JPS5837330B2 - Shiketsu Yokora Genfun Matsuno Seizou Hohou - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing hemostatic collagen powder from a stock solution obtained by solubilizing a collagen material in water as molecular collagen that does not contain telopeptides.

Bleeding during injury or surgery primarily results from the cutting of blood vessels, and the purpose of hemostasis can usually be easily achieved by ligating the blood vessels.

However, bleeding in cases such as amputation or rupture of the liver, spleen, etc. is generally caused by the cutting of multiple tubules, which is called oozing, and it is virtually impossible to stop the bleeding by ligating as described above.

Therefore, there has been a lack of suitable hemostatic means other than compression, and there has been a strong desire to develop an effective hemostatic agent.

Regarding the occurrence of blood clots in blood vessels, morphologically it has been known for a long time that there are red thrombus and white thrombus.

The former is often seen on the venous side where blood flow is slow, and the latter is often seen on the arterial side.

Although both ultimately lead to serious consequences as coagulation progresses in blood vessels and blood flow is inhibited, it has been revealed that there are major differences in the mechanisms of their formation.

In other words, red blood clots are mainly directly related to red blood cells and fibrins, whereas white blood clots are caused by platelets in the blood when the endothelial cell layer of the artery is damaged for some reason and the outer collagen layer is exposed. It is generally accepted that this process begins with adhesion and aggregation of collagen.

As a result of basic research on the interaction between various types of collagen and platelets, it has been found that deamination or acetylation of the ibusilone-amino group in the collagen molecule, or blocking with dinitrofluorobenzene, etc., improves the interaction between platelets. It is known that the interaction property decreases significantly, and it has been found that even if the carboxyl group is blocked by esterification or the like, the interaction property does not change much.

Furthermore, it has become clear that even if the telopeptide portion of the collagen molecule is removed by the action of pepsin or the like, the coagulation rate is slightly slowed, but there is no significant change in the coagulation ability.

The present inventors have focused on the significance of using collagen as a hemostatic agent in consideration of the above-mentioned circumstances and facts, and have conducted extensive research to arrive at the present invention.

Collagen is considered to be an essentially useful material as a hemostatic agent, based on the knowledge that it serves as a promoter of white thrombi as described above.

However, if collagen is used as a hemostatic agent, it may be necessary to inject a large amount into a living body, and therefore, other effects on the living body, particularly antigenicity, become a problem.

Another important issue was molding it into the most effective form as a hemostatic material.

Collagen is generally known as a protein with relatively little antigenicity.

Furthermore, it is known that the part responsible for the antigenicity of collagen is mainly located at the terminal end of the molecule, particularly in the telopeptide part, which acts as a major factor in insolubilizing collagen.

Therefore, without cleaving the main peptide bonds of the collagen molecule, only the telopeptide part is solubilized by selective digestion, and further, by repeated precipitation and re-dissolution using its fiber regeneration ability, it is purified and purified. We have discovered that purified collagen (so-called atelocollagen) with enhanced strength is a material that fully meets the purpose of the invention.

Examples of materials for carrying out the present invention include Aspergillus niger variety macrosporus (Asp.
ergillus niger. var. macr-o
For example, a proteolytic enzyme produced by S. sporus and having an optimum pH in the acidic range and solubilized by treating cowhide (hereinafter referred to as oxygen-solubilized collagen) can be used.

Also, collagen molecularly solubilized by the action of sodium sulfate, amines and caustic soda, or by the action of amines and caustic soda (hereinafter referred to as alkali-solubilized collagen) can be similarly utilized.

The purity of these solubilized collagens is 97 or higher, the tyrosine content, which is a measure of the remaining amount of telopeptide moieties, is less than 1 per 1000 amino acid residues, and the precipitate titer, which is a measure of the strength of antigenicity. (Complete adj
(immunized with uvant) is 21-2.

On the other hand, in the case of acid-soluble collagen containing telopeptide, the tyrosine content was 4-5 per 1000 amino acid residues, and the precipitate titer was 24-5.

Therefore, the antigenicity of the material obtained as described above is so weak that it poses no clinical problem when used as a hemostatic agent, and the content of impurities other than collagen is extremely low and essentially It will be understood that since it is dissolvable and absorbable in vivo, it satisfies the purpose of the present invention.

Collagen solubilized by the above method is usually pn
Although it is prepared as a slightly acidic aqueous solution of about 2 to 3, it is possible to use this as a stock solution to form collagen products in various forms such as gels, films, powders, and fibers.

As a result of examining the use of collagen products in various forms as the hemostatic agent of the present invention, it was found that a powdered collagen product most suited the purpose of the present invention.

In other words, powdered products inherently have good adhesion and adhesion to living tissue, and are also highly water-absorbing, so they have excellent compatibility with wound surfaces even when used on fairly wet areas. Furthermore, it has the advantage that the amount used can be adjusted or added as appropriate.

In the case of powder, if the particle size is excessively coarse, the coverage of the wound surface will be poor, and conversely, if the particle size is too fine, small agglomerates are likely to occur, resulting in uneven distribution on the wound surface, etc. Be looked at.

It can normally be used as a 30-400 mesh powder, but
Powders of 50 to 100 mesh are particularly preferred.

Next, the outline of the method for producing collagen powder for hemostasis will be explained.

Although it is not impossible to coagulate the raw collagen solution by adding a conventional coagulant or precipitant, it is necessary to adjust the pH of the raw collagen solution to adjust the isoelectricity of the dissolved collagen. An effective method is to control the precipitation by adjusting the temperature to a certain point.

The purpose can be achieved by adjusting the pH to 7 to 9 in the case of enzyme-solubilized collagen, and between 4 to 5.7 in the case of alkali-solubilized collagen.

An alternative method is to dialyze the stock solution against a 0.03-0.2 molar saline solution or a 0.02 molar aqueous solution of Na2HPO4.

In either case, collagen precipitates as fine fibrous aggregates.

The precipitate thus obtained is separated after washing with water, dried, and then ground to a particle size of 30 to 400 mesh, preferably 50 to 100 mesh, using a Wiley type grinder, for example.

If the moisture content of the powder obtained at this time is high and the temperature rises excessively, thermal denaturation of collagen may occur.

Particular care must be taken since the blood clotting ability of denatured collagen is significantly reduced.

The obtained powder is sterilized, for example, by ethylene oxide gas treatment, and then packaged aseptically and opened as necessary for use.

In use, in the case of a normal bleeding wound, the atelocollagen powder of the present invention can be uniformly spread over the wound surface, and a sufficient effect can be obtained by simply applying the powder lightly if necessary.

However, when blood is particularly violently discharged, such as when an artery is severed, the atelocollagen powder alone may be washed away by the blood flow.

In such a case, the atelocollagen powder of the present invention is sprinkled onto a cotton-like material made of stable spun fibers with a fineness of about 1 to 5 deniers obtained by wet spinning the above-mentioned atelocollagen aqueous solution as a spinning dope and compressed onto the wound surface. It is extremely effective to use it.

In such cases, if necessary, it is possible to suture and fix this mixture to the wound surface using sutures, which can be expected to have both the effects of compressing blood vessels and the essential coagulation effect of collagen. can.

This will be explained in detail below using examples.

Example 1 Enzyme-solubilized collagen with a purity of 98.3%, a tyrosine content of 0.8 per amino acid too residue, and a precipitated titer of 21°8 was dialyzed against 0.05 mol of saline to regenerate fine fibers. It was precipitated, separated, washed with water, and then dried.

It was then ground with a Wiley grinder to obtain a 70 mesh powder.

Example 2 Alkaline solubilized collagen with a purity of 99.0%, a tyrosine content of 0.5 per 1000 amino acid residues, and a sedimentation titer of 21.2 was prepared at a pH of 4. 8 to form a fine fibrous precipitate, which was separated, washed with water, and dried.

The mixture was then ground using a Wiley grinder to obtain a 100 mesh powder.

Claims (1)

[Claims] 1 Adjust the pH of an aqueous solution of molecularly oxygen- or alkali-solubilized collagen without substantially telopeptide to around the isoelectric point of dissolved collagen, or adjust the aqueous solution to 0.03-0.2 mol of collagen. For hemostasis, the collagen is precipitated by dialysis against saline or a 0.02 molar dibasic sodium phosphate aqueous solution, the precipitate is collected, dried, and then ground to a particle size of 30-400 mesh. Method for producing collagen powder.