JPS5951304B2 - antithrombotic material - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an antithrombotic material, and more particularly to an antithrombotic material using an ethylene-vinyl alcohol copolymer as a carrier.

Intrinsic blood coagulation that occurs when using artificial organs or catheters, etc., the initial reaction is initiated by the activation of coagulation factor factor II due to contact between blood and the surface of a foreign body, and the well-known cascade of activation of coagulation factors occurs. As a result of this process, Xa, thrombin, etc. are produced, and ultimately a fibrin network is formed.

The action of these activated coagulation factors is gradually inhibited by the inhibitory substance antitropin III in the blood, but it is well known that the rate of inhibition is significantly accelerated by the coexistence of heparin.

Therefore, when there is clinical concern about blood coagulation, it has become a common practice to administer heparin.

However, when using artificial organs such as a heart-lung machine or an artificial kidney, a large amount of heparin must be administered, but in such cases, there are many side effects such as a marked tendency for bleeding; It becomes more pronounced with long-term use of the organ.

Therefore, in recent years, heparin has been immobilized on a suitable water-insoluble polymer and blood is brought into contact with it.
A method has been proposed for preventing blood coagulation by keeping heparin in a state where it does not mix with the blood and allowing it to capture thrombin and the like in the blood.

However, materials that only have heparin immobilized do not have satisfactory anticoagulant properties.

As a result of extensive research, the present inventors have discovered that a material in which heparin and antitropin III are immobilized in a coexisting state on a carrier exhibits a significantly high anticoagulant effect.

In other words, when heparin is immobilized, for example, when it is immobilized simultaneously with an antibody as a complex while maintaining the coexistence state with anti-1-herobin III, it is much more effective than when heparin or antithrombin III is immobilized alone. It has been found that it exhibits an excellent anticoagulant effect (see Japanese Patent Application Laid-Open No. 54-244785).

It is known that heparin and antithrombin III cooperate to exert an anticoagulant effect.

Therefore, even if only heparin is immobilized, when blood is brought into contact with it, it is thought that the antithrombin III present in it naturally cooperates with the immobilized heparin to exert an anticoagulant effect. It was getting worse.

The above-mentioned findings of the present inventors are contrary to such conventional thinking, and show that the anticoagulant effect can be effectively exerted by immobilizing the two while maintaining their coexistence.

By the way, when developing an antithrombotic material, it becomes possible to provide a highly practical material only when the following conditions are met.

In other words, (1) the antibody should have excellent antithrombotic function, (2) the carrier material should have good blood compatibility, and (3) the antibody should have excellent chemical stability and mechanical properties. This is clear from the above findings.

Also, a statement similar to this is published in the 7th Journal of the Society for Artificial Organs.
Volume 1, No. 193, 1978 and Japanese Patent Publication No. 54-2447
This is published in Publication No. 8.

However, the materials used as carriers in these documents include agarose, polyacrylamide, Textlan, cellulose, polyvinyl alcohol, and polyhydroxyethyl methacrylate-1, and all of them are water-soluble or water-swellable substances. However, the mechanical strength and chemical stability are not satisfactory.

Therefore, the present inventors conducted further research to find a material that satisfies conditions (2) and (3).

Polymer materials conventionally used as antithrombotic materials for medical equipment include polyethylene, polypropylene, polytetrafluoroethylene, natural rubber,
Hydrophobic polymers such as synthetic rubber and hydrophilic polymers such as polyvinyl alcohol, polyhydroxyethyl methacrylate, and polyacrylamide are well known.

Hydrophilic polymers have poor mechanical strength, and methods of coating hydrophilic polymers on materials made of natural rubber or synthetic rubber are also used, but there are problems with adhesion to the materials, and eventually It is also insufficient as an antithrombotic material.

On the other hand, with hydrophobic polymers, there remains a difficulty in immobilizing heparin and antithrombin III, which are essential in the present invention, in coexistence.

However, ethylene-vinyl alcohol copolymer (hereinafter abbreviated as EVOH) is a material with excellent blood compatibility, chemical stability, and mechanical strength, and is difficult to immobilize anticoagulants. It has been found that this material can be easily immobilized using ordinary protein immobilization techniques and is extremely excellent as a carrier for antithrombotic materials.

The present invention was completed based on this knowledge, and
The gist thereof lies in an antithrombotic material in which heparin and antithrombin III are immobilized by the cyanogen bromide method, in which an ethylene-vinyl alcohol copolymer is used as the immobilizing carrier.

The EVOH used in the present invention can have a composition ratio of ethylene and vinyl alcohol that can be changed depending on the form of use as an antithrombotic material, and usually has an ethylene content of 10 to 5.
A range of 0 mol% is preferred.

When the ethylene content exceeds 50 mol%, the affinity for blood decreases, resulting in poor blood compatibility.

Furthermore, when the ethylene content is less than 10 mol%, the mechanical strength decreases.

EVOH can be formed into a film by dissolving it in an alcohol/water mixed solvent and immersing it in cold water, and the surface becomes porous, so the surface area becomes extremely large.

Therefore, it is extremely advantageous for immobilizing heparin and antithrombin III.

When EVOH is activated and used, activation is most easily carried out by the action of cyanogen bromide, but the method is not limited to this.

As a method for immobilizing heparin and antithrombin III on EVOH, a conventional protein immobilization method can be adopted [Japan Biochemical Research Institute, "Biochemistry Experiment Course 5 Enzyme Research Methods (Part 2)", Volume VI (1975) and references cited therein].

There are no particular restrictions on the amounts of heparin and anti-I/kutonbin III that should be immobilized on EVOH, but usually EVOH
By immobilizing 1 to 50 μg of heparin and 1 to 50 μg of antithrombin III per cm 2 of area, an artificial medical material exhibiting good anticoagulant effect can be obtained.

Although there is no particular restriction on the fixed ratio of heparin and antithrombin III, it is generally appropriate to keep it in the range of 1:1 to 10:1 (weight ratio).

Although heparin and antithrombin III may be immobilized separately, it is easier and more advantageous to immobilize them at the same time.

The antithrombotic material of the present invention can be prepared in an appropriate form such as powder, sheet, or tube depending on the purpose of use, and when it is brought into contact with blood, thrombin and other substances are easily captured and the blood is absorbed. Coagulation is prevented.

The material in which thrombin has been captured can be washed with, for example, an acetic acid aqueous solution to remove thrombin, and as a result, the thrombin-capturing function is reactivated.

Next, the present invention will be specifically explained with reference to Reference Examples and Examples.

Reference Example I Hemolytic Nee of EVOH Fill a test tube with a 2% solution of EVOH (vinyl alcohol content 70 mol%) in dimethyl sulfoxide (DMSO), drain the excess EVOH solution, and then remove the solvent by evaporation at 150°C. to form an EVOH coating on the inner wall of the test tube.

Repeat this operation three times to obtain a sample.

The EVOH-coated test tubes, glass test tubes, and polyethylene test tubes thus obtained were filled with citrated blood (a 9:1 mixture of blood and an isotonic solution containing 3.08% sodium citrate and sodium chloride). Add 1 ml of each mixture (volume ratio) mixture, and shake back and forth 75 times/min at 37°C.

After shaking for 3 or 6 hours, centrifuge at 3,000 rpm for 15 minutes, add 200 μm of supernatant plasma to 3 ml of coloring reagent for hemoglobin measurement (manufactured by Wako Pure Chemical Industries), leave at room temperature for 10 minutes, and measure the absorbance at 541 nm. The amount of free hemoglobin in plasma was quantified by measuring , and the degree of hemolysis was investigated.

Cyan in the coloring test solution combines with hemoglobin in red blood cells to form cyan-bonded hemoglobin, which absorbs at 541 nm.

Calculate the amount of free hemolysis from the absorbance using the following conversion formula, and use the degree of hemolysis as a guide.

Free hemoglobin amount (mg/di) =16X146.5 (absorbance) The results are shown in Table 1.

From this result, it is understood that EVOH is a material with little hemolytic effect.

Reference Example 2 Blood Compatibility of EVOH Blood was collected from the vein of a knee rabbit using a syringe, and mixed with physiological isotonic saline containing 0.308% sodium citrate at a ratio of 9:1.
(volume ratio).

1 ml each of this blood mixture was transferred to (1) a glass test tube, (
2) Polyethylene test tube, (3) silicone coated test tube (KC-88 manufactured by Shin-Etsu Chemical Co., Ltd.), and (4) EVO
Add to H (vinyl alcohol content 70 mol%) coated test tube and shake at 25°C.

100 μl are taken at given time intervals and added to 200 μl of isotonic saline containing phospholipids (0.1% by weight) and 25 mM calcium ions.

The time from the time of addition until blood coagulation occurred was measured and defined as the coagulation time.

Clotting times were determined by the Baltimore Biochemical Laboratory (Baltimore Biochemical Laboratory).
Fibrometer 60H manufactured by Boratory (USA)
z# 60415).

The results are shown in Table 2.

Example IA Add isopropanol-water mixed solvent (6:4) to a Separate flask equipped with a cooling device, stir at 70-80°C, and then dissolve EVOH (vinyl alcohol content 70%) to make a 20% by weight solution. get.

The resulting solution is cast onto a glass plate and immersed in water at 0°C to obtain a porous film with a thickness of 50μ.

B: Suspend about 1 g (dry weight) of EVOH cut into pieces about 1 mm wide in 100 ml of water, and add 5-1 ON thorium hydroxide solution while stirring to maintain the temperature at 11-12.

Add cyanogen bromide solution (4-24g 780-480m
While adding 1), add 5-ION sodium hydroxide solution to keep the volume at 11-12, and continue until no drop in consistency is observed.

During this time, the liquid temperature is maintained at around 20°C by adding ice.

The reaction is completed in 8-12 minutes.

After the reaction is completed, it is immediately filtered through a glass filter, and excess cyanogen bromide is washed away with cold water 11 to obtain activated EVOH.

1 g of C-activated EVOH is suspended in 5 ml of 0.1 M sodium bicarbonate solution, and a solution of 100 mg of heparin and 20 mg of antithrombin III dissolved in 10 ml of 0.1 M sodium bicarbonate solution is added thereto, and the mixture is stirred overnight at 4°C.

To block excess active groups on activated agarose, 1
After adding 10 ml of M ethanolamine solution (pH 8,0) and stirring at 4°C for 1 hour, the mixture was washed with a 2M sodium chloride solution and a 0.15M thorium chloride solution in sequence, and heparin and antithrombin III immobilization material (hereinafter referred to as I
-ATIII-HEP for short.

). This material was formulated with 3 mg heparin and 8 mg antithrombin III per gram of carrier.

By the same method, 6 heparin per 1 g of activated EVOH was added.
Heparin-immobilized material (hereinafter referred to as I-H
EP) and antithrombin III immobilized material (hereinafter abbreviated as I-ATIII) in which 116 mg of antithrombin was immobilized per 1 g of activated EVOH were obtained.

However, since antithrombin III does not have an anticoagulant effect unless it is fixed under the protective effect of heparin, when preparing the latter material, 8 times the amount of acetylated heparin relative to antithrombin III should be added to the reaction system. By allowing anti-I and rhombin III to coexist, the activities of anti-I and rhombin III are protected, and only these are immobilized on the activated EVOH.

In addition, activated EVOH is treated as above without the addition of heparin and antitropin and serves as a control.

D Bovine citrated plasma (a 9:1 (volume ratio) mixture of blood and 3.8% thorium citrate solution was added to 300 Orp.
0.2 ml of supernatant obtained by centrifugation at m for 15 minutes
Place in a small glass test tube and warm to 37°C.

Add a certain amount of immobilization material to this and immediately add 0.2 ml of 1740 M calcium chloride solution and 0.0 ml of physiological isotonic saline.
．． Add 5 ml, shake gently in a 37°C constant temperature bath,
The time taken for plasma to coagulate was measured.

The results are shown in Table 3. Example 2 I-AT III-HEP prepared in Example 1, ■
-ATIII and ■-HEP each 10m each sound 0mg, 0
．． Add to 0.2 ml of 15M 1-lium chloride solution.

0.1 ml of 0.15 M sodium chloride solution for the I-ATIII-HEP suspension, 0.1 ml of heparin solution (165 units/ml) for the I-ATIII suspension, I
- Antithrombin III solution (1 m
g/ml) o, 1 ml each, and to this, 0.2 ml of thrombin solution (0.1 ml of which shows activity with a clotting time of 10 seconds in a clotting time measuring system) was added, and 3
Shake for 30 minutes in a constant temperature bath at 7°C.

Next, 0.1 ml of supernatant containing residual L. rhombin
Acacia solution (15% acacia saline, 1% calcium chloride saline, imidazole buffer and physiological saline 2:1:1: 5
(Volume ratio mixture) 0.2 ml and stirred,
Leave it in a constant temperature bath at 28°C for 3 minutes.

To this was added a standard fibrinogen solution (commercially available fibrinogen was dissolved in 0.15M sodium chloride solution, adjusted to 0.5M sodium hydrogen phosphate solution, pH 7.2,
1% solution with 15M sodium chloride solution)0,
The remaining thrombin activity in the supernatant was determined by adding 1 ml of the supernatant and measuring the clotting time.

In addition, activated EVOH on which heparin and antitropin III are not immobilized (active group masked with albumin) is used as Control I Roll, and this is
Antithrombin III solution (1 mg/ml) suspended in 0.2 ml of 1M chloride solution
1 ml was added, and the same operation as above was performed.

The results are shown in Table 4.

From this result, it is understood that a system in which both heparin and antithrombin III are immobilized exhibits a significantly stronger anticoagulant effect than a system in which either one is immobilized and the other is used in solution. .

In addition, in the above-mentioned coagulation test, when the anticoagulant effect of the immobilized material was compared without using heparin and antithrombin III solutions, that is, I-ATI
A certain amount of each of II-HEP, I-ATIII and ■-HEP was added to the thrombin solution and incubated at 37°C for 20
After shaking for a minute, the residual thrombin activity in the supernatant was compared according to the clotting time. Assuming that the residual l-thrombin activity in the control was 100%, [when using HEP, the residual thrombin activity was 55%] Under the conditions, -ATIII had a residual thrombin activity of 48%;
■-AT■■I-HEP showed residual thrombin activity of 3%.

In addition, I-ATP-HEP did not show any decrease in anti-thrombin activity even after being stored in a constant temperature bath at 37°C for 4 weeks.
Maximum activity was reached in 30 seconds in reaction with thrombin solution.

Example 3 A Vinyl alcohol content is 90.70.50.40
and 30 mol% of EVOH or polyethylene was coated on the inner wall of a test tube with an inner diameter of 12 mm, and after activation with cyanogen bromide in the same manner as in Example 1, antithrombin I
1 mg of II and 3 mg of heparin dissolved in 5 ml of 0.1 M sodium bicarbonate solution are added.

This was left at 4°C overnight, and antithrombin lll-
Use it as a heparin complex immobilized sample.

Since polyethylene does not have a hydroxyl group, antithrombin III and heparin cannot be immobilized at the same time, but the same procedure is performed as a control.

B Add 0.1 ml of 0.15 M thorium chloride solution and Doulombin solution (0.1 ml of which shows activity with a clotting time of 10 seconds in the clotting time measuring system) to each test tube, and place in a constant temperature bath at 37°C. Shake for 30 minutes and perform the reaction.

Take 0.1 ml of supernatant containing residual 1 to rhombin,
It is added to 0.2 ml of acacia liquid heated to 28°C, stirred, and then left to stand in a constant temperature bath at 28°C for 3 minutes.

0.1 ml of standard fibrinogen solution was added to this, and the clotting time was measured to determine the residual l.thrombin activity in the supernatant.

The results showed that the residual thrombin activity in the control was reduced to 1
It is shown in Table 5 as 00%.

Claims (1)

[Scope of Claims] 1. An antithrombotic material in which heparin and antitropin III are immobilized by a cyanogen bromide method, characterized in that an ethylene-vinyl alcohol copolymer is used as the immobilizing carrier. 2. The antithrombotic material according to claim 1, wherein the ethylene-vinyl alcohol copolymer contains 10 to 50 mol% of ethylene moiety. 3 Hehan and antitropin III at 1:1-10
The antithrombotic material according to claim 1, wherein the antithrombotic material is regulated at a ratio of :1 (weight ratio). 4. The antithrombotic material according to claim 1, wherein 1 to 50 μg of heparin and 1 to 50 μg of antitropin III are immobilized per cm2 of carrier area. 5. The antithrombotic material according to claim 1 or 2, wherein the carrier is activated prior to immobilization.