JPS624232A - Hemostatic agent - Google Patents

Abstract

PURPOSE:To provide a hemostatic agent composed of a chitin sponge immobilized with a blood coagulant and having specific wet strength. CONSTITUTION:A hemostatic agent having a wet strength of >=1g/mm<2> can be produced by immobilizing a blood coagulant (e.g. fibrinogen, prothrombin, etc.) to a spongy chitin {poly(N-acetyl-D-glucosamine) or its derivative {e.g. [N-acetyl-6-0-(2'-hydroxyethyl)-D-glucosamine], poly[N-acetyl-6-0-(ethyl)-D-gluco samine], etc. produced by treating the exoskeleton, etc., of crustacean or insect with hydrochloric acid and then with sodium hydroxide and separating and purifying the produced protein and calcium component}}. When the wet strength is lower than 1g/mm<2>, sufficient hemostatic function cannot be attained. The agent can be applied effectively for stopping the bleeding caused by lesion, bleeding during or after operation, bleeding from the superior digestive tract and other bleeding from a part which is difficult to be ligated.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a hemostatic agent, and more specifically, to bleeding associated with trauma, intraoperative or postoperative bleeding, osseous bleeding, bladder bleeding, Bleeding after tooth extraction, epistaxis, upper gastrointestinal bleeding,
Obstetric and gynecological bleeding, bleeding due to wounds, adhesions and bone stumps,
The present invention relates to a hemostatic agent that can be effectively used to stop bleeding that is difficult to ligate, such as bleeding from a cut surface of a parenchymal organ.

(Prior art) (Problems to be solved by the invention) Regarding the chitin sponge used in the present invention, US Patent No. 39
On page 8, line 48 of the specification of No. 88411, a sponge is specified as an example of a chitin molded article, but the specific method for producing it is not described.

Furthermore, the first chitin and chitosan international conference report (1977
), pages 296 to 305 of ``Applications of chitin and chitosan in promoting wound healing'' exemplify the use of chitin sponges, and page 300, lines i16 to 2.
Line 4 describes that a sponge is manufactured by mixing sodium sulfate with a chitin solution obtained in a process similar to the cellulose viscose method and then eluting the sodium sulfate, but the mechanical properties etc. nothing is written down.

On the other hand, developing effective surgical hemostatic agents has been a major challenge in various medical fields.

For example, gelatin sponge containing thrombin (U.S. Patent No. 2,558,395 1, Japanese Patent Publication No. 31-4644)
However, since gelatin sponges do not undergo gelatin denaturation, when applied to a wound surface that requires hemostasis or a surgical field, they are quickly dissolved by the infiltration of bleeding fluids and body fluids. Not only does the sponge detach from the local area, but the combined drugs, such as thrombin, also flow out before they can exert their effects. If they rub against each other, the formed fibrin film may come off due to frictional force, and a complete hemostasis effect cannot be expected after all.

Therefore, the wound surface requires surgical hemostatic agents to stop the bleeding.

Or, when applied to the surgical field, it maintains a certain shape even in the presence of bleeding or body fluid infiltration, and protects the wound surface so that the fibrin coating formed by thrombin will not fall off due to frictional force. There has been a desire for a surgical hemostatic agent that can be used in a variety of situations.

Therefore, Special Publication No. 26-5648, No. 26-5649
No. 28-2995, No. 35-16496, No. 44
No. 22829 and No. 54-41111 disclose that gelatin is denatured with a protein denaturing agent such as formalin and then foamed.

The porous sponge uses the fact that this sponge is poorly soluble to absorb bleeding blood.

Does it promote hemostasis by releasing coagulation factors from blood components?

Alternatively, a hemostatic agent has been proposed in which a blood coagulant contained in a sponge is slowly released to the wound surface to promote hemostasis. Although such gelatin sponges do not have the above-mentioned drawbacks such as detachment of the fibrin coating and leakage of the ingredients, they immediately become moist due to bleeding blood and body fluids, and the strength of the gelatin sponge in such a moist state is extremely low, making it difficult to stop bleeding. It has the disadvantage that it is very difficult to handle, loses its shape, maintains a certain shape, and the formed fibrin coating comes off due to frictional force. In this way, when using a hemostatic sponge, blood and body fluids can cause
Therefore, when using a hemostatic sponge, the wet state is low, the handleability is very poor, the sponge loses its shape, and it cannot maintain a constant shape.

A hemostasis sponge cannot avoid getting wet with blood, etc. Therefore, in order to reliably stop bleeding, the sponge needs to maintain its shape even in a wet state and have enough strength to protect the wound surface. That is, there has been a long-awaited development of a product that overcomes the drawbacks of conventional hemostatic sponges, such as their strength decreasing when wet and their shape being unable to be maintained.

(Means for Solving the Problems) As a result of intensive research to develop a hemostatic agent that has excellent strength even in a wet state, the present inventors found that 1g7
It has a wet strength of more than mm".

The present invention was achieved by discovering that a chitin sponge on which a blood coagulant is immobilized is excellent as a hemostatic agent.

That is, the present invention is a hemostatic agent comprising a chitin sponge on which a blood coagulant is immobilized, and having a wet strength of at least 1 kg/mm''.

Wet strength in the present invention refers to the tensile strength of a hemostat after immersing it in physiological saline maintained at 20° C. for 60 minutes in a wet state using a tensile tester.

The obtained measured value (g) is divided by the cross-sectional area (+nm2) of the hemostatic agent and is displayed. The measurement conditions vary depending on the form and size of the hemostatic agent, but for example, if the hemostatic agent is in the form of a sheet, it is cut into 10 mm width, moistened, and then pulled at an initial length of 50 mm and a pulling speed of 2 QOmm/min. Adopt. The measurement environment is a temperature of 20° C. and a relative temperature of 60%. For example, if a sheet-shaped hemostatic agent with a dry thickness of 8 mm is cut into 10 mm width pieces and immersed in physiological saline maintained at 20°C for 60 minutes, the apparent tensile strength measured immediately is 300 g. The wet strength is 300/(10X8)=3.75 (g/mm2).

The hemostatic agent of the present invention needs to have a wet strength of at least 1 g/mm", preferably at least 3 g/mm", more preferably at least 5 g/mm". If the wet strength is less than 1 g and 7 mm, it will not function adequately as a hemostatic agent.

In the present invention, chitin refers to poly(
N-acetyl-D-glucosamine) or derivatives thereof. Examples of chitin derivatives include those in which acetylglucosamine groups are partially deacetylated, etherified products, esterified products, carboxymethylated products, hydroxyethylated products, 0-ethylated products, etc. Preferred specific examples include poly[N -Acetyl-6-〇-(2'-hydroxyethyl)-D-glucosamine copoly [N-acetyl-6-0-(ethyl)-〇-glucosamine] and the like.

The hemostatic agent of the present invention can be obtained by molding chitin into a sponge shape and then fixing a blood coagulant thereon.

Chitin sponge has a porosity measured in a dry state (X1
00%; B: volume of pores contained in the porous molded body per unit weight, A: total volume of the porous molded body per unit weight) is 90%
The above-mentioned molded object 1 means a wet-formable molded object such as a sheet, rod, or fiber shape.

Such a chitin sponge can be manufactured, for example, by the following method.

The purified chitin is mixed with a known chitin solvent such as a mixture of dimethylacetamide and lithium chloride, a mixture of N-methylpyrrolidone and lithium chloride, and a mixture of trichloroacetic acid and a halogenated hydrocarbon at a concentration of about 0.5 to 15% by weight. to obtain dope. A water-soluble polymer 2 that is solid at room temperature, such as polyvinyl alcohol, polyethylene glycol, or polypropylene glycol, is added to this dope.

Agar, water-soluble starch, protein, etc., preferably polyvinyl alcohol or agar powder, are uniformly dispersed in the dope. At that time, the ratio of chitin dope to water-soluble polymer was 115
It is preferable to select from the range of ~5/l (weight ratio). The chitin dope mixed with a water-soluble polymer is coagulated in a coagulating liquid while maintaining a desired shape by pouring it into a form or extruding it through a slit-like gouer. As the coagulating liquid, for example, water, alcohols such as methyl alcohol, ethyl alcohol, propyl alcohol, and butyl alcohol, or ketones such as acetone are preferably used. After the solvent has been sufficiently removed and coagulation has been completed, the figurine is treated with water or an aqueous solution. In general, water-soluble polymers are difficult to remove from this coagulated material at low temperatures, so
Treatment at a high temperature of 25° C. is preferred, and 1 hour is preferably 30 minutes or more. The molded product that has been thoroughly washed after processing has a sponge-like shape with a high moisture content. To obtain a dried product from this state, normal room temperature drying may be performed, but freeze-drying is preferably used. It will be done. That is, it is preferable to freeze the molded product at a temperature of about -40°C while containing water, and then freeze-dry it at a temperature of 10°C.

The chitin sponge in the present invention may be one obtained by treating the chitin sponge obtained as described above in an alkaline solution. Preferred conditions for performing the alkali treatment are 9, for example, using a 5-60 W/V% aqueous solution of hydric soda at a temperature of 10-120°C for 1 minute or more, and the alkali-treated chitin sponge is heated at 25°C.
, which does not dissolve when immersed in a 2V/V% acetic acid aqueous solution.

Examples of the blood coagulant in the present invention include blood coagulation factors such as fibrinogen, prothrombin, tissue thromboplastin, calcium ions, proaccelelin, procompartin, antihemophilic factor, Christmas factor, Stewart factor, and plasma thromboplastin precursor.

Examples include Hagemann factor, fibrin stabilizing factor, prekallikrene, polymeric kininogen, sodium alginate, and ferric chloride.

Thrombin is particularly preferably used in the present invention. Thrombin can be isolated from the blood of humans, cows, pigs, etc., but it is preferable to use human thrombin when applying to one person.

The blood coagulant in the present invention can be immobilized by binding to or adsorbing onto the chitin sponge. For attaching blood coagulants to chitin sponges 1 eg O, Zaborsky.

Immobilized Enzymes”CRCPr
Conventionally known covalent bonding methods and ionic bonding methods such as those described in Ess. .

When producing the hemostatic agent of the present invention, a blood coagulant can be bonded to a chitin sponge in the following manner, for example. That is, if the blood coagulant has a functional group capable of forming a covalent bond or an ionic bond, such as an amino group or a carboxyl group, a chitin sponge is treated with a solution containing these functional groups. Immobilization by binding can be performed. At this time, if the chitin sponge has no or only a few functional groups that can covalently or ionically bond with the functional groups of the blood coagulant, those functional groups may be introduced into the chitin sponge in advance by chemical reaction. Thereafter, blood clotting agents can be attached to such chitin sponges.

If the blood coagulant does not have a functional group, it can be used after introducing a functional group through a chemical reaction as in the case described above, but in many cases, blood This method is not preferred since it would impair the medicinal properties of the coagulant. In addition, for l to be covalently bonded, it is preferable to use a dehydration condensation agent such as dicyclohexylcarposiimide, 1-cyclohexyl-5-(2-morpholinoethyl)-carposiimide-metho-P-1-luenesulfone. .

In addition, when manufacturing the hemostatic agent of the present invention, a blood coagulant can be adsorbed onto a chitin sponge by physical adsorption as follows. That is, the blood coagulant can be physically adsorbed by dissolving or suspending the blood coagulant in a solvent that can wet the chitin sponge and treating the chitin sponge with this solution. This adsorption method is preferably employed in the present invention because it is effective for any blood coagulant, is easy to use, and rarely impairs its properties as a drug.

In order to produce the hemostatic agent of the present invention, in addition to the method of binding or adsorbing a blood coagulant to a chitin sponge as described above, it is possible to first bind or adsorb a blood coagulant to chitin itself before processing it into a sponge. It is manufactured by adsorbing it and then processing it into a sponge.

When producing the hemostatic agent of the present invention, pharmaceuticals such as anti-inflammatory agents, antibacterial agents, antibiotics, hormones, albumin, α1-antiplasmin, α2
- protease inhibitors such as macroglobulin;
Blood proteins such as ceruloplasmin, haptoglobulin, and cold insoluble globulin can be immobilized on chitin sponges. Antiplasmin is an inhibitor of plasmin, a fibrinolytic enzyme.

Therefore, by inhibiting plasmin, fibrinolytic, ie, fibrinolytic activity is suppressed. Therefore, a hemostatic agent in which antiplasmin is immobilized together with a blood coagulant can promote fibrin production by suppressing fibrinolytic activity. Examples of antiplasmin include aprotinin extracted from cow lungs, natural substances such as pepstatin, leupepsin, antipain, and chymostatin isolated from microbial culture solutions, ε-aminocaproic acid, tranexatic acid, and gabexate mesylate. However, antipain is particularly preferably used.

(Example) Next, the present invention will be explained in more detail with reference to Examples.

Example 1. Comparative example 1. Comparative Example 2 Chitin powder (manufactured by Kyowa Yushi Co., Ltd.) was ground into 100 mesoyu pieces,
IN-+1cI at 4°C for 1 hour, heated in 3% NaOH solution for 3 hours at 90-100"C, washed repeatedly with water, and dried. The obtained chitin was treated at room temperature with 8% lithium chloride. After dissolving the solution in a dimethylacetamide solvent containing 2% by weight to obtain a clear liquid, it was filtered under pressure using a 1480 mesh stainless steel net, and further decomposed under reduced pressure while stirring. A chitin dope was obtained by foaming.This chitin dope 100
g to polyvinyl alcohol (Poval IF-17OG
S, manufactured by Unitika Chemical Co., Ltd.: degree of polymerization 170. Saponification degree of 95% or more) was added thereto and stirred for 1 hour in a beaker equipped with a stirring blade to prepare a uniform dispersion.

This dispersion was cast onto a glass plate to a thickness of 10 mm, and then immersed in water at about 25°C to sufficiently coagulate and wash to obtain a sheet.Next, after immersing this sheet in water, A sheet-like chitin sponge was obtained by treating in a boiling state for 5 hours. This sponge was sterilized in an autoclave for 15 minutes, then frozen at -40°C, and then freeze-dried at 10°C for 15 hours to obtain a chitin sponge with a thickness of 8 mm.

This chitin sponge (50mmX 70mmX 8mm
) 125 units/m1 of tropin [concentrated dry preparation of human thrombin (manufactured by Green Cross)] in physiological saline solution 28n+j! The mixture was frozen at -40°C, and then freeze-dried at 10°C for 15 hours to obtain a sheet-like hemostatic agent on which thrombin was immobilized. The porosity of this hemostatic agent was 99.2%.

This hemostatic agent was cut into 10 mm widths, dried and immersed in physiological saline for 1 hour.
Using an M-II type tensile tester, the initial tensile length was 50 mm.
The measurement was carried out under conditions of a tensile speed of 200 mm/min, a temperature of 20° C., and a relative humidity of 60%.

As Comparative Example 1, a sponge was prepared by the method described in the 1st Chitin and Chitosan International Conference Bulletin (1977), and then thrombin was immobilized in the same manner as in Example 1. Measurement was carried out in the same manner as in 1. That is, chitin powder (manufactured by Kyowa Oil Co., Ltd.) was ground into 100 meshes, treated with IN-HCl at 4°C for 1 hour, and further heated at 90-100°C in 3% NaOtl solution for 3 hours.
and repeated washing with water. 100 g of the obtained chitin was immersed in a 300 mβ 40 W/W% aqueous sodium hydroxide solution at 11 to 13°C for 2 hours and at 0 to 5°C for 10 hours to obtain alkaline chitin. The material was squeezed to about 3 times the weight of the raw material, excess sodium hydroxide aqueous solution was removed, and the mixture was pulverized with a mixer.

The alkaline chitin was placed in a separating funnel, the inside of which was evacuated, and then left at -20°C for 10 hours. Next, 50 g of carbon disulfide was injected using the internal vacuum, and xanthogenization was carried out at 30° C. for 15 hours with occasional stirring. Furthermore, it was dissolved in an aqueous sodium hydroxide solution previously cooled to 0°C so that the alkaline concentration was 4.5χ and the chitin concentration was 5%, and once it had completely melted, it was kept at -20°C for 5 hours and frozen.

Next, the temperature was raised to 0 to 5°C over about 3 hours to dissolve and obtain a completely uniform chitin viscose solution.

Add 30g of sodium sulfate to 50g of chitin viscose.
was added and stirred for 1 hour in a beaker equipped with a stirring blade to prepare a uniform dispersion. Transfer this dispersion liquid onto a glass plate.
It was cast to a thickness of 0 mm, and then immersed in a 5% sulfuric acid solution at about 25°C to regenerate chitin. Next, this sheet was treated with deionized water to elute sodium sulfate to obtain a sheet-like sponge. This sponge was sterilized in an autoclave for 15 minutes, then frozen, and then freeze-dried at 10° C. for 15 hours to obtain a sponge with a thickness of 8 mm.

This sponge was treated in the same manner as in Example 1 to obtain a sheet-like sponge on which thrombin was immobilized. The strength of this sponge was measured in the same manner as in Example 1.

As shown in Table 1, the strength of Comparative Example 1 in a wet state was extremely low, the handleability in a wet state was very poor, the product lost its shape, and it could not function as a hemostatic agent. There wasn't. on the other hand.

The hemostatic agent made of a chitin sponge according to the present invention has high strength, is easy to handle even in a wet state, maintains a constant shape, and is optimal as a hemostatic agent.

Table 1 Examples 2 and 3 A dispersion of chitin dope and polyvinyl alcohol (Poval [IF-170GS) prepared in the same manner as in Example 1 was poured into a Φ41 plastic syringe with a cut off tip (
Example 2) or a plastic syringe with a diameter of 8 mm with the tip cut off (Example 3), extruded into water at 25°C, thoroughly solidified and washed,
), a football-shaped ball (Example 3) was obtained. Then,
The rondos and spheres were immersed in water and then treated in a boiling state for 5 hours to obtain a rondo-shaped chitin sponge (Example 2) and a ball-shaped chitin sponge (Example 3).

After sterilizing this sponge in an autoclave for 15 minutes,
Freeze and freeze-dry at 10°C for 15 hours to obtain Φ3
．． 5 mm, l = 45nv+ (Example 2),
φ7mm, I! = 13 mm (Example 3) A chitin sponge was obtained. 1 for each of these chitin sponges
25 units/ml thrombin (concentrated dry preparation of human thrombin [Midori Juji]) in physiological saline solution 0.4
ml (Example 2), and 0.5 nl (Example 3), were frozen at -40°C, and then freeze-dried at 10°C for 15 hours to form a rod-shaped hemostat with immobilized thrombin. (Example 2, porosity 99.1%) and a football-shaped hemostatic agent (Example 3, porosity 99.1%) were obtained.

This hemostatic agent was cut into a thickness of 2IIlffi, dried and immersed in physiological saline at 20°C for 60 minutes, and then tested using a Toyo Baldwin UTM-n type tensile tester at an initial tension length of 5 mm and a tension speed of 200 mm/min. m i
n.

The results measured under conditions of a temperature of 20° C. and a relative humidity of 60% are shown in Table 2.

Table 2 Example 4 Poval UF-170GS 50g used in Example 1
A sheet-shaped hemostatic agent (porosity: 98.0%) on which thrombin was immobilized was obtained in the same manner as in Example 1, except that 60 g of agar powder (manufactured by Wako Pure Chemical Industries, Ltd.) was used instead.

Next, the dry strength and wet strength of this sheet-shaped hemostatic agent were measured in the same manner as in Example 1.

The results are shown in Table 3.

Table 3 Examples 5 and 6 Poval UP-170G 350g used in Example 1
A rod-shaped hemostatic agent with immobilized thrombin (Example 5. Porosity 98.0%) was prepared in the same manner as in Example 2.3, except that 60 g of agar powder (manufactured by Wako Pure Chemical Industries, Ltd.) was used instead. )
and football-shaped hemostatic agent (Example 6゜porosity 98.0
%) was obtained.

The dry strength and wet strength of this hemostatic agent were measured in the same manner as in Example 2.3.

The results are shown in Table 4.

Table 4 Example 7 Sheet-shaped hemostatic agent with immobilized thrombin (porosity 99.0%) in the same manner as in Example 1 except that N-methylpyrrolidone was used instead of dimethylacetamide as the solvent.
I got it.

Next, the dry strength and wet strength of this sheet-shaped hemostatic agent were measured in the same manner as in Example 1.

The results are shown in Table 5.

Table 5 Example 8.9 A rod-shaped hemostatic agent with immobilized thrombin (Example 8. Porosity 99.0%) and 7) Ball-shaped hemostatic agent (Example 9. Porosity: 99.0%) were obtained.

The dry strength and wet strength of this hemostatic agent were measured in the same manner as in Example 2.3.

The results are shown in Table 6.

Table 6 Example 10 N-methylpyrrolidone was used instead of dimethylacetamide as the solvent, and Poval UF-170GS 50
A sheet-like hemostatic agent (porosity: 98.0%) on which thrombin was immobilized was obtained in the same manner as in Example 1, except that 60 g of agar powder (manufactured by Wako Pure Chemical Industries, Ltd.) was used instead of g.

Next, the dry strength and wet strength of this sheet-shaped hemostatic agent were measured in the same manner as in Example 1.

The results are shown in Table 7.

Table 7 Example 11.12 N-methylpyrrolidone was used instead of dimethylacetamide as the carrier, and Poval mouth F-170GS 5
60g of powdered agar (manufactured by Wako Pure Chemical Industries, Ltd.) instead of 0g
A rond-like hemostatic agent with immobilized thrombin (Example 11. Porosity 98
%) and football-shaped hemostatic agent (Example 12. Porosity 9
8%).

The dry strength and wet strength of this hemostatic agent were measured in the same manner as in Example 2.3.

The results are shown in Table 8.

Table 8 Reference Examples 1 and 2.3 Each hemostatic agent obtained in Example 1.2.3 theoretically contains 3500 units of thrombin (Example 1), 50 units (Example 2), 62. 5 units (Example 3)
It contains. Table 9 shows the results of measuring the changes in thrombin activity of these three different hemostatic agents over time. The numbers in Table 1 indicate the number of thrombin units.

Table 9 In addition, the number of thrombin units was measured by the following method. That is, in physiological saline 10 times the volume of the hemostatic agent,
After extraction at 37° C. for 20 minutes, the time was calculated using a conventional thrombin time measurement method.

Reference Example 4 The hemostatic effect of the hemostatic agent produced in Example 1 was visually evaluated using its constituent components chitin sponge, thrombin, and the hemostatic agent produced in Comparative Example 1 as controls.

Specifically, a female rabbit (female) weighing 2.0 to 2.5 kg was anesthetized with thiopencool (24 mg/kg), the hair of the dorsal position was shaved, and the surface of the dorsal muscle was punctured to cause bleeding. 10 pins (commercially available: length 3cm) with a diameter of approximately 8m11
I bundled them so that there were 1.

In addition, the length of the needle was adjusted so that the depth of the needle was 5 to 6 mm when piercing the back muscle. Using such a puncture device, the same spot on the back muscle was punctured three times to cause bleeding. After bleeding, the hemostatic agent obtained in Example 1 (15 mm x 15 mm x 8
n+m) + chitin sponge (15mm x 15m)
mX8+nm) and the hemostatic agent obtained in Comparative Example 1 (15
mm x 15 mm x 8 mm). Since the thrombin contained in the hemostatic agent of the present invention (15 na + x 15 mm x 8 mm) is 180 units, 180 units of powdered thrombin as a control was sprinkled on the puncture site.

Hemostatic agent treatment obtained in Example 1 and hemostatic agent treatment obtained in Comparative Example 1 on the same rabbit at the same time for the purpose of eliminating individual differences between rabbits.

Five treatments were performed: chitin sponge treatment, thrombin treatment, and no treatment, and the positional relationship of the five treatments was changed to the rabbit side in order to eliminate deviations due to differences in the puncture locations on the dorsal muscle. Next, the hemostatic agent obtained in Example 1, the hemostatic agent obtained in Comparative Example 1, a chitin sponge, and powdered thrombin were placed on the puncture wound surface, and
After a few minutes, the hemostatic agent obtained in Example 1, the hemostatic agent obtained in Comparative Example 1, and the chitin sponge were removed (however, the powdered thrombin was left as is), depending on the degree of clot formation. The hemostatic conditions of the hemostatic agent obtained in Comparative Example 1, chitin sponge, and powdered thrombin were compared with an untreated control. The state of the puncture wound was determined by the hemostatic agent obtained in Example 1,
The chitin sponge was found to have a hemostatic effect when applied to the puncture wound.

The hemostatic agent obtained in Comparative Example 1 became wet with the bleeding blood, and when pressure was applied to fix the hemostatic agent to the puncture site, the hemostatic agent collapsed into several fragments and formed fibrin. The coating dislodged and additional hemostatic agents failed to protect the puncture wound.

Simply sprinkling powdered thrombin on the puncture wound did not immediately stop the bleeding; thrombin was sometimes washed away, and bleeding continued for some time. The condition of the stab wound after 5 minutes is as in Example 1.
A blood clot was formed at the puncture site using the hemostatic agent obtained in 1, and no bleeding was observed even after the hemostatic agent was removed. Comparative example 1
Some of the fragments of the hemostatic agent obtained in 1 were detached from the puncture wound, and no blood clot formation was observed in the puncture wound.

No blood clots were formed on the chitin sponge, and most of the blood that had bled into the sponge was absorbed. When the chitin sponge was removed, slight bleeding was observed.

Powdered thrombin was less effective because it was washed away by the flowing blood.

Reference Example 5 The hemostatic effect of the hemostatic agent produced in Example 1 was evaluated based on the amount of bleeding using its constituent components, chitin sponge and thrombin, as controls. As in the case of Example 2,
The hemostatic agent of the present invention at the puncture site.

Chitin sponge, powdered thrombin treatment, hemostat,
Blood volume and hemostatic agent absorbed by the chitin sponge.

Blood clots formed with powdered thrombin were combined and the blood loss was measured by the cyanmethemoglobin method.

That is, in advance, add Van Kampen reaction solution (0.05 g of potassium cyanide, 0.20 g of potassium ferricyanide, 5terox-SR0.5m#) to a test tube.
Dissolve in 30M phosphate buffer (pH 7,2) and add 100%
A hemostatic agent that absorbed blood and a blood clot formed by the hemostatic agent were collected with tweezers and placed in a test tube to dissolve. Similarly, blood-absorbed chitin sponges and blood clots formed with powdered thrombin were placed in test tubes and dissolved. After standing for 5 minutes, the absorbance of 5401 was measured using a spectrophotometer to determine the blood volume. As an untreated control, flowing blood was collected into a test tube containing a VanKanpen reaction solution and measured.

The results were as shown in Table 10.

Table 10 Bleeding amount (g/di) Here, (untreated bleeding amount - hemostatic agent bleeding amount), (untreated bleeding amount - chitin sponge bleeding amount), (untreated bleeding amount - powdered thrombin bleeding amount), respectively. Considering the hemostatic effect of a hemostatic agent, chitin sponge, and powdered thrombin, the hemostatic effect is in the order of hemostatic agent>chitin sponge>powdered thrombin, and the effect of the hemostatic agent is greater than the sum of the effects of the chitin sponge and powdered thrombin. (The synergistic effect of these components was observed.

Reference Example 6 The hemostatic agent produced in Example 1 was subjected to a mutagenic test using microorganisms in accordance with the Industrial Safety and Health Act. the result,
All strains used were negative regardless of the presence or absence of metabolic activity.

Reference Example 7 Regarding the hemostatic agent produced in Example 1, the tissue absorbability within the animal body was examined.

That is, 10 domestic rabbits each weighing 12.0 to 2.5 kg (♀
), and after anesthesia with thiobental (24 mg/kg), a hemostatic agent (10 mm x 10n++ n x 8 mm) was implanted into the dorsal muscle. After that, the hemostatic agent was sutured (when the incision was made after 30 days, the hemostatic agent had been decomposed and absorbed in 8 birds.
In the case of feathers, most of them were decomposed, with only slight traces of chitin sponges visible.

Reference Example 8 The hemostatic agent produced in Example 1 was subjected to an acute toxicity test in mice.

i.e. 1 DDY hermaphrodite mouse weighing 15-17 g 1 dose 1
0 animals were used. Such mice are kept at room temperature of 20±2°C.

Solid feed (C1e
a CA-1) and water ad libitum. First, the sample was suspended in sterile physiological saline and homogenized using a homogenizer (15,000 rpm, 2
m1n) L/, was used as the test solution. The dose of the test solution is 0.1 m7 per mouse body weight! It was prepared and injected subcutaneously. After drug administration, mice were individually housed and behavioral changes of the mice were observed continuously for 3 hours and then every hour for 12 hours. During this time.

No feed or water was given. After that, 24 days after drug administration.
．． The behavior of the mice at 48 and 72 hours was observed, and the LDSO after 72 hours was determined. Hemostatic agent 2.0
No significant changes were observed in the general symptoms of mice after subcutaneous injection of 00 mg/kg. However, 3,00
At 0 mg/kg, slight piloerection and decreased locomotor activity were observed 4 hours after administration, but both sexes recovered after a few hours. At increased doses of 5,000 and 7.000 mg/kg, piloerection was observed all over the body 2 hours after administration in both males and females, and decreased spontaneous movement and mild irregular breathing appeared.

However, the symptoms of intoxication started to recover after 5 hours, the side surface returned to normal after 24 hours, and even after 72 hours, no deaths were observed (Effects of the Invention) The hemostatic agent of the present invention It has a wet strength of Ig/mm'' or higher, making it an extremely practical hemostatic agent for hemostasis in various medical fields compared to the previously known Svonzel and Gelathrombin.

Especially bleeding associated with trauma, intraoperative or postoperative bleeding.

Bone bleeding, bladder bleeding, tooth extraction bleeding, epistaxis, upper gastrointestinal bleeding, obstetric and gynecological bleeding, bleeding from wounds, bleeding from adhesions, bone stumps, cut surfaces of parenchymal organs, etc. that are difficult to ligate. It has a remarkable effect on stopping bleeding. Furthermore, there is no need to remove the chitin sponge after hemostasis has been achieved, and its pharmacological action is that it strongly adheres to the surface of the wound, exhibiting a hemostasis effect almost equivalent to that of fibrin. Furthermore, when such a hemostatic agent is embedded within a tissue or a body cavity, it biodegrades and is absorbed within about one month.

Furthermore, chitin, the raw material, has long been known to have healing effects, and is expected to promote wound healing.

Claims (1)

[Claims] (1) A hemostatic agent comprising a chitin sponge on which a blood coagulant is immobilized and having a wet strength of at least 1 g/mm^2.