JP3043096B2 - Antithrombotic medical material, medical device, and method for producing antithrombotic medical material - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an antithrombotic medical material used for, for example, an artificial blood vessel or a catheter, a medical device using the same, and a method for producing the antithrombotic medical material. .

[0002]

2. Description of the Related Art In recent years, with the progress of medical technology, various medical instruments applied to places that come into direct contact with blood, for example,
Artificial blood vessels, vascular catheters, extracorporeal circulation circuits, and the like are widely used.

[0003] In particular, the use of artificial blood vessels has been increasing with the recent increase in vascular diseases, and there is a long-awaited desire to provide more improved artificial blood vessels. That is, in the artificial blood vessel having a large diameter having an inner diameter of 8 mm or more, almost satisfactory physical properties (eg, operability, antithrombotic property, tissue healing property, etc.) are obtained, but on the other hand, many applications are expected to be expanded. At present, satisfactory physical properties have not been achieved for artificial blood vessels having a small diameter.

For example, a method of chemically bonding an anticoagulant such as heparin to the surface of a material in order to improve antithrombotic properties (JP-B-51-103190), 1,2-diphenyl-3 or 5-dioxypyra A method of adding a compound having synthetic fibrinolytic activity such as a lysone derivative to a material (Japanese Patent Publication No. 52-142772) is known.

Further, in order to improve tissue healing after transplantation, a method of applying collagen to the surface of a substrate (Japanese Patent Publication No.
No. 1-58196), a method of providing a gelatin layer cross-linked with an isocyanate (JP-A-62-258666).
), A method of copolymerizing proteins such as albumin and gelatin into dacron using glutaraldehyde (Japanese Patent Laid-Open No.
No. 4-63588) is known.

[0006] On the other hand, the present inventors have first made it possible to react thrombin with fibrinogen on the inner surface of a tubular body to form a fibrin coat substantially containing no blood cell component, thereby obtaining antithrombotic properties and tissue. A technique for obtaining an artificial blood vessel having both excellent healing properties is disclosed (JP-A-2-305575).

[0007]

The anticoagulant property of fibrin is largely related to the ratio of the amount of fibrinogen to thrombin used during fibrin formation. The optimum ratio of the amount of fibrinogen is 1 mg of fibrinogen to thrombin. It is known to be 0.3-0.5 units.
That is, if the amount of thrombin charged during fibrin formation is too small, fibrinogen cannot be completely converted to fibrin, and a fragile fibrin gel will be formed. Also, if the amount of thrombin charged is too large,
The blood clotting ability of thrombin reduces the anticoagulant ability of fibrin.

As a result of further studies on the above-mentioned technology, the present inventors have found that even if fibrinogen and thrombin are reacted at an optimal charge ratio to form a complete fibrin gel, thrombin in the obtained fibrin gel is not affected. The activity was found to remain. Therefore, by reducing the thrombin activity remaining in the fibrin gel, assuming that it can exhibit more excellent antithrombotic properties,
The present invention has been completed.

Accordingly, an object of the present invention is to provide a novel antithrombotic medical material having remarkably excellent antithrombotic properties and a method for producing the same.

[0010]

The above object is achieved by the following antithrombotic medical material, a method for producing the antithrombotic medical material, and a medical device. (1) An antithrombotic medical material comprising a fibrin gel containing thrombin whose activity has been reduced by heat treatment as a main component. (2) an antithrombotic medical device, comprising: a step of contacting and reacting thrombin with fibrinogen to form a fibrin gel; and a step of subjecting the fibrin gel to a heat treatment to reduce the activity of residual thrombin. Material manufacturing method. (3) The heat treatment is performed at 40 to 80 ° C. for 5 minutes to
3. The method for producing an antithrombotic medical material according to the above 2, which is performed for 24 hours. (4) The heat treatment is performed at 55 to 65 ° C. for 15 to 6
4. The method for producing an antithrombotic medical material according to the above item 3, which is performed for 0 minutes. (5) The ratio of the charged amounts of fibrinogen and thrombin is 1 mg of fibrinogen and 0.1% of thrombin.
The method for producing an antithrombotic medical material according to any one of the above items 2 to 4, which is 3 to 5 units. (6) The method for producing an antithrombotic medical material according to any one of (2) to (5) above, wherein the fibrin gel is heat-treated while impregnated with a protective solution. (7) The method for producing an antithrombotic medical material according to the item 6, wherein the protective solution is distilled water, an electrolyte solution, or a saccharide solution. (8) The antithrombotic medical material according to the above 1, or the above 2 to 7
A medical device having at least a surface in contact with blood formed by the antithrombotic medical material produced by any one of the production methods described above.

[0011]

The method for producing an antithrombotic medical material according to the present invention comprises:
Contacting and reacting thrombin with fibrinogen to produce a fibrin gel, and subjecting the fibrin gel to a heat treatment to reduce the activity of the remaining thrombin, so that thrombin is charged. An antithrombotic medical material with excellent mechanical properties and antithrombotic properties by forming a complete fibrin gel with sufficient amount to form a fibrin gel and then reducing the blood clotting ability of the remaining thrombin. Material can be provided.

It is also conceivable that thrombin is charged in a sufficient amount for the formation of fibrin gel to form a complete fibrin gel, and then a thrombin activity inhibitor is acted on to reduce the thrombin activity. There is a concern about the stability in vivo and the side effects of the activity inhibitor, which should be distinguished from the present invention.

Hereinafter, the present invention will be described in detail.

The fibrinogen used in the present invention is at least one selected from purified fibrinogen, plasma, and cryoprecipitate.
It is preferable to use a starting material of any kind.

To contact and react thrombin with fibrinogen, use a test tube or a plate, etc.
Thrombin and fibrinogen may be contacted and reacted, or when forming a fibrin gel coating on the surface of the substrate, first coat the surface of the substrate with a fibrinogen solution, and then contact the thrombin solution with the fibrinogen solution, It can be performed by reacting. The concentration of the fibrinogen solution is preferably 1% by weight or more. Further, it is preferable that calcium ions are added to the thrombin solution in advance.

This contact reaction is preferably carried out under a neutral condition using a buffer solution or the like. The ratio of the amount of thrombin charged to fibrinogen is preferably 0.3 to 5 units of thrombin per 1 mg of fibrinogen. That is, in the present invention, after the fibrin gel is formed, the activity of the remaining thrombin is reduced by heat treatment, so that thrombin is brought into contact with fibrinogen in a sufficient amount as described above and reacted, and the mechanical strength is excellent. A complete fibrin gel can be formed.

As the substrate, various substrates can be used according to the use of the medical device described later. For example, when the medical device is an artificial blood vessel, a synthetic polymer material having excellent mechanical performance such as nylon, polyester, polypropylene, polyethylene, polyurethane, silicone, polytetrafluoroethylene, or a living body such as a natural blood vessel or ureter. A tissue of origin may be used. In addition, the use of medical instruments,
Depending on the type, polyvinyl chloride, polyethylene, polypropylene, nylon, ethylene-vinyl acetate copolymer, polyvinyl chloride-polyurethane copolymer, polyvinyl chloride- (ethylene-vinyl acetate copolymer) copolymer , Silicone rubber, polypropylene, polycarbonate, high-density polyethylene, acrylic resin and the like can also be used.

As such a substrate, from the viewpoint of tissue healing (easy cell invasion) and fibrin gel adhesion,
A porous material is desirable. In addition, in order to improve the adhesion of the fibrin gel to the surface of the substrate, a substrate whose surface has been made hydrophilic by treatment with acid, plasma, ozone, or the like may be used.

After forming the fibrin gel in this way, the fibrin gel is subjected to a heat treatment. The condition of this heat treatment depends on the amount of the obtained fibrin gel, but it is usually desired to perform the heat treatment at 40 to 80 ° C. for 5 minutes to 24 hours. That is, when the temperature is lower than 40 ° C., a long time is required to reduce the activity of thrombin,
On the other hand, if the temperature exceeds 80 ° C., the fibrin gel itself may be deteriorated and the anticoagulant activity may be lost. More preferably, it is desired to be performed at 55 to 65 ° C. for 15 to 60 minutes. Specifically, such a heat treatment is performed by immersing the fibrin gel in a thermostatic bath set at the above temperature.

The heat treatment of the fibrin gel is preferably performed with the fibrin gel impregnated with a protective solution. By impregnating the protective solution, the activity of fibrin gel is not reduced by the heat treatment, and only the activity of thrombin can be suitably reduced. As the protective solution, an electrolyte solution such as distilled water or physiological saline, or a saccharide solution such as a sorbitol solution or a trehalose solution can be used. Specifically, the protective solution is filled in a thermostatic bath, the temperature of the protective solution is adjusted to 40 to 80 ° C., and fibrin gel is placed in the protective solution for 5 minutes to 24 hours.
This is performed by soaking for a time.

Thus, a medical material containing fibrin gel containing thrombin whose activity has been reduced by the heat treatment as a main component is obtained.

The fibrin gel thus obtained is preferably subjected to partial hydrolysis by an enzyme such as plasmin. This partial hydrolysis of plasmin further improves the antithrombotic properties of fibrin gel. Specifically, the enzyme reaction is performed by bringing a fibrin gel into contact with a hydrolase such as a plasmin solution, performing partial hydrolysis for a predetermined time, and then adding a trypsin inhibitor or an anti-radiation solvent to stop the enzyme reaction. The preferred range of the contact between the plasmin solution and the fibrin gel and the reaction time varies depending on the plasmin concentration in the plasmin solution, the temperature of the plasmin solution, and the like. For example, when the plasmin concentration of the solution is 0.05 units / ml and the temperature is 37 ° C. In this case, it is desired that the time is about 5 to 15 minutes.

Next, if necessary, the antithrombotic medical material according to the present invention can be obtained through operations such as washing and sterilization.

Next, the medical material of the present invention will be described.

That is, in the medical material of the present invention, at least a surface that comes into contact with blood is formed by the antithrombotic medical material obtained as described above.

Specific examples of such medical instruments include various artificial organs such as artificial blood vessels, artificial hearts, artificial kidneys, artificial lungs and other artificial organs, but are not limited thereto. For example, a gas exchange membrane, a dialysis membrane, or the like that is the main body of these devices or a part of these devices, and further, various tubes that constitute a blood extracorporeal circulation circuit, a connector that connects the tubes, a catheter inserted into an artery and artery, and a bubble. Traps, blood bags, chambers, centrifugal pumps,
Blood transfusion or blood collection devices, such as an indwelling intravascular catheter, a syringe, a blood collection tube, a blood bag, and their accessories can also be included.

In such a medical device, a fibrin film may be formed on the surface that comes into contact with blood as described above, and heat treatment may be performed to give heat-treated fibrin, or a heat-treated fibrin gel may be separately formed. This may be applied to the surface that comes into contact with blood by a method such as coating.

Hereinafter, the present invention will be described more specifically with reference to examples.

[0029]

EXAMPLES (Example 1) Human fibrinogen preparation (Ka
bi Vitrum, grade L) in distilled water to remove fibronectin and plasminogen, a gelatin-cellulofine column (manufactured by Seikagaku Corporation) and a lysine-Sepharose 4B column (manufactured by Pharmacia) To remove fibronectin and plasminogen. The fibrinogen eluted from the column was precipitated with ethanol, dissolved in 10 mM Hepes buffer (pH 7.4) containing 150 mM sodium chloride, and filtered through a 0.8 μm filter. The concentration of the fibrinogen solution is determined by the coagulation time measurement time method (trade name Data
-Fi, manufactured by Dade) and adjusted to 30 mg / ml.

The fibrinogen solution was treated with 150 mM
10 mM Hepes buffer containing sodium chloride (p
H7.4) was readjusted to 5 mg / ml, 400 μl was dispensed into a test tube, and 60 μl of a 50 unit / ml thrombin solution (manufactured by Mochida Pharmaceutical Co., Ltd.) containing calcium ions was added to prepare a fibrin gel. Further, after sufficiently washing the fibrin gel with distilled water, 3 ml of distilled water heated to 60 ° C. was added, and
It is treated for 1, 2, 3, 5 hours in a constant temperature bath at 0 ° C.
Heat treated fibrin gels 1-4 were made.

[Measurement of Thrombin Activity] The thrombin activities of the obtained heat-treated fibrin gels 1 to 4 were measured as follows. 50 mM T in fibrin gels 1-4
400 ml of a ris-HCl buffer (pH 8.4) was added, and the mixture was heated at 37 ° C. for exactly 3 minutes.
ml Chromogenic substrate HD-Phenylalanyl-L-pipecoryl L-arginyl-P-nitroanilide (trade name Test Team, S-2238, manufactured by Daiichi Pure Chemicals) 100 m
After heating at 37 ° C. for exactly 5 minutes, 1 ml of a 2% aqueous citric acid solution was added, and the reaction was stopped.
The absorbance was measured at. The measurement was performed using two samples for each heat treatment time, and the thrombin activity of unheated fibrin was calculated as 100. Table 1 shows the results.

[Table 1]

From the results shown in Table 1, it was confirmed that the heat treatment of the fibrin gel significantly reduced the thrombin activity.

The fibrin gel obtained in the same manner as in the example except that no heat was applied was allowed to stand at room temperature for 72 hours, and the remaining activity of thrombin was measured. As a result, no decrease in the activity was observed.

[Measurement of Activation Effect of Plasminogen Activator] The activation effect of plasminogen activator on the heat-treated fibrin gels 1 to 4 was measured as follows. 50 mM Tris-H was added to fibrin gels 1-4.
0.08mg adjusted with Cl buffer (pH 8.8)
/ Ml Glu-plasminogen (BioPool)
200 ml, 200 ml of 1.74 mM chromogenic substrate (S-2251, manufactured by Daiichi Kagaku) and 400 ml of 80 unit / ml tissue plasminogen activator (manufactured by BioPool) were dispensed and heated at 37 ° C. for about 20 minutes. After that, 1 ml of a 2% aqueous citric acid solution was added to stop the reaction, and the absorbance was measured at 405 nm. The measurement was performed using two samples for each heat treatment time. Table 2 shows the results.

[Table 2]

From the results shown in Table 2, it was confirmed that the fibrin gel according to the present invention exhibited the same plasminogen factor activating effect as the non-heat-treated fibrin gel and was not altered.

[SDS-polyacrylamide gel electrophoresis of heat-treated fibrin gel] Heat-treated fibrin gel 1
Regarding No. 4, an SDS-polyacrylamide gel electrokinetic image was examined by the following method. Heat treated fibrin gel 1
4, 2% mercaptoethanol, 2% SDS, 9M
10 mM Tris-HCl buffer containing Urea (p
H7.4) was dissolved in 2 ml and subjected to SDS-polyacrylamide gel electrophoresis. As a result, even in the fibrin gel that had been subjected to the heat treatment, the same electrokinetic image was obtained as in the case of the fibrin gel that had not been subjected to the heat treatment, and it was confirmed that the fibrin molecules did not change at all due to the heat treatment.

Example 2 The fibrinogen solution prepared in Example 1 was mixed with a final concentration of 140 mM sodium chloride,
Readjust to contain 0M calcium chloride, 2 mg concentration
/ Ml of fibrinogen solution. Put this solution in 35
400 μl into a 100 mm glass Petri dish, and 100 uni
13.6 μl of t / ml thrombin (manufactured by Mochida Pharmaceutical Co., Ltd.) was added to prepare a fibrin gel. The fibrin gel thus prepared was placed in a thermostatic bath at 60 ° C. for three pieces each, 15, 30,
Soak for 60, 180 minutes, heat treated fibrin gel 5-8
It was created.

[Measurement of Blood Contact Phase Activation Reaction] 0.5 ml of canine citrated plasma was dispensed into petri dishes containing heat-treated fibrin gels 5 to 8, and allowed to stand at 4 ° C. for 4 hours. Thereafter, the plasma in each petri dish was collected in a tube, and kallikrein generated by activating the blood contact phase was quantified.
Quantitation was performed by adding 1 ml of 50 mM Tris-HCl buffer (pH 8.0) containing 0.1 M sodium chloride and 0.1 mM Z-Phe-Arg-MCA (manufactured by Peptide Research Laboratories) to 100 μl of the collected plasma at 37 ° C. After heating for 1 hour, the fluorescence intensity was measured at an excitation light of 380 nm and a measurement wavelength of 460 nm. In addition, the amount of kallikrein was similarly determined for non-heat-treated fibrin gel. Table 3 shows the results.

[Table 3]

From the results shown in Table 3, it was confirmed that the heat treatment at 60 ° C. for 15 to 60 minutes effectively suppressed the blood contact phase activation reaction.

Example 3 A polyester tubular body having an inner diameter of 4 mmφ and a length of 10 cm was immersed in 30 ml of the fibrinogen solution prepared in Example 1, and then a 50 unit / ml thrombin solution containing calcium ions (manufactured by Mochida Pharmaceutical Co., Ltd.) ), And the surface of the tubular body was coated with fibrin gel. Further, a 3.8 mmφ stainless steel rod was inserted into the lumen of the tubular body, and fibrin gel was pressed on the surface of the tubular body.
Further, the fibrin-coated tubular body was allowed to stand at 37 ° C. overnight, and then heat-treated in a thermostatic bath at 60 ° C. for 5 hours to produce an artificial blood vessel.

Each of the obtained artificial blood vessel and a control artificial blood vessel obtained in the same manner except that the heat treatment was not performed was cut into pieces each having a size of 5 cm, and these were transplanted to the left and right of the canine carotid artery by end-to-end anastomosis. After circulating blood for a period of time,
The inner state was compared. As a result, remarkable thrombus adhesion due to the residual activity of thrombin was observed on the inner surface of the control artificial blood vessel that was not heat-treated. In contrast, thrombosis hardly adhered to the inner surface of the heat-treated artificial blood vessel according to the present invention, and it was confirmed that the artificial blood vessel had good blood compatibility.

[0042]

As described in detail above, the antithrombotic medical material of the present invention is capable of contacting thrombin with fibrinogen,
Reacting to produce a fibrin gel, and subjecting the fibrin gel to a heat treatment to obtain a production method having a step of reducing the activity of the remaining thrombin, so that the thrombin activity remaining in the fibrin gel is reduced, It has the effect of exhibiting excellent antithrombotic properties.

Claims (8)

(57) [Claims] 1. An antithrombotic medical material comprising a fibrin gel containing thrombin whose activity has been reduced by heat treatment as a main component. 2. Contacting thrombin with fibrinogen,
A method for producing an antithrombotic medical material, comprising: a step of forming a fibrin gel by reacting; and a step of subjecting the fibrin gel to a heat treatment to reduce the activity of remaining thrombin. 3. The method according to claim 1, wherein the heat treatment is performed at 40 to 80 ° C. for 5 hours.
The method for producing an antithrombotic medical material according to claim 2, which is performed for a period of from minutes to 24 hours. 4. The method according to claim 1, wherein the heat treatment is performed at 55 to 65 ° C.
The method for producing an antithrombotic medical material according to claim 3, which is performed for 5 to 60 minutes. 5. The method for producing an antithrombotic medical material according to claim 2, wherein the ratio of the charged amount of fibrinogen and thrombin is 0.3 to 5 units of thrombin per 1 mg of fibrinogen. 6. The method for producing an antithrombotic medical material according to claim 2, wherein the fibrin gel is heat-treated while impregnated with a protective solution. 7. The method for producing an antithrombotic medical material according to claim 6, wherein said protective solution is distilled water, an electrolyte solution, or a saccharide solution. 8. The antithrombotic medical material according to claim 1 or the antithrombotic medical material produced by the method according to any one of claims 2 to 7, wherein at least a surface that comes into contact with blood is formed. Medical equipment.