Biodegradable medical adhesive or sealant composition

Abstract

The present invention provides a biodegradable medical adhesive or a sealant composition containing an oxidized glycosaminoglycan and a polyamine. The composition of the present invention exhibits improved effects in biodegradation, coating property, gelation time, hemostatic capacity, adhesive force, moisture absorptive capacity and the like, and thus can be applied to various medical uses in which a medical adhesive or sealant can be used, such as biotissue adhesion, filling, coating, adhesion prevention, wound covering, leakage prevention and hemostasis.

Claims

1. A biodegradable medical adhesive or sealant composition, comprising: (a) a first component containing two or more different oxidized glycosaminoglycans obtained by oxidation through the introduction of a formyl group; and (b) a second component containing a polyamine having two or more amino groups, the pH of the second component in an aqueous solution phase being 8.5-11.0: wherein the oxidized glycosaminoglycans are selected from the group consisting of oxidized hyaluronic acid, oxidized chondroitin sulfate, oxidized chondroitin, oxidized dermatan sulfate, oxidized heparan sulfate, oxidized heparin, and oxidized keratan sulfate, and wherein the polyamine is selected from the group consisting of polylysine, putrescine, cadaverine, spermidine, spermine, protamine, and polyethylenimine (PEI).

2. The composition of claim 1, wherein the degree of oxidation (%) of the oxidized glycosaminoglycan is 10-99.5%, the degree of oxidation (%) being calculated by the following equation: $\mathrm{Degree}\mathrm{of}\mathrm{oxidation}\left(\%\right)=\frac{\mathrm{number}\mathrm{of}\mathrm{moles}\mathrm{of}\mathrm{CHO}}{\mathrm{mumber}\mathrm{of}\mathrm{mols}\mathrm{of}\mathrm{oxidized}\mathrm{glycosaminoglycan}}?100.$

3. The composition of claim 1, wherein the two or more different oxidized glycosaminoglycans are oxidized hyaluronic acid and oxidized chondroitin sulfate.

4. The composition of claim 3, wherein the degree of oxidation of the oxidized hyaluronic acid is 10-40%, and the degree of oxidation of the oxidized chondroitin sulfate is 10-55%.

5. The composition of claim 1, further comprising a drug having an amine group.

6. A method for performing adhesion, filling, coating, anti-adhesion, wound covering, and hemostasis, on biological tissues, the method comprising a step of applying the biodegradable medical adhesive or sealant composition of claim 1 to biological tissues in need of adhesion, filling, coating, anti-adhesion, wound covering, and hemostasis.

7. The composition of claim 1, wherein the polyamine is selected from the group consisting of polylysine, protamine, and polyethylenimine (PEI).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 illustrates analysis results of oxidized hyaluronic acid using an FT-IR spectrometer.

(2) FIG. 2 shows an image illustrating the gelation state and gelation time of mixtures that use a second component having an amino group.

(3) FIG. 3 shows images illustrating gelation evaluation results of mixtures of a first component and a second component mixed at different weight ratios.

(4) FIG. 4 shows images illustrating the comparison of gelation time between adhesive and sealant compositions of the present invention and an existing adhesive composition (LYDEX).

(5) FIG. 5 shows a graph illustrating the comparison of adhesive strength between adhesive and sealant compositions of the present invention and an existing adhesive composition (LYDEX).

(6) FIG. 6 shows images illustrating results (of mucosal adhesive ability and hemostatic ability) when a bleeding site after gastric mucosectomy is coated with the adhesive and sealant composition of the present invention or an existing adhesive composition (LYDEX).

(7) FIG. 7 shows images illustrating the gelation state, gelation time, and gelation depending on pH, of a mixture of an oxidized glycosaminoglycan and a polyamine.

(8) FIGS. 8 to 11 illustrate comparative test results between an adhesive and sealant composition of the present invention and an existing hemostatic agent (Arista?AH) on a hepatolobectomy model, a nephrectomy model, a gastric mucosectomy model, and a vascular hemorrhage model.

(9) FIG. 12 shows a graph illustrating quantitative results of FIGS. 8 to 11.

MODE FOR CARRYING OUT THE INVENTION

(10) Hereinafter, the present invention will be described in detail with reference to examples. These examples are only for illustrating the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

EXAMPLES

Example 1: Preparation of Medical Adhesive (1)

(11) (1) Preparation of Oxidized Hyaluronic Acid (CHO-HA; First Component)

(12) 1 g or 3 g of hyaluronic acid (HA) with a molecular weight of 7 kDa, 150 kDa, 1400 kDa, or 3000 kDa was dissolved in 150 ml of sodium periodate (NaIO.sub.4) in water. Here, the concentration and reaction conditions of sodium periodate was varied as shown in tables 1 to 4 to make varying degrees of oxidation (degree of substitution (DS), %). A reaction flask was allowed to react at 15-70? C. for 3-48 h. The reaction material was dialyzed with distilled water for 24 h using a dialysis membrane with a molecular weight cut-off of 1-100 kDa. Here, the obtained oxidized hyaluronic acid was freeze-dried for 4 days, followed by pulverization, and then passed through a 500 ?m-sized mesh to give oxidized hyaluronic acid with a diameter of about 500 ?m or smaller.

(13) ##STR00001##

(14) As a result of analysis of the oxidized hyaluronic acid using an FT-IR spectrometer (Cary 640, Agilent Technologies, USA), the substituents were confirmed at 4000-400 cm.sup.?1 (resolution 4 cm.sup.?1) (FIG. 1).

(15) In order to investigate the degree of oxidation of hyaluronic acid, 17.5 g of hydroxylamine hydrochloride and 6 ml of 0.05% methyl orange were mixed in 994 ml of distilled water to prepare a 0.25 M hydroxylamine hydrochloride solution, which was then titrated to pH 4. 0.1 g of oxidized hyaluronic acid was dissolved in 25 ml of the solution, and then titrated to pH 4 with 0.1 mM sodium hydroxide. The degree of oxidation (%) was calculated by the following equation, and the results are shown in tables 1 to 4.

(16) $\begin{array}{cc}\mathrm{Degree}\mathrm{of}\mathrm{oxidation}\left(\%\right)=\frac{\mathrm{number}\mathrm{moles}\mathrm{of}\mathrm{CHO}}{\mathrm{number}\mathrm{of}\mathrm{mols}\mathrm{of}\mathrm{oxidized}\mathrm{HA}}?100=\frac{\begin{array}{c}\mathrm{concentration}\mathrm{of}\mathrm{sodium}\mathrm{hydroxide}?\\ \mathrm{volume}\mathrm{of}\mathrm{soduim}\mathrm{hydroxide}?{10}^{-3}\end{array}}{\frac{\mathrm{weight}\mathrm{of}\mathrm{oxidized}\mathrm{hyaluronic}\mathrm{acid}}{\begin{array}{c}\mathrm{amount}\mathrm{of}\mathrm{hyaluronic}\\ \mathrm{acid}\mathrm{repeating}\mathrm{unit}\end{array}}}?100& \mathrm{Equation}1\end{array}$

(17) TABLE-US-00001 TABLE 1 HA HA Oxidant Reaction Reaction M.W. DS weight concentration temperature time (kDa) (%) (g) (mM) (? C.) (h) 7 67.4 3.1 15.8 40 24 75.9 3.1 15.7 40 24 79.3 1.0 15.8 40 24

(18) TABLE-US-00002 TABLE 2 HA HA Oxidant Reaction Reaction M.W. DS weight concentration temperature time (kDa) (%) (g) (mM) (? C.) (h) 150 4.0 1.0 2.6 RT. 24 5.7 1.0 2.6 40 6 7.2 1.1 2.6 RT. 6 11.6 1.0 2.6 40 24 16.8 3.1 7.9 40 24 17.2 1.0 5.3 RT. 6 19.5 1.0 7.8 RT. 6 36.5 3.0 15.7 40 24 37.4 3.1 15.8 40 24 48.4 3.1 23.4 40 24 82.2 1.0 15.7 40 24

(19) TABLE-US-00003 TABLE 3 HA HA Oxidant Reaction Reaction M.W. DS weight concentration temperature time (kDa) (%) (g) (mM) (? C.) (h) 1400 2.2 1.1 2.5 RT. 3 8.3 1.1 2.8 40 3 3.9 1.0 2.6 RT. 24 7.6 1.1 2.6 RT. 6 9.2 1.0 3.8 RT. 6 9.7 1.0 2.7 40 6 9.9 1.0 5.3 RT. 6 11.3 1.0 3.8 RT. 6 14.0 1.0 5.2 RT. 6 14.3 1.0 2.6 40 24 14.8 3.1 8.0 40 6 15.4 1.0 7.8 RT. 6 16.5 1.0 3.8 RT. 6 17.4 3.1 7.9 40 24 17.7 1.0 5.3 RT. 6 18.0 1.0 9.6 RT. 6 19.5 1.0 11.1 RT. 6 21.0 1.0 11.0 RT. 6 21.6 1.0 12.7 RT. 6 21.9 1.0 9.4 RT. 6 22.8 1.0 7.8 RT. 6 23.2 1.1 7.9 RT. 6 23.6 1.0 12.5 RT. 6 24.7 1.0 11.1 RT. 6 27.0 1.0 9.5 RT. 6 30.9 1.0 12.7 RT. 6 40.8 3.1 15.8 40 24 47.7 3.1 23.5 40 24 82.6 1.1 15.7 40 24

(20) TABLE-US-00004 TABLE 4 HA HA Oxidant Reaction Reaction M.W. DS weight concentration temperature time (kDa) (%) (g) (mM) (? C.) (h) 3000 4.4 1.0 2.6 RT. 24 4.6 1.1 2.6 RT. 6 4.7 1.1 2.5 40 6 19.8 3.0 7.9 40 24 23.3 1.1 2.6 40 24 43.4 3.0 15.9 40 24 82.2 1.1 15.7 40 24

(21) (2) Second Component Having Two or More Amino Groups

(22) Out of various amino group-containing polyamines, chitosan, protamine, PEI, polylysine, spermine, spermidine, and albumin were typically used as the second component. The powders, which were obtained by adjusting 5 wt % or more of polyamine solutions to pH 8.5, 9.0, 9.5, and 10 using pH adjusters (acid, acidic salt, base, basic salt), and then freeze-drying the solutions in the same manner as in the oxidized hyaluronic acid/oxidized chondroitin sulfate, was used.

(23) The gelation degree and gelation time of the listed amino group-containing polyamines were evaluated. As a result, albumin, basic polylysine (BPL), and PEI were excellent in view of the gelation rate and gel safety (FIG. 2). Of these, BPL was used for the following tests.

Example 2: Evaluation of Physical Properties

(24) (1) Evaluation of Gelation

(25) The first and second components obtained in example 1 were mixed at different weight ratios (1:1, 2:1, 4:1, 8:1). The degree of gelation of the mixed components was confirmed by sprinkling water.

(26) As a result, the 8:1 mixture of the first component and the second component was partially changed into a liquid after 10 min, and the 2:1 mixture and the 4:1 mixture had relatively low gel elasticity compared with the 1:1 mixture (FIG. 3). In addition, the mixture preparation using a hyaluronic acid with a molecular weight of 3,000 kDa was gelated, but less elastic. As for hyaluronic acid with a molecular weight of 150 kDa and 1,400 kDa, the mixture preparation was gelated regardless of the degree of substitution, but when the degree of substitution was around 10% (10-19%), the mixtures showed a shorter gelation time and excellent elasticity.

(27) Based on the above results, the first component (oxidized hyaluronic acid having a degree of substitution of 10%, obtained by introducing an aldehyde group into hyaluronic acid with a molecular weight of 150 kDa or 1,400 kDa) and the second component were mixed at a weight ratio of 1:1, and this mixture was used for the following tests.

(28) (2) Evaluation of Gelation Time

(29) The components mixed in a 2 ml tube were small divided into 30 mg, which was then collected in a tube cap. 120 ?l of water was sprinkled thereon within 1 s, and then the gelation time was measured. LYDEX consumed 10 s or more in obtaining a solidified gel, in spite of employing a smaller amount (80 ?l) compared with the hyaluronic acid mixture preparation. On the other hand, the oxidized hyaluronic acid mixture preparation showed a short gelation time of within 2-3 s (FIG. 4 and table 5).

(30) TABLE-US-00005 TABLE 5 Gelation time (s) Sample Mean SD LYDEX 10 0.00 CHO-HA 150 kDa 2.87 0.62 CHO-HA 1,400 kDa 2.95 0.58

(31) (3) Evaluation on Adhesive Strength

(32) 800 ?l of water was sprinkled on 100 mg of the mixed components, and then the adhesive strength was measured using a Texture Analyzer. As a result, LYDEX showed a mean adhesive strength of about 53.6 gf in spited of employing a smaller amount (500 ?l) compared with the oxidized hyaluronic acid mixture preparation. On the other hand, the oxidized hyaluronic acid mixture preparations were measured to have the mean adhesive strength values of 65.9 gf and 67.5 gf, respectively (FIG. 5 and table 6).

(33) TABLE-US-00006 TABLE 6 Adhesive strength (gf) Sample Mean SD LYDEX 53.60 18.58 CHO-HA 150 kDa 65.86 11.32 CHO-HA 1,400 kDa 67.51 2.22

(34) (4) Evaluation of Absorption Power

(35) Absorption power was evaluated for LYDEX, and CHO-HA 150 kDa (DS 10%) and CHO-HA 1,400 kDa (DS 10%) mixed with the second component. 30 mg of each sample was placed on a petri dish (?60), and weighed. Distilled water, which was previously warmed at 37? C., was added to the sample, and here, the weight of the distilled water was 30 times (30 g) the weight of the sample, considering the absorption power of the product. The resultant material was left in a thermostat at 37? C. for 30 min, and then the petri dish was overturned for 30 s to measure the weight. The absorption power was calculated by the following equation.

(36) $\begin{array}{cc}\mathrm{Absorption}\mathrm{power}\left(\%\right)=\frac{\begin{array}{c}\mathrm{Sample}\mathrm{weight}\left(\mathrm{mg}\right)\mathrm{after}30\min -\\ \mathrm{Initial}\mathrm{weight}\left(\mathrm{mg}\right)\end{array}}{\mathrm{Initial}\mathrm{weight}\left(\mathrm{mg}\right)}?100& \mathrm{Equation}2\end{array}$

(37) As a result, it was verified that the absorption power of the oxidized hyaluronic acid mixture preparation (CHO-HA 1,400 kDa) was excellent by about 5-fold compared with an existing LYDEX formulation (table 7).

(38) TABLE-US-00007 TABLE 7 Absorption power (%) Sample Mean SD LYDEX 4.2 0.1 CHO-HA 150 kDa 17.6 3.5 CHO-HA 1,400 kDa 19.9 0.4

Example 3: In Vivo Evaluation

(39) (1) Animals

(40) Three male rabbits (New Zealand White; Orient Bio, Seongnam, Korea) weighing 2-3 kg were used for a test. All animal breeding and test procedures were conducted according to the guidelines of the Experimental Animal Research Committee of Inha University.

(41) (2) Gastric Hemorrhage Inducing Animal Model

(42) The rabbit mucosectomy-induced gastric hemorrhage model was constructed as follows. Rabbits were fasted for 24 h prior to the surgery, then anesthetized with an intramuscular injection of a mixture of ketamine (4.2 mg/kg) and xylazine (11.7 mg/kg). The upper part of the belly was incised to expose the stomach, and a 5-7 cm incision was made along the greater curvature. 200 ?l of isotonic saline was injected into the submucosal layer of the stomach, and then the swollen gastric mucosa was resected using surgery scissors. The diameter of the resected part was around 7-10 mm.

(43) (3) Mucosal Adhesive Ability and Hemostatic Ability

(44) Approximately 0.5 g of a mixture preparation (mixture of the first component and the second component at a weight ratio of 1:1) was coated on the resected bleeding gastric mucosa of the rabbit, which is bleeding. As a result, as shown in FIG. 6, the gelation of the mixture preparation occurred through the reaction of the mixture preparation and the blood immediately after the mixture preparation was coated, and the bleeding time was shortened compared with the non-treatment group. In addition, the mucosal adhesive ability of the composition of the present invention was confirmed (FIG. 6).

Example 4: Preparation of Medical Adhesive (2)

(45) (1) Preparation of Oxidized Hyaluronic Acid and Oxidized Chondroitin Sulfate (First Component)

(46) 3 g of hyaluronic acid (Shandong Bloomage Freda Biopharm Co., Ltd) with a molecular weight of 1,400 kDa was dissolved in 150 ml of distilled water. Then, as shown in table 1, sodium periodate (molecular weight: 213.89) was added, and a reaction flask was allowed to react with stirring at 40? C. for 24 h. Then, the solution after the reaction was dialyzed with distilled water for 48 h (using a dialysis membrane with a molecular weight cut-off of 12000-14000), and then freeze-dried.

(47) 3 g of chondroitin sulfate (Yantai Dongcheng Biochemical Co., Ltd) with a molecular weight of 5,000-50,000 was dissolved in 15 ml of distilled water. Then, as shown in table 2, sodium periodate (molecular weight: 213.89) was added, and the mixture was allowed to react with stirring at room temperature for 18 h. Then, the solution after the reaction was dialyzed with distilled water for 48 h (using a dialysis membrane with a molecular weight cut-off of 12000-14000), and then freeze-dried.

(48) In the following test, the oxidized hyaluronic acid and the oxidized chondroitin sulfate were used as the first component.

(49) The degrees of substitution (degrees of oxidation) of hyaluronic acid and chondroitin sulfate were confirmed through NaOH titration. Specifically, 17.5 g of hydroxylamine hydrochloride and 6 ml of 0.05% methyl orange were mixed in 994 ml of distilled water to prepare a 0.25 mol/l hydroxylamine hydrochloride solution, which was then titrated to pH 4. 0.1 g of oxidized hyaluronic acid or chondroitin sulfate was dissolved in 25 ml of the solution, and then titrated to pH 4 with 0.1 mol/l sodium hydroxide. The degree of substitution (%) was calculated by the following equation, and the results are shown in tables 8 to 9.

(50) $\begin{array}{cc}\mathrm{Degree}\mathrm{of}\mathrm{oxidantion}\left(\%\right)=\frac{\mathrm{number}\mathrm{of}\mathrm{moles}\mathrm{of}\mathrm{CHO}}{\mathrm{number}\mathrm{of}\mathrm{mols}\mathrm{of}\mathrm{oxidized}\mathrm{glycosaminoglycan}}?100=\frac{\mathrm{concentration}\mathrm{of}\mathrm{sodium}\mathrm{hydroxide}?\mathrm{volume}\mathrm{of}\mathrm{soduim}\mathrm{hydroxide}?{10}^{-3}}{\frac{\mathrm{weight}\mathrm{of}\mathrm{oxidized}\mathrm{glycosaminoglycan}}{\begin{array}{c}\mathrm{amount}\mathrm{of}\mathrm{glycosaminoglycan}\\ \mathrm{repeating}\mathrm{unit}\end{array}}}?100& \mathrm{Equation}3\end{array}$

(51) TABLE-US-00008 TABLE 8 HA Oxidant Reaction Reaction DS weight concentration temperature time (%) (%) (mM) (? C.) (h) 3.7 1.0 0.8 40 24 7.4 1.0 1.7 40 24 15.2 1.0 2.6 40 24 17.7 1.0 3.3 40 24 21.8 1.0 4.1 40 24 35.6 1.0 5.3 40 24 52.8 1.0 7.8 40 24 96.4 1.0 15.6 40 24

(52) TABLE-US-00009 TABLE 9 HA Oxidant Reaction Reaction DS weight concentration temperature time (%) (%) (mM) (? C.) (h) 9.8 1.0 2.2 RT 18 16.6 1.0 2.8 RT 18 20.8 1.0 3.4 RT 18 29.2 1.0 4.3 RT 18 33.9 1.0 5.6 RT 18 47.2 1.0 7.3 RT 18 60.0 1.0 8.4 RT 18 99.2 1.0 11.0 RT 18

(53) (2) Preparation of Second Component Having Two or More Amino Groups

(54) Out of various amino group-containing polyamines, chitosan, protamine, PEI, polylysine, spermine, spermidine, and albumin were typically used as the second component. In order to investigate gelation depending on pH, the powders, which were obtained by adjusting pH in an aqueous solution phase to several ranges (5.5-6.4, 6.5-7.4, 7.5-8.4, 8.5-9.4, 9.5-10.4, and 10.5-11), and then freeze-drying the solution in the same manner as in the oxidized hyaluronic acid/oxidized chondroitin sulfate, were used as the second component. The oxidized hyaluronic acid/oxidized chondroitin sulfate as the first component and a polyamine were mixed. As a result, it was verified that the gelation occurred only at pH of 8.5-11, regardless of the type of polyamine. For example, the gel was formed when the pH of poly-L-lysine was 8.5, but the gel was not formed when the pH thereof was 5.6. In the present test, the formation or not of a gel was determined by the transparency of the gel (transparent; gelation, opaque: non-gelation, FIG. 7).

Example 5: Verification of Optimal Ratio

(55) (1) Establishment of Optimal Conditions According to Molecular Weight and Ratio

(56) The powder states of oxidized chondroitin sulfate and oxidized hyaluronic acid with a molecular weight of 150-3,000 (1:1) were mixed with a polyamine (PA; selecting and using polylysine of pH 8.5-8.9) according to the degree of oxidation at different mixing ratios, and then the physical properties were verified. 200 ?l of sterile distilled water was added to 50 mg of the powders, which were obtained by mixing according to the degree of oxidation at different mixing ratios, and the degree of the sterile distilled water absorbed was verified by the naked eye. The solubility was verified by evaluating the moisture absorption power to be good (+++) when the powder starts to absorb the sterile distilled water within 10 s, moderate (++) within 30 s, and bad (+) over 60 s. In addition, it was verified whether the gelation occurred when the sterile distilled water was added, and the time for gelation was determined. It was verified whether the formed gel was again liquefied, and the time for liquefaction was determined. The results thus verified are shown in the following tables (tables 10 and 11).

(57) As shown in table 11, almost all combinations of the first component and the second component showed desirable results in view of moisture absorption power and the gelation time. Of these, the mixture preparation of 10-50% oxidized chondroitin sulfate+10-40% oxidized hyaluronic acid (150, 1,400 or 3,000 kDa) and the polyamine at a mixing ratio of 1:1 (sample #: 3, 4, 12, 14, 17, 26, 29) showed the best performance (moisture absorption power: +++; and the gelation time: within 30 s). Based on these results, tests were conducted using #14 (named UI-SAH) in the following examples.

(58) TABLE-US-00010 TABLE 10 Sample DS (%) Sample DS (%) Sample DS (%) Sample DS (%) Oxi-CS1 11.7 Oxi-HA1400-1 16.0 Oxi-HA150-1 14.6 Oxi-HA3000-1 15.9 Oxi-CS2 99.2 Oxi-HA1400-2 35.6 Oxi-HA150-2 33.0 Oxi-HA3000-2 33.1 Oxi-CS3 60.8 Oxi-HA1400-3 52.8 Oxi-HA150-3 49.3 Oxi-HA3000-3 48.7 Oxi-CS4 42.2 Oxi-HA1400-4 96.4 Oxi-HA150-4 99.4 Oxi-HA3000-4 95.4

(59) TABLE-US-00011 TABLE 11 Gela- Lique- Moisture tion faction absorption time time No. Sample power (s) (min) 1 Oxi-CS1 + Oxi-HA150K-1 + PA ++ 50 2 Oxi-CS2 + Oxi-HA150K-1 + PA ++ 16 3 Oxi-CS4 + Oxi-HA150K-1 + PA +++ 25 4 Oxi-CS1 + Oxi-HA150K-2 + PA +++ 25 5 Oxi-CS2 + Oxi-HA150K-2 + PA ++ 10 6 Oxi-CS4 + Oxi-HA150K-2 + PA ++ 38 7 Oxi-CS1 + Oxi-HA150K-3 + PA ++ 20 7 Oxi-CS2 + Oxi-HA150K-3 + PA ++ 15 8 Oxi-CS4 + Oxi-HA150K-3 + PA ? 9 Oxi-CS1 + Oxi-HA150K-4 + PA ++ 25 10 Oxi-CS2 + Oxi-HA150K-4 + PA ++ 15 11 Oxi-CS4 + Oxi-HA150K-4 + PA ? 12 Oxi-CS1 + Oxi-HA1400K-1 + PA +++ 25 13 Oxi-CS2 + Oxi-HA1400K-1 + PA ++ 20 14 Oxi-CS4 + Oxi-HA1400K-1 + PA +++ 25 15 Oxi-CS1 + Oxi-HA1400K-2 + PA ++ 25 16 Oxi-CS2 + Oxi-HA1400K-2 + PA ++ 15 17 Oxi-CS4 + Oxi-HA1400K-2 + PA +++ 25 18 Oxi-CS1 + Oxi-HA1400K-3 + PA ++ 26 19 Oxi-CS2 + Oxi-HA1400K-3 + PA ++ 12 20 Oxi-CS4 + Oxi-HA1400K-3 + PA ++ 23 21 Oxi-CS1 + Oxi-HA1400K-4 + PA +++ 34 22 Oxi-CS2 + Oxi-HA1400K-4 + PA ++ 10 23 Oxi-CS4 + Oxi-HA1400K-4 + PA +++ 33 24 Oxi-CS1 + Oxi-HA3000K-1 + PA +++ 32 25 Oxi-CS2 + Oxi-HA3000K-1 + PA ++ 11 26 Oxi-CS4 + Oxi-HA3000K-1 + PA +++ 28 27 Oxi-CS1 + Oxi-HA3000K-2 + PA ++ 27 28 Oxi-CS2 + Oxi-HA3000K-2 + PA ++ 11 29 Oxi-CS4 + Oxi-HA3000K-2 + PA +++ 30 30 Oxi-CS1 + Oxi-HA3000K-3 + PA ++ 30 31 Oxi-CS2 + Oxi-HA3000K-3 + PA ++ 10 32 Oxi-CS4 + Oxi-HA3000K-3 + PA +++ 35 33 Oxi-CS1 + Oxi-HA3000K-4 + PA +++ 50 34 Oxi-CS2 + Oxi-HA3000K-4 + PA ++ 10 35 Oxi-CS4 + Oxi-HA3000K-4 + PA +++ 35

Example 6: Construction of Rat Hemorrhage Model and Evaluation of Hemostatic Action

(60) (1) Hepatolobectomy Model

(61) The male SD rat weighing 200-300 g was anesthetized with an intraperitoneal injection of a mixture of ketamine and Rompun, and then the upper part of the center of the belly was incised by about 3-4 cm in a vertical or horizontal direction. The hepatic lobe was exposed through a gap of the incised belly using wet gauze, and the hepatic artery and portal vein were ligated with vascular clips. The site, which was about 1 cm away from the edge of the hepatic lobe, was incised using surgery scissors, and then coated with UI-SAH 50-100 mg. As a control, Arista? AH (Medafor Inc., USA) was used for coating. After the coating, the clips used for ligation were removed to verify whether bleeding occurred, and then the bleeding amount was measured using sterile gauze.

(62) (2) Nephrectomy Model

(63) The male SD rats weighing 200-300 g was anesthetized with an intraperitoneal injection of a mixture of ketamine and Rompun, and then the right part of the belly was incised by about 3-4 cm in a vertical direction. The kidney was exposed through a gap of the incised belly using wet gauze, and the renal vein and artery were ligated with vascular clips. The site, which was about 1 cm away from the edge of the kidney, was incised using surgery scissors, and then coated with UI-SAH 50-100 mg. As a control, Arista? AH (Medafor Inc., USA) was used for coating. After the coating, the clips used to ligation were removed to verify whether bleeding occurred, and then the bleeding amount was measured using sterile gauze.

(64) (3) Gastric Mucosectomy Model

(65) The male SD rat weighing 200-300 g was fasted for 24 h, and was anesthetized with an intraperitoneal injection of a mixture of ketamine and Rompun, and then the upper part of the center of the belly was incised by about 3-4 cm in a vertical or horizontal direction. The stomach was exposed through a gap of the incised belly using wet gauze, and the curved portion of the stomach, which has less vessels, was incised by about 3 cm in a horizontal direction to expose the stomach lining. 100 ?l of isotonic saline was injected into the stomach lining, and then the stomach lining was resected to have a circular shape with a diameter of about 5 mm, followed by coating with UI-SAH 50-100 mg. As a control, Arista? AH (Medafor Inc., USA) was used for coating. It was verified whether bleeding occurred at the coated site, and the bleeding amount was measured using sterile gauze.

(66) (4) Portal Vein Hemorrhage Model

(67) The male SD rat weighing 200-300 g was anesthetized with an intraperitoneal injection of a mixture of ketamine and Rompun, and then the upper part of the center of the belly was incised by about 5-6 cm in a vertical or horizontal direction. The portal vein was exposed after the other organs were moved to the left through a gap of the incised belly. Two sites above and below the portal vein were ligated using vascular clips. The portal vein was punched using a 18-gauge needle, and then coated with UI-SAH 50-100 mg. As a control, Arista? AH (Medafor Inc., USA) was used for coating. After the coating, the clips used for ligation were removed to verify whether bleeding occurred, and then the bleeding amount was measured using sterile gauze.

(68) (5) Test Results

(69) Test results are shown in FIGS. 8 and 12. As shown in FIG. 12, the hemostatic effect of the composition of the present invention was more excellent than the control (Arista? AH).

(70) Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only for a preferred embodiment and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.