JP2711112B2 - Medical materials and medical instruments - Google Patents

Description

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

<Conventional Technology> Conventionally, various antithrombotic materials for use in artificial lungs, catheters, artificial hearts, and the like have been devised. One of the methods is to fix heparin on the substrate surface. The method includes a method in which heparin is ionically bonded to the substrate and a method in which heparin is covalently bonded to the substrate.

<Problems to be solved by the invention> However, in ionic bonding, heparin is detached when coming into contact with blood, or it is too strongly bound to a sulfate group that is important for the expression of heparin activity. However, in the case of covalent bonding, the bonding portion with AT-III was used as the bonding point with the substrate, and the surface activity of heparin was not sufficient. Attempts have also been made to crosslink heparin with glutaraldehyde after heparin is ionically bonded to the cationic surface. However, since aldehyde groups mainly react only with primary amino groups, etc. Since there was almost no primary amino group and the substrate could not be shared with the substrate without a primary amino group, the sustainability of the effect was insufficient.

Therefore, an object of the present invention is to provide a medical material and a medical device which are excellent in antithrombotic properties and which solve the above problems.

<Means for Solving the Problems> The present inventors have found that it is difficult to obtain sufficient antithrombotic properties even when trying to fix heparin on the surface of a base material as in the prior art, or to use a useful site for this fixation. In view of the fact that they are used, as a result of repeated studies, they succeeded in fixing while leaving useful sites, and have reached the present invention.

That is, heparin used is one in which the N-sulfate group is partially desulfated and primary aminated.

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

This method of preparation is optional, but includes hydroxyethyl methacrylate (HEMA) and methyl methacrylate (MM
It is preferable to introduce a functional group capable of binding the above-mentioned heparin onto a substrate via a compound containing A). This functional group is preferably a primary amino group or an epoxy group.

Hydroxyethyl methacrylate (HEMA) and methyl methacrylate (MMA) exist separately in each segment, and the functional group has a structure that exists in the segment containing hydroxyethyl methacrylate (HEMA) as a component. Good to use.

As the compound containing hydroxyethyl methacrylate (HEMA) and methyl methacrylate (MMA), a compound containing 30% or more of hydroxyethyl methacrylate (HEMA) is preferably used.

Such a substrate and heparin are prepared, and heparin is immobilized on the surface of the substrate. Immobilization of the compound having the functional group and heparin on the base material is performed directly or by covalent bonding via a coupling agent. As the coupling agent, a compound having two or more aldehyde groups such as glutaraldehyde can be used.

The medical material obtained by the above-mentioned manufacturing method can be used as various antithrombotic materials, and this antithrombotic material can be used particularly for medical instruments having a portion that comes into contact with blood. Examples of the medical material include hollow fibers and tubes, and examples of the medical device include an artificial lung, an artificial heart-lung circuit, a catheter, and an artificial heart.

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

 Hereinafter, the present invention will be described in detail.

The medical material of the present invention comprises a substrate having a functional group such as an amino group, preferably a primary amino group, on which N-
Heparin in which a part of sulfate group is desulfated and primary aminated is fixed.

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

The substrate may be properly used depending on the application, but polypropylene, polyvinyl chloride, polyurethane or the like is generally used.

In many cases, the substrate itself generally does not have the above functional group. In such a case, the functional group is introduced into the substrate. Although there are various introduction methods, it is preferable to adopt the following method.

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

The limitation is due to the high blood and biocompatibility and safety of the polymer itself, the relatively easy synthesis of the polymer, and the simple coating.

Here, the polymer containing HEMA and MMA is preferably in the form of a block copolymer, each of which forms a segment (referred to as an A and a B segment) and is bonded. In this polymer, each segment preferably has a structure that exists separately. The reason for this is that the segments including the MMA having good water resistance can be brought into close contact with the base material by forming and joining the segments in this manner.
Here, the A and B segments are HEMA and
A fragment (portion) mainly containing MMA.

And the said functional group is made to exist in the segment A which makes a hydroxyethyl methacrylate (HEMA) a component. As the functional group, a primary amino group, an epoxy group, and the like can be considered.

In order to have the above functional group, a compound having a functional group such as glycidylmethyl methacrylate (GMA) may be provided in the HEMA segment in addition to HEMA. In this case, the epoxy group in GMA is reacted with a compound having two or more primary amino groups, and
It is preferred to introduce a secondary amino group.

Similarly, in segment B having MMA as a component, M
Compounds other than MA may be included.

However, hydroxyethyl methacrylate (HEMA)
And methyl methacrylate (MMA) containing 30% hydroxyethyl methacrylate (HEMA)
It is preferred to contain the above. This is because when the amount of HEMA is reduced, the surface becomes hydrophobic, and the introduced functional group hardly appears on the surface during the reaction in an aqueous solution.

Next, heparin to be fixed to the above-mentioned substrate having a primary amino group will be described.

Heparin is widely known as a compound showing antithrombotic properties,
It has an N-sulfuric acid site called NHSO ₃ Na. As described above, a problem arises when heparin is directly fixed on the surface of the base material.

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

The degree of primary amination is preferably such that the amount of primary amino groups is 5 to 25% of all amino groups in heparin. More preferably, it is 10 to 20%, and still more preferably 10 to 15%. Here, the amount of primary amino groups in heparin is defined as N
-Includes both the primary amination by desulfation of the sulfuric acid site and that of heparin itself. If the amount of the primary amino group in heparin is less than 5%, it is difficult to fix the primary amino group to the substrate, and if it exceeds 25%, the activity of heparin decreases, so it is preferable that the amount is 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. 5.5 NH ₂ SO ₄ 0.4 ml in 10 ml of this heparin solution
And reacted at 95 ° C, 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. The results are shown in FIG.

Further, the reaction was carried out under conditions (2% heparin 0.04N HCl 95 ° C) in which all sulfoamino groups in heparin are desulfated in the literature, and the increase in the amount of amino groups over time was measured in the same manner.

 The results are shown in FIG.

(Note 1) Ninhydrin reagent: 2 g of ninhydrin and 0.3 g of tohydrindantin are dissolved in 75 ml of methyl cellosolve, and 4N
Add 25 ml of sodium acetate (pH 5.5).

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 the amino group is quantified as the degree of color development of leucine.

In FIG. 1, the mark ○ indicates the anticoagulant property of blood against coagulation factor 2, and the mark ● indicates the anticoagulant property of blood against coagulation factor 10. There is a coagulation factor that does not express its anticoagulant property unless heparin is in a high molecular weight range, and a coagulation factor that expresses its anticoagulant property even in a low molecular weight range,
FIG. 1 shows that the anticoagulant activities of heparin in both high and low molecular weight regions are almost the same, taking the second and tenth factors representative of the coagulation factors as examples.

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

Next, the immobilization of the base material having a functional group obtained as described above and heparin which has been partially aminated and primary aminated by desulfation of the N-sulfuric acid site will be described.

The immobilization of the base material and heparin can be performed by using a compound having at least two aldehyde groups as an example of a coupling agent and reacting a primary amino group with an aldehyde group. Examples of such aldehyde compounds include glutaraldehyde.
As the coupling agent, polyethylene glycol diglycidyl ether or the like may be used.

Further, the substrate and heparin can be directly bonded. At this time, the functional group should be an epoxy group, an aldehyde group, or the like that can bond to an amino group.

As described above, the medical material in which heparin having a primary amination by immobilizing a part of the N-sulfate group on the base material having the above functional group is immobilized has an antithrombotic property utilizing the antithrombotic property of heparin. Material, which can be used for at least a part of a medical device, such as an artificial lung, a heart-lung machine catheter, an artificial heart, etc., which comes into contact with blood.

In particular, when a porous membrane having a large number of pores (for example, a hollow fiber) is treated as described above as a gas exchange membrane, a hollow fiber having antithrombotic properties can be obtained. When used in the lung, an artificial lung with excellent antithrombotic properties can be obtained.

Further, it is preferable to previously fill fine particles having a smaller diameter than the fine pores in the fine pores of the porous membrane used for the artificial lung. The reason is that the gas exchange membrane is porous and hydrophobic, so that the polymer cannot be uniformly coated on the gas exchange membrane, and therefore, the antithrombotic property cannot be sufficiently exerted. Is made hydrophilic, and plasma may leak from the pores during long-time circulation.

Japanese Patent Application Laid-Open No. 62-6437 discloses a method for filling fine particles into a porous membrane.
It should be as described in No. 4. Here is a brief description.

A dispersion liquid of fine particles having a diameter smaller than the pores is allowed to flow through the porous membrane so that the fine particles are clogged in the pores.

Examples of the material of the fine particles include 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. The average diameter of the fine particles is 0.003 to 1.0 μm, preferably
It is about 0.003 to 0.5 μm.

The fine particles are formed into a dispersion and applied to the gas exchange membrane. Any dispersion medium may be used as long as it is stable to the fine particles and the gas exchange membrane. For example, water, alcohols and the like 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 flowing the dispersion to form a gas exchange membrane. It is necessary to make the surface hydrophilic.

When the gas exchange membrane is a hollow fiber, the fine particles can be suitably filled by passing a suitably pressurized fine particle dispersion from inside the hollow fiber.

<Example> Hereinafter, the present invention will be described specifically with reference to examples.

I. Introduction of Functional Group into Substrate (Example 1) Polymers P1 to P4 having various compositions shown in Table 1 below were prepared,
The microporous polypropylene was coated on a flat membrane. The adjustment of the polymer solution was performed by diluting a 15% polymer solution of methyl cellosolve with a methanol or methanol: acetone = 9: 1 solution to a 2.5% polymer solution.

Where hydroxyethyl methacrylate (HEMA)
Glycidylmethyl methacrylate (GMA) is bonded to the segment containing, and an epoxy group is introduced, and acrylic acid (AA) is introduced to the segment containing methyl methacrylate (MMA).

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.

Primary amino groups among all amino groups in the obtained heparin include 11 amino acids including those originally possessed by heparin and those primary-aminated by desulfating the N-sulfuric acid site.
%Met.

III Immobilization and evaluation of heparin on the substrate (Example 3) The membrane prepared according to Example 1 was adjusted to pH10.
It was immersed in 1% ethylene diamine and 1.7% PGD-10 (polyethylene glycol diamine) at 45 ° C. for 24 hours.

Subsequently, a 0.5%, pH 4.5 acetate buffer solution was prepared for the partially desulfated heparin reacted in Example 2 and the heparin before the reaction, and the membrane was immersed in this solution at 45 ° C. for 24 hours. Subsequently, it was immersed in a 2.5% glutaraldehyde pH 4.5 acetate buffer solution at room temperature for 24 hours. Then 1% NaBH ₄ , pH 1
0 It was immersed in a carbonate buffer solution at room temperature for 4 hours.

After immersing the treated membranes in 0.01N hydrochloric acid and staining with toluidine blue, the membrane immersed in heparin that was not desulfated was hardly stained, but a part of the polymers P2 to P4 Those immersed in desulfated heparin were stained reddish purple. However, polymer
None of P1 was stained. After immersion in a borate buffer solution of pH 9 for 15 hours to remove the ion-bound heparin and staining in the same manner, there was no change except that the membrane immersed in heparin was not stained at all.

(Example 4) Ion-binding property between base material and heparin in neutral region The membrane prepared according to Example 1 was adjusted to pH10.
It was immersed in 1% ethylene diamine and 1.7% PGD-10 (polyethylene glycol diamine) at 45 ° C. for 24 hours.

Subsequently, a 0.5%, pH 4.5 acetate buffer solution was prepared for the partially desulfated heparin reacted in Example 2 and the heparin before the reaction, and the membrane was immersed in this solution at 45 ° C. for 24 hours. Subsequently, the cells were immersed in a pH 7.4 phosphate buffer solution for 24 hours at room temperature and stained in the same manner. As a result, almost no staining was observed.

From this, it was considered that the base material and heparin did not ion-bond around neutral.

(Example 5) Antithrombotic property of heparin-immobilized tube A polyamide tube having an inner diameter of 1.4 mm was coated with the polymer shown in Example 1 and partially desulfated heparin prepared in Example 2 in the same manner as in Example 3. Was immobilized.
For these tubes, add 15
After washing for 2 hours and further washing with a phosphate buffer solution of pH 7.4 for 2 hours, the surface antithrombin activity of each tube was measured. Specifically, a heparin-fixed tube was cut to 56 cm, and thrombin 0 to 10 U / cc (4% Alb saline solution) was cut into 0.5 cm.
Inject ml and make contact with inner surface with rotary mixer for 15min. Thereafter, the thrombin concentration of the inner solution is measured, and the amount of the thrombin adsorbed on the inner surface is calculated. After washing the thrombin-adsorbing tube with a saline solution, 1.0 ml of 0.6 mM S-2238 is flowed through the tube at 2 ml / min, and 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 of the color development of S-2238 with respect to the amount of thrombin adsorbed on the inner surface is prepared.

Next, ATIII 1u / c was added to the tube to which thrombin had been adsorbed.
After the addition of c and incubation, S-2238 is similarly colored with thrombin remaining on the inner surface, and the thrombin remaining on the inner surface is calculated from the color development degree and a calibration curve.

FIG. 3 shows a change in the amount of residual thrombin on the inner surface when the incubation time of ATIII is changed. FIG. 3 shows the results. From the above, those dyed in reddish purple had activity.

FIG. 3 shows that the polymer having a glycidyl group introduced therein has surface heparin activity, and among them, those having a high HEMA content have high activity.

IV Fixation and evaluation of heparin in oxygenator (Example 6) Table 1 shows a polypropylene hollow fiber oxygenator (membrane area 0.8 m ² ) having an inner diameter of 200 µ, a thickness of 25 µ, a porosity of 45%, and an average pore diameter of 700 mm. The polymer P2 shown was coated, and this membrane was immersed in 1.7% PGD-10 (polyethylene glycol diamine) adjusted to pH 10 at 45 ° C. for 24 hours.

Subsequently, a partially desulfated heparin 0.5%, pH 4.5 acetate buffer solution reacted in Example 2 was prepared, and this membrane was immersed in this solution at 45 ° C. for 24 hours. Subsequently, it was immersed in a 2.5% glutaraldehyde, pH 4.5 acetate buffer solution at room temperature for 24 hours. Subsequently, it was immersed in 1% NaBH ₄ , pH 10 carbonate buffer solution at room temperature for 4 hours to obtain an artificial lung A.

On the other hand, for the same artificial lung, ethanol was flowed in from the blood inlet to hydrophilize the gas exchange membrane, and after replacement with distilled water, colloidal silica / water dispersion having an average particle size of 0.0125 μm was flowed into the gas exchange membrane. Filtered and filled the pores 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. After that, the same processing as in the artificial lung A
I got

In addition, a similar oxygenator without coating the polymer shown in Table 1 is referred to as oxygenator C for comparison.

(Example 7) Evaluation of antithrombotic property Artificial lung A in which heparin was fixed according to Example 6,
For B and C, femoral arteriovenous AV in 25 kg of dogs
Shunting was performed and blood was circulated at a maximum of 400 ml / min.

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

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

On the other hand, in the oxygenator C, circulation was disabled in 2 hours due to thrombus formation in the oxygenator. In addition, in the oxygenator A, plasma leakage was observed after 6 hours, but in the oxygenator B, plasma leakage was not observed after 12 hours.

<Effects of the Invention> In the present invention, a predetermined functional group is introduced into a base material in advance, and heparin is also partially desulfated at the N-sulfuric acid site to undergo primary amination to form a primary amination. Since heparin is fixed to the base material by bonding the functional groups directly or via a coupling agent, the resulting medical material is used as an antithrombotic agent in a medical device such as an artificial lung. Properties and plasma leakage were significantly improved, and it became possible to withstand prolonged use.

[Brief description of the drawings]

FIG. 1 is a graph showing the desulfation of the N-sulfate site of heparin and the change in activity. FIG. 2 shows the first by desulfation of the N-sulfate site of heparin.
4 is a graph showing a change in the amount of primary amino groups. FIG. 3 is a graph showing the surface heparin activity of the sample (medical material) obtained in Example 5.

 ──────────────────────────────────────────────────続 き Continuation of front page (72) Inventor Hiroshi Omura 5-3-1 Mt.

Claims (18)

(57) [Claims] 1. A medical material comprising heparin immobilized on a substrate, wherein the functional material is introduced onto the substrate via a compound containing hydroxyethyl methacrylate (HEMA) and methyl methacrylate (MMA). A medical material, wherein a group and a primary amino group of heparin are 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. The medical material according to claim 1, wherein heparin is a primary aminated amino acid by partially desulfating a part of the N-sulfate group. 4. The hydroxyethyl methacrylate (HEMA) and methyl methacrylate (MMA) are present separately in each segment, and the functional group is present in a segment containing hydroxyethyl methacrylate (HEMA) as a component. 2. The medical material according to claim 1, wherein the medical material has a structure. 5. The compound containing hydroxyethyl methacrylate (HEMA) and methyl methacrylate (MMA), wherein the compound containing hydroxyethyl methacrylate (HEMA)
The medical material according to claim 1, which contains 30% or more of 6. The medical material according to claim 1, which is used as an antithrombotic material. 7. A method according to claim 1, wherein at least a portion in contact with blood is provided.
A medical device formed from the medical material according to any one of claims 1 to 6. 8. The method according to claim 1, wherein at least a portion in contact with blood is provided.
7. A hollow fiber formed from the medical material according to any one of items 1 to 6. 9. A method comprising the steps of: (a) introducing a functional group onto a substrate via a compound containing hydroxyethyl methacrylate (HEMA) and methyl methacrylate (MMA); With a secondary amino group,
A method for producing a medical material, wherein the medical material is directly or covalently bonded via a coupling agent. 10. The step (a) comprises, after coating a compound containing hydroxyethyl methacrylate (HEMA) and methyl methacrylate (MMA) and having an epoxy group on a base material, The method according to claim 9, which is carried out by reacting a compound having a primary amino group. 11. The step (b) comprises the step of adding N to heparin as a functional group.
The method according to claim 9 or 10, wherein the method is carried out by directly or covalently bonding heparin which has been primary aminated by desulfation of a part of a sulfate group or via a coupling agent. 12. The method according to claim 9, 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. The method according to claim 9, wherein the step (a) and the step (b) according to any one of claims 9 to 13 are performed on the contact surface of the substrate constituting the medical device with the blood. A method for producing a medical device, comprising a step of imparting antithrombotic properties. 15. An artificial lung using a porous membrane having a large number of pores as a gas exchange membrane, wherein a blood circulation surface is provided.
7. An artificial lung formed of the medical material according to any one of claims 6 to 6. 16. The artificial lung according to claim 15, wherein a large number of fine particles having a smaller diameter than the fine pores are filled in the fine pores of the porous membrane. 17. The oxygenator according to claim 16, wherein the fine particles are silica. 18. The method according to claim 9, wherein a blood flow surface of a porous membrane having a large number of pores used as a gas exchange membrane is used as a base material.
13. A process for producing an artificial lung, comprising performing the steps (a) and (b) according to any one of the above (13) to (13).