JPH0638851B2 - Medical materials and instruments - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION <Field of Industrial Application> The present invention relates to a medical material and a medical device having excellent antithrombotic properties.

<Prior Art> Conventionally, various antithrombogenic materials have been devised for use in artificial lungs, catheters, artificial hearts, and the like. One method is to fix heparin on the surface of the base material. Examples of the method include a method of ionically binding heparin to a substrate (Japanese Patent Publication No. 54-18317) and a method of covalently binding heparin to a substrate (Japanese Patent Publication No. 55-46741).

<Problems to be Solved by the Invention> However, in ionic bonding, heparin is dissociated when it comes into contact with blood, or it binds too strongly to the sulfate group, which is important for the expression of heparin activity, so the surface activity is not sufficient. In addition, in the covalent bond, most of the binding site with AT-III (antithrombin III) was used as the binding point with the substrate, and the surface activity of heparin was not sufficient.

In addition, a method of coating a prepolymer is often used to fix heparin, but it has a defect that the polymer peels off and cracks occur at a site where elastic properties are required.

Therefore, it is an object of the present invention to provide a medical material and a medical device having excellent antithrombotic properties that solve the above problems.

<Means for Solving the Problems> The inventors of the present invention cannot easily obtain sufficient antithrombogenicity even if they attempt to immobilize heparin simply on the surface of the substrate as in the conventional art, or many of the useful sites cannot be immobilized. As a result of repeated studies in view of the fact that it will be used for this purpose, the present invention has been succeeded by fixing with most of the useful site left, and also by stably fixing heparin on the elastic body.

In the medical material of the present invention, in the substrate, a functional group capable of binding heparin is introduced onto the substrate directly by ozone treatment or via one or more coupling agents. This functional group is preferably an amino group, an aldehyde group or an epoxy group.

Such a substrate and heparin are prepared, and heparin is fixed on the surface of the substrate. Immobilization of the functional group introduced on the substrate and heparin is carried out directly or by covalent bonding via one or more coupling agents. 1
When using a coupling agent of a kind, a compound having two or more aldehyde groups or an epoxy group can be used as the coupling agent.

When a plurality of types of coupling agents are used, a coupling agent composed of a compound having two or more functional groups such as amino groups is preliminarily bonded to the above-mentioned functional group introduced onto the substrate to form an amino group on the substrate. After introducing a group or the like, it is preferable to bond heparin to the substrate using a coupling agent composed of a compound having two or more aldehyde groups or epoxy groups. Further, when coupling heparin, a coupling agent is used. Is preferably added to the reaction system at the same time as heparin or after the introduction of heparin.

By doing so, since the coupling agent reacts in a state where heparin is present in the vicinity of the base material at a high density, the coupling agent can effectively act and more heparin is fixed on the base material. .

In particular, when a coupling agent that introduces an amino group in the former coupling agent is used, it shows almost the same reactivity in the reaction system as the amino group of heparin, and thus the latter coupling agent is more efficient. Heparin can be fixed to the base material by the agent.

When the functional group of the coupling agent that directly binds to heparin or the functional group introduced into the substrate is an aldehyde group, heparin is desulfurized by partially desulfurizing the N-sulfate group of heparin to give a primary amino group. It is preferable to use the compound.

The medical material obtained by the above-described manufacturing method can be used as various antithrombotic materials, and this antithrombotic material can be used particularly for medical devices having a portion in contact with blood. Examples of the medical material include hollow fiber and tube, and examples of the medical device include artificial lung, artificial heart-lung circuit, catheter, artificial heart and the like.

Further, it is preferable to use a porous membrane having a large number of pores for both the hollow fiber and the gas exchange membrane of the artificial lung, and it is preferable that the pores are filled with fine particles such as silica having a diameter smaller than the pores in advance.

The present invention will be described in detail below.

The medical material of the present invention is obtained by fixing heparin directly or via one or more coupling agents to a base material having a functional group such as an epoxy group or an aldehyde group. In the case of using a coupling agent, when heparin has a functional group of an aldehyde group, heparin is desulfated from a part of N-sulfate group from the viewpoint of reactivity to be a primary amine. It is preferable to use heparin.

First, the base material having the functional group will be described.

As the base material, polypropylene, polyurethane, polyvinyl chloride, etc. are generally used, although they may be used properly depending on the application.

In many cases, the substrate itself generally does not have a functional group capable of binding to heparin directly or via a coupling agent. In such a case, the functional group is introduced into the base material. There are various introducing methods, but the following method is preferable.

In the present invention, ozone oxidation is used to introduce the functional group. Generally, when an organic compound is ozone-oxidized, various functional groups, for example, highly reactive functional groups such as aldehyde, ketone and epoxy are generated.

Therefore, it was devised to subject the substrate to ozone treatment and immobilize heparin by using these highly reactive functional groups introduced on the surface. Although it is possible to directly bond a functional group to these functional groups, there are also problems such as steric hindrance, and the method of introducing a spacer (coupling agent) into these functional groups and binding heparin is easy. Moreover, it is also useful in terms of expression of heparin activity on the surface.

Next, the ozone treatment method will be described.

Ozone treatment is carried out by oxidizing O ₂ with an ozone generator to produce O ₃ /
O ₂ gas may be brought into contact with the base material. Although ozone may be dissolved in a solvent and used, the solvent used is preferably water, acetic acid, trichlorofluoroethane, carbon tetrachloride or the like in order to prevent the ozone from being oxidized.

The ozone treatment conditions vary depending on the material such as concentration, reaction time and reaction temperature.

For example, even under optimum conditions for one material, the functional group cannot be sufficiently introduced into another material, or the reaction is too strong and the material deteriorates too much. As a guide, when contacting in a gaseous state, the concentration is 1 to 20 mg / m, 50 to 1000 ml / m
flow rate of in, 0.5-120 m at reaction temperature of 0-70 ° C
It is possible to select the optimum condition suitable for the material from among the reaction times of in.

By these methods, it is possible to directly introduce a functional group into an elastic body such as a polyurethane or a polyamide / polyether copolymer.
Heparin can be easily bound.

Next, heparin for fixing the above-mentioned substrate having a functional group will be described.

Heparin is widely known as an antithrombotic compound, and NH
And a N- sulfate site of SO ₃ Na. Heparin can be fixed on the surface of the substrate as it is, but especially when the functional group of the partner that binds to heparin is a functional group that cannot be sufficiently fixed only by the amino group originally possessed by heparin like an aldehyde group. , A part of the N-sulfate site of heparin is desulfated for primary amination.

In this case, the degree of primary amination is preferably 5 to 25% of the total amount of primary amino groups in heparin. It is more preferably 10 to 20%, further preferably 10 to 15%. Here, the amount of the primary amino group in heparin includes both the primary amino group obtained by desulfating the N-sulfate site and the primary amino group possessed by heparin itself. If the amount of the primary amino group in heparin is less than 5%, it becomes difficult to fix it on the substrate, and if it exceeds 25%, the activity of heparin decreases, so it is preferable to set it to 5 to 25%.

Desulfation of the N-sulfate site of heparin can be performed as follows. A specific example will be described.

Commercially available heparin was dissolved in distilled water to prepare a 10% heparin solution. To 10 ml of this heparin solution, 0.4 ml of 5.5N sulfuric acid was added, reacted at 95 ° C, and sampled over time. The increase in the amount of amino groups was measured by the ninhydrin method (Note 1) and the heparin activity was measured by the synthetic substrate method. did. The results are shown in Fig. 1.

Further, in the literature, the condition that all sulfaamino groups in heparin are desulfated (2% heparin, 0.04N hydrochloric acid, 9%
The reaction was carried out at 5 ° C.), and the increase in the amount of amino groups over time was similarly measured.

Results are shown in FIG.

(Note 1) Ninhydrin reagent: Nimbidrin 2 g and hydrin danthine 0.3 g are dissolved in methyl cellosolve 75 ml, and 25 ml of 4N sodium acetate (pH 5.5) is added.

0.5 ml of ninhydrin reagent is added to 0.75 ml of the sample and heated in boiling water for 15 minutes. After quenching, 5 ml of 25% ethanol is added and the absorbance is measured at 570 nm. The quantification of amino groups is quantified as the color development of leucine.

In FIG. 1, the circles show the anticoagulant property of blood against the second factor of coagulation, and the black circles show the anticoagulant property of blood against the tenth coagulation factor. There are coagulation factors that do not exhibit their anticoagulant properties unless heparin is in a high molecular weight region, and coagulation factors that exhibit their anticoagulant properties even in a low molecular weight region.
Therefore, in FIG. 1, the second representative of each coagulation factor is shown.
It was shown that the anticoagulant activity of heparin against both high and low molecular weight regions was almost the same, taking Factor 10 and Factor 10 as examples.

As can be seen from FIGS. 1 and 2, the primary amino group in heparin increases with the incubation time, but as shown in FIG. 1, the heparin activity gradually decreases. Therefore, it is necessary to carry out primary amination of the N-sulfate site of heparin in a region where heparin activity is not inappropriately reduced.

Specifically, the leucine equivalent of heparin is 0.05 to 0.1.
It is preferably 8 (μmol / 10 mg), more preferably 0.05 to 0.13 (μmol / 10 mg). That is, the leucine equivalent is 0.05 (μmol / 1
If the amount is less than 0 mg), the amount of primary amino groups is not sufficient, and the amount of heparin fixed on the surface of the base material decreases, resulting in 0.18
If it is higher than (μmol / 10 mg), the heparin activity will decrease, which is not preferable.

Next, fixing of the base material having a functional group obtained as described above and heparin will be described.

The immobilization of the base material and heparin can be carried out by using a coupling agent having a compound having at least one, preferably two or more aldehyde groups or epoxy groups, and binding the amino group with these functional groups. Glutaraldehyde etc. can be mentioned as such an aldehyde compound. As the coupling agent, polyethylene glycol diglycidyl ether which is an epoxy compound may be used in addition to the aldehyde compound. In these cases, the functional group obtained by the ozone treatment on the substrate has a coupling agent having two or more primary amino groups (for example,
It is preferable to previously couple (polyethylene glycol diamine, polyethylene imine), which will be described below.

First, a coupling agent having two or more primary amino groups in the introduced functional group on the substrate is coated with a coupling agent solution if the substrate is molded, or in the solution. When the base material is liquid, the coupling agent is mixed by mixing with a coupling agent to introduce an amino group into the base material. For example, the reaction is carried out by bringing the base material into contact with a coupling agent having two or more primary amino groups in a reaction liquid such as water, lower alcohol, chloroform, or acetone. In case of, pH 4 ~
12, the temperature is 0 to 80 ° C., and the reaction time is preferably 10 minutes to 24 hours. That is, if the pH is less than 4, the binding property of the coupling agent to the surface of the base material may be deteriorated, and the base material may be deteriorated depending on the material. If the pH is higher than 12, the deterioration may be caused depending on the base material. There is a risk of this being undesirable. If the temperature is lower than 0 ° C, the reactivity is lowered, and if the temperature is higher than 80 ° C, the primary amino group may be modified, which is not preferable.

If the reaction time is less than 10 minutes, the reaction will not proceed sufficiently, and if it is longer than 24 hours, the pH will be extremely low or high, or the temperature will be extremely high. And there is a risk that the amino group may be modified, which is not preferable.

Examples of the coupling agent having two or more amino groups include polyethyleneimine (PEI), polyethylene glycol diamine, ethylene diamine and tetramethylene diamine.

Further, the base material used in the present invention may be any one having a reactive group capable of binding to the amino group at the terminal, such as carboxymethyl cellulose and glycidyl methacrylate-methyl methacrylate copolymer. In addition, polyvinyl chloride, vinyl chloride-vinyl acetate copolymer, vinyl chloride-ethylene copolymer, vinyl chloride-
Vinylidene chloride copolymer, polyvinyl chloride-urethane copolymer, polyvinyl chloride-acrylonitrile copolymer,
Vinyl chloride-methyl methacrylate copolymer, and a soft polyvinyl chloride modified product comprising the above polymer and a plasticizer, polyurethane, polycarbonate, polystyrene,
Acrylonitrile-butadiene-styrene (ABS),
With respect to acrylonitrile-styrene resin, polymethylmethacrylate, etc., these can be used by easily converting them into a functional group which reacts with an amino group by subjecting them to ozone oxidation, partial hydrolysis or the like.

After introducing the amino group into the base material in this manner, the amino group and the amino group of heparin are bonded by a coupling agent having two or more aldehyde groups or epoxy groups, and heparin is fixed to the base material. A coupling agent having two or more epoxy groups will be described as an example. The coupling agent may be any compound as long as it is a compound having two or more epoxy groups, but a water-soluble compound is preferable from the viewpoint of being advantageous in the reaction. Specifically, Denacol EX-421, 512, 521, 611, 61
2,614,614B or a diepoxy compound, 1,4-butanediol diglycidyl ether, Denacol EX-313, 810, 811, 851, 8
21, 830, 832, 841, 861 (manufactured by Nagase Kasei Co.) and the like. Furthermore, due to the difference in the reactivity of the epoxy, Denacol EX-313, 421, 512, 52
1, 810, 811, 821, 851 and the like are more preferable.

Furthermore, the amino group on the substrate and the amino group of heparin, which are covalently bonded by the above-described method, are respectively bonded to the epoxy groups of the compound having two or more epoxy groups. And the coupling agent and heparin may be mixed and reacted at the same time,
First, the amino group of the base material and the functional group of heparin may be reacted with each other and lightly bonded, and then reacted with the epoxy group of the coupling agent.

For example, when mixing and reacting at the same time, the reaction conditions are pH 2.0 to 10.0, reaction time 1 hr to 200 h
The reaction temperature is preferably 15 to 80 ° C.

When the pH is within this range, the stability of heparin is preferable.

If the reaction time is less than 1 hr, the reaction is not sufficient,
If it exceeds r, the decomposition of heparin and the reduction of the surface activity of heparin fixed on the surface due to excessive crosslinking are not preferable.

If the reaction temperature is less than 15 ° C, the reaction does not proceed sufficiently,
If it exceeds the above range, the stability of heparin is lowered, which is not preferable.

As the solvent, for example, water can be used.

When the amino group of the base material is first reacted with the functional group of heparin to form an ionic bond between them and then the compound having an epoxy group is subjected to a crosslinking reaction, the reaction between the base material and heparin is first performed. As conditions, pH 2.0 ~
A value of 5.0 is recommended.

If the pH is less than 2.0, the stability of heparin will decrease and the pH will be 5.0.
If it exceeds the above range, the positive charge on the surface of the base material is reduced, and the amount of bound heparin is reduced, which is not preferable.

The reaction temperature is preferably 0 to 80 ° C. If the temperature is lower than 0 ° C, the ionic bond rate is remarkably reduced, and if it exceeds 80 ° C, the stability of heparin is lowered, which is not preferable.

The reaction time is preferably 10 to 1500 minutes. 1
If it is less than 0 minutes, the ionic bond is insufficient, and if it exceeds 1500 minutes, the ionic bond amount is completely saturated, and no new bond is formed.

As a reaction method, a heparin solution may be dipped in or applied to the substrate.

When the substrate is liquid, it may be mixed with the heparin solution.

Further, when the reaction product of the base material and heparin is cross-linked with the compound having an epoxy group, the pH may be either alkaline or acidic. However, in the present invention, the surface having an amino group is not charged unless it is under acidic condition, and thus heparin is promptly released from the surface, so that effective crosslinking cannot be achieved unless it is acidic, which is not preferable.

The concentration of the compound is preferably 0.01 to 2 mol / l in terms of the content of epoxy groups for efficient crosslinking.

The reaction temperature is preferably 15 to 80 ° C. If it is lower than 15 ° C, the reaction is not sufficient, and if it exceeds 80 ° C, the stability of heparin is lowered, which is not preferable.

The reaction time is preferably 1 hr to 200 hr. 1 hr
If it is less than 200 hr, the reaction is not sufficient, and if it exceeds 200 hr, heparin is decomposed and the surface activity of fixed heparin is lowered due to excessive crosslinking, which is not preferable.

As a reaction method, when the reaction product of the base material and heparin is already molded, it may be immersed in the compound or applied.

In the case of a liquid before molding, it may be mixed with a solution of the compound.

In the product obtained by this reaction, heparin is trapped in the network structure in which the amino group of the base material reacts with the epoxy group of the compound to form a network structure.

Two fixing states are conceivable when the amino group of the base material and the amino group in heparin are crosslinked with the epoxy group.

However, as a result of the heparin elution experiment, heparin elution was not observed at all from the initial stage, and it is considered that most of the heparin elution was due to this.

By using an epoxy compound instead of an aldehyde for the cross-linking, a complicated process post-treatment using a reducing agent or the like is not necessary, and a simpler treatment of contacting with ethanolamine or the like can be performed.

In addition, since it is possible to remove ion-binding heparin by using ethanolamine or the like under alkali, it is excellent in preventing bleeding tendency due to mass desorption of ion-binding heparin during use. Becomes

Further, when a hydrophilic epoxy compound is used, the treatment can be carried out in an aqueous system, and the surface becomes better compatible with blood, so that it becomes possible to express a larger surface activity.

In addition, since it is not necessary to desulfate the sulfoamino group of heparin in advance, it not only simplifies the process, but also does not cause fluctuations in heparin titer between batches due to subtle differences in reaction conditions and high potency without lowering the titer. Valence heparin can be immobilized stably.

Further, the medical device, using the same resin as exemplified in the antithrombotic material, using the molded one as a substrate, may be reacted with heparin and a coupling agent later, the antithrombotic material The sheet-shaped product may be attached to the surface of the molded article of the medical device.

Further, the base material and heparin can be directly bonded.
At this time, as a functional group, an epoxy group and an aldehyde group capable of binding to an amino group serve as common bonding points.

As described above, the medical material in which heparin is immobilized on the base material having the functional group is an antithrombotic material utilizing the antithrombotic property of heparin, and this is various medical devices such as artificial lungs and heart-lung machines. It can be used in a circuit, a catheter, an artificial heart, etc., at least in a portion in contact with blood. In particular, when it is used for an artificial cardiopulmonary circuit or an indwelling catheter in the body, an artificial cardiopulmonary circuit or an indwelling catheter having excellent antithrombotic properties and stable in bending, bending and clamping can be obtained. Further, when a porous membrane having a large number of pores (eg, hollow fiber) is treated as a gas exchange membrane as described above, a hollow fiber having antithrombogenicity is obtained, and the porous membrane (eg, hollow fiber) is artificially produced. When used in the lungs, an artificial lung with excellent antithrombotic properties can be obtained.

Further, it is preferable to previously fill the pores of the porous membrane used for the artificial lung with fine particles having a diameter smaller than the pores. The reason is,
If the gas exchange membrane is porous and hydrophobic and functional groups do not appear due to ozone treatment, a polymer that allows functional groups to appear must be coated in advance.
In that case, the polymer cannot be uniformly coated on the gas exchange membrane, so that the antithrombogenicity cannot be sufficiently exerted, and since the membrane becomes hydrophilic due to the fixing of heparin, the plasma from the pores is circulated during a long circulation. This is to prevent leaks that may occur.

Japanese Patent Laid-Open No. 62-64 discloses a method for filling fine particles into a porous film.
No. 374. Here is a brief description.

A dispersion liquid of fine particles having a diameter smaller than the pores is flowed through the porous film so that the fine particles are exactly clogged in the pores.

Examples of the material of the fine particles include silica, alumina, zirconia, magnesia, barium sulfate, calcium carbonate, silicate, titanium oxide, silicon carbide, carbon black, white carbon, and other inorganic substances, or polystyrene latex, styrene rubber ( Polymer latex such as SBR latex and nitrile rubber (NBR) latex may be used, but silica is particularly preferable.

The average diameter of the fine particles is 0.003 to 1.0 μm,
It is preferably about 0.003 to 0.5 μm.
The fine particles are made into a dispersion liquid and applied to the gas exchange membrane. Any dispersion medium may be used as long as it is stable with respect to the fine particles and the gas exchange membrane. For example, water, alcohols, etc. are used. However, when the dispersion medium is water, when the gas exchange membrane is hydrophobic, alcohols such as ethanol and isopropanol are brought into contact with the surface of the gas exchange membrane before the dispersion liquid is flowed. It is necessary to make the surface hydrophilic.

When the gas exchange membrane is a hollow fiber, fine particles are suitably filled by passing an appropriately pressurized fine particle dispersion liquid from the inside of the hollow fiber.

<Examples> The present invention will be specifically described below with reference to Examples.

A. When a compound having two or more aldehyde groups is used as a coupling agent I. Introduction of Functional Group to Substrate (Example 1) P which is a block copolymer of nylon and polyether
EBAX 6333 SAOO, 2533 SAOO (At
Ochem) and solvent-soluble polyurethane (NKY
A sheet of -9LH / Japan Polyurethane) (hereinafter referred to as A, B, and C, respectively) was prepared. Using an ozone generator (Nippon Ozone Co., Ltd.), these sheets were placed at 0.8 l / min
_Under the conditions of ₂ and 50 ° C., A was treated for 10 minutes and B and C were treated for 20 minutes (hereinafter referred to as A1, B1, and C1).

The change in the surface elemental composition ratio before and after the ozone treatment was measured by ESCA. The results are shown in Table 1.

Further, the surface aldehyde was confirmed with a Schiff reagent. As a result, those treated with ozone (A1, B1,
C1) was stained from red purple to blue purple, but those not treated with ozone (A, B, C) were not stained.

II Preparation of partially desulfated heparin (Example 2) Commercially available heparin was dissolved in distilled water to prepare a 10% heparin solution. 10 ml of this heparin solution was placed in 0.4 ml of 5.5N sulfuric acid and incubated at 97 ° C. for 10 minutes.

The primary amino groups in all the amino groups in the obtained heparin include those which are originally possessed by heparin and those which are primary aminated by desulfating the N-sulfate site.
It was 1%.

III Immobilization and Evaluation of Heparin on Substrate (Example 3) Sheets prepared according to Example 1 (A, B, C, A)
1, B1, C1) 1.7% PGD- adjusted to pH 10
10 (polyethylene glycol diamine) or 0.5
% Polyethyleneimine (PEI) (BASF) 45
It was soaked at ℃ for 24 hours. Subsequently, 0.5% pH 4.5 acetic acid buffer solution was prepared for the partially desulfated heparin and heparin before the reaction, which were reacted in Example 2, and this membrane was immersed in this solution at 45 ° C. for 24 hours. Then 2.5
It was immersed in a% glutaraldehyde pH 4.5 acetate buffer solution at room temperature for 24 hours. Then, it was immersed in a 1% NaBH ₄ , pH 10 carbonate buffer solution at room temperature for 4 hours.

As a result of soaking the treated sheets in 0.01N hydrochloric acid and dyeing with toluidine blue, the sheets not subjected to the ozone treatment (A, B, C) were hardly dyed.

In addition, in the case of ozone treatment (A1, B1, C1),
Those reacted with heparin before the reaction were only slightly stained, but those reacted with partially desulfated heparin were stained purple to magenta in all of A1, B1 and C1.

In addition, the sheets (A, B, and
C, A1, B1, and C1) were similarly treated except for the step of reacting PEI and PGD-10. After ozone treatment, only partially desulfated heparin was reacted with blue. It was stained purple.

(Example 4) Antithrombotic property of heparin-immobilized tube The polymer B shown in Example 1 was used to prepare a tube having an inner diameter of 1.4 mm. Further, a tube having an inner diameter of 1.4 mm was produced by coating the polymer C on the inner surface of polyurethane. Then, the inside of these tubes was subjected to ozone treatment under the same ozone treatment conditions as in Example 1, and then the partially desulfated heparin obtained in Example 2 was immobilized as in Example 3 (hereinafter, B2 and B2, respectively). C2, B3, C3). In addition, the above tubes were also treated with heparin fixation without ozone treatment (hereinafter, respectively, B4, C4, B5, C5).
And).

Here, B2, C2, B4, and C4 are polyethylene glycol diamines, and B3, C3, B5, and C5 are polyethylene imines, which are used as coupling agents for the base material and heparin.

For these tubes, use a borate buffer solution of pH 9 for 15
After washing for an hour and further with a phosphate buffer solution of pH 7.4 for 2 hours, the surface antithrombin activity of each tube was measured. The specific method is to use a heparin-fixed tube with a length of 56 cm.
Then, 0.5 ml of thrombin 0 to 10 U / cc (4% Alb saline solution) is injected, and the inner surface is brought into contact with the rotary mixer for 15 minutes. After that, the thrombin concentration of the inner solution is measured, and the amount of adsorbed thrombin on the inner surface is calculated. After washing the thrombin adsorption tube with saline, 0.6 mn S-2
Flow 238 1.0 ml at 2 ml / min through the tube,
The liquid coming out of the tube is dropped into 0.2 ml of 50% acetic acid to stop the reaction. The absorbance of the reaction solution is measured, and a calibration curve for the color development of S-2238 with respect to the amount of adsorbed thrombin on the inner surface is prepared.

Next, add ATIII 1U to the tube that adsorbed thrombin.
/ Cc was added and incubated, then S-2
238 is colored with the remaining thrombin on the inner surface, and the remaining thrombin on the inner surface is calculated from the degree of color development and the calibration curve.

FIG. 3 shows changes in the amount of thrombin remaining on the inner surface when the incubation time of ATIII was changed. The results are shown in FIG. From the above, those treated with ozone were active.

The tube was further washed with plasma for 30 minutes to wash the heparin washed out in the plasma, and then the Chandler's loop test was performed.

The results are shown in Table 2. From this, it can be seen that the effect of fixing heparin is exhibited in actual blood by the ozone treatment.

The method and significance of the Chandler's Loop test are as follows.

By measuring the time until fresh blood that has not been anticoagulated is coagulated, it is possible to evaluate the antithrombotic level of the material that is exposed to the fresh blood.

The tube was cut into 20 cm pieces, and 88 μm of fresh blood collected from the rabbit ear vein was put into the tube and looped to 8 r. p.
m. Rotate with.

Then, measure the time it takes for the blood to coagulate and start spinning with the tube.

From the above results, it can be seen that the ozone treatment fixes heparin and imparts antithrombogenicity.

(Example 5) B3, C3, B4, C4 and B5, C obtained in Example 4
Each tube of 5 was cannulated into the left and right femoral veins of a 15 kg miscellaneous dog, and 5 ml / hr of lactated Ringer was placed inside.
While flowing, it was left for 8 hours, then taken out and the degree of thrombus formation in the tube was visually observed. As a result, B3,
In C3, almost no thrombus was formed both inside and outside the nub, but in B4, C4, B5, and C5, the inside was almost blocked, and many thrombus adhered to the outer surface. The effect of immobilizing heparin by ozone treatment was clear. Met.

Immobilization and evaluation of heparin in IV artificial lung (Example 6) Hollow fiber of polypropylene hollow fiber type artificial lung (membrane area 0.8 m ² ) having an inner diameter of 200 μm, a wall thickness of 25 μm, a porosity of 45% and an average pore diameter of 700 Å After coating a polyether / polyamide block copolymer on the above, the same ozone treatment as in Example 1 was performed, and 0.5% P adjusted to pH 10 was further added.
It was immersed in EI (polyethyleneimine) at 45 ° C. for 24 hours.

Subsequently, a partially desulfated heparin 0.5%, pH 4.5 acetic acid buffer solution reacted according to Example 2 was prepared, and this hollow fiber was immersed in this solution at 45 ° C. for 24 hours. Then 2.
In 5% glutaraldehyde, pH 4.5 acetate buffer solution,
It was immersed at room temperature for 24 hours. Then 1% NaBH ₄ , pH 1
Oxygenator A was obtained by immersing in 0 carbonate buffer at room temperature for 4 hours.

On the other hand, in a similar artificial lung, ethanol was introduced from the blood inlet to make the gas exchange membrane hydrophilic, and after replacement with distilled water, colloidal silica / average particle size 0.0125 μm /
The water dispersion was introduced, and the gas exchange membrane was filtered to fill the pores with silica. Then, distilled water was introduced to sufficiently remove the silica / water dispersion liquid remaining inside the gas exchange membrane, and then dried. Then, the same treatment as that for the artificial lung A was performed to obtain the artificial lung B.

A similar artificial lung that has not been subjected to ozone treatment is referred to as an artificial lung C for comparison.

(Evaluation experiment of antithrombotic property) With regard to artificial lungs A, B and C, femoral arteriovenous AV shunt was performed in 25 kg of dogs, max 400 ml / mi.
Blood was circulated at n.

As a result, in both the artificial lungs A and B, the flow rate did not decrease with the increase in circulating pressure loss for 8 hours.

The artificial lung A showed a decrease in the flow rate after 9 hours, but the artificial lung B did not show a decrease in the flow rate even after 12 hours.

On the other hand, in the artificial lung C, the circulation became impossible in 2 hours due to the formation of thrombus in the artificial lung.

In the artificial lung A, plasma leakage was observed from 6 hr, but in the artificial lung B, plasma leakage was not seen even after 12 hr.

B. When Two or More Epoxy Compounds are Used as Coupling Agents (Example 7) Soft Vinyl Chloride Tube (NO.CBT65 manufactured by Terumo Corp.)
0) was coated with a solvent-soluble polyurethane (NKY-9LH / Nippon Polyurethane). This tube was placed in an ozone generator under conditions of 0.8 l / min O ₂ 50 ° C.
It was processed for 0 min. The tube was immersed in 0.05% polyethyleneimine (PEI) at 45 ° C for 20 hours, and then 0.5% heparin (pH 4.0, 0.1M acetic acid buffer).
It was soaked at 45 ° C. for 4 hours.

In contrast to this, a polyepoxy compound (Denacol EX
-421 (manufactured by Nagase Kasei) and a diepoxy compound (Denacol EX-313 (manufactured by Nagase Kasei)) at pH 4.
Dissolve it in 0, 10 mM acetic acid buffer to make an aqueous solution of 5% polyepoxy compound and 10% diepoxy compound.
The water-insoluble matter is precipitated by centrifugation at 3000 rpm for 10 minutes, the supernatant is separated, and the above-mentioned treated tubes are respectively immersed, and designated as I and II. As a comparative control, 10 mM acetic acid buffer (pH 4.0) containing no epoxy compound was dipped in the above treatment tube and designated as III.

These were reacted at 45 ° C. for 20 hours and 44 hours to give I
-20 (hr), I-44 (hr), II-20 (h
r) and II-44 (hr). After that, pH 10, 1M
It was immersed in ethanolamine for 16 hours and then washed with water. Toluidine Bull-staining was performed on these samples, and the results are shown in Table 3.

Example 8 A sheet of solvent-soluble urethane (NKY-9LH) was prepared by dipping. This sheet was treated as in Example 7, heparin was fixed to this sheet, and the same polyepoxy compound and diepoxy compound as in Example 7 were dipped for 20 hr or 44 hr, epoxy treated, and then treated with 1M ethanolamine. pH 10.0, 1M ethanolamine immersion for 16 hours
Then, it was further immersed in 1 M sodium chloride at pH 10 to remove the ion-binding heparin.

About these, the sheet at each operation is
The elemental composition ratios of N, O, C and S were measured. The results are shown in Table 4.

(Example 9) A solvent-soluble polyurethane (NKY-9LH) was coated on the inner surface of a soft vinyl chloride tube having an inner diameter of 1.4 mm, treated in the same manner as in Example 7, and I-44 (hr) and II-20 (h).
r) and II-44 (hr) were prepared as heparinized tubes. The antithrombogenicity of this tube was evaluated by the Chandra loop method. In addition, III of Example 7 was used as a comparative control. This was carried out as it was, or after incubation for 24 hr three times with 6% albumin and further washing the excess adsorbed heparin thoroughly. The results are shown in Table 5.
Shown in.

From the above results, immobilization of heparin on the epoxy compound imparted antithrombotic properties. Further, it is considered that the excess heparin is almost removed by the treatment with pH 1 and 1M ethanolamine.

(Example 10) Solvent-soluble polyurethane (NKY-9LH) was coated on the inner and outer surfaces of a soft vinyl chloride tube having an inner diameter of 1.4 mm and an outer diameter of 2 mm, and I-44 (hr) was applied in the same manner as in Example 7.
A heparinized tube was prepared according to (Epoxy compound 0% -44 hr treatment as Comparative Control III). This is cannulated into the left and right femoral arteries and veins of a 15 kg miscellaneous dog, and the inside is flushed with 5 ml / hr lactated Ringer's for 8 h.
After being left for a while, the tube was taken out and the degree of thrombus formation in the tube was visually observed. As a result, I-44 showed almost no thrombus, while III-44 had many thrombi both inside and outside.

<Effects of the Invention> In the present invention, a predetermined functional group is introduced into the substrate in advance,
Since the functional groups of these base material and heparin are bonded directly or via one or more coupling agents to fix heparin to the base material,
The obtained medical material, the antithrombotic property in the medical devices such as artificial lungs and cardiopulmonary circuits, and the stability of heparin fixed to the substrate are remarkably improved, and heparin efflux even during blood perfusion. There is little danger of it being able to withstand long-term use.

[Brief description of drawings]

FIG. 1 is a graph showing changes in the desulfation and activity of the N-sulfate site of heparin. Fig. 2 shows the result of desulfation of the N-sulfate site of heparin.
It is a graph which shows the change of the amount of primary amino groups. FIG. 3 is a graph showing the surface heparin activity of the sample (medical material) obtained in Example 4.

Claims (3)

[Claims] 1. A medical material comprising heparin immobilized on a substrate, wherein the functional group contained in the oxide on the surface of the substrate and the amino group of heparin are directly or at least one cup. A medical material characterized by being covalently bonded via a ring agent. 2. The method according to claim 1, wherein at least a portion in contact with blood is included.
A medical device formed from the medical material according to 1. 3. A functional group is introduced onto a substrate by (a) treating the substrate with ozone, and (b) the functional group and heparin are directly or through at least one coupling agent. A method for producing a medical material, which comprises covalently bonding.