JPS6022943B2 - Antithrombotic artificial medical materials - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an antithrombotic artificial medical material, and more particularly to a blood-compatible antithrombotic artificial medical material in which an anticoagulant is bound to a silicone coating material or a silicone resin.

As is well known, the normal flow of blood in a living body is essential for maintaining life.

Therefore, preventing blood coagulation during surgical operations and blood coagulation caused by contact between blood and various artificial medical materials, including artificial organs, has become an important medical topic. In surgical operations, large doses of heparin have been used to inhibit the action of trompin on fibrinogen and prevent blood coagulation. Bleeding causes side effects that can lead to death, such as mesenteric hemorrhage.

Therefore, in recent years, heparin has been immobilized on a suitable water-insoluble polymer compound, and blood is brought into contact with the heparin, thereby keeping the heparin from being mixed into the blood and allowing it to capture tropine. Methods have been proposed to prevent blood clotting. In the field of artificial medical materials,
Attempts have been made to immobilize anticoagulants on materials to impart antithrombotic properties.

Furthermore, materials with excellent blood compatibility, that is, materials that do not recognize foreign substances in blood and suppress thrombus formation, have been developed. However, in the method of immobilizing an anticoagulant on a material, the types of functional groups that serve as binding sites are limited depending on the material, and therefore the immobilization methods that can be used are limited. In addition, many materials suffer from the disadvantage that they do not have sufficient functional groups to provide sufficient antithrombotic properties.
On the other hand, if an artificial medical material without immobilized anticoagulants is used, if the coagulation system is activated for some reason and the level of coagulation in the blood increases, it will no longer be effective. Even if a material has sufficient blood compatibility, it must have the necessary strength and flexibility to be used as an artificial organ or artificial medical material, and it is difficult to select a material that can be used. . The present inventors have conducted repeated research to develop an antithrombotic artificial medical material that has the necessary physical properties as an artificial medical material and has excellent blood compatibility. They found that an artificial medical material on which heparin and antithrombin M are immobilized satisfies these properties, and completed the present invention. Silicone has excellent blood compatibility, as it has traditionally been used to coat instruments used in experiments and clinical tests that involve blood.

Moreover, silicone can be applied to the surface of various substances and easily formed into a coating by heat treatment or by curing with moisture in the air at room temperature. In the present invention, any type of silicone can be used as long as it can immobilize heparin-antithrombin m, such as silicone represented by the formula:

Simultaneous immobilization of heparin and antithrombin m as anticoagulants has superior antithrombotic properties than when either one is used alone, and even if the carrier material used for immobilization has a small number of functional groups, it is sufficient. It can impart strong antithrombotic properties.

Therefore, even when using silicone with a small number of functional groups, it is possible to impart remarkable antithrombotic properties, further expanding the range of selection. Heparin-antithrombin m can be immobilized on the silicone surface by applying ordinary protein immobilization techniques [Published by the Japanese Biochemical Society,
See “Biochemistry Experiment Course 5 Enzyme Research Methods (Part 2)” Part W: Immobilized Enzymes (1973 King) and the cited references therein]
. Depending on the type of silicone, various functional groups such as hydroxyl groups, carbonyl groups, amino groups, etc. can be introduced, and any immobilization method such as covalent bonding or ionic bonding can be employed. Depending on the immobilization method employed, the carrier material may need to be activated before its use. Various conventional methods are known for this purpose, and an appropriate method can be selected from them. For example, a method in which cyanogen bromide is used is one example. Although there is no particular restriction on the ratio of heparin and antithrombin to be immobilized on the carrier material, good results can be obtained by immobilizing antithrombin and heparin at a ratio of 1:1 to 1:10 (weight ratio).

According to the present invention, since the surface of the material is coated with silicone having blood compatibility, a substance that is not blood compatible or has weak compatibility can be effectively used as an artificial medical material.

Therefore, the range that can be selected as an artificial organ or artificial medical material is expanded, and it is possible to provide an artificial medical material that has more appropriate physical properties (strength, flexibility, etc.) and is easier to administer. For example, polymeric materials such as nylon and vinyl chloride can also be used as carriers for the antithrombotic artificial medical material of the present invention. For example, in the case of using a vinyl chloride tube as the material, a silicone coating 2 is formed on the inner wall of the vinyl chloride tube 1, and a layer of heparin and antithrombin m is formed on the surface of the coating 2, as shown in the figure. 3 can be immobilized to produce the antithrombotic artificial medical material of the present invention.

Further, as shown in Fig. 2, a silicone coating 2 is formed on the surface of the rod-shaped material wing and used as a carrier to immobilize heparin and antithrombin m. It is schematically shown in Figure 3.

Heparin (HEP) and antithrombin m (AT) are
It coexists and is immobilized on the surface of the silicone coating 2 coated on the damage body material 1'. Next, the present invention will be explained in more detail with reference to Examples.

Example 1 This example shows that proteins can be effectively immobilized on silicone coatings.

■ Clean the nylon polymer mesh with water and dioxane and leave it to dry indoors.

Silicone x-22-052 and formula: CH3Si(OCO
A silicone solution is prepared by mixing a crosslinking agent represented by CH3)3 at a ratio of 100:5 (weight ratio) with 2 to 5 times the volume of dioxane. After soaking the dried mesh in the silicone solution, let it air dry for about 30 minutes, and heat each powder at 10°C to complete curing.
．． After washing with 1M sodium bicarbonate buffer (pHil. 0), cut into approximately 3 x 10 pieces. The buffer solution 30
Place as much of the cut mesh as possible in a 0M solution, and add cyanogen bromide to pH 10 to 15 minutes with a 200% sodium hydroxide solution (immediately before addition) while stirring.
(adjusted to 0), then 2. Add Cape Sodium Hydroxide solution and pHil. While adjusting the temperature to 0 to 11.5, perform the cascade for 5 to 7 minutes. Remove the mesh from the liquid and wash with 0.1M sodium bicarbonate at 4°C. Immediately place the mesh activated by step [C} and [B} in 40°C of 0.1M sodium bicarbonate at 4°C, and add thrombin 100% IH to this.
Add unit.

Incubate at low temperature (400℃) for 2 hours to immobilize thrombin. After this, excess thrombin and adsorbed thrombin are removed by washing with 2M sodium chloride solution (400) and water (400). The silicone-coated mesh with immobilized thrombin is stored at 4°C in 0.1 sodium chloride solution. ■ The activity of immobilized thrombin was determined by the method of Morita et al.
The Journal of Biochemistry (J.
Biochem) No. 82, page 1495, 1977)
Measure as follows.

That is, 0.05 mmol of the compound of formula: 100 mmol of sodium chloride and 10 mmol of calcium chloride
A 50 mmol Tris-HCl buffer solution (pH = 8.0) containing 1 mmol of thrombin was prepared and the solution was kept at about 2600.
4. Add 1 and worship the rope.

At each hour, 1.0% of the reaction solution was sampled, and 1.0% of the reaction solution was collected with 1.0% of 17% acetic acid.
Add [5]. Then, the compound [1
] is decomposed to produce a compound represented by the formula: (lower AMC
The crab light intensity was measured using a field photometer under the conditions of excitation at 38 m and crab light at 46 m.

The amount of AMC produced was used as a measure of thrombisoactivity, and the results are shown in Table 1. Table 1 Examples 2 and 3 In this example, a silicone coating was formed on a nylon polymer mesh and a vinyl chloride tube, and as in Example 1, it was shown that proteins were immobilized.

Wind Polymer mesh and PVC pipe (inner diameter 0.6
Fox, length 30 arcs) were washed with water and dioxane and dried at room temperature. Silicone Prepare a silicone solution by diluting with

After soaking the dried polymer mesh and PVC pipe in this solution, air dry for 30-60 minutes, then
Heat for 30 minutes at noon to harden. After cooling at room temperature,
0.40℃ Hydrolyze by soaking in hydrochloric acid and stirring for 10 to 30 minutes. The protective group for the hydroxyl group of silicone is removed to form a hydroxyl group that can be used for immobilization. After this, wash with water and dry. The polymer mesh is activated by a procedure similar to that of Example 1.

The vinyl chloride pipe is activated by flowing the same cyanogen bromide solution as in Example 1'B}. Thrombin was immobilized using the same procedure as in Example 1 for the C1 polymer mesh. PVC tube is thrombin 100 skin IHunit 0.1
A solution of 40 M sodium bicarbonate is refluxed and immobilized. Breast Thrombin-immobilized mesh 11.99 was added to Compound [1] in a 0.1 mmol buffer solution (prepared in the same manner as in Example 1).

After reacting with 4' and 270, thrombin activity was measured by the method described in Example 1 (2).

The results are shown in Table 2. 2 In the case of a vinyl chloride pipe, after flowing 25.7 μl of a buffer solution containing 0.075 mmol of compound [1] (prepared in the same manner as in Example 1) at a flow rate of 3.85'/min, Thrombin activity was measured by the method described in .

The results are shown in Table 3. The average value of the AMC production rate in Examples 1 to 3 in Table 3 is
Shown in the table.

The results in Table 4 and above show that proteins can be effectively immobilized on the silicone coating, and it is understood that silicone is effective as an immobilization material.

Example 4 Heparin and antithrombin m (approximately 1:1 to 1:10, weight ratio) are immobilized by the procedure of Example IC to a silicone-coated nylon mesh prepared by the procedures of Example 1 A and B. However, hevarin and antithrombin m are used instead of thrombin).

Immobilized silicone coated nylon mesh 8.4 flow of heparin and antithrombin M
Hunit's Tris-hydrochloric acid 50 mmol "Sodium chloride 100 mmol and calcium chloride 10 mmol solution (pH 8.0) was immersed in it and reacted at 30 ° C. The residual thrombin activity in the solution was measured as follows. did.

In other words, "Collect each Q.05 secretion of the reaction solution every hour,
This was added to 1.0% of a 0.1 mmol buffer solution of compound [1] overflowed to 370°C, and after shaking at 370°C for 10 minutes, 1.5% of 17% acetic acid was added to stop the reaction. The thrombin activity of this solution was measured by measuring the light intensity (excitation: 38 m, fluorescence: 46 m) using a light photometer. The results are shown in Table 5. 5

[Brief explanation of drawings]

FIG. 1 is a cross-sectional view of an example of the antithrombotic artificial medical material of the present invention using a vinyl chloride tube, where 1 shows the vinyl chloride tube, 2 shows a silicone coating, and 3 shows a layer of heparin and antithrombin m. . FIG. 2 is a cross-sectional view of an example of the antithrombotic artificial medical material of the present invention using a rod-shaped material, where 1' indicates the material, 2 indicates the silicone coating, and 3 indicates the heparin and antithrombin m layers.
FIG. 3 is a diagram schematically showing the fixed I-Q state of heparin and antithrombin m, where 1' represents material 2 coated with silicone, HEP represents heparin, and AT represents antithrombin m. Figure 1 Figure 2 Figure 3

Claims (1)

[Claims] 1 Heparin and antithrombin on a silicone carrier
An antithrombotic artificial medical material characterized by immobilizing III. 2. The antithrombotic artificial medical material according to claim 1, wherein the silicone-based carrier is a silicone resin. 3. The antithrombotic artificial medical material according to claim 1, wherein the silicone-based carrier is a carrier material coated with silicone.