JPS6052825B2 - Manufacturing method for anticoagulant medical materials - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing a medical material, particularly a medical material having anticoagulant properties.More specifically, the present invention relates to a method for producing a medical material having anticoagulant properties.More specifically, collagen has anticoagulant properties. The present invention relates to a method for producing a novel biocompatible medical material by immobilizing an enzyme.

In recent years, research has been actively conducted to replace some biological organs with organs made of synthetic polymer materials, and some of these have already been put into practical use as artificial blood vessels, artificial bones, etc.

However, when organs made of synthetic polymer materials are used as part of a living body, it is extremely difficult to obtain sufficient and long-term compatibility with the living body due to the vastly different materials. . In contrast, collagen,
In other words, collagen originally exists as a part of living tissues of animals. In other words, collagen is the main protein of connective tissue, and is found in skin, bones, blood vessels, locks, teeth, ocular membranes, cornea, and vitreous. It exists in almost every tissue in the body, has excellent affinity with surrounding living tissues, has no antigenicity, is easy to purify, is strong and flexible, and is one of the few products on the market. Since it is a relatively inexpensive material that can be easily obtained, it is recommended as a material for use in living organs.
Far superior to synthetic polymer materials.

However, when collagen is used as it is as part of a living organ, there is a drawback that it causes a blood coagulation reaction.

So, if this drawback can be removed, as mentioned earlier,
Collagen can be said to be the most promising material as a medical material. As a result of extensive research into ways to prevent the blood clotting effect of collagen, the present inventors discovered that collagen has enzymes that have anticoagulant properties, such as fibrinolytic enzymes such as urokinase and streftokinase, and trypsin. We have found that by immobilizing proteolytic enzymes such as proase in the presence of cyanogen bromide or glutaraldehyde, a medical material with excellent anticoagulant properties can be obtained.

The collagen used in this invention may be any commercially available collagen, and soluble collagen, insoluble collagen, and a mixture of both may be selected and used.

However, when using soluble collagen that has been solubilized with enzymes such as pepsin and protase as a raw material,
For handling purposes, it is desirable to use it after making it insolubilized by crosslinking or the like. As the fibrinolytic enzyme, commercially available urokinase, streptokinase, etc. are most preferred, but other proteolytic enzymes such as trypsin, pronase, etc. can also be used for the purpose of the present invention. When immobilizing enzymes, one type or any mixture of two or more of these enzymes is used, and the enzymes are immobilized on collagen in the presence of a fixative. As the fixing agent, cyanogen halides such as cyanogen fluoride, cyanogen chloride,
Examples include cyanogen bromide and cyanogen iodide; cyanogen fluoride and cyanogen chloride are difficult to handle as they are gases at room temperature, making it difficult to obtain commercially available products; be.

On the other hand, cyanogen iodide has the disadvantage of being inferior to cyanogen bromide in its ability to immobilize enzymes. Cyanogen bromide is particularly preferred because the reaction operation is simple, the amount of enzyme immobilized is large, the immobilization power is strong, and the reaction active site of the enzyme is hardly masked. Also,
Polyfunctional aldehydes can also be used, for example glyoxal, malondialdehyde, succindialdehyde, glutaraldehyde, adipine dialdehyde,
Examples include maleic dialdehyde, which has a large amount of enzyme immobilized and has a strong immobilizing power, but glutaraldehyde is particularly preferred. In addition, hexamethylene diisocyaner. , isocyanates such as hexamethylene diisothiocyanate, benzidines such as bisdiazobenzidine, N,N
"Polymethylene biiodoacetamide, N,N" ethylene bismaleimide, synchrohexylcarbodiimide, Utsudowa. Code reagent K1, ethyl chloroformate, etc. can also be used as a fixative for the purpose of the present invention. However, compared to the above-mentioned cyanogen bromide and glutaraldehyde, these are considerably inferior in enzyme immobilization ability, and in particular have a drawback in that the rate of masking the reaction active site of the enzyme is high. To determine the fibrinolytic activity of urokinase immobilized on collagen, that is, its anticoagulant or antithrombotic properties, commercially available standard fibrin plates can be used without using animal blood or conducting animal experiments. It is convenient to measure the dissolution area using

Next, typical examples of the method according to the present invention will be specifically described.

Example 1 After thoroughly loosening 11.65f of collagen prepared from a cow Achilles key and adding 20e of water to it, (a) y/
20' of an aqueous solution of cyanogen bromide was added.

Then, a 2N Na0H aqueous solution was added dropwise, and PHll. O~1
1.3 and held for 8 minutes. After suction and filtration, the collagen was cooled with ice at 0.1M. Washed with pH 9.0 sodium carbonate buffer (400e). Suction again? After 2
0e of sodium carbonate buffer was added, followed by 10e of urokinase aqueous solution (480,000 (1) units), and the mixture was stirred in an ice water bath for 4 hours. After suctioning again, 0.1M,
．． It was suspended in Tris buffer 20e at pH 9.0. After standing for 3 days, the activity of urokinase was measured according to the following procedure.

That is, urokinase-fixed collagen 0.20
0.1M.m9 and 0.39T1t9 respectively. ．． P
0.1 ml of H7.4 Tris buffer and 1 tin unit/
Add 0.1 ml of plasminogen and incubate at 37°C.
After incubation for an hour, 6 μe of supernatant was added to each sample.
The sample was placed on a fibrin plate manufactured by Mochida Pharmaceutical Co., Ltd., and the dissolved area was measured after 3 hours. To obtain a calibration curve, a mixture of urokinase and plasminogen was used as a control. In this plate method, the vertical axis represents the dissolution area,
Since a linear relationship is established when the logarithm of the concentration of urokinase is plotted on the horizontal axis, the concentration of urokinase fixed in collagen can be determined from the control experiment. The results measured in this way are shown below. Looking at the above table, it can be seen that 1111 g of collagen immobilized with urokinase corresponds to urokinase at about 35 pupils/ml (equivalent titer), and collagen with extremely excellent anticoagulant properties was obtained.

Example 2 Same collagen &74 as used in Example 1
f sufficiently loosened, 0.1M. ．． It was suspended in an aqueous glutaraldehyde solution 20e with a concentration of 12% using a sodium borate buffer solution with a pH of 8.5, and the two powders were stirred at 0°C.

After suction offshore, 0.1M at 0℃. ．． Washing was performed for 5 minutes using 100e of boric acid buffer having a pH of 7.5. Add urokinase aqueous solution 2' (1920000 energy) to this, and
The mixture was stirred for 4 hours. Suction this thing and pass it off to 0.05M.
．． ．． It was washed with 100' of phosphate buffer at pH 7.0.

After suction and filtration, add 0.1 M 1 PH 8.0 Tris buffer 12
It was suspended at 0e. After standing for 3 days, the amount of urokinase was measured using the same method as in Example 1. As a result, it was found that 1Tng of collagen immobilized with urokinase corresponded to about 2 levels/ml of urokinase (equivalent titer).

Although this is inferior to that obtained in Example 1, it can be sufficiently used as a medical material. Example 3 To a solution in which cyanogen bromide 1k9 was dissolved in 20e of water, 70f of collagen obtained from a cow Achilles Ken (suspended in 10f of water) was added, and the pH was adjusted to 11.0 to 11.3.

The solution was added to a Buchner funnel, filtered with suction, and then washed with 20e of 0.1M aqueous sodium bicarbonate solution. Add to this an aqueous solution of Streftkinase 2e (10000000
After 3 hours, aspirate on a Büchner funnel and add 0.1 M Tris buffer (pH 7.
4) Washed with 10e. This was suspended in 10' Tris buffer, stored at 4°C, and washed the next day, the day after, and one week later with 10' Tris buffer on a Büchner funnel to remove nonspecifically adsorbed streftokinase. Removed. Before measuring the fibrinolytic capacity, aspirate it with a Bützfner funnel, weigh it while still wet, and add 1 force tin unit/m.
After mixing 0.1 ml of plasminogen and culturing at 4 rc for 1 hour, 10 μe of the supernatant was taken and placed in the holes of a fibrin plate manufactured by Mochida Pharmaceutical Co., Ltd. to examine the dissolution area. I got good results. 2 From the above results, it can be seen that 1 m9 of collagen immobilized with strepkinase corresponds to (equivalent titer) to the strepkinase in ml of the 1 bullet position.

Claims (1)

[Claims] 1. A method for producing an anticoagulant medical material, which comprises immobilizing enzymes having anticoagulant properties on collagen, singly or as a mixture thereof, in the presence of cyanogen bromide or glutaraldehyde.