JPH0246856A - Medical material and medical equipment - Google Patents

Abstract

PURPOSE:To obtain a medical material which possesses the superior antithrombosis performance by previously introducing the prescribed functional groups onto a basic material and partially processing Haparin to form the primary amino compound by the desulfurization of the N-sulfuric acid part and joining the functional groups of the basic material and Heparin directly or through coupling agents. CONSTITUTION:A medical material which is prepared by fixing Heparin onto a basic material is used as antithrombosis material. In this material, the functional group (primary amino group) which is introduced onto the basic material through a compound containing hydroxyethyl methacrylate(HEMA) and methyl methacrylate(MMA) and the primary amino group of Heparin are covalent- bonded directly or through coupling agents. Further, Heparin is the primary amino compound after the desulfurization of a part of the N-sulfuric acid group, and HEMA and MMA exist in separation to each segment, and the functional group exists in the segment consisting of HEMA.

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention relates to medical materials and medical instruments with excellent antithrombotic properties.

<Prior Art> Various antithrombotic materials have been devised for use in artificial lungs, catheters, artificial hearts, and the like. One such method is to fix heparin on the surface of a base material. This method includes a method in which heparin is ionically bonded to a substrate, and a method in which heparin is covalently bonded to a substrate.

<Problem to be solved by the invention> However, with ionic bonding, heparin may come off when it comes into contact with blood, or it may bond too tightly with the sulfate group, which is important for the expression of heparin's activity, so the surface activity may not be sufficient. Moreover, in the case of covalent bonding, the bonding site with A T -Ill was used as the bonding point with the base material, and the surface activity of heparin was not sufficient. Attempts have also been made to ionicly bond heparin to a cationic surface and then crosslink it with glutaraldehyde, but since aldehyde groups mainly react only with primary amino groups, etc., There are almost no grade amino groups, and the base material has 111 grade amino groups.
;If there is no L-amino group, it cannot be shared with the base material,
The durability of the effect was insufficient.

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

Means for Solving the Problems> The present inventors have discovered that even if they simply try to fix heparin on the surface of a base material as in the past, it is difficult to obtain sufficient antithrombotic properties, or that it is difficult to obtain sufficient antithrombotic properties, or that it is difficult to obtain sufficient antithrombotic properties by simply fixing heparin on the surface of a base material, or that it is difficult to obtain sufficient antithrombotic properties by fixing heparin on the surface of a base material. As a result of repeated research, we succeeded in fixing the device while leaving the useful parts intact, resulting in the present invention.

That is, in heparin, one whose N-sulfate groups are partially desulfated and primary aminated is used.

The base material is prepared to have a functional group, preferably a primary amino group, on its surface.

This preparation method is arbitrary, but hydroxyethyl methacrylate (HEMA) and methyl methacrylate)
It is preferable to introduce a functional group that binds the heparin onto the substrate via a compound containing -(MMA). This functional group is preferably a primary amino group or an epoxy group.

Hydroxyethyl methacrylate (HEMA) and methyl methacrylate (MMA)
is present separately in each segment, and the functional group is preferably present in the segment containing hydroxyethyl methacrylate (HEMA).

Hydroxyethyl methacrylate (HEMA) and methyl methacrylate (MMA)
Compounds containing hydroxyethyl methacrylate (
It is preferable to use one containing 30% or more of HEMA).

Such a base material and heparin are prepared, and the heparin is fixed on the surface of the base material. The immobilization of the compound having the above functional group and heparin on the substrate is carried out directly or by covalent bonding via a coupling agent. The coupling agent has two or more aldehyde groups such as glutaraldehyde. Compounds can be used.

The medical material obtained by the above manufacturing method can be used as various antithrombotic materials, and this antithrombotic material can be used particularly for medical instruments having parts that come into contact with blood. Examples of medical materials include hollow fibers and tubes, and examples of medical instruments include artificial lungs, artificial heart-lung circuits, catheters, and artificial hearts.

Further, it is preferable to use a porous membrane having a large number of pores for both the hollow fiber and the oxygenator, and it is preferable to fill the pores in advance with fine particles such as silica having a smaller diameter than the pores.

The present invention will be explained in detail below.

The medical material of the present invention includes heparin obtained by desulfating a part of the N-sulfate groups of heparin to form a primary amine on a base material having a functional group such as an amino group, preferably a primary amino group. It is fixed.

First, the base material having the above functional group will be explained.

The base material may be selected depending on the purpose, but polypropylene, polyvinyl chloride, polyurethane, etc. are generally used.

This base material itself generally does not have the above-mentioned functional groups in many cases. In such a case, the above functional group is introduced into the base material. Although there are various methods of introduction, it is preferable to use the method described below.

In the present invention, it is preferable to use a polymer containing hydroxyethyl methacrylate (hereinafter referred to as HEMA) and methyl methacrylate (hereinafter referred to as MMA) as the substance for introducing the functional group into the base material. be.

The reason for limiting this is that the polymer itself has high blood and biocompatibility and safety, is relatively easy to synthesize, and can be easily coated.

Here, the polymer containing HEMA and MMA is preferably in the form of a block copolymer, each of which is bonded to form a segment (referred to as A and B segment, respectively). In this polymer, each segment preferably has a structure in which it exists separately. The reason for this is that by forming segments and bonding them together, MMA has good water resistance.
This is because the segment containing the segment can be brought into close contact with the base material. Here, the A and B segments refer to fragments (parts) mainly containing HEMA and MMA, respectively.

The functional group is present in segment A containing hydroxyethyl methacrylate (HEMA). As this functional group, a primary amino group, an epoxy group, etc. can be considered.

In order to have the above-mentioned functional group, the HEMA segment may contain a compound having a functional group such as glycidyl methyl methacrylate (GMA) in addition to HEMA. In this case, it is preferable to introduce primary amino groups onto the surface by reacting the epoxy groups in GMA with a compound having two or more primary amino groups.

Similarly, in segment B containing MMA,
Compounds other than MMA may also be included.

However, hydroxyethyl methacrylate (HEMA
) and methyl methacrylate (MMA), hydroxyethyl methacrylate (HEMA)
It is preferable to contain 30% or more of. This is because when the amount of HEMA decreases, the surface becomes hydrophobic, and the introduced functional groups become less likely to appear on the surface during reaction in an aqueous solution.

Next, heparin for immobilization on the base material having the above-mentioned primary amino group will be described.

Heparin is widely known as a compound that exhibits antithrombotic properties, and N
It has an N-sulfuric acid site called HSO3Na. As mentioned above, problems arise when heparin is directly fixed onto the surface of a base material.

Therefore, in the present invention, a part of the N-sulfuric acid site is desulfated to undergo primary amination.

The degree of primary amination is preferably such that the amount of primary amino groups is 5 to 25% of the total amino groups in heparin.
More preferably 10-20%, still more preferably 10-1
5% is good. Here, the amount of primary amino groups in heparin includes both those obtained by desulfating the N-sulfate site to form Mx class amination, and those possessed by heparin itself. If the amount of primary amine groups in heparin is less than 5%, it becomes difficult to be fixed to the substrate, and if it exceeds 25%, the activity of heparin decreases, so it is preferable to keep it in the range of 5 to 25%.

Desulfation of the N-sulfate site of heparin can be performed as follows. This will be explained by giving a specific example.

Commercially available heparin was dissolved in distilled water to prepare a 10% heparin solution. 5.58+ for 10m2 of this heparin solution
(Added 4 ml of 25040, reacted at 95°C, sampled over time, and measured the increase in the amount of amino groups using the ninhydrin method (Note 1) and heparin activity using the synthetic substrate method. The results are shown in Figure 1. show.

In addition, in the literature, conditions are said to desulfate all sulfamino groups in heparin (2% heparin 0.04NHCIL9
The reaction was carried out at 5° C.), and the increase in the amount of amino groups over time was measured in the same manner.

The results are shown in Figure 2.

(Note 1) Ninhydrin reagent: Dissolve 2 g of ninhydrin and 0.3 g of tohydrin dantin in 75 mft of methyl cellosolve, and add 25 ml of 4N sodium acetate (pH 5.5).
Add.

Sample 0.75mjZ with ninhydrin reagent 0. 5m1L
Add and heat in boiling water for 15 minutes.

After quenching, add 5 ml of 25% ethanol and
Measure the absorbance. The amino group is quantified numerically as the degree of color development of leucine.

In FIG. 1, the mark Q indicates the anticoagulant property against the second coagulation factor of blood, and the mark ・ indicates the anticoagulant property against the tenth coagulation factor of blood. There are coagulation factors that do not exhibit anticoagulant properties unless heparin is in a high molecular weight range, and coagulation factors that exhibit anticoagulant properties even when heparin is in a low molecular weight range. Taking factor 10 as an example, this means that the anticoagulant activity of heparin in both high and low molecular weight ranges is almost the same.

As seen in FIGS. 1 and 2, the number of primary amine groups in heparin increases with incubation time, but as seen in FIG. 1, heparin activity gradually decreases. Therefore, it is necessary to aminate the N-sulfate site of heparin in a region where heparin activity is not inappropriately reduced.

Next, the fixation of the base material having the functional group obtained as described above with heparin in which some of the N-sulfuric acid sites have been desulfated to form a primary amine will be described.

The aforementioned immobilization of the base material and heparin can be carried out by using a compound having at least two aldehyde groups as an example of a coupling agent, and by reaction between a primary amino group and an aldehyde group. Examples of such aldehyde compounds include glutaraldehyde.

In addition to this, polyethylene glycol diglycidyl ether or the like may be used as the coupling agent.

Moreover, the base material and heparin can also be directly bonded.

At this time, the functional group should be an epoxy group, an aldehyde group, etc. that can bond with an amine group.

In this way, a medical material in which heparin, which is primary aminated by desulfating a portion of the N-sulfate groups, is immobilized on a base material having the above-mentioned functional groups has antithrombotic properties that utilize the antithrombotic properties of heparin. This material can be used in various medical devices, such as artificial lungs, heart-lung catheters, artificial hearts, etc., at least in the parts that come into contact with blood.

In particular, porous membranes with many pores (
For example, if a hollow fiber is treated as described above, a hollow fiber with antithrombotic properties can be obtained, and if the above porous membrane (for example, a hollow fiber) is used in an oxygenator, an oxygenator with excellent antithrombotic properties can be obtained. .

Further, it is preferable that the pores of a porous membrane used in an oxygenator are filled in advance with fine particles having a smaller diameter than the pores. The reason for this is that because the gas exchange membrane is porous and hydrophobic, it is not possible to uniformly coat the gas exchange membrane with the polymer, which prevents it from exhibiting sufficient antithrombotic properties. This is because plasma becomes hydrophilic, which may cause plasma to leak from the pores during long-term circulation.

Regarding the filling of fine particles into a porous membrane, see JP-A-62-64.
It is preferable to do as described in No. 374. I will explain briefly here.

A dispersion of fine particles having a diameter smaller than the pores is flowed through the porous membrane so that the fine particles clog the pores.

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

In addition, the average diameter of the fine particles is 0.003 to 1.0 μm
, preferably about 0.003 to 0.5 μm.

The fine particles are made into a dispersion and applied to the gas exchange membrane. As the dispersion medium, any medium may be used as long as it is stable to the fine particles and the gas exchange membrane; for example, water, alcohols, etc. are used. However, if the dispersion medium is water and the gas exchange membrane is hydrophobic, alcohols such as ethanol or isopropanol may be brought into contact with the surface of the gas exchange membrane before the dispersion is allowed to flow through the gas exchange membrane. It is necessary to make the surface of the membrane hydrophilic.

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

methyl methacrylate)-(M
Acrylic acid (AA) was introduced into the segment containing MA).

Table 1 Polymer composition <Example> The present invention will be specifically described below with reference to Examples.

(2) Introduction of functional groups into base material (Example 1) Polymers P1 to P4 having various compositions shown in Table 1 below were prepared and coated on a flat film of microporous polypropylene. To prepare the polymer liquid, use methyl cellosolve 1.
A 5% polymer solution was diluted to a 2.5% polymer solution with methanol or methanol:acetone=9:1-8.

Here, hydroxyethyl methacrylate (HEMA
) Glycidyl methyl methacrylate (GMA) was bound to the segment containing +1. Preparation of partially desulfated heparin (Example 2) Commercially available heparin was dissolved in distilled water to prepare a 10% heparin solution. 5.5 mL of this heparin solution
It was placed in 0.4 mu of N sulfuric acid and incubated at 97°C for 10 minutes.

Of all the amino groups in the obtained heparin, the primary amino groups are:
11% including what heparin originally has and what is primary aminated by desulfation of the N-sulfate site
Met.

Immobilization and evaluation of heparin on the m group M (Example 3) Membranes prepared according to Example 1 with 0, 1% ethylenediamine, and 1.7% PGD-1 adjusted to pH 10.
0 (polyethylene glycol diamine) at 45°C, 24
Soaked for an hour.

Subsequently, the partially desulfated heparin reacted according to Example 2 and the heparin before reaction were added at 0.5%, pH 4,5.
Prepare an acetate buffer solution, and place this membrane in this solution at 45°C.
Soaked for 24 hours. Subsequently, it was immersed in a 2.5% glutaraldehydro H4,5 acetic acid buffer solution for 24 hours at room temperature. Subsequently, it was immersed in a 1% N&BHa, pH 10 carbonate buffer solution at room temperature for 4 hours.

After immersing the membrane subjected to these treatments in 0.0 IN hydrochloric acid,
As a result of staining with toluidine blue, the membranes immersed in non-desulfated heparin were hardly stained, but the membranes immersed in partially desulfated heparin of polymers P2 to P4 were stained reddish-purple. . However, polymer P] was not dyed. In addition, after immersing in a boric acid buffer solution of pH 9 for 15 hours to remove ionically bound heparin, the results were similarly stained.
There was no change in the membrane soaked in heparin except that it was no longer stained.

(Example 4) Ionic binding of heparin to the substrate in the neutral region The membrane prepared according to Example 1 was adjusted to pH 10 with 0, 1% ethylenediamine, and 1.7% PGD-1.
0 (polyethylene glycol diamine) at 45°C, 24
Soaked for an hour.

Subsequently, partially desulfated heparin reacted according to Example 2 and 0.5% of heparin before reaction, f)) 14
．． Prepare a 5-acetic acid buffer solution and place this membrane in this solution for 45-50 min.
℃ for 24 hours. Subsequently, the samples were immersed in a pH 7.4 phosphate buffer solution at room temperature for 24 hours and then stained in the same manner. As a result, almost no staining was observed in either case.

This suggests that there is no ionic bond between the base material and heparin in the vicinity of neutrality.

(Example 5) Anti-thrombotic effect of heparin-immobilized tube A polyamide tube with an inner diameter of 1.4 mm was coated with the polymer shown in Example 1, and a partially desulfated product was prepared in the same manner as in Example 3 in Example 2. Hebarin was immobilized. These tubes were washed with a boric acid buffer solution of pH 9 for 15 hours and further washed with a phosphate buffer solution of pH 7.4 for 2 hours, and then the surface antithrombin activity of each tube was measured. The specific method is to cut a heparin-fixed tube into 56 cm pieces and add thrombin 0 to 10 U/
0.5 mu of c c (4% AJZb saline solution) is injected and brought into contact with the inner surface using a 15 mtn rotary mixer. Thereafter, the thrombin concentration of the internal fluid is measured, and the amount of thrombin adsorbed on the internal surface is calculated. After washing the thrombin adsorption tube with saline, add 0.6mM S-22381, 0mJ.
1 L was allowed to flow through the tube at a rate of 2 mj2/min, and the liquid coming out of the tube was dropped into 0.2 ml 1 L of 50% acetic acid to stop the reaction. The absorbance of the reaction solution is measured, and a calibration curve of color development of S-2238 with respect to the amount of thrombin adsorbed on the internal surface is created.

Next, A T II was added to the tube adsorbed with thrombin.
After incubating with IIU/cc, S-2238 is similarly colored with thrombin remaining on the inner surface, and the amount of thrombin remaining 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 inside the cells when the incubation time of AT Ill was changed. The results are shown in FIG. From the above, those stained reddish-purple were active.

From FIG. 3, it can be seen that polymers into which glycidyl groups have been introduced have surface heparin activity, and among them, those with a high HEMA content have high activity.

! ■ Fixation and evaluation of heparin in the oxygenator (Example 6)
) Inner diameter 200μ, wall thickness 25μ, porosity 45%, average pore diameter 7
00 people polypropylene hollow fiber oxygenator (membrane area 0.
8 m2) was coated with polymer P2 shown in Table 1,
This membrane was immersed in 1.7% PGD-10 (polyethylene glycol diamine) adjusted to pH 10 at 45° C. for 24 hours.

Subsequently, an acetic acid buffer solution containing 0.5% partially desulfated hebarin reacted in accordance with Example 2 and having a pH of 4.5 was prepared, and the membrane was immersed in this solution at 45° C. for 24 hours. Next is 2.
It was immersed in a 5% glutaraldehyde, pH 4,5 acetate buffer solution for 24 hours at room temperature. followed by 1% NaBH4,
An artificial lung A was obtained by immersing it in a pH 10 carbonate buffer solution 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 then replaced with distilled water.
An aqueous dispersion was introduced and filtered through a gas exchange membrane, and the pores were filled with silica. Next, distilled water was introduced to sufficiently remove the silica/water dispersion remaining inside the gas exchange membrane, and then drying was performed. Thereafter, the same treatment as the artificial lung A was performed to obtain an artificial lung B.

In addition, a similar oxygenator not coated with the polymer shown in Table 1 is designated as oxygenator C for comparison.

(Example 7) Antithrombotic evaluation Artificial lungs A and B fixed with heparin according to Example 6
and C, femoral artery and vein A-
Perform V shunt, max 400 m 11
/min blood was circulated.

As a result, in both artificial lungs A and B, there was no decrease in flow rate associated with an increase in pressure drop during circulation for 8 hours.

In oxygenator A, a decrease in flow rate was observed after 9 hours, but in oxygenator B, no decrease in flow rate was observed even after 12 hours.

On the other hand, in oxygenator C, circulation became impossible within 2 hours due to thrombus formation in the oxygenator. In addition, plasma leakage was observed from 6 hours in artificial lung A, but in artificial lung III.
In the snake, no plasma leakage was observed even after 12 hours.

FIG. 3 is a graph showing the surface heparin activity of the sample (medical material) obtained in Example 5.

<Effects of the Invention> In the present invention, predetermined functional groups are introduced into the base material in advance,
Heparin is also partially desulfated at the N-sulfate site to undergo primary amination, and these base materials and the functional groups of heparin are bonded directly or via a coupling agent to convert heparin into a base material. Because of this, the resulting medical material has significantly improved antithrombotic properties and plasma leakage properties in medical devices such as oxygenators, making it durable for long-term use.

Claims (18)

[Claims] (1) A medical material in which heparin is fixed on a base material, and a functional group introduced onto the base material via a compound containing hydroxyethyl methacrylate (HEMA) and methyl methacrylate (MMA). A medical material characterized in that a primary amino group of heparin is covalently bonded directly or via a coupling agent. (2) The medical material according to claim 1, wherein the functional group is a primary amino group. (3) In heparin, some of the N-sulfate groups are desulfated and become
The medical material according to claim 1, which is aminated. (4) The hydroxyethyl methacrylate (HEM
A) and methyl methacrylate (MMA) are present separately in each segment, and the functional group is present in the segment containing hydroxyethyl methacrylate (HEMA) as a component. material. (5) The hydroxyethyl methacrylate (HEM
A) and methyl methacrylate (MMA)-containing compounds include hydroxyethyl methacrylate (HEMA)
) The medical material according to claim 1, containing 30% or more of. (6) The medical material according to any one of claims 1 to 5, which is used as an antithrombotic material. (7) Claims 1 to 6 include at least a portion that comes into contact with blood.
A medical device formed from the medical material according to any one of the above. (8) Claims 1 to 6 include at least a portion that comes into contact with blood.
A hollow fiber formed from the medical material according to any one of the above. (9) (a) Hydroxyethyl methacrylate (HEMA)
and methyl methacrylate (MMA) on the substrate via a compound, (b) this functional group and the primary amino group of heparin,
A method for producing a medical material, characterized by covalently bonding directly or via a coupling agent. (10) In step (a), hydroxyethyl methacrylate (HEMA) and methyl methacrylate (M
10. The method according to claim 9, which is carried out by coating the compound containing MA) and having an epoxy group, and then reacting the compound with a compound having two or more primary amino groups. (11) Step (b) is carried out by covalently bonding heparin, which has been primary aminated by desulfating a portion of the N-sulfate groups of heparin, to the functional group directly or via a coupling agent. The manufacturing method according to claim 9 or 10. (12) The method according to claim 9 or 11, wherein the coupling agent is a compound having at least two aldehyde groups. (13) The method according to claim 12, wherein the compound having at least two aldehyde groups is glutaraldehyde. (14) By applying the steps (a) and (b) according to any one of claims 9 to 13 to the blood contact surface of the base material constituting the medical device, the contact surface is antithrombotic. 1. A method for manufacturing a medical device, characterized by including a step of imparting sex. (15) In an artificial lung using a porous membrane having a large number of pores as a gas exchange membrane, the blood circulation surface is as claimed in claims 1 to 6.
An artificial lung characterized by being formed of the medical material according to any one of the above. (16) Item 15, wherein the pores of the porous membrane are filled with a large number of fine particles having a smaller diameter than the pores.
Artificial lung as described. (17) The artificial lung according to claim 16, wherein the fine particles are silica. (18) Claims 9 to 1, wherein the blood circulation surface of a porous membrane having a large number of pores used as a gas exchange membrane is used as a base material.
3. A method for producing an artificial lung, which comprises performing step (a) and step (b) according to any one of 3 above.