JPH0566148B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides an embolic material for use in the treatment of wounds such as cuts and abrasions, burn surfaces, surgical wounds, ulcers on the body surface, tooth extraction sockets, and vascular occlusion therapy. or regarding injection materials used for puncture therapy,
More specifically, the present invention relates to a fibrin formation promoting material that can effectively promote the production of stabilized fibrin for a long period of time.

The present inventors have developed blood coagulation factor (hereinafter “F
” is abbreviated as “. ) and trond-immobilized materials are superior as materials for wound treatment because they can promote the formation of stabilized fibrin compared to materials in which only trond is immobilized, that is, materials that only promote the formation of non-stabilized fibrin. I discovered that there was a problem and proposed it first (Japanese Patent Application Laid-open No. 58163-1983)
However, we continued our research into using oxidized cellulose, which is commonly used as a hemostatic agent, as a material, and found that if oxidized cellulose was used directly to immobilize F and throbin, it would have an adverse effect on promoting the production of stabilized fibrin. It was found that

In view of the current situation, the present inventors have continued to study in order to solve the above-mentioned problems. As a result, if oxidized cellulose is neutralized and F and throbin are immobilized, the ability to promote the formation of stabilized fibrin can be improved. It was discovered that this property was maintained well, and the present invention was developed based on this finding.

That is, the present invention neutralizes a structure made of oxidized cellulose and has properties such as a fiber aggregate, sponge, powder, monofilament, film, or microcapsule, and then immobilizes F and thrombin on the structure. The present invention provides a fibrin formation promoting material capable of effectively promoting the production of stabilized fibrin for a long period of time.

Until now, regarding the use of oxidized cellulose as a carrier for immobilization after neutralization, for example, US Pat. It has been shown that it can prevent the deactivation of However, there is no precedent for treating oxidized cellulose with F, and furthermore, there is no precedent for considering the ability to promote the formation of stabilized fibrin as in the present invention.

The oxidized cellulose used in the present invention refers to cellulose materials such as wood pulp, cotton, cotton linters, ramine, jute paper, hemp, regenerated cellulose, and rayon, which are prepared by using ozone, hydrogen peroxide, periodate, nitrogen dioxide, etc. Although it refers to cellulose oxidized by a known method, in the present invention, cellulose in which 10 to 30% of the -CH ₂ OH groups are oxidized to -COOH groups is preferably used because it has good bioabsorption.
Particularly preferably used is one with a content of 15 to 25%.

In the present invention, such oxidized cellulose is used after being neutralized, and neutralization here means converting the acid group of oxidized cellulose -COOH into a salt. To neutralize the oxidized cellulose, the oxidized cellulose may be treated with a metal hydroxide such as sodium or calcium, or various salts such as sodium, calcium, or ammonium, such as an acetate salt or a carbonate salt. If the degree of neutralization is too high, the structure will become water-soluble and it will be difficult to immobilize it, while if it is too low, it will be difficult to obtain good sustained release properties, so the structure will become gel-like and lose its shape. It is preferable to carry out the neutralization in such a manner that the pH of the neutralized oxidized cellulose when immersed in water is between 3 and 7, preferably between 5 and 6. Neutralization may be performed on the structure, or the material may be neutralized before being processed into the structure, and then the structure may be formed.

Trond used in the present invention is a protease that can convert fibrinogen to fibrin. Thrombin is degraded from blood of humans, cows, pigs, etc., but when applied to human treatment, it is preferable to use human thrombin.

The F-fibrin stabilizing factor used in the present invention is a factor that directly acts on non-stabilized fibrin and is involved in the generation of isopeptide bonds between fibrin molecules. F is isolated from the blood or placenta of humans, cows, etc., but when applied to human treatment, it is preferable to use human-derived F.

F and thrombin can be immobilized by binding or adsorbing to neutralized oxidized cellulose. To combine, e.g. O. ZaborsKy, “Immobilized Enzymes” CRC
Conventionally known covalent bonding and ionic bonding methods such as those described in J. Press, 1973 can be employed.

To covalently bind F and thrombin to neutralized oxidized cellulose, dicyclohexyl-bodiimide, 1-cyclohexyl-3-(2
A method using a dehydration condensation agent such as -morpholinoethyl)-carboimide-meth-toluenesulfonate, a method in which oxidized cellulose is treated with a polyaldehyde such as glutaraldehyde or dialdehyde starch to introduce a formyl group, and then treated with F and trompine. Method, instead of polyaldehyde, bromic cyanide is used to create iminocarbonate groups, bromoacetyl groups, polymaleic anhydride groups such as maleic anhydride copolymers are used to form acid anhydride groups, and polyisocyanates such as toluene diisocyanate and hexamethylene diisocyanate are used to form isocyanates. Epoxy groups can be introduced into structures using polyglycidyl derivatives such as ethylene glycol, tetoemethyl glycol, and glycerin, and chlortriazinyl groups can be introduced into structures using cyanuric chloride. using F
and a method of combining trobin, etc. can be adopted.

In order to ionically bond F and trobin to neutralized oxidized cellulose, the carboxyl group of oxidized cellulose may be used, or as mentioned above, formyl group, iminocarbonate group, bromoacetyl group, acid anhydride, and isocyanate group may be used. , epoxy groups, and chlorotriazinyl groups are aminated by treatment with polyamines such as polyethyleneimine, and carboxylated by treatment with aminocarboxylic acids such as glycine. F and thrombin can be ionically bonded to a structure into which amino groups and carboxyl groups, which are ion exchange groups, have been introduced.

When F and thrombin are adsorbed onto neutralized oxidized cellulose by a physical adsorption method, an entrapment method, etc., it can be carried out as follows. That is, F is used as a solvent that can wet the neutralized oxidized cellulose.
F and thrombin can be physically adsorbed by dissolving F and thrombin and treating the structure with this solution. The entrapment method is a method in which F and thrombin are wrapped in a fine lattice of gel so that they cannot be released.

When F and thrombin are immobilized by any of the above methods, F and thrombin may be immobilized at the same time, or F and thrombin may be immobilized first.
may be immobilized first and then thrombin may be immobilized, or vice versa. Further, it is also possible to separately produce a product with immobilized F and a product with immobilized trobin, and then uniformly mix or stack them.

Furthermore, in producing the material of the present invention, calcium ions involved in F activation can be immobilized on the structure. Furthermore, when producing the material of the present invention, pharmaceuticals such as antiplasmin agents, bactericides, antibiotics, hormones, etc. can be immobilized on the structure together with F and thrombin, if necessary. Antiplasmin is an inhibitor of plasmin, a fibrinolytic enzyme, and therefore suppresses fibrinolytic activity, ie, fibrinolytic activity, by inhibiting plasmin. Therefore, a material in which antiplasmin is immobilized together with F and thrombin can promote fibrin production by suppressing fibrinolytic activity. Examples of antiplasmin include aprotinin extracted from bovine lungs, natural substances such as pepstitan isolated from microbial culture fluid, leupepsin, antivaine, and chymostatin, ε-aminocaproic acid, tranexamic acid, and gabexate mesylate. but,
In particular, ε-aminocaproic acid and tranexamic acid are preferably used.

The material of the present invention can be used to protect wounds such as cuts, abrasions, burns, ulcers on the body surface, tooth extraction sockets, anastomoses, etc., or as an embolization material used for vascular occlusion therapy, or for puncture therapy. Used in injection materials, it can effectively promote the production of stabilized fibrin for a long period of time. Therefore, when the material of the present invention is used as a wound protection agent, it has the effect of significantly accelerating wound healing and at the same time alleviating local pain, and when used as an embolization agent, it has the effect of rapidly and firmly It does not form a thrombus or cause blood vessel recanalization, and when used as an injection material, it enables rapid and reliable hemostasis.

EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples. The blood is human plasma thrombin from Midori Juji Co., Ltd., and the F is F.
A concentrated dry formulation was used. The F formulation has an F activity equivalent to 60 times the amount present in 4 ml of fresh human plasma per bottle.

Further, a test for the ability to form stabilized fibrin was conducted as follows. That is, a structure in which F and throbin were immobilized was treated with an aqueous solution containing Ca ⁺⁺ after immobilization and 12 months later to activate F, and then the structure was placed in a 0.5% fibrinogen aqueous solution. It was left at 37°C for 10 minutes. Thereafter, it was confirmed whether the fibrin produced on the surface of the structure was stabilized fibrin. Non-stabilized fibrin dissolves in 1% monochloroacetic acid, but stabilized fibrin does not dissolve in 1% monochloroacetic acid, so this can be confirmed by examining the solubility of the fibrin film formed as described above in 1% monochloroacetic acid. I went there.

Example 1 20 mg of Oxycel cotton (oxidized cellulose cotton type, Sankyo Co., Ltd.) was immersed in 20 ml of 0.5M calcium acetate aqueous solution at room temperature for 2 hours, washed with ethanol and water, and air-dried to obtain neutralized Oxycel cotton. I got it.

The entire amount of this product was dissolved in 2 ml of fibrogamine aqueous solution (concentrated dry preparation of blood coagulation factor, Hoechst Co., Ltd., dissolved in 4 ml of water in 1 bottle) and thrombin (human thrombin Midori, Midori Juji Co., Ltd., dissolved in 5 ml of water in 1 bottle). After immersing it in a mixture of 2 ml at 0℃ for 2 minutes, -
Freeze-drying was performed at 60°C for 24 hours to obtain immobilized neutralized Oxycell cotton with immobilized fibrogamine and thrombin.

Using the immobilized neutralized Oxycel cotton obtained in this way, the above-mentioned fibrin formation ability test was conducted after immobilization and after 12 months, and it was found that the fibrin formed on the surface was mixed with both 1% monochloroacetic acid and 1% monochloroacetic acid. It was stabilized fibrin that was insoluble in water. Furthermore, the rate and amount of stabilized fibrin formed were similarly rapid and large both immediately after immobilization and after 12 months.

Comparative Example 1 Immobilized Oxycel cotton was obtained in exactly the same manner as in Example 1, except that the treatment with the calcium acetate aqueous solution was not performed.

Using this material, we conducted a fibrin-forming ability test similar to that in Example 1 immediately after immobilization and after 12 months, and found that stabilized fibrin was formed immediately after immobilization, but the test in Example 1 The formation rate was slow and the amount formed was small compared to Although fibrin was formed after 12 months, it was found to be non-stabilized fibrin soluble in 1% monochloroacetic acid, indicating that the F activity had been lost.

Claims (1)

[Claims] 1. After neutralizing a structure made of oxidized cellulose in the shape of a fiber aggregate, sponge, powder, monofilament, film, microcapsule, etc., blood coagulation factor and thrombin are immobilized on the structure. A method for producing a fibrin formation-promoting material capable of effectively promoting the production of stabilized fibrin for a long period of time, characterized by: