JPS5915121B2 - Manufacturing method for medical polymer materials - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION 10. The present invention relates to a method for producing a medical polymer material, particularly a method for producing a regenerated cellulose-based medical polymer material having anticoagulant properties.

It is known to obtain medical polymeric materials with anti-blood coagulation properties by immobilizing heparin on polysaccharides such as cellulose15.

For example, after treating a cellulose membrane with bromine cyanide,
A method of immobilizing heparin is proposed in Transactions of the American Society for Internal Organs, Vol. 22, p. 654 (1976);20 it is also A method for immobilizing heparin by treating cellulose is disclosed in JP-A-53-57288.
However, in these conventional heparinization methods for cellulose, heparin is directly bonded and immobilized to the cellulose substrate, so the amount of heparin immobilized is small and the mobility of the immobilized heparin is low. Due to these problems, it has been difficult to obtain practical anticoagulant properties.

Furthermore, the processing agents used in the process of hepar-30 phosphorization were highly toxic, and a lot of effort was required for cleaning them before they could be used as medical materials. As a result of various studies regarding the above-mentioned problems, the present inventors reacted cellulose, particularly regenerated cellulose, with 35 glycidyl acrylate or glycidyl methacrylate to create a graft copolymer having regenerated cellulose as the main chain. Next, by reacting heparin to the epoxy group of this graft copolymer and bonding and immobilizing it, a surprisingly large amount of heparin can be immobilized and a medical polymer with excellent anticoagulant properties can be obtained. It has been found that molecular materials can be obtained.

The present invention is based on this knowledge, and its gist is to react regenerated cellulose with glycidyl acrylate or glycidyl methacrylate in the presence of a polymerization initiator to produce a graft copolymer, and to add heparin to this graft copolymer. The purpose is to react with heparinization.
The graft polymerization rate of the graft copolymer obtained by the method of the present invention varies depending on the use, but is generally 0.
The reaction is preferably carried out at a concentration of 1 to 90%, preferably 0.5 to 30%.

Typical examples of regenerated cellulose used in the present invention include cuprammonium rayon, viscose rayon, and saponified cellulose.

Copper ammonia rayon is preferred as the material for hollow fibers for artificial kidneys. Although any of the two polymerizable monomers used in the present invention, glycidyl acrylate or glycidyl methacrylate, is effective, glycidyl methacrylate is preferred industrially because of its ease of handling.

When making a graft copolymer, it is preferable to use regenerated cellulose that has been molded in advance into the desired object or a shape close to the desired object.

Such molded articles include hollow fibers, flat membranes, filaments, and the like.
The reason why it is preferable to use molded products in this way is due to the relationship with the solvent during polymerization.

For example, in the case of cuprammonium cellulose, the solvent used is limited to cuprammonium. However, it is extremely difficult to graft-polymerize glycidyl acrylate or glycidyl methacrylate in a copper ammonia solution. Even if graft polymerization were possible, the epoxy group is likely to ring open under such conditions, making it extremely difficult to fix heparin in the subsequent second step. Therefore, except in special cases, it is advantageous for the regenerated cellulose to be a molded product. When performing graft polymerization of molded products, use ceric ammonium nitrate or dibasic sulfate as a polymerization initiator.
Cerium salts such as cerium ammonium, hydrogen peroxide/
Ferrous sulfate type, hydrogen peroxide/ferric sulfate type, sodium periodate, permanganate acid 18, tributyl boron, etc. can be used, but from the viewpoint of toxicity, ease of cleaning, catalytic ability, etc. , cerium salts are most preferred. The graft polymerization temperature can range from a temperature at which freezing of the polymerization solution does not occur (nearly 0°C) to about 90°C, but by lowering the ring-opening rate of the epoxy group, heparin can be made more For the purpose of efficient bonding and prevention of gelation of the graft polymer, it is desirable to carry out the graft polymerization at as low a temperature as possible, and the generally preferred range is 0 to 60°C, particularly preferably 0 to 60°C. It is in the range of 40°C.

When performing graft polymerization in an aqueous solvent, the concentration of glycidyl acrylate or glycidyl methacrylate is preferably within a range that dissolves in water, for example, glycidyl methacrylate is within about 1% (at room temperature). The reason for this is that when it is water-insoluble at high concentrations, homopolymers are often formed significantly, reducing the efficiency of graft polymerization, and that most of the epoxy groups in the homopolymers ring open and stick to the surface of the molded product. However, fixation of heparin becomes extremely difficult. The graft polymerization rate depends on the temperature, the concentration of monomer and polymerization initiator, the hydroxyl group concentration of the regenerated cellulose as the base,
It can change depending on time etc.

Since the optimum polymerization rate differs depending on the use of the molded article, the above parameters are set by experiment accordingly.
The molded article or polymer that has been graft-polymerized in this way is then reacted with heparin.

After washing the catalyst and unreacted monomers, the molded product subjected to graft polymerization in water is immersed in an aqueous solution of heparin and left at room temperature or slightly heated to react with heparin. Fixed.

What is the reaction temperature for heparin fixation? Although it is possible to heat up to about 90°C, the lower the temperature, the better to take a longer time.

Considering decomposition of heparin, prevention of structural changes in regenerated cellulose, economical fixation speed, etc., between room temperature and 70℃,
The temperature is preferably 30'C to 50C for about 1 hour to 48 hours, more preferably about 5 to 20 hours. The heparin concentration during the reaction should be as high as possible. The reason is,
This is thought to be because heparin is a polymer, and the higher the concentration, the fewer the bonding points to the graft copolymer per molecule of heparin and the higher the density of immobilization. In this way, the mobility of immobilized heparin will be increased, and it can be expected to prevent a decrease in biological activity. Possible heparin concentration range is 1-5
0%, preferably about 5 to 40%. In the case of heparin immobilization, when a tertiary amine such as pyridine or acetic acid is used as a catalyst, the amount of heparin immobilized is slightly higher than when no catalyst is used, or is at the same level.

However, considering the trouble of cleaning the catalyst, it is practical to fix it without a catalyst.

The medical polymer material produced by the method of the present invention has a large amount of heparin immobilized because glycidyl acrylate or glycidyl methacrylate graft-polymerized to regenerated cellulose has one epoxy group for each repeating unit. obtain.

Therefore, it has extremely high anticoagulant properties. Furthermore, since an epoxy group is used to fix heparin, the reaction can be carried out under extremely mild reaction conditions. This is very effective in maintaining the activity of biological substances. Furthermore, the heparin immobilized by the method of the present invention has extremely high mobility and can be in a nearly free state, resulting in highly biologically active heparin. Next, Examples will be shown to specifically explain the present invention, but before that, test methods in Examples of the present invention will be summarized.

(1) Semi-quantitative analysis of heparin Heparin is stained with azure A, and its blue-purple color is due to the fact that azure A forms a complex with a sulfate group, and its maximum absorption wavelength ranges from 654 to 5337.
It is known that this corresponds to a shift near Itm.

(For example, Biosimica et Biosimica Acta 1
84, p. 646 (1969)) Applying this, a transparent heparinized film was stained with 0.025% Azure A staining solution (PH = 3.5 to 3.6) at room temperature for 10 minutes, and then stained for 10 minutes. By washing with running water for a minute, air drying, and measuring the absorbance at a wavelength of 570 nm, it is possible to compare the amount of heparin immobilized between the same heparinized sample films. This can be understood by the correspondence between the absorbance of the heparinized sample and the human plasma coagulation time ratio, the canine whole blood coagulation time, the sulfur element content, etc., as shown in Example 2 below. The absorbance was set to a wavelength of 570 nm because the maximum absorption peak is below 550 nm in a wet state, but this shifts to around 570 nm in a dry state. However, samples fixed with heparin must be
% (W/V) saline solution twice for 10 hours each time, and weakly bound heparin that is not a covalent bond was extracted out of the system.

2) Elemental analysis The amount of heparin immobilized was determined by measuring the sulfur content.

The method involves burning about 1 gram of a sample in a pump, and dissolving the decomposition products in a 10 ml solution of water containing several drops of hydrogen peroxide.
Completely absorb in N caustic soda solution, acidify with approximately 18% by weight aqueous hydrochloric acid solution, heat and filter, add heated barium chloride solution to the filtrate to convert sulfur to barium sulfate, weigh the sulfur content, and calculate the sulfur content. . (3) Measurement of clotting time ratio Behring Werke A.G.
) standard human plasma was kept at 37°C for 5 minutes in a 10 mm (1) test tube, extracted with 25% saline as shown in test method (1), and washed with physiological saline. 6.0 cr of heparinized film was immersed in this.

After 30 minutes, the film is removed, and immediately 0.2 ml of a 0.1 molar calcium chloride solution is added and the coagulation time is measured in the same manner as the usual Lee-White method.

Separately, a film that is not heparinized is also measured at the same time, and the blank coagulation time is taken as a blank coagulation time.The ratio with the coagulation time of the heparinized film is taken as the coagulation time ratio. The blank and test samples are averaged at n-3. (4) Measurement of coagulation time using canine whole blood Apply the Riichi-White method to a flat membrane, extract with 25% saline as shown in Test 1 (1), and heparinize by washing with physiological saline. Place the film on a glass plate and place the 2.0 in the center.
It was held down with a silicone gasket having a thickness of 5 mm with a hole of (:FlLφ), and was further pressed down and tightened with a glass plate having a hole of the same diameter.

Next, after draining the first 0.5 ml from the dog's jugular vein, collect about 5 ml of blood, and send 0.5 ml of blood to the above 1 sample film.
Drop 1 ml at a time, spread it evenly on the film, and tilt it every 5 minutes for the first 20 minutes, then every 2 minutes after that, and measure the time until the blood coagulates and stops moving. do. Each of the three dogs was measured once, and the average value is shown. This method uses LindhOlmCel (LindhOlmCel).
1). (5) Measurement of mass transfer coefficient This method uses about 50 d of test substance solution (urea 100Tr19/Dj or vitamin B, 21O~/De, both concentrations in physiological saline) on both sides of the sample film.
Then, create a sealed room filled with physiological saline, continue stirring at a constant speed for 30 minutes at 37°C, find out the amount of solute in the test substance solution transferred to the physiological saline from the concentration, and then Calculate the mass transfer coefficient U (CT!L/Sec) using the formula.

Here, A is the effective transmission area (Cd) of the measurement film, V
A and B are the volumes of the test solution and physiological saline (d) (-
50), t1, and T2 are the measurement start time and end time (S
ee) ΔC1, ΔC2, (~/Dj) are T and T, respectively
2 shows the solute concentration difference between the test solution and physiological saline in No. 2.

In addition, the concentrations of urea and vitamin Bl2 were determined by the Micon method and 268m7!, respectively. It was determined by measuring the absorbance of L. (
6) Measurement of water permeation rate Create a room with one side of the film closed and the other side open, apply pressure from the closed side, and set the pressure difference between the open side and the open side to 3011 mmHg. Leave it to stand for 30 minutes, measure the amount of water that passed through the film during that time, measure the effective permeation area in advance, and express it in the unit of (ml/HOUrllllLHg-i), which is the unit of water permeation rate. Calculate so that

Measurement is performed three times every 30 minutes, and the average value is taken as the average value. (7) Determination of epoxy groups Measured by the hydrochloric acid pyridine method.

That is, a film containing about 100 mf as a polymer calculated from the graft polymerization rate of a film obtained by graft polymerizing glycidyl acrylate or glycidyl methacrylate was added to 100 m of a 0.015N hydrochloric acid pyridine solution.
1 (equivalent to about twice the theoretical amount of epoxy), heated under reflux for 3 hours, and then back titrated with a 1/10N caustic soda methanol solution using phenolpthalene as an indicator.

An equal volume of distilled water was added to the pyridine solution to clarify the endpoint before titration. The difference from the theoretical amount of epoxy groups calculated from the consumption amount of caustic soda was expressed as a percentage of the theoretical amount and was defined as the ring opening rate. Example 1 In a 500ml Mitsuro separable flask, add 30ml of distilled water.
0ml and 1.42 grams of glycidyl methacrylate (commercially available product purified by distillation) and stirred with a magnetic stirrer for about 30 minutes.

I dried this in advance in a phosphorus pentoxide desiccator for 3 days to determine the bone dry weight, and found that it was 50m7 with a thickness of 20μ! L
1.00 g of copper ammonia cellulose film (weighed each film in advance) of φ was introduced. Furthermore, 0.
Add 5 ml of 1N nitric acid, connect the aspirator suction port, nitrogen supply port, and temperature measurement port to the flask using the three tubes. First, use the aspirator to perform nitrogen replacement.
With a slight supply of nitrogen into the flask, 0.58 g of ceric ammonium nitrate was added, nitrogen was replaced once more, stirring was continued for 30 minutes, and the reaction was then carried out. After stopping, the film was transferred to a beaker containing 500 ml of distilled water, and the distilled water was changed 5 times to wash the film. One part was washed with acetone and dried under vacuum, and the graft polymerization rate was measured. On the other hand, the first part is 10
% (W/V) of heparin soda solution at 50°C for 20 hours, then the film was taken out and washed with tap water for one day and night, followed by extraction with 25% saline as shown in Test Method (1) of the main text. The cells were washed thoroughly with physiological saline and stored in fresh saline (physiological saline; hereinafter the same). Heparinized samples were washed with distilled water and stained with Azure A (Test method (1).
)), human plasma clotting time ratio (test, test method (3)), elemental analysis (
Test method (2)) was conducted, and in all Examples, heparin soda was 1501. U. / Wa's glycidyl methacrylate is a commercially available product.
The product was purified by vacuum distillation at ℃. From the above tests, the graft polymerization rate of this heparinized sample was 37.5%.
, the absorbance of Azure A staining is 155, the human plasma clotting time is CO (does not clot at 37°C for more than 2 days), and the sulfur content is 0.
．． It was 60% by weight. The human plasma coagulation time in a control (untreated sample) tested under the same conditions was 7 minutes. Example 2 In the same manner as shown in Example 1, under the conditions shown in Table 1,
Copper ammonia cellulose film (20 μm thickness, 50
mmφ) was graft-polymerized with glycidyl methacrylate, similarly heparinized and washed, and the sample was stored in saline.

The above samples were tested according to Test Methods (1) to (7), and the results are also shown in Table 1. Yu, J-1, J
-5 and J-7 were subjected to elemental analysis using test method (2), and the sulfur content was trace and 0.55, respectively.
% by weight, 0.70% by weight, and therefore J-1 has almost no heparin soda bound, consistent with the results of human plasma coagulation time ratio, canine whole blood coagulation time, staining, etc. I understood.

This is probably because gelation of the glycidyl methacrylate polymer occurred during graft polymerization. In addition, in Table 1,
J-0 is an untreated copper ammonia cellulose film, and J-0' is a copper ammonia cellulose film directly immobilized with heparin and then washed. Regarding the human plasma coagulation time ratio, only J-0 shows the time.
The other values indicate multiples of JO of 6.5 minutes, and when the value is 1, it represents the same state as in the first embodiment. Example 3 Copper ammonia cellulose film (20 μm thickness,
50 mmφ), glycidyl acrylate was graft-polymerized at 25°C for 50 minutes using ceric ammonium nitrate (Test A) and ferrous sulfate (Test B) as polymerization catalysts. The sample was then heparinized and subjected to the same washing procedure.

Samples were stained with Azure A and human plasma clotting time ratio (Trial 1)
The results of tests conducted using experimental methods (1) and (3)) are shown in Table 2, and it can be seen that glycidyl acrylate also has the same function as glycidyl methacrylate. Example 4 A module (hollow fiber) in which 800 hollow fibers of copper ammonia cellulose (outer diameter 230μ, inner diameter 200μ) were filled in a cylindrical polycarbonate container with a dialysate inlet and both ends were fixed with urethane adhesive. Effective length of 12
0mm) was created.

After completely purging with nitrogen, add 500 ml of water, 2.5 gr of glycidyl methacrylate, 1.0 gr of ceric ammonium nitrate, and 2 ml of nitric acid (1/10N) to a flask that can be sealed, and the above small module was also completely purged with nitrogen. The flask was connected to the flask by a circuit, and the fluid was circulated through the module using a roller pump in a sealed device for 50 minutes at room temperature at a flow rate of 50 ml/m.

After that, rinse with distilled water 31, wash, and reduce to 10%.
A soda solution was filled into the hollow fibers and circulated at 50° C. for 10 hours, the heparin soda solution was removed, and the fibers were stirred with tap water for one day. After washing, the module was then circulated with 500 ml of 25% saline for 10 hours, and then thoroughly washed with saline. When the module thus obtained was dyed with Azure A, it became deep blue-purple.

In addition, the module was quickly filled with dog blood collected by the method described in Test Method (4) to prevent air from entering from one side of the module. After that, only the hollow fibers were cut and removed from the module, kept in a polyethylene bag at room temperature for 3 hours, and then examined for the presence of blood clots.The heparinized fibers were not clotted, but the untreated hollow fibers were found to be clotted. The blood inside him had clotted. Example 5 A copper ammonia cellulose film (thickness 2
Glycidyl methacrylate was added to 01tm150Ryuφ) at 40°C, 20°C, and 4°C for 30 minutes, 60 minutes, and 18 minutes, respectively.
Graft polymerize for 0 minutes, immediately wash thoroughly with distilled water, and incubate for 10 minutes.
% heparin soda aqueous solution (W/V) at 50° C. for 10 hours.

Thereafter, it was washed in the same manner as in Example 1, further washed with distilled water, and tested according to experimental methods (1) and (3). In addition, a sample that had only been subjected to graft polymerization was sufficiently washed with distilled water, and the epoxy groups were quantified using test method (7). The results are shown in Table 3, and it can be seen that the lower the temperature, the lower the ring opening rate. Prescription
Comparative Example 1 The following test was carried out in accordance with the method described in Transaction American Society, Artificial Internal Organs, Vol. 22, p. 654 (1976).

(However, heparin extraction using concentrated saline was not included in the original method.) Copper ammonia cellulose film (20 μm 1
Five films each (thickness: 50 mmφ) were placed in 500 m of a 0.2 molar soda solution of 10% cyanogen bromide.
1 (PH-7.3) at 4°C for 30 minutes, changing the solution and repeating the operation four times. Soaked in 100ml of sodium morphoborate solution at 4℃ for 3 days and nights,
The specimen was washed with running water for 2 hours using distilled water from Step 1, then immersed in 25% saline for 10 hours, thoroughly washed with distilled water, and then stored in physiological saline.

Comparative Example 2 The following test was conducted in accordance with Example 1 of JP-A-53-57288.

However, heparin extraction using concentrated saline is not included in the original method.
Add 0.05 mol of sodium metaperiodate to 100 ml of distilled water, keep it at 5°C, and immerse the copper ammonia cell Danko film (20 μm thick, 50 m7! Lφ) for 30 minutes, and then add enough distilled water. Wash with 17% (W/
V, 250001. U. /ml) of heparin aqueous solution (pH adjusted to -4.0 with sulfuric acid) at 40°C for 30 minutes.

After the heparin reaction, the film was washed in the same manner as in Comparative Example 1 and stored in saline. The films of Comparative Examples 1 and 2 and J-5 of Example 1 were tested using test methods (1), (3), and (
When compared with method 4), the results are shown in Table 4, proving the superiority of the method of the present invention.

The human plasma coagulation time and canine whole blood coagulation time were measured without any treatment of cuprammonium cellulose, and the results were 7.5 minutes and 8 minutes, respectively.

Claims (1)

[Claims] 1. A medical polymer material comprising reacting regenerated cellulose with glycidyl acrylate or glycidyl methacrylate in the presence of a polymerization initiator, and reacting the reaction product obtained by the reaction with heparin. Production method. 2. The manufacturing method according to claim 1, wherein the regenerated cellulose is a molded article, and the molded article is reacted with glycidyl acrylate or glycidyl methacrylate. 3. The manufacturing method according to claim 2, wherein the molded article is a hollow fiber. 4. The manufacturing method according to claim 2, wherein the molded product is a flat membrane. 5. The manufacturing method according to claim 1, wherein the reaction between regenerated cellulose and glycidyl acrylate or glycidyl methacrylate is carried out in an aqueous solvent using ceric ammonium nitrate as a polymerization initiator. 6. The manufacturing method according to claim 1, wherein the reaction between regenerated cellulose and glycidyl acrylate or glycidyl methacrylate is carried out at a temperature above the freezing point of the polymerization solution and below 60°C. 7. The manufacturing method according to claim 1, wherein the heparin reaction is carried out in an aqueous solution at 10 to 70°C.