JPS6040862B2 - antithrombotic material - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an antithrombotic material, and more specifically, it immobilizes albumin on the surface of the material to suppress the adhesion of platelets, and also immobilizes the albumin with an anticoagulant and/or a fibrinolytic system activating enzyme. The present invention relates to antithrombotic materials that are immobilized to reduce blood coagulation activity and thus synergistically enhance antithrombotic properties.

Intrinsic blood coagulation that occurs when using artificial organs or catheters, etc., the initial reaction is initiated by the activation of coagulation system factors due to contact between blood and the surface of a foreign body, and as a result of the cascade activation of coagulation system factors, Xa and thrombin are generated,
The final result is the formation of a fibrin network.

The action of these activated coagulation system factors is gradually inhibited by the inhibitory substance antithrombin m in the blood, but the coexistence of heparin significantly enhances the inhibitory effect.

Therefore, when there is clinical concern about blood coagulation, it is common practice to administer heparin. For this reason, when using artificial organs such as an artificial heart arm or an artificial kidney, large amounts of heparin must be administered, but in such cases, there are many side effects such as a marked tendency for bleeding. , these side effects become even more significant with long-term use of artificial organs. Therefore, in recent years, heparin has been immobilized on a suitable water-insoluble polymer carrier and blood is brought into contact with the carrier to keep heparin from being mixed into the blood while also removing thrombin, etc. in the blood. A method of trapping blood to prevent blood coagulation has been proposed.

However, only immobilizing heparin on a carrier does not exhibit satisfactory anticoagulant properties. For this reason, the present inventors first immobilized heparin and antithrombin m when simultaneously immobilizing them on a carrier while maintaining a coexistence state. It has been discovered that the anticoagulant effect is much superior to that of the anticoagulant (Tsubaki Seki Sho No. 54-24478).

In this way, a material in which heparin and antithrombin M are immobilized while maintaining coexistence can act on the blood coagulation factors Xa and thrombin, inactivate them, and exhibit excellent pore coagulation effects. However, since immobilized heparin antithrombin m acts stoichiometrically with these blood coagulation factors, the anticoagulant effect of this material is finite, and therefore the binding with a corresponding amount of activated coagulation factors is limited. There still remains a problem in that it does not have a blood coagulation limiting effect. On the other hand, carriers for immobilizing anticoagulants have traditionally been made of hydrophobic polymers such as polyethylene, polypropylene, ethylene-vinyl alcohol copolymers, polytetrafluoroethylene, natural rubber, synthetic rubber, polyvinyl alcohol, Hydrophilic polymers such as polyacrylamide gel are well known, but ``hydrophobic polymers generally have poor blood compatibility required for antithrombotic materials, and hydrophilic polymers tend to have poor mechanical strength. In addition, immobilization of anticoagulants generally requires complex operations.

However, because blood tubes and artificial organs in particular require high mechanical strength, ``hydrophobic polymers are often used as carriers in the past. Since there are generally few functional groups for immobilizing blood substances, it is difficult to immobilize an amount of anticoagulant sufficient to obtain a sufficient anticoagulant effect. As a result of intensive research to determine the various problems mentioned above in conventional antithrombotic materials, it was found that if albumin is immobilized on the material, a large amount of anticoagulant can be produced by utilizing the many functional groups of albumin. We have discovered that not only can albumin be easily immobilized, but also that the immobilized albumin suppresses the adhesion of platelets to the material, and in combination, it is possible to obtain an excellent antithrombotic material. It is something that

Therefore, the antithrombotic material according to the present invention is characterized in that an anticoagulant and/or a fibrinolytic eel activating enzyme are immobilized on albumin immobilized on the surface of the material.

In the present invention, both hydrophobic polymers and hydrophilic polymers as described above can be used as materials, and in addition, they are substances that do not have a substantial harmful effect on living organisms, and albumin is immobilized. Any material can be used.

Therefore, examples of polymers that can be used in the present invention include polyethylene, polypropylene, polyisobutylene, polymethylbentene, polystyrene, natural rubber, chloroprene rubber, polyvinyl chloride, polyvinyl fluoride, polytetrafluoroethylene, polychlorinated vinylidene, polyvinyl acetate, polyvinyl alcohol, ethylene-vinyl alcohol copolymer, polyacrylonitrile,
Polyglycolic acid, polyvinylpyrrolidone, polyvinyl formal, polyvinyl butyral, acetal resin,
Examples of acrylic resin, polyacrylamide, polycarbonate, polysulfone, polyethylene terephthalate, polyethylene naphthalate polyamide, and polyimide include cellulose, nitrocellulose, cellophane, collagen, gelatin, and polysaccharides. The method of immobilizing albumin onto the material is not limited at all, and any conventionally known protein immobilization method can be used.

Albumin forms a secondary carrier for immobilizing anticoagulant substances and/or fibrinolytic system activating enzymes, and "albumin itself does not have any anticoagulant activity, so the immobilization method and conditions are A desired amount of albumin can be immobilized by selecting any of these methods. Preferably, the immobilization is performed by a covalent bonding method. An anticoagulant and/or a fibrinolytic immobilizing enzyme is immobilized using a large number of carboxyl groups and amino groups.The anticoagulant and fibrinolytic immobilizing enzyme to be used are not particularly limited, but preferably Heparin and antithrombin plates are immobilized in coexistence as anticoagulants, and urokinase and streptokinase are immobilized as the green lytic-immobilized enzymes. Particularly preferably, heparin, antithrombin m, and urokinase are immobilized. According to such a system, the coagulation inhibiting function by heparin antithrombin m and the fibrinolytic function by fibrinolytic system activating enzyme have different mechanisms of action. However, compared to the immobilization of both systems separately, the antithrombotic effect is significantly prolonged.In the present invention, anticoagulant and anticoagulant system activating enzyme are added to the material. The bonding method is not particularly limited, and appropriate methods such as "covalent bonding method" and "ionic bonding method" may be used.

For example, if the above-mentioned substance is reacted with a material together with a carbodimide reagent or a Woodward reagent, it can be easily immobilized by a covalent bond, but it is not limited to these methods. Furthermore, in order to ionicly bond the above-mentioned substance to the material, the material may be immersed in an aqueous solution of the above-mentioned substance. The antithrombin melon used in the present invention is mainly derived from cows, but may also be derived from other sources such as dogs and rabbits.

In addition, fibrinolytic system activating enzymes are mainly derived from humans. Kinase is used, but streptokinase may also be used. There are no particular restrictions on the amounts of heparin, antithrombin m, and fibrinolytic system activating enzyme to be immobilized on the material, but usually 0.1 to 100 heparin and 0.1 to 100 antithrombin blood per material area. 100 Butta and line lacquer-based activating enzyme 0.1
By immobilizing the material from 2004 to 2004, an artificial medical material exhibiting good anticoagulant effect can be obtained.

There are no particular limitations on the immobilization ratio of heparin-antithrombin m. and fibrinolytic system activating enzyme. Although heparin-antithrombin m and fibrinolytic system activating enzyme may be immobilized separately, it is easier and more advantageous to immobilize them simultaneously. The antithrombotic material according to the present invention can be prepared in any form, such as particulate, sheet, or tubular, by forming the material into a desired shape in advance depending on the purpose of use, and by contacting it with blood. , thrombin, etc. are easily captured, and even a small amount of blood clots can be re-dissolved by the activation of the paulytic system such as urokinase, which prevents blood coagulation and restores normal blood flow. can get.

The thrombin-capturing function of the material that has captured thrombin can be reactivated by washing it with, for example, an IN acetic acid solution. As described above, the antithrombotic material of the present invention has an anticoagulant and/or a fibrinolytic competitive enzyme immobilized on a material on which albumin is immobilized.

That is, since many functional groups of albumin are utilized, anticoagulants and/or fibrinolytic enzymes can be easily immobilized in large amounts under quiet conditions, and the albumin has high antithrombotic properties. materials can be obtained. In particular, ``Even if the material itself has only a few functional groups, albumin is used as a secondary carrier, so a material with high antithrombotic properties can be obtained regardless of the type of material. When immobilizing, there is no need to consider the stability of the enzyme, and the immobilization conditions can be strict, so a large amount of albumin can be immobilized.Also, for example, regenerated cellulose or ethylene-vinyl alcohol Copolymers have many hydroxyl groups as functional groups, but even if the hydroxyl groups are activated with cyanogen bromide, it is difficult to immobilize a sufficient amount of anticoagulant or fibrinolytic activating enzyme. However, according to the present invention, even in the case of these materials, a large amount of albumin can be immobilized due to the functional group of albumin.Since the albumin immobilization material has a large number of carpoxyl groups and amino/groups, it has a large amount of immobilization. Another great advantage is that when immobilizing blood clotting substances and fibrinolytic system activating enzymes, it is possible to immobilize them in large quantities while maintaining their high activity under extremely indeterminate conditions. In antithrombotic materials, albumin immobilized on the material has the effect of suppressing the adhesion of platelets to the material. It synergistically exhibits high antithrombotic properties in conjunction with the effects of anticoagulants.

In particular, as described above, if an anticoagulant and a fibrinolytic competitive activating enzyme are immobilized in coexistence, thrombus formation is significantly suppressed. The present invention will be explained below with reference to Examples, but the present invention is not limited to these Examples in any way.

Example 1 (Immobilization of urokinase on a polyamide carrier) ○} Immobilization {a} Immobilization of polyamide albumin An aromatic polyamide tube (inner diameter 1 side L outer diameter 2 willow) was cut into approximately 5 pieces in length, After washing with water, it was immersed in an IN hydrochloric acid/methanol solution at a temperature of 30 qo for 30 minutes, and was partially hydrolyzed and activated.

After washing with water, soaking for 15 minutes in two acetate buffers (5 hM, PH 4.8) containing 10 mmol of ethyldimethylaminopropylcarbodiimide hydrochloride (EDC);
After cooling, 25M of the same acetate buffer as above containing albumin at a concentration of 2% by weight was added. After adjusting the pH to 4.8 by sequentially adding 0.1N hydrochloric acid or an aqueous sodium hydroxide solution while holding a candle at room temperature, the mixture was left overnight to immobilize albumin on the polyamide. After this, water, 2M sodium chloride aqueous solution, dish hM Tris-HCl buffer (pH 9)
．． 4). 'b} Immobilization of urokinase onto polyamide The polyamide activated in the same manner as above was placed in an acetate buffer (pH 4) containing 10 mmol of EDC.
．． 8) After soaking in 25' and boiling for 15 minutes, add 25 ml of the same potassium phosphate buffer as above containing 50 units of urokinase, and adjust the pH to the same as 'a'. Urokinase was immobilized directly onto the polyamide by overnight concentration.

This was followed by washing with water and a 2M aqueous sodium chloride solution. 'c- Immobilization of urokinase onto albumin-immobilized polyamide Urokinase was immobilized on the albumin-immobilized polyamide obtained in {a) in exactly the same manner as {b}.

(2' Measurement of immobilized protein amount Each sample obtained as above was immersed in a crucian carp hydrochloric acid aqueous solution,
Hydrolysis was carried out by flooding for 2 hours at a temperature of 5.5°C.

Proteins in the supernatant liquid were quantified according to Yamawhira's method. As shown in Table 1, by immobilizing urokinase on albumin-immobilized polyamide, the amount of urokinase immobilized can be significantly increased compared to when urokinase is immobilized directly on polyamide. Table 1 {3} Measurement of enzyme activity Next, the activity of the immobilized urokinase obtained above was measured by a synthetic substrate method.

CIutaryl-GIycyl-Ar as a synthetic substrate
gjnyl-me ratio y-co mountain maryl-amide
was added to 5 hM Tris-HCl buffer (0.1
MNaC1, 1 skin MCaC12 was dissolved in PH8.0 (final concentration 0.1mM).

Immobilized urokinase was soaked in this solution 2' and 370
After reacting for 3 minutes at a temperature of 0.0 m, the reaction was stopped by adding 1 wt. AmjM-methyl-Commarin (AMC) released by
The concentration of was measured.

As the results are shown in Table 2, compared to urokinase directly immobilized on polyamide, the urokinase shown in Table 2 immobilized on albumin-immobilized polyamide is significantly more sensitive to the amount of urokinase immobilized in Table 1. Shows high activity.

Furthermore, when immobilized urokinase was allowed to act on the natural substrate plasminogen and the activated plasmin activity was measured by a synthetic substrate method, urokinase immobilized on albumin-immobilized polyamide showed high activity.

That is, the synthetic substrate ten-butyl-osha-rbo
nyl-gutari11$yl-lysyl-MCA
O. The same Tris-HCl buffer as above containing 2 hM 0
．． Fixed in the 5 precepts. After impregnating the kinase, 0.5' of plasminogen solution was added and reacted at 370°C for 1 hour. After terminating the reaction by adding 2.0% of a 17% by weight aqueous acetic acid solution, the concentration of free AMC was measured in the same manner as above. The results are shown in Table 3. Table 3 Example 2 (Immobilization of urokinase on polyimide carrier) ○} Immobilization [a} Immobilization of albumin on polyimide 1, 2,
After washing a small piece of a polyimide film with an imidoyelization ratio of almost 100% obtained by co-condensation of 3,4-butanetetracarboxylic acid and diamine with water, Na2HP04-Na
OH buffer (Na2HP04 0.15M50', N
aOHO. IM15 Otsu, soaked in Kanji 11.0), 40
It was partially hydrolyzed and activated by soaking at qo for 90 minutes.

Albumin was immobilized on this polyamide carrier by the same method as in Example 1'a}. {b} Immobilization of urokinase onto polyimide After activating the polyimide in the same manner as above, urokinase was directly immobilized in the same manner as in Example 1'b}.

{c} Immobilization of urokinase onto alpumiso-immobilized polyimide Urokinase was immobilized on the albumin-immobilized polyimide obtained in {a} in the same manner as in Example 1'b}.

'2} Measurement of enzyme activity The activity of the immobilized urokinase obtained as described above was measured using a synthetic substrate in the same manner as in Example 1.

As the results are shown in Table 4, the immobilized urokina of the present invention
ze exhibits significantly high activity. ' Table 4 Example 3 (Co-immobilization of beparin, antithrombin m and urokinase on ethylene-vinyl alcohol copolymer carrier) {1) Ethylene-pinyl alcohol co-immobilization with immobilized vinyl alcohol content of 70 mol% Polymer (hereinafter simply referred to as copolymer).

) was shredded into pieces of about 1 side width, about 19% dry weight was suspended in 100% water, and the pH was maintained at 11-12 by adding 5~ION sodium hydroxide solution under Takaazusa. Ta.
Add cyanogen bromide solution (4-249/80-480 [
) while adding 5-1 side sodium hydroxide solution to maintain the pH at 11-12 until no decrease in pH was observed. During this time, add ice to lower the liquid temperature by 2.
It was kept around 0o0. The reaction was completed in 8-12 minutes.
After the reaction was completed, the mixture was immediately filtered through a glass filter, and excess cyanogen bromide was removed by washing with one sleeve of cold water to obtain an activated copolymer. One day of this activated copolymer (with a surface area of about 80) was then suspended in five portions of 0.1 M sodium bicarbonate solution, to which were added 0.5 g of heparin, 10 g of antithrombin, and 10 g of urokinase. A solution of 10% of 1M sodium bicarbonate solution was added, and the mixture was heated at 4°C overnight. To block excess active groups in the activated copolymer, 10 IM ethanolamine solution (Book 8.0) was added, and after incubation at 4°C for 1 hour, 2M sodium chloride solution and 0.19M sodium chloride solution were added. The material was washed sequentially with water to obtain a heparin-antithrombin m and urokinase immobilized material (hereinafter abbreviated as 1-HEP-ATm-UK). This material contains 0.5 meta heparin per 1 carrier;
antithrombin ml. 3 Butsuya and urokinase 1.
0 Buddha and evening were fixed. A heparin-immobilized material (hereinafter abbreviated as 1-HEP) in which an activated copolymer LC-Heparin 2.0 was immobilized was prepared using a similar method.

), antithrombin m2.5 per 1 activated copolymer
〃Antithrombin m immobilized material (hereinafter abbreviated as 1-ATm) on which antithrombin m is immobilized, urokinase immobilized material on which urokinase 2.0 is immobilized on activated copolymer 1 (hereinafter referred to as 1-ATm) , 1-UK) and a heparin-antithrombin m-immobilized material (hereinafter referred to as heparin-antithrombin m-immobilized material) in which heparin 1.0 and antithrombin m2.5 were simultaneously immobilized per activated copolymer 1. , 1-HEP-ATm) was obtained. Separately, albumin was immobilized on an activated copolymer at a ratio of 3.1 mm/h (hereinafter abbreviated as 1 mm), and this albumin-immobilized carrier was treated in the same manner as in Example 1. Palin and antithrombin m were immobilized (hereinafter abbreviated as 1-AI day EP-ATm). In this material, 2.8 m of heparin and m6.0 m of antithrombin were immobilized per carrier. Unusually, heparin is added to the albumin-immobilized carrier.
4butsuta, antithrombin m4.1muta, and urokinase 2.7sanyu were immobilized (hereinafter referred to as [1N-HEP-A
It is abbreviated as Tm-UK.

). In the above immobilization procedure, antithrombin m does not cause anticoagulation unless it is immobilized under the protective action of heparin. By coexisting acetylated heparin in the reaction system, antithrombin m
This alone was immobilized on the activated copolymer while preserving the activity of Additionally, the activated copolymer was treated as above without the addition of heparin and antithrombin m, and served as a control. ■ Measurement of anticoagulant effect Citrate-added blood count of cows (a mixture of blood and 3.8% sodium citrate solution in a volume ratio of 9:1 at 300 pm)
The upper part of the supernatant solution (0.2) obtained by centrifugation for 1 minute was placed in a small glass test tube and humidified to 370 ml.

A certain amount of immobilization material is added to this, and immediately 1/40M calcium chloride solution is added. Physical and physiological isotonic saline 0.5
Z was added, and the rice bran was gently stirred in a constant temperature bath at 37° C., and the time until the blood bran coagulated was measured. The results are shown in Table 5.

As is clear from the above results, rather than directly immobilizing an anticoagulant or an aliphatic system activating enzyme on a carrier, it is possible to immobilize albumin on a carrier. Moreover, by immobilizing heparin, antithrombin m, and urokinase in coexistence, antithrombotic properties can be synergistically enhanced.

Example 4 (Adhesion behavior of platelets to materials) Heparin and antithrombin m were immobilized on the albumin-immobilized materials obtained in Examples 1, 2, and 3 by the method described in Example 1.

Heparin and antithrombin m shown in Table 6 in each material
The amount of immobilization is shown in Table 6.

Blood collected from the upper limb vein of an adult dog using sodium citrate as an anticoagulant (final concentration 0.38%) was collected in 8 tubes for 6 days.
After centrifugation for 1 minute, a few drops of the obtained polycythemia 4 and plate blood rings were gently dropped onto each of the above sample pieces. After leaving the sample pieces at room temperature for 3 minutes, the sample pieces were placed in physiological saline and washed up and down two or three times. to remove non-adherent platelets.

Next, 1% glutaraldehyde solution (0.1M, pH 7)
．． Platelets were immobilized by soaking them in a phosphate buffer solution (dissolved in phosphate buffer solution No. 4) and reacting at a temperature of 40 qo for 2 hours. Table A: The sample was dehydrated and dried using an alcohol system, and its surface condition was observed using a scanning electron microscope.

Table 7 shows the number of platelets adhesion per 0.016 ribs of each sample piece. From this result, it is clear that by immobilizing albumin on the carrier, the adhesion of platelets to the antithrombotic material is significantly suppressed.

Claims (1)

[Claims] 1. An antithrombotic material characterized in that (a) heparin and antithrombin III, and (b) urokinase or streptokinase are immobilized on albumin immobilized on the surface of the material.