range, a synergistic effect of the pharmaceutical composition with the thrombin, i.e., improved hemostatic and wound healing effects compared to those of thrombin itself, can be achieved.

[0030] Hereinafter, the constitution for defining the synergistic effect of the pharmaceutical composition with the thrombin, that is, the effect improved over the hemostatic and wound healing effects of thrombin itself, will be described.

[0031] When 200 μl of blood and a 15 mg sample of the pharmaceutical composition are mixed and the blood clotting time is measured as the time until blood stops flowing, the pharmaceutical composition of the present invention can achieve a synergistic effect in which the blood clotting time appears to be shorter than the blood clotting time of a sample consisting of the thrombin measured under the same conditions.

[0032] For example, the blood clotting time of the pharmaceutical composition made according to one embodiment of the present invention may be less than 50 seconds, for example, 30 seconds or less, and particularly for example, 25 seconds or less.

[0033] The pharmaceutical composition of the present invention comprises the steps of mixing 200 μl of blood and 15 mg of a sample of the pharmaceutical composition, incubating the mixture at 37° C. for 10 minutes, and then adding 1 ml of purified water to stop the blood coagulation reaction for 5 minutes; removing the thrombus in the state where the blood coagulation reaction has been stopped, and collecting a supernatant containing red blood cells hemolyzed by the purified water; and adding 1 ml of purified water to the collected supernatant, collecting the remaining red blood cell hemolyzed solution, and measuring the OD 540 The optical density (OD) at 540 nm was measured by a method comprising the steps of: 540 ) is the OD of the sample consisting of the thrombin measured under the same conditions. 540 It is possible to achieve a synergistic hemostatic effect that appears to be lower than the values.

[0034] For example, the optical density (OD 540 ) may be less than 1.0, for example, 0.9 or less, for example, 0.8 or less, for example, 0.7 or less, and for example, 0.6 or less.

[0035] A synergistic effect can be achieved in which the weight of a clot measured by a method comprising the steps of mixing 200 μl of blood and 15 mg of a sample of the pharmaceutical composition, reacting at 37° C. for 10 minutes, adding 1 ml of purified water to stop the blood coagulation reaction for 5 minutes, isolating the coagulated clot in a state where the blood coagulation reaction has been stopped and fixing it with 10% formalin for 20 minutes, immersing the coagulated clot in purified water and washing it for 5 minutes, and then measuring the weight after drying for 24 hours, appears to be heavier than the weight of the clot in a sample consisting of the thrombin measured under the same conditions.

[0036] For example, the thrombus weight of the pharmaceutical composition implemented according to one embodiment of the present invention may be greater than 8.0 mg, for example greater than 10.0 mg, for example greater than 15.0 mg, for example greater than 20.0 mg, and for a specific example greater than 30.0 mg.

[0037] For example, the thrombus weight of the pharmaceutical composition implemented according to one embodiment of the present invention may be more than twice, for example more than three times, and for a specific example more than four times, the thrombus weight of a sample consisting of the thrombin measured under the same conditions.

[0038] The pharmaceutical composition may further comprise a third and / or fourth component.

[0039] The pharmaceutical composition may further comprise a third component which is a wound treatment component.

[0040] For example, the third component may include at least one of the wound treatment substances epidermal growth factor, keratinocyte growth factor, and basic fibroblast growth factor. The third component may be included in a therapeutically useful amount, which may be readily determined by one of skill in the art.

[0041] The pharmaceutical composition may further comprise the fourth component, which is a component that improves the hemostatic effect.

[0042] For example, the fourth component can include at least one inorganic substance selected from the group consisting of calcium chloride, calcium phosphate, calcium hydroxide, calcium silicate, and calcium sulfate.

[0043] For example, the first component, the second component, and the fourth component may be contained in an amount of 40 to 75 parts by weight, 5 to 30 parts by weight, and 10 to 40 parts by weight, for example, 50 to 70 parts by weight, 10 to 20 parts by weight, and 10 to 30 parts by weight, based on 100 parts by weight of the pre-composition before the addition of thrombin. If the amounts are within the above numerical ranges, the hemostatic effect due to the appropriate composition of the physical hemostatic effect and the chemical hemostatic factor can be improved.

[0044] The pharmaceutical composition of the present invention may include a powder form, and the pharmaceutical preparation formed from the pharmaceutical composition may include a powder form. In this case, the "powder form" includes any form of powder, for example, a pharmaceutical powder form and a pharmaceutical granule form (i.e., granules) obtained by a conventional formulation method.

[0045] When the pharmaceutical composition and / or pharmaceutical preparation is prepared in a powder form, it has suitable flowability, which allows easy passage through a catheter and application, and the additional effect of minimizing loss by forming a barrier at the application site while exhibiting high mucosal adhesion through instantaneous gelation can be achieved.

[0046] The pharmaceutical composition of the present invention can be administered into the digestive tract via an endoscope (ie, an endoscope catheter) to exert hemostatic and wound healing effects on bleeding digestive tract ulcers.

[0047] The present invention will be described in more detail below with reference to examples. However, the examples are for illustrating the present invention and the scope of the present invention is not limited to these examples.

[0048] (Example of preparation) In order to confirm the hemostatic and wound healing effects of the pharmaceutical preparation according to the present invention, conventional commercially available hemostatic agents and wound dressings were prepared as follows.

[0049] [Table 1]

[0050] Also, a powder preparation (S3') containing 70 to 95 parts by weight of a mucoadhesive polymer and 5 to 30 parts by weight of a moisture absorbent per 100 parts by weight of the pharmaceutical composition according to the above-mentioned embodiment of the present invention was prepared.

[0051] (Preparation Example 1) In Preparation Example 1, as an example of the present invention, a composition S3' was prepared by mixing 90 parts by weight of hydroxyethyl cellulose (HEC), 5 parts by weight of sodium starch glycolate, and 5 parts by weight of croscarmellose sodium.

[0052] (Production Example 1) For S1, S2, and S3' according to Preparation Example 1, 0.080 parts by weight of thrombin powder having high activity extracted from bovine plasma relative to the total weight of the pharmaceutical composition was added to a glass vial and mixed uniformly using a shaker to prepare powdered samples, and the compositions containing thrombin powder were indicated as "-1." That is, S1-1 was prepared as a powdered composition containing 0.080 parts by weight of thrombin in S1 relative to the total weight of S1-1, S2-1 was prepared as a powdered composition containing 0.080 parts by weight of thrombin in S2 relative to the total weight of S2-1, and S3'-1 was prepared as a powdered composition containing 0.080 parts by weight of thrombin in S3' relative to the total weight of S3'-1.

[0053] At this time, the thrombin activity of the added solution was 624 IU / mg, and the content was controlled to be 500 IU / g in all cases.

[0054] Furthermore, a thrombin solution sample (S4) was prepared by dissolving the same thrombin in purified water to give a concentration of 100 IU / ml.

[0055] (Experimental Example 1) Experimental Example 1-1: Blood clotting time measurement After mixing 200 μl of blood and 15 mg of each sample in a 2 ml microtube, the time until blood stopped flowing was measured as the blood coagulation time.

[0056] At this time, in the cases of S1-1, S2-1, and S3'-1, the treatment was carried out so that the thrombin content was 7.5 IU, and the thrombin content of each sample was controlled to be equal to 500 IU / g.

[0057] In the case of S4, 200 μl of blood and 75 μl of the S4 sample were placed in a 2 ml microtube to measure the blood clotting time, and the blood was treated to have the same thrombin content of 7.5 IU as in the cases of S1-1, S2-1, and S3'-1.

[0058] The experimental results are shown in Table 2 below and in FIGS.

[0059] [Table 2]

[0060] Referring to S1, S1-1, S2, S2-1, S3, and S3'-1 in Table 2 and Figures 1 to 6, it was confirmed that when thrombin is added to a conventional product, the blood clotting time is shortened and the hemostatic effect is improved.

[0061] Meanwhile, by comparing (S1-1, FIG. 2), (S2-1, FIG. 4), and (S3'-1, FIG. 6), it was confirmed that the blood clotting time was much shorter in the pharmaceutical composition containing thrombin according to one embodiment of the present invention than in the case of combining thrombin with other compositions known as hemostatic components.

[0062] Furthermore, comparing (S3'-1, Figure 6) with (S4, Figure 7), it was found that the pharmaceutical composition containing thrombin according to one embodiment of the present invention (S3'-1) had a shorter blood clotting time than the same amount of thrombin alone (S4).

[0063] The reason why the synergistic effect of the shorter blood clotting time of the pharmaceutical composition containing thrombin according to an embodiment of the present invention was observed compared to thrombin itself at the same concentration in an embodiment of the present invention is believed to be because thrombin, which exerts a hemostatic effect by directly acting on the physiological blood clotting mechanism, exerts a synergistic effect with the biocompatible polymeric substance of the pharmaceutical composition according to an embodiment of the present invention, which is composed of a hygroscopic polymer and a mucoadhesive polymeric substance and can exert a physical hemostatic effect by forming a barrier through a gelation reaction.

[0064] Experimental Example 1-2: Evaluation of thrombus formation To carry out the clot weight and red blood cell hemolysis tests, 200 μl of blood and 15 mg of each sample were mixed in a 2 ml microtube and reacted at 37° C. for 10 minutes, after which 1 ml of purified water was added to stop the blood coagulation reaction.

[0065] Experimental Example 1-2-1: Measurement of red blood cell hemolysis After each sample was added, the blood coagulation reaction was stopped, and the supernatant containing red blood cells hemolyzed with purified water was collected, removing the blood clots.

[0066] Then, 1 ml of purified water was added again and the remaining red blood cell lysate was collected and the OD 540 was measured, where OD 540 Hemolysis was measured using an ELISA instrument as absorbance at 540 nm, which can be quantitatively evaluated.

[0067] The experimental results are shown in Table 3 and FIG.

[0068] [Table 3]

[0069] Referring to Table 3 and FIG. 8, when thrombin was added to a conventional product, the red blood cell hemolysis was higher (S1-1) or at the same level (S2-1) as that caused by thrombin itself, whereas the red blood cell hemolysis of the pharmaceutical composition containing thrombin according to one embodiment of the present invention (S3'-1) was not only compared to the conventional product but also at a level much lower than that of thrombin itself.

[0070] Therefore, it can be confirmed that the pharmaceutical composition (S3'-1) containing thrombin according to one embodiment of the present invention has excellent early hemostatic effect within 5 minutes and has a rapid tissue regeneration effect, thereby having excellent hemostatic effect and wound healing effect in bleeding gastrointestinal ulcers.

[0071] The reason why a synergistic effect was observed as in the blood clotting time evaluation result, in which the red blood cell hemolysis value of the pharmaceutical composition according to one embodiment of the present invention was much lower than that of thrombin itself at the same concentration, is believed to be because thrombin, which exerts a hemostatic effect by directly acting on the physiological blood clotting mechanism, exerts a synergistic effect with the biocompatible polymer of the pharmaceutical composition according to one embodiment of the present invention, which is composed of a hygroscopic polymer and a mucoadhesive polymer and can exert a physical hemostatic effect by forming a barrier through a gelation reaction.

[0072] Experimental Example 1-2-2: Thrombus weight measurement After the addition of each sample, the blood coagulation reaction was stopped, and the coagulated clots were separated and fixed in 10% formalin for 20 minutes.

[0073] Thereafter, the coagulated thrombus was immersed in purified water for 5 minutes to wash, and then the weight was measured after drying for 24 hours.

[0074] The experimental results are shown in Table 4 below and in FIGS.

[0075] [Table 4]

[0076] Referring to Table 4 and Figures 9 to 12, it was confirmed that when thrombin was added to conventional products, the degree of early thrombosis formation due to thrombin itself was low (S1-1, S2-1), whereas the pharmaceutical composition containing thrombin according to one embodiment of the present invention (S3'-1) showed a much higher level of early thrombosis formation than thrombin itself.

[0077] Therefore, it can be seen that the pharmaceutical composition containing thrombin according to one embodiment of the present invention can rapidly coagulate blood at the wound site within 5 minutes, providing rapid hemostatic and wound healing effects as well as preventing rebleeding.

[0078] The reason why the pharmaceutical composition according to one embodiment of the present invention exhibited a synergistic effect of showing a much higher degree of early thrombosis formation than thrombin itself is believed to be because thrombin, which exerts a hemostatic effect by directly acting on the physiological blood coagulation mechanism, is composed of a hygroscopic polymer and a mucoadhesive polymer substance and can exert a physical hemostatic effect by forming a barrier through a gelation reaction, and thus a synergistic effect is exhibited as thrombin exerts its hemostatic effect prior to the gelation reaction of the polymer.

[0079] (Production Example 1-2) In the S3' of Preparation Example 1, 0.003 parts by weight, 0.016 parts by weight, 0.080 parts by weight, 0.401 parts by weight, 0.802 parts by weight, and 1.603 parts by weight of thrombin were added to each glass vial and mixed uniformly to prepare powder-form samples (S5 to S10). That is, S5 was prepared as a powder-form composition containing 0.003 parts by weight of thrombin relative to the total weight of S5, S6 was prepared as a powder-form composition containing 0.016 parts by weight of thrombin relative to the total weight of S6, S7 was prepared as a powder-form composition containing 0.080 parts by weight of thrombin relative to the total weight of S7, S8 was prepared as a powder-form composition containing 0.401 parts by weight of thrombin relative to the total weight of S8, S9 was prepared as a powder-form composition containing 0.802 parts by weight of thrombin relative to the total weight of S9, and S10 was prepared as a powder-form composition containing 1.603 parts by weight of thrombin relative to the total weight of S10.

[0080] The content of thrombin added at this time is as shown in Table 5 below, and the same thrombin having an activity of 624 IU / mg was used in all of S5 to S10.

[0081] Experimental Example 1-3: Blood clotting time measurement After mixing 200 μl of blood and 15 mg of each sample in a 2 ml microtube, the time until blood stopped flowing was measured as the blood clotting time. The blood clotting time (s) was recorded as the average value after three measurements.

[0082] The experimental results are shown in Table 5 below and in FIGS.

[0083] [Table 5]

[0084] 13 to 19, it was confirmed that the blood clotting time was shortened as the thrombin content increased in the pharmaceutical composition according to an embodiment of the present invention.

[0085] In addition, in comparison with S4, it was confirmed that when the thrombin content was 0.016 parts by weight or more relative to the total weight, the blood clotting time was significantly shorter than that due to thrombin itself.

[0086] In other words, when the thrombin content in the pharmaceutical composition of the present invention is 0.016 parts by weight or more based on the total weight, it was confirmed that thrombin, which exhibits a physiological hemostatic effect, exhibits a synergistic effect with the biocompatible polymer substance of the pharmaceutical composition according to one embodiment of the present invention, which exhibits a physical hemostatic effect.

[0087] (Preparation Example 2) In Preparation Example 2, as an example of the present invention, compositions S11, S12 and S13 were prepared by mixing hydroxyethyl cellulose (HEC), sodium starch glycolate and croscarmellose sodium in the composition ratios shown in Table 6 below.

[0088] [Table 6]

[0089] (Production Example 2) In Preparation Example 2, S11, S12, and S13 were prepared by adding thrombin powder having high activity extracted from bovine plasma to glass vials in the composition ratios shown in Table 7 below based on the total weight of the pharmaceutical composition, and mixing them uniformly using a shaker to prepare powdered samples, and the compositions containing thrombin powder were indicated as "-1." That is, 11-1 was prepared as a powdered composition containing 0.080 parts by weight of thrombin in S11-1 based on the total weight of S11-1, 0.401 parts by weight of thrombin in S12-1 based on the total weight of S12-1, and 0.802 parts by weight of thrombin in S13-1 based on the total weight of S13-1.

[0090] At this time, the thrombin activity added was controlled to be equal to 624 IU / mg, and the contents (IU / g) were controlled to be 500 for S11-1, 2500 for S12-1, and 5000 for S13-1.

[0091] Thrombin solution samples S14, S15, and S16 had an activity of 624 IU / mg and were prepared by dissolving in purified water to the concentrations shown in Table 8. Specifically, the thrombin solution samples were prepared to have concentrations (IU / ml) of 100 for S14, 500 for S15, and 1000 for S16.

[0092] [Table 7]

[0093] [Table 8]

[0094] Experimental example 2: Blood clotting time measurement After mixing 200μl of blood and 15mg of each sample in a 1.5ml microtube, the time until blood stopped flowing was measured as the blood clotting time. At this time, the blood clotting time (s) was recorded as the average value after three measurements. At this time, 75μl of the thrombin solution samples S14, S15, and S16 were added to proceed with the blood clotting reaction.

[0095] The experimental results are shown in Table 9 below and in FIGS.

[0096] [Table 9]

[0097] 25 to 30, it was confirmed that the blood clotting time was shortened as the thrombin content increased in the pharmaceutical composition according to an embodiment of the present invention.

[0098] Furthermore, it was confirmed that S11-1, S12-1, and S13-1 all exhibited shorter blood clotting times than when the same amount of thrombin itself (S14, S15, and S16) was treated with blood in the same amount as the amount of thrombin treated in the blood in each example.

[0099] In other words, it was confirmed that the powder preparations (S3', S5 to S10, S11, S12, S13) containing 70 to 95 parts by weight of mucoadhesive polymer and 5 to 30 parts by weight of hygroscopic agent per 100 parts by weight of the pharmaceutical composition of the present invention exhibit a synergistic effect between thrombin, which exhibits a physiological hemostatic effect, and the biocompatible polymer substance of the pharmaceutical composition according to one embodiment of the present invention, which exhibits a physical hemostatic effect.

[0100] Thus, it was confirmed that the powder-form pharmaceutical composition according to one embodiment of the present invention was not only more effective in all experiments than other commercially available products to which thrombin had been added, but also more effective in all experiments than thrombin itself.

[0101] Experimental Example 3: In-vivo test The following in vivo tests were performed at Asia University Hospital.

[0102] <1> Test animal preparation and drug treatment Twelve male pigs (Sus scrofa (porcine)) aged 4-5 months and weighing 35-40 kg were used in the study. Since the pigs used in the experiment have a bleeding tendency similar to that of humans, aspirin and heparin were administered before and during the experiment as follows.

[0103] (1) An antiplatelet agent (aspirin, 200 mg / day) was crushed and mixed with feed and orally administered to the pigs for 5 days prior to the experiment.

[0104] (2) For 3 days prior to the experiment, the animals were fed the minimum amount of feed (100 g / head) required for administration of the antiplatelet agent, and were kept fasting on the day of the experiment.

[0105] (3) Before the experiment, animals were given a total anesthesia by intramuscular injection of a mixture of animal muscle relaxant and general anesthetic (zoletil + xylazine, 6:4), and then given respiratory anesthesia using isoflurane and a respiratory anesthesia machine.

[0106] (4) Heparin (200 IU / kg) was administered intravenously immediately before the endoscopic procedure, and was continuously administered using a Cylinder pump during the experiment (mixed with 10% glucose and administered at a rate of 10,000 IU / hr).

[0107] <2> Bleeding gastric ulcer model A bleeding gastric ulcer model was created using endoscopic mucosal resection (EMR) in the following order.

[0108] (1) A double-channel upper endoscope (GIF-2T260M; Olympus Medical Systems, Tokyo, Japan) was inserted into the oral cavity of anesthetized animals and entered the gastric cavity, after which a 6-mm sizer was attached to the gastrointestinal mucosal tissue.

[0109] (2) The boundaries of areas that would become ulcers of 20 mm in size were displayed using APC based on the sizer size.

[0110] (3) Isotonic saline was injected into the submucosal layer.

[0111] (4) Mucosal resection was performed using endoscopic mucosal resection and exfoliation biopsy (grasp-and-snare) technique. -The mucosa was pulled with forceps and captured with a snare. -An electric current was passed through the snare to remove the mucosal tissue.

[0112] (5) If bleeding was not induced, bleeding was induced by tearing the exposed submucosal blood vessels with forceps.

[0113] (6) Using this method, two ulcers were created in one pig, one in the upper part of the stomach and one in the lower part, spaced at least 5 cm apart to prevent mutual influence.

[0114] <3> Application of test substances The above-mentioned S1, S3 and S3'-1 samples were prepared as test substances, and the dosage was set at 3 g each.

[0115] According to the treatment protocol randomly assigned in advance, four types of treatment were performed: no treatment, Endoclot (S1 sample), CGGEL (S3 sample), and CGGEL_Thrombin (S3'-1 sample). When performing powder spray treatment, it was sprayed on the entire ulcer and applied sufficiently so that the base of the ulcer was no longer visible.

[0116] <4> Measurement of evaluation variables Archive of hemostasis was defined as no visible bleeding or visible outward flow of blood from the artificial ulcer.

[0117] Each artificial ulcer was checked for hemostasis up to 5 minutes after bleeding induction and application of the test substance, and then checked again after 1 hour. The animals were then allowed to recover in their individual cages but were not allowed to eat for 24 hours. Follow-up endoscopies were performed 24 hours, 48 ​​hours, 72 hours, 1 week, and 2 weeks after the procedure to check for rebleeding at the artificial ulcer site and the healing status of the ulcer.

[0118] The occurrence of rebleeding was judged by the Forrest classification based on endoscopic observation, and rebleeding was defined as occurring if endoscopic findings corresponded to Forrest classification I.

[0119] To evaluate the degree of ulcer healing and the effect of mucosal regeneration, the change in ulcer area was recorded at each endoscopic observation. A standard 6 mm sizer was placed on the ulcer to measure (estimate) the ulcer area. The ulcer area was measured and calculated using a Java-based image processing program (Image J, National Institutes of Health, USA).

[0120] Six pigs were observed one week after the procedure, and the remaining six pigs were observed two weeks after the procedure. At the end of the observation period, the pigs were euthanized and their digestive tract tissues were removed. The removed tissues were fixed in 10% formalin solution, and H&E staining and immunochemical staining (CD31) were performed. The specimens fixed in formalin solution were dehydrated, cleared, and infiltrated using an automatic tissue processor, and then embedded in paraffin. They were then sliced ​​to a thickness of 4 μm using a rotary slicer. The sliced ​​tissues were placed on slides to prepare unstained slides. All tissues were deparaffinized and moisturized, and one of them was stained with H&E using Harris-Hematoxylin (YD Diagnostics, Korea) and eosin Y (Showa, Japan).

[0121] <5> Sacrifice and specimen collection All animals were euthanized according to the experimental schedule for specimen collection. To sacrifice the experimental animals, total anesthesia was induced by intramuscular injection of 0.1ml / kg of a 1:1 mixture of Zoletil and Xylazine, and then euthanasia was induced by intramuscular administration of 1ml of succipharm. The transplantation site of each animal was then incised, and the gastrointestinal mucosa from the transplanted site was completely removed and fixed in 10% neutral buffered formalin solution.

[0122] <6> Experimental Results Early hemostasis success rate The rates were 0% in the no treatment group (FIG. 20), 66.7% in the endoclot treatment group (FIG. 21), and 16.7% in the CGGEL treatment group (FIG. 22), whereas they were 100% in the CGGEL_Thrombin treatment group (FIG. 23).

[0123] Mucosal regeneration area 24, the CGGEL(EGF)-treated group and the CGGEL(Thrombin)-treated group showed significantly increased mucosal regeneration in the tissue on day 14 compared to the tissue on day 7. In particular, the area of ​​mucosal regeneration in the CGGEL(Thrombin)-treated group on day 14 was approximately three times greater than that in the CGGEL(EGF)-treated group, indicating that CGGEL(Thrombin) has the most excellent ulcer treatment effect.

Claims

1. A pharmaceutical composition for hemostasis, comprising: a first component comprising a water-soluble copolymer that adheres to gastrointestinal mucosal tissue; a second component comprising a water-absorbent material; thrombin.

2. The first component includes at least one of hydroxyethyl cellulose (HEC), sodium alginate, chitosan, hydroxypropyl cellulose (HPC), and polyethylene oxide (PEO); The pharmaceutical composition for intra-gastrointestinal hemostasis according to claim 1, wherein the second component comprises at least one of croscarmellose sodium (Ac-di-sol), sodium starch glycolate, calcium silicate, and crospovidone.

3. A pharmaceutical composition for intra-digestive hemostasis as described in claim 1, wherein, when 200 μl of blood and a 15 mg sample of the pharmaceutical composition are mixed and the blood clotting time is measured as the time until blood stops flowing, the blood clotting time is shorter than the blood clotting time of a sample consisting of the thrombin measured under the same conditions.

4. mixing 200 μl of blood with 15 mg of a sample of the pharmaceutical composition, reacting at 37° C. for 10 minutes, and then adding 1 ml of purified water to stop the blood coagulation reaction; A step of collecting a supernatant containing red blood cells hemolyzed by purified water in a state where the blood coagulation reaction has been stopped; 1 ml of purified water was added to the collected supernatant, and the remaining red blood cell hemolyzed solution was collected and analyzed for OD. ５４０ and measuring The optical density (OD) at 540 nm was measured by a method consisting of ５４０ ) is the OD of the sample consisting of the thrombin measured under the same conditions. ５４０ The pharmaceutical composition for gastrointestinal hemostasis according to claim 3, wherein the value of

5. mixing 200 μl of blood with 15 mg of a sample of the pharmaceutical composition, reacting at 37° C. for 10 minutes, and then adding 1 ml of purified water to stop the blood coagulation reaction; In a state where the blood coagulation reaction has been stopped, a step of isolating the coagulated thrombus and fixing it with 10% formalin for 20 minutes; A step of immersing the coagulated thrombus in purified water for 5 minutes, and then measuring the weight after drying for 24 hours; 4. The pharmaceutical composition for intra-digestive hemostasis according to claim 3, wherein the weight of the thrombus measured by a method comprising the steps of: (a) measuring the weight of the thrombus in a sample containing said thrombin under the same conditions;

6. 3. The pharmaceutical composition for intra-gastrointestinal hemostasis according to claim 2, wherein said first component is composed of hydroxyethyl cellulose (HEC) and said second component is composed of sodium starch glycolate and croscarmellose sodium.

7. The pharmaceutical composition for intra-gastrointestinal hemostasis according to claim 6, wherein the thrombin is contained in an amount of 20 to 10,000 IU / g.

8. 2. The pharmaceutical composition for gastrointestinal hemostasis according to claim 1, further comprising a fourth component consisting of at least one inorganic substance selected from the group consisting of calcium chloride, calcium phosphate, calcium hydroxide, calcium silicate, and calcium sulfate.

9. The pharmaceutical composition for hemostasis in the gastrointestinal tract according to any one of claims 1 to 8, wherein the composition is administered in powder form into the gastrointestinal tract and exerts a hemostatic effect on bleeding gastrointestinal ulcers.

10. A pharmaceutical composition for treating wounds, comprising: a first component comprising a water-soluble copolymer that adheres to gastrointestinal mucosal tissue; a second component comprising a water-absorbent material; thrombin.

11. The composition according to any one of claims 1 to 8, The composition is a pharmaceutical composition for gastrointestinal hemostasis and wound healing, which is administered in powder form into the gastrointestinal tract to perform hemostatic and wound healing effects on bleeding gastrointestinal ulcers.

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