JPH06254151A - Anti-thrombogenic material - Google Patents

Abstract

PURPOSE:To obtain the antithrombogenic material which can suppress thrombus for a long period of time in a stable manner by fixing carphobindin I on a solid carrier. CONSTITUTION:Carphobindin I as biological active substance is fixed on a solid carrier. The carphobindine I (CPB-1) is the protein which is separated and refined from a human placenta, and joined with the tissue factor and the acidic phospholipid in the presence of Ca<++>, and suppresses the coagulation of blood. As the solid carrier for fixing CPB-I, is selected the natural or synthetic high polymer which has the functional group such as amino group, carboxyl group, alcoxycarbonxyl group, hydroxy group, acyloxy group, iscocyanate group, ureid group, and halogen, at the terminal or at a part of a side chain. Such material can suppress thrombosis for a long period of time in a stable manner.

Description

Detailed Description of the Invention

[0001]

The present invention relates to an antithrombotic material,
Specifically, calphobindin I (annexin V) [Ca
lphobindin-I (Annexin V)]-immobilized antithrombotic material.

[0002]

BACKGROUND OF THE INVENTION Advances in medicine and engineering today have increased the demand for artificial materials for direct contact with biological components, especially blood. The requirements for such materials are:
It is to prevent blood components from adhering to the surface, especially when blood adheres and forms a thrombus, the blood flow is cut off at that site, or it peels off from the surface of the material and blocks the blood vessels required for various organs. The problem arises.

In order to solve these problems, many antithrombotic materials have been studied so far, and the surface is made hydrophilic (J. Polym. Sci., 66, 363).
-375, 1979), or as a microphase-separated material composed of hydrophobic and hydrophilic surfaces (artificial organ, 11, 1167).
-1170, 1982) using physiologically active substances or using vascular endothelial cells (Annals of the Ne
w York Academy Science, 28
3, 317-331, 1977).

However, when the surface is made hydrophilic, there is a problem that although the adsorption energy of the adhering molecules is eliminated and thrombus formation is suppressed, the life of circulating platelets is shortened. In addition, the microphase separation material has both advantages of suppressing the adhesion of thrombus on the hydrophilic surface and not damaging blood components, and promoting the adhesion of blood components on the hydrophobic surface but not damaging blood components. Although it was taken in,
Strict control of the phase separation process is difficult in practice. Therefore, both had no significant clinical effect.

Further, a material utilizing endothelial cells is intended to utilize the thrombus-inhibiting action of a living body by transplanting vascular endothelial cells on the surface of the material, based on the viewpoint that it is difficult to suppress thrombus with an artificial material in the long term. However, the question is whether the transplanted endothelial cells can withstand blood pressure, blood flow, or work as normal endothelial cells, and it has not been clinically reached.

On the other hand, as an antithrombotic material utilizing a physiologically active substance, a heparin sustained-release material (Am. Soc. Artif. Int. Organ) which suppresses blood coagulation is used.
s. , 24,736-745,1978), an antithrombin III-heparin complex immobilization material (J. Biom).
ed. Mater. Res. , 14, 619-630,
1980), thrombomodulin-immobilized material (Japanese Patent Application Laid-Open No. 6-68242
1-82760), a urokinase-immobilized material that promotes thrombolysis (artificial organ, 9,870-873, 19).
80), a prostacyclin-immobilized material that suppresses platelet adhesion (Thromb. Res., 46, 685-69).
5,1987) and the like are being studied. Among them, the heparin sustained-release material imitates the heparin secretion phenomenon of the blood vessel wall, has been applied to catheters and bypass tubes, and has been clinically confirmed to have an inhibitory effect for about three months.

[0007] However, there are problems that heparin is depleted to prevent thrombosis in the long term, and that when many surfaces come into contact with blood, the amount of elution increases and the coagulation factor in blood is seriously affected. . Urokinase-immobilized materials are used in various vascular catheters and drainage tubes. Since they are enzymes, they have the property that their effects last for a long time even if they are fixed, but they are actually inactivated or exist in blood. There is a problem that the fibrinolytic enzyme is remarkably activated. The prostacyclin-immobilized material has an action of suppressing platelet aggregation, but has a problem that it is inactivated or inhibits the aggregation action of circulating platelets.

[0008]

SUMMARY OF THE INVENTION Therefore, an object of the present invention is to provide an antithrombotic material on which a physiologically active substance having a new action without the above-mentioned drawbacks is immobilized.

[0009]

In view of such circumstances, the present inventors have conducted diligent research, and as a result, an antithrombotic material having calphobindin I as a physiologically active substance immobilized on a solid carrier is stable for a long period of time. The inventors have found that thrombosis is suppressed and completed the present invention.

That is, the present invention provides an antithrombotic material comprising calphobindin I immobilized on a solid carrier.

Calphobindin I (Annexin V) [Calphobindin-I (Anne
xinV)] (hereinafter simply referred to as “CPB-I”) is a protein separated and purified from human placenta, and Ca ⁺⁺
Binds to tissue factor (TF) and acidic phospholipids in the presence of
It has been shown to inhibit blood coagulation (Th
romb. Res. , 48, 449-459, 198.
7). Further, recently, a structural gene of placenta-derived CPB-I has been introduced into yeast, and recombinant CPB-I (rCPB-I), which has no difference in physicochemical properties and anticoagulant activity, has been mass-produced. (J. Biochem.,
102, 1261-1273, 1987).

The solid carrier for immobilizing CPB-I is a natural or synthetic polymer, which has an amino group, a carboxyl group, an alkoxycarbonyl group, a hydroxy group or an acyloxy group at the terminal or a part of the side chain. , Isocyanato group, ureido group, is not particularly limited as long as it has a functional group such as a halogen, specifically ethylene-
Vinyl acetate copolymers, celluloses, polyvinyl alcohol, polyethylene terephthalate, polyesters such as poly (meth) acrylates, nylons, polyurethanes, polyamides, silicones, halogen-containing resins, copolymers of these polymers with other polymers, and Examples thereof include materials coated with these polymers.

The means for immobilizing CPB-I and the solid carrier is
There is no particular limitation, and any known means can be used, but it is preferable to covalently bond the amino group or carboxyl group of CPB-I and the functional group of the carrier directly or via a spacer.

As shown in FIG. 1, CPB-I consists of 319 amino acids, and its N-terminus is acetylated with alanine. When covalently fixing, CPB-I has 22 amino acid fixing sites in one molecule of lysine (K), and a carboxyl group of C-terminal, aspartic acid (D) and glutamic acid (E). There are 53.

The spacer used in the immobilizing means is not particularly limited as long as it is capable of covalently linking the functional group of the above-mentioned polymer and the amino group or carboxyl group of CPB-I. For example, cyanide bromide activation method. , Water-soluble carbodiimide (Water soluble c
arbodimide (WSC) activation method, mixed acid anhydride method, active ester method using N-hydroxysuccinimide and the like, amino acid method via glycine azide and the like.

Next, an immobilization method suitable for each carrier will be illustrated. (1) In the case of ethylene-vinyl acetate copolymer (EVA), cellulose, polyvinyl alcohol (PVA). Using a mixed acid anhydride method, a spacer is introduced by reacting methyl vinyl ether-maleic anhydride (MVE-MA) with a carrier, followed by drying and reacting with a solution of CPB-I, which is fixed. (2) Saturated polyester For example, in the case of polyethylene terephthalate, the following method is exemplified. a. The terminal hydroxyl group is fixed in the same manner as the above cellulose. b. After fixing the terminal carboxyl group by polyamine condensation reaction, the amino group of CPB-I is fixed to the remaining amino group via an acid anhydride (spacer). Examples of the polyamine used here include diamines such as ethylenediamine and hexamethylenediamine, and polyamines such as polyvinylamine and polyethyleneimine.

(3) Nylon For example, in the case of nylon 6.6, the following method is exemplified. a. The amino group of CPB-I is fixed to the terminal amino group via an acid anhydride (spacer). b. The terminal carboxyl group is fixed in the same manner as in (2) b.

(4) Polyurethane After introducing a polyamine into the terminal isocyanate group, the amino acid of CPB-I is fixed via an acid anhydride. (5) In the case of polymethylmethacrylate (PMMA) The method using an acid anhydride is impossible because PMMA dissolves in a solvent that dissolves the acid anhydride. Therefore, it can be fixed according to the following formula.

[0019]

[Chemical 1]

(6) In the case of glass, metal, ceramics, or polymer having no functional group CPB-polymer as described in (1) to (5) above is coated or copolymerized with the polymer having a functional group. Fix I.

The acid anhydride used in the above method is a methyl vinyl ether-maleic anhydride copolymer (MVE).
Besides -MA), a copolymer of ethylene, styrene, propylene or methyl methacrylate and maleic anhydride can also be used.

[0022]

The antithrombotic material of the present invention can suppress thrombus stably for a long period of time. Therefore, application as various medical materials for internal or external use is expected.

[0023]

The present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

Reference Example 1 (1) Preparation of ethylene-vinyl acetate copolymer (EVA, ethylene 75%) film EVA (molecular weight 25,000: converted to polystyrene by GPC) was dissolved in chloroform at 7 wt% to prepare a glass petri dish ( (45 mm) was put in 5 ml portions and air dried for 3 days. Further, after vacuum drying for 6 hours or more to completely remove residual chloroform, it was cut into 30 × 30 mm and used. (2) Preparation of Maleic Anhydride-Methyl Vinyl Ether Copolymer 35 g (0.36 mol) of maleic anhydride and 16.6 g (0.36 mol) of methyl vinyl ether were dissolved in purified benzene, and AIBN was 1 wt% with respect to the monomer. Polymerization reaction was carried out at 60 ° C. for 1 hour while bubbling with nitrogen. After completion of the reaction, the mixture was poured into diethyl ether to precipitate the copolymer, washed twice with diethyl ether, and dried in vacuum. Next, Soxhlet extraction was performed overnight with diethyl ether, followed by vacuum drying and purification. Molecular weight 20,000 (polystyrene equivalent by GPC)

Example 1 Immobilization of CPB-I on a carrier for affinity chromatography (1) Immobilization of amino group CNBr activated Sepharose
After swelling 10 mg of 4B with 1 mM HCl for 15 minutes, washing-swelling was performed 3 times with 1 mM HCl on a Kiriyama funnel. Next, the coupling buffer (0.5M N
After washing with 0.1 M NaHCO ₃ containing aCl, pH 8), the mixture was immediately put into a protein coupling buffer solution prepared in advance and reacted overnight at 4 ° C. with gentle rotation. After the reaction was completed, the amount of immobilized protein was determined from the supernatant. Next, the carrier is 0.1 M Tris-HCl (pH 8)
The mixture was gently rotated at room temperature for 2 hours to block the remaining active groups, and the excess adsorbed protein was washed away with 0.1 M acetate buffer (pH 4) containing 0.5 M NaCl and coupling buffer. Stored in water (pH 8). (2) Immobilization of carboxyl group 10 mg of AH-Sepharose 4B was swelled and shrunk three times with 0.5 M NaCl on a Kiriyama funnel, and then washed with physiological saline (pH 5) to prepare in advance. The protein was placed in a physiological saline solution (pH 5) containing 10 mg / ml of the condensing agent WSC and reacted at 4 ° C. overnight with gentle rotation. After the reaction was completed, the amount of fixed protein was measured from the supernatant, and excess protein, urea derivative and unreacted condensing agent were washed away with physiological saline (pH 5), and then saline (pH 8)
Saved inside.

Example 2 Immobilization of CPB-I on EVA film: EVA prepared
The film (30 × 30 mm) was put in a 15% NaOH methanol solution and saponified at 60 ° C. for 2 hours, and then 50% 1
Put in 2% aminoacetal solution containing N HCl at 60 ° C
The reaction was carried out for 6 hours to introduce an amino group on the surface. Then 4
% MVE-MA acetone solution was reacted at room temperature for 1 hour to introduce a spacer, and vacuum dried overnight. After drying, the proteins were fixed by allowing them to react in a physiological saline solution (pH 4) of various concentrations for 3 days at 4 ° C. The fixed amount was measured from the supernatant after the reaction, and the film was washed with water and then stored in physiological saline (pH 8).

Example 3 Measurement of CPB-I on cotton yarn A cotton yarn was cut into 90 cm pieces and dried in a vacuum, and then placed in a 2% aminoacetal solution and reacted at 60 ° C. for 7 hours to introduce an amino group on the surface and then vacuum. Dried. Next, 4% MVE-MA
A spacer was introduced by reacting in an acetone solution at room temperature for 3 hours. After vacuum drying, the protein was immobilized by allowing it to react in a physiological saline solution (pH 4) of various concentrations for 3 days at 4 ° C. The fixed amount was measured from the supernatant after the reaction, and the cotton thread was stored in ion-exchanged water.

Test Example 1 Measurement of the amount of fixed CPB-I (Examples 1 to 3) BCA prepared in advance by a conventional method was 50 μl of a protein solution.
Protein Assay Reagent (PIE
(Manufactured by RCE) and incubated at 37 ° C. for 30 minutes, and the absorbance at 550 nm was measured. The calibration curve was obtained from the absorbance of BSA, and the concentration of the protein solution was converted from this.
The results are shown in Tables 1 to 4.

[0029]

[Table 1]

[0030]

[Table 2]

[0031]

[Table 3]

[0032]

[Table 4]

Test Example 2 In Vitro Antithrombotic Test (1) Calcium Re-addition Method (Carrier for Affinity Chromatography) The CPB-I immobilized on the carrier for affinity chromatography in (1) and (2) of Example 1 was tested. It was confirmed by calcium re-addition whether or not it had coagulation activity. Specifically, the following method was used.

100 μl of a carrier on which protein was immobilized in a silicone-treated glass test tube diluted to an arbitrary concentration with 1 ml of physiological saline (pH 8), rabbit ppp 100 μm
Add 1 ml and incubate at 37 ℃ for 3 minutes, then add 25mM Ca
100 μl of Cl ₂ solution was added, and the fibrin formation time was visually measured while shaking at 37 ° C.

The test results are shown in FIG. From FIG. 2, it is seen that the coagulation extension time by the calcium re-addition method when immobilized (substantial coagulation extension time after subtracting the measured value when BSA was fixed) exceeded 100 μg / ml for both amino group and carboxyl group fixation. It can be seen from the striking point that it exhibits a remarkable anticoagulant activity. The rCPB-I concentration at a coagulation extension time of 200 sec was 0.5 μg / ml when it was not fixed, whereas it was approximately 120 to 150 μg / ml when an amino group or a carboxyl group was fixed, and the same anticoagulant activity was obtained. The resulting concentration of P. This is considered to be a decrease in the activity of rCPB-I due to immobilization, but this comparison shows that there is no freedom of rCPB-I because the immobilization state is directly immobilized on the carrier and the plasma in the immobilization state and the immobilization state is It is not exact because the mixed state inside is different.

(2) Imai-type antithrombotic test Next, in consideration of application to catheters, artificial blood vessels, artificial organs and the like as antithrombotic materials, rCPB-in Example 2 was used.
It was examined whether or not I fixed to EVA film had anticoagulant activity. The test method was as follows. A protein-immobilized EVA film was cut into 20 × 20 mm, and ACD added blood collected from rabbit ear vein on the film surface 25
0 μl, 25 μl of 0.1 M CaCl _{2 was} added, and the same film was covered thereon, and incubated at 37 ° C. for 10 minutes to cause a clotting reaction. After the reaction was completed, the blood clot was taken out from the film surface with a spatula, immersed in water for 5 minutes, fixed with formalin for 5 minutes, further immersed in water for 30 minutes, air-dried overnight, and weighed.

FIG. 3 shows the amount of blood clots as a result of the Imai-type antithrombotic test when rCPB-I was immobilized on an EVA film. The amount of blood clot is r compared to 16.7 mg of BSA fixed.
CPB-I fixation was as small as 5.5 to 7.4 mg, and anticoagulant activity was confirmed, but differentiation by the fixed amount of rCPB-I was not possible. It is considered that this is because it is not suitable to obtain a subtle difference in the Imai-type antithrombotic test, and then the same sample was used to evaluate by calcium re-addition method.

(3) Calcium re-addition method (EVA film) In the calcium re-addition method, the film is cut into 5 × 5 mm and BS
After confirming that there was no effect on the coagulation time even if the number of A-immobilized films was changed, it was carried out using a film having a fixed amount of 140 μg, which was the most fixed amount. Specifically, in a silicone-treated glass test tube, a protein-fixed EVA film cut into 5 × 5 mm pieces, rabbit ppp 2
Add 00 μl and incubate at 37 ℃ for 3 minutes, then add 25 mM
200 μl of CaCl ₂ solution was added, and the fibrin formation time was visually measured while shaking at 37 ° C.

The results are shown in FIG. 4. The coagulation time was hardly increased even when the concentration of BSA was fixed and the concentration thereof was increased, whereas that of rCPB-I fixed was 300 to 600 sec due to the increase of the concentration. Significantly prolonged clotting time. Test Example 3 In Vivo Antithrombotic Test From the above results, it was confirmed that when rCPB-I was immobilized with high efficiency via a spacer, it had a remarkable anticoagulant activity in vitro. An attempt was made to confirm anticoagulant activity. The in vivo experiment was performed by inserting a cotton thread fixed with rCPB-I into the rat abdominal vena cava and comparing the amount of thrombotic proteins formed in the cotton thread after a certain period of time. At this time, the rCPB-I was fixed to the cotton thread by using the mixed acid anhydride method as in the EVA film.

The details were tested as follows. Wist
An er rat (male, 8 weeks old, average weight: 292 g) was anesthetized with pentobarbital, and a 10 mm to 15 mm protein-fixed cotton thread passed through a needle (3-2) was put into the abdominal vena cava and allowed to stand for 1 hour. did. After a while, Heparin 2
A rapid injection of 00 U / 0.2 ml from the vena cava was performed and the mixture was allowed to stand for 2 minutes to prevent the generation of thrombus that occurs when the cotton thread is taken out. After cutting the aorta and exsanguinated the rat, the cotton thread was taken out from the vena cava, washed with physiological saline, lightly squeezed with paper, and the length existing in the blood vessel was measured to measure 1N NaO.
It was immersed in H 2. In order to completely dissolve the thrombus adhering to the cotton thread, leave it immersed in 1N NaOH at 50 ° C 1
Incubate for 0 minutes and adjust the amount of protein to B by BCA reagent.
It was calculated in terms of SA. Prior to the in vivo test, the same in vitro immobilized cotton thread was used for in vitro antithrombogenicity by the calcium re-addition method as follows.

In vitro test In a silicone-treated glass test tube, protein-fixed cotton thread cut into 10 cm and 200 μl of rabbit ppp were placed and incubated at 37 ° C. for 3 minutes, and then 25 mM CaCl ₂
200 μl of the solution was added, and the fibrin formation time was visually measured while shaking at 37 ° C.

The results are shown in FIG. As shown in FIG. 5, 21.1 μg of cotton thread introduced with MVE-MA only
/ Mm, due to an increase in the fixed amount of rCPB-I.
The result was that the protein amount was 0 to 15.6 μg / mm, which was remarkably small. This result is in agreement with the in vitro experimental result by the calcium re-addition method using the same sample, and the rCPB-I
It was confirmed that when the amino group of was highly efficiently immobilized via a spacer, it had excellent anticoagulant activity in in vitro and in vivo systems.

Test Example 4 Temperature stability test (by calcium re-addition method) The stability of the antithrombotic material of the present invention was tested as follows. Cotton thread-MVEMA-rCPB-I (Unit fixed amount 1.68
8 μg / mm) was cut into 10 mm pieces and put into a siliconized glass test tube with 10 mm thread and 200 μl rabbit ppp 3
After incubation at 7 ° C for 3 minutes, 20 mM CaCl ₂ 2
00 μl was added, and the fibrin formation time was visually measured while shaking at 37 ° C. FIG. 6 shows the coagulation extension time (substantial coagulation extension time from which BSA was immobilized) was subtracted until 24 hours passed. (Data is the average value of 3 points.) From this, the activity was maintained for 70 to 120 seconds by leaving it at 4 ° C. and 37 ° C. (ion exchange water).

[Brief description of drawings]

FIG. 1 shows the amino acid sequence of CPB-I.

FIG. 2 is a diagram showing anticoagulant activity when immobilized on a carrier for affinity chromatography.

FIG. 3 is a diagram showing the results of an Imai type antithrombotic test when immobilized on an EVA film.

FIG. 4 is a diagram showing anticoagulant activity by a recalcification method when immobilized on an EVA film.

FIG. 5 is a graph showing anticoagulant activity in vitro and in vivo when immobilized on a cotton thread.

FIG. 6 is a view showing temperature stability of cotton yarn-MVEMA-rCPB-I.

Claims (1)

[Claims] 1. An antithrombotic material comprising calphobindin I immobilized on a solid carrier.