JPS5811225B2 - Method for producing anticoagulant block copolymer - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing a medical polymer material, and more specifically, the present invention relates to a method for producing a medical polymer material, and more specifically, a block copolymer comprising a conjugated diene compound and an aromatic vinyl compound into which a hydroxyl group has been introduced has an anti-blood coagulation effect. The present invention relates to a method for producing an anti-coagulant block copolymer which has been endowed with anti-coagulant properties.

Several attempts have already been made to impart anticoagulant properties to a block copolymer (hereinafter simply referred to as block copolymer) comprising a conjugated diene compound and an aromatic vinyl compound.

For example, the Journal of Biomedical Materials Research by E. W. Merrill et al.
10, pp. 33-45, 1976), styrene-
There is a suggestion that heparin can be bonded to a butadiene-styrene block copolymer by reacting peracetic acid to introduce a hydroxyl group into the butadiene moiety.

Also, in the Journal of Biomedical Materials Research (vol. 13, pp. 347-364, 1976) by M.B. Sefton et al., there is a report on the styrene-butadiene-styrene block copolymer into which the above-mentioned hydroxyl groups have been introduced. It has been shown that heparinization can be achieved by applying polyvinyl alcohol together with geltaraldehyde or formaldehyde to fix heparin on the surface of the membrane, thereby imparting anticoagulant properties.

However, since all of these methods of fixing heparin use aldehydes, the amount of heparin that is effectively fixed by covalent bonds is small, and even though it initially appears that a large amount of heparin has been fixed, it does not affect the blood. When brought into contact with heparin, most of it is easily eluted, resulting in a problem in that the amount of heparin decreases and the activity also decreases.

Furthermore, when using a highly toxic substance such as glycuraldehyde, it must be thoroughly cleaned so that virtually no residue remains, which requires a great deal of time and expense.

The inventors of the present invention overcame the above-mentioned problems and worked diligently to create a block copolymer with even better anticoagulant properties. If you create a graft polymer with a structure in which the vinyl monomer is included in the graft polymer chain to the base polymer,
The method of the present invention was completed based on the finding that heparin can be easily and effectively immobilized on the graft strands.

That is, the present invention requires glycidyl acrylate or glycidyl methacrylate in the presence of a polymerization initiator to a block copolymer consisting of a conjugated diene compound and an aromatic vinyl compound, in which the double bond portion is hydroxylated. This is a method for producing an anti-blood coagulant block copolymer, which comprises graft polymerizing one or more vinyl monomers as one component, and binding heparin to the graft polymer.

In the method of the present invention, hydroxyl groups are introduced into a block copolymer as a substrate, and glycidyl acrylate (hereinafter referred to as GA) or glycidyl methacrylate (hereinafter referred to as GMA) is introduced into the block copolymer.
Although it is possible to introduce only the GA present in the graft chain, it is more preferable to introduce it in combination with other vinyl monomers to form a polymer graft polymer chain consisting of two or more vinyl monomer components. Alternatively, heparin is bonded to the epoxy group of GMA.

The anti-blood coagulant block copolymer produced by the method of the present invention is different from conventional methods in which it is bonded directly to a substrate or via a low-molecular substance such as gel tar aldehyde, etc., in a surprisingly large amount. It became possible to immobilize heparin in a highly active state.

Typical block copolymers used in the present invention include a block portion consisting of one component of aromatic vinyl compounds such as styrene, α-methylstyrene, vinyltoluene, etc., or a mixed component thereof; It is a copolymer consisting of one component of conjugated diene compounds such as 1, 3-butadiene and isoprene, or a block portion consisting of a mixed component thereof.

Here, if the polymer portion of the aromatic vinyl compound is A and the polymer portion of the conjugated diene compound is B, the block copolymer is generally represented by the following formulas (1) to (4).

C in formula (4) is a coupling agent such as silicon tetrachloride, sulfuryl chloride, di(chloromethyl)benzene, and n is a positive integer of 1 to 5.

The molecular weight of the block copolymer is usually 10,000 or more.

This lower limit is determined based on the practical strength when the block copolymer is made into a film.

Also, the ratio of A and B is A:B = 10:90 to 90:1
It is possible to achieve a weight ratio of about 0 (weight ratio).

A supports the strength of the block copolymer, and B gives elasticity and contributes to hydroxyl group-introduced heparinization. Therefore, in order to balance practical strength and the possible range of heparinization, the above A:B The ratio is determined.

Preferred block copolymer components are combinations of styrene and butadiene, or styrene and isoprene.

When carrying out the method of the invention, it is desirable to heparinize the block copolymer molded article.

This is because the solvent for the block copolymer into which hydroxyl groups have been introduced and the solvent for heparin (almost none exist outside Mercury) are not necessarily the same, and it is also possible to use heparin without impairing the physical properties of the block copolymer. This is because in order to fix it, it is sufficient to introduce hydroxyl groups only near the surface of the molded article.

Furthermore, it is practically unnecessary to fix heparin inside the molded article, and expensive heparin is wasted.

Molded products include those in the form of membranes or tubes, or those coated on the surface of medical instruments, such as those coated with a block copolymer on the part of a blood bag that comes into contact with blood.

Various known methods can be used to introduce hydroxyl groups into a polymer having double bonds.

These methods involve adding peracid to a solution of a polymer with double engravings, such as polybutadiene, polyisoprene, etc., and holding the solution at a temperature ranging from room temperature to 50°C for several hours or more. There are methods such as hydrolyzing acid residues with alkali, or adding formic acid and hydrogen peroxide to a solution to generate performic acid and converting double bonds into hydroxyl groups. may also be used.

When performing such treatment, in the case of molded products, hydroxyl groups are introduced using an organic acid as a solvent and a double bond cleaving agent such as peracid, and then organic acid residues are removed using a caustic soda solution such as alcohol. It is better to hydrolyze.

GA or GMA used in the present invention is used as a vinyl monomer component that is graft-polymerized onto a substrate in order to bond heparin to its epoxy groups.

In this case, when graft polymerizing only GA or GMA to a block copolymer into which hydroxyl groups are introduced (hereinafter abbreviated as hydroxyl block copolymer), the epoxy groups of GA or GMA are ring-opened. As a result, they tend to bond with each other, which tends to cause gelation of the grafted chains.

Therefore, in order to prevent the amount of heparin binding from decreasing, or from excessively binding epoxy groups to heparin and reducing heparin activity, it is necessary to use other vinyl monomers other than GA or GMA, such as acrylamide and N-vinylpyrrolidone. It is desirable to add GMA or GA as a component of the graft copolymer to reduce the proportion of GMA or GA present in the graft chain.

Although the results are almost the same whether GA or GMA is used in the graft copolymerization of the present invention, from an industrial standpoint, it is desirable to use GMA, which has excellent stability.

The vinyl monomer other than GA or GMA used in the graft copolymerization in the present invention is preferably a hydrophilic vinyl monomer, but it is also possible to mix a slightly hydrophobic vinyl monomer.

Examples of these vinyl monomers include acrylic acid or its salts, acrylamide, 2-hydroxyethyl methacrylate, acrylonitrile, N-vinylpyrrolidone, sodium styrene sulfonate, methyl acrylate, methyl methacrylate, vinyl acetate. , sodium allylsulfonate, etc., and one component or two or more of these components may be used as a mixed component.

The polymerization initiator used in the graft polymerization in the present invention may be any initiator as long as it causes a compound having a hydroxyl group to initiate graft polymerization, such as a hydroxylized glue copolymer.

Examples of such polymerization initiators include cerium salts such as ceric ammonium nitrate and ceric ammonium sulfate, hydrogen peroxide-ferrous sulfate systems, and hydrogen peroxide-ferrous sulfate systems.
Ferric sulfate, sodium periodate, tributyl boron, etc., but from the viewpoint of toxicity, ease of cleaning, polymerization initiation ability, and ease of handling, ceric ammonium nitrate, hydrogen peroxide-sulfuric acid, etc. Iron is preferred.

Examples of solvents for carrying out the graft polymerization of the present invention include aqueous,
In particular, it is most preferable to use water from both handling and economical points of view, but when there is a problem with the solubility of the vinyl monomer or when the content of aromatic vinyl compounds in the block copolymer is high, When the weight ratio is exceeded, a mixed solvent such as water-acetone, water-methyl alcohol, water-ethyl alcohol, water-dimethylacetamide, water-dimethylformamide, etc. can be used.

The mixing ratio is arbitrary, but in the case of a hydroxylated block copolymer with an aromatic vinyl compound content exceeding 50 parts, water/water/
Acetone or a composition with a water/alcohol ratio of 1 or less is desirable.

The reaction temperature in the graft copolymerization of the present invention is preferably as low as possible.

The reason for this is that the lower the graft polymerization temperature, the greater the amount of heparin bound at the same grafting rate (the percentage of the weight of the graft polymer bonded to the hydroxylated block copolymer relative to the base hydroxylated block copolymer). To become.

However, the lower the temperature, the longer the reaction time. Taking these into consideration, the practical reaction temperature range is from around 0°C, which is the freezing point of the reaction solution, to 70°C, preferably from about 5°C to 40°C.

The amount of graft polymer per unit surface area of the molded article can be determined using the amount of bound heparin after the heparinization reaction as the main indicator.

Generally, when graft polymerizing only GMA or GA, 1.5 x 1010-4 (/cm2) to 3.5 x 1
The amount of heparin binding reaches the maximum at a surface graft chain amount of 0-4 (gr/cm2), but when other vinyl monomers are introduced through graft copolymerization, the maximum heparin binding amount is even higher. Shift in the direction indicating the amount of surface graft chains.

It varies depending on the type of other vinyl monomer co-grafted with GA or GMA, but generally D is 3X10-4~
10101010X1O-4 (often maximum at about 7).

Factors that affect the amount of graft polymerization include reaction temperature, type and amount of hexamer, concentration of polymerization initiator, reaction time, etc.

The reaction charge ratio in the graft copolymerization reaction of the present invention is based on 100 parts by weight of water as a reaction solvent.
It will look like this:

GA or GMA is in the range of 0.001 to 1.2 parts by weight, the lower limit is determined by the range in which heparin can be substantially fixed even when reacted simultaneously with other vinyl monomers, and the upper limit is determined by the solubility in water. It will be done.

Polymerizable vinyl monomers other than GA or GMA are GM
The amount charged varies depending on the reaction rate ratio with A or GA,
Generally, heparin binding occurs at about 500 times the amount of GMA or GA, so the amount is 0.01 to 600 parts by weight relative to the solvent.

However, this upper limit is determined by the solubility in the solvent (actually, in many cases, solubility takes precedence).

The polymerization initiator varies depending on its type.

In the case of ceric ammonium nitrate and hydrogen peroxide, it has been empirically determined that 0. OO1mmol/l~5mmol
l/l range, and nitric acid with ceric ammonium nitrate or ferrous sulfate with hydrogen peroxide are at their respective starting points.

It is best to use about 5 times the amount of the drug.

Since the amount of the hydroxylated block copolymer is often reacted in the form of a molded article, it is appropriate to express it in terms of the reaction surface area relative to the weight of the monomers charged.

The lower limit of the reaction surface area is arbitrary, but the upper limit is empirically determined as 6 X 1O-3 (gr 7cm2) (
The reaction surface area is approximately the same as the reaction surface area.

The graft polymerization reaction of the present invention can be stopped by taking out the reactant from the reaction tank and washing it with a large amount of water, but after stopping the reaction, it is immediately washed thoroughly with water and used for the next heparin binding reaction.

Since the epoxy groups of GA or GMA ring open when left in water for a long time even at room temperature, it is necessary to carry out the heparin binding reaction as quickly as possible.

The heparin used in the heparin binding reaction may be commercially available one derived from pig intestinal mucosa, one derived from bovine lung, or the like.

Usually, these are used in the form of powdered heparin sodium of about 100 to 200 (units/mg), and the block copolymer that has undergone graft polymerization is immersed in this dissolved in water, and the temperature is adjusted to 30 to 80°C. Preferably, the temperature is maintained at 50 to 70°C, and the reaction is carried out for about 1 hour to 48 hours.

The reaction temperature at this time is preferably lower in order to suppress the decomposition and decrease in activity of heparin, and higher in order to increase the reaction temperature and binding amount.

In addition, the heparin solution used in the reaction should have a pH of 5 from the viewpoint of activity.
~9 is good.

The higher the concentration of heparin aqueous solution, the more heparin coagulates, but if the concentration is too high, the viscosity will become too high and it will be difficult to immerse the molded product. With consideration, 1st section ~ 50% (W/■
) Desirably, the ratio is about 5 to 30.

For the heparin binding reaction, a tertiary amine, an acid catalyst, etc. can be used as a catalyst, but considering the degree of promotion of the heparin binding reaction and cleaning of residual catalyst, it is advantageous to carry out the reaction without a catalyst.

There is no particular restriction on the amount of the molding reactant in the heparin solution, and the upper limit is the amount that can be immersed.

The heparin-immobilized block copolymer produced in this manner has extremely high heparin activity because heparin is bound under mild conditions.

In addition, since heparin is bound to the polymer graft chain, the amount of heparin bound is large.

Furthermore, each substance used in the steps up to heparin fixation has low toxicity and can be easily washed away, making it ideal as a medical polymer material.

For example, a medical tube made entirely of a block copolymer and having heparin fixed by the method of the present invention only on the inner surface that comes into contact with blood, or a medical tube that is made of a block copolymer and has heparinized on the inner and outer surfaces that come into contact with blood, or on the inner or outer surface that comes in contact with blood. Block copolymers can be coated to modify the surfaces of medical catheters and other medical molded products that come into contact with blood.
The method of the invention can be applied.

EXAMPLES Examples will be shown below to explain the present invention in more detail, and the main types of data in these examples were determined by the following method.

That is, the amount of graft polymerization was determined from the weight change before and after the graft polymerization reaction.

In the case of molded products, the graft weight per unit area (gr/c
m2).

In addition, the amount of heparin fixed is determined by first using an acid such as hydrochloric acid,
At a high temperature of 00℃ or higher, the sulfate groups in heparin are released,
A certain amount of it is reacted with barium chloride in a ceratin solution to form barium sulfate, the light transmittance is determined using visible light with a wavelength of 50 mm, and the sulfur concentration is determined using the nephelometric method using a calibration curve determined in advance. Yes.

In addition, in order to measure the anticoagulant property, the following Lindholm test (modified method) was performed.

That is, the heparinized test sample was placed in a 5cm x 5c
The sample is cut into pieces of about 1.0 m in size (usually film-like samples) and immersed in 500 ml of 25% saline at a rate of about 1 gr, and the saline solution is changed and the process is repeated twice.

After that, wash thoroughly with physiological saline, place it on a thick glass plate, and drill a hole of 1.9 cm in the center to make it 5 m thick.
Hold down with a silicone gasket of size m, then set a glass plate with holes of the same size so that the holes match the silicone gasket holes, and press and tighten from above.

Next, after pouring out the first 0.5 ml from the dog's neck, replace only the syringe, collect 5 ml of blood, drop 0.5 ml per sample film, and spread it evenly on the film. The device was tilted at 45° every 5 minutes for the first 20 minutes, and every 2 minutes after 20 minutes, and the time until the blood coagulated and stopped moving was measured.

Three dogs were measured using this method, and the average value was used as the Lindholm test blood clotting time.

[Production of block copolymer]

The following block copolymers were produced.

(1) Styrene-butadiene-styrene (hereinafter 5-
B-8) block copolymer.

In a nitrogen gas atmosphere, 1.5 mmol of n-butyllithium per 100 gr of monomer was used in toluene, 20 parts by weight of styrene was polymerized, then 60 parts by weight of 1,3-butadiene was added and polymerized, and finally 20 parts by weight of styrene was added to the mixture, and more than 99% of the styrene was polymerized.

Next, the polymer was poured into a large amount of methanol to precipitate it, filtered, and dried under vacuum.

The obtained block copolymer was a 5-B-8 triblock copolymer having a molecular weight of 69.000 and a styrene content of 40%.

(2) Styrene-in-prene-styrene (hereinafter S
-I-8) A block copolymer was produced in the same manner as in block copolymer (1), using isoprene in place of 1,3-butadiene.

The obtained block copolymer was a 5-I-8 type triblock copolymer with a molecular weight of 76,000 and a styrene content of 38%.

(3) Styrene-butadiene-styrene-butadiene (hereinafter abbreviated as 5-B-8-B) block copolymer.

1.5 mm o of n-butyllithium per 100 gr of total monomer in n-hexane under nitrogen gas atmosphere
1, 40 parts by weight of a monomer mixture of 1,3-butadiene and styrene in a weight ratio of 50:50 was polymerized to 99% or more at 60°C, and this active copolymer solution was added with 1,
A 15% by weight n-hexane solution containing 60 parts by weight of a monomer mixture of 3-butadiene and styrene in a weight ratio of 65:35 was added, and after copolymerizing more than 99% of the solution, methanol was added to stop the reaction, and a large amount of The reaction solution was poured into methanol, and the block copolymer was precipitated and dried under vacuum.

The obtained block copolymer had a molecular weight of 65,000. It was a 5-B-8-B type quaternary copolymer with a styrene content of 40%.

(4) Star block copolymer (hereinafter abbreviated as star block copolymer) In a nitrogen gas atmosphere, add n-butyllithium as active lithium to a 15% by weight cyclohexane solution containing 30 parts by weight of styrene, and remove all monomers. 3 mmol per 100 gr
was added, and after 99% or more of it was polymerized, 70 parts by weight of 1,3-butadiene was further added to this active solution, and 99% of it was polymerized.
% or more was confirmed to have polymerized.

At this point, add 0.75 mm of silicon tetrachloride per total monomer.
After completing the polymerization by adding ol, the polymer was precipitated in methanol, filtered, and dried under vacuum.

The resulting polymer had a styrene content of 35%. It is a type of block copolymer. Ivy.

Example 1 20% of the various block copolymers of (1) to (4) above
A film with a thickness of 100 μm was made on a glass plate by a toluene solution straw solvent evaporation method, and the film was immersed in a hydroxyl group introduction reaction solution having the following formulation at 40° C. for 20 hours.

Mixture> Glacial acetic acid 70ml Distilled water 10ml Peracetic acid (10% aqueous solution) 10ml Concentrated sulfuric acid 2.7ml! The film taken out has an infrared spectrum of 3450cm-
The absorption near 1720 cm-1 confirmed that a large amount of hydroxyl groups had been introduced, and the carbonyl absorption near 1720 cm-1 was observed.

The above film was washed with water and immersed in a methanol solution of IN NaOH at room temperature for a day and night, then thoroughly washed with water and stored in distilled water.

In the infrared spectrum of this alkali-treated film, carbonyl absorption near 1720σ21 completely disappears,
The absorption near 3450 cm-1 was deep.

A graft copolymerization reaction was performed on this film by the following method.

In other words, 300ml of distilled water in a 500m1 4 sealed bone flask.
ml, GMAo, 3gr, N-vinylpyrrolidone 2
Add gr to dissolve it completely, cut out 10 pieces of the above-mentioned 5cm diameter film, and add nitric acid (1/1O
Add 3 ml of N) and 0.6 gr of ceric ammonium nitrate as a polymerization initiator, immediately seal the entire flask, reduce the pressure, return to normal pressure, and repeat the method of introducing nitrogen three times to fully replace the flask with nitrogen. .

The temperature was brought to 20° C. and left for 2 hours with slight stirring.

After that, remove the film from the flask, wash it in a large amount of water, and immediately add 20% (W/W) heparin soda.
v) It was immersed in an aqueous solution and held at 60°C for 2 hours to form heparin.

The heparinized film was washed with copious amounts of water and then stored in physiological saline at 4°C.

The thus obtained film was measured for the weight of the graft, the amount of heparin fixed, etc., and was further subjected to a Lindholm test using a glass plate as a control.

The results are shown in Table 1 below.

From the results in Table 1, it can be seen that the method of the present invention is extremely effective in improving the anticoagulant properties of block copolymers.

Example 2 A film was made using the 5-B-S block copolymer of (1) in the same manner as in Example, hydroxyl groups were introduced, and then the graft copolymer was heparinized.

During graft copolymerization, experiments were carried out by varying the type and amount of polymerization initiator, solvent, monomer, and reaction temperature and time.

Table 2 shows the results of measurements such as the amount of graft polymerization, the amount of heparin immobilized, and the Lindtholm test blood coagulation time.

These results demonstrated the effectiveness of the technique of imparting anticoagulant properties of the present invention.

Example 3 Using the block copolymer of (1), a tube with an inner diameter of 2.0 mmφ and an outer diameter of 40 mmφ was manufactured using an extruder.

(However, when applying it to an extrusion molding machine, block copolymer 1
05 parts by weight of phenyl-β-naphthylamine was added to 00 parts by weight, and after forming a tube, it was left immersed in a methanol solution and extruded.

) This tube was cut into a sample length of 1 m and filled with an acetic acid solution of peracetic acid having the same composition as shown in Example 1. After being left at 30°C for 2 days, it was washed with water and treated with IN NaOH.
was filled with methanol solution and left at room temperature for one day.

After this, the inner surface of the tube was washed with copious amounts of water.

Nitrogen substitution was carried out in the same manner as in Example 1, and in the same nitrogen atmosphere, a graft polymerization solution having the composition shown in Table 3 was introduced into the above-mentioned tube, both ends of which were sealed, and the tube was left at 20° C. for 3 hours.

After that, take out the internal graft polymerization solution and add 1 portion of distilled water.
The tube was then filled with a 15% (W/■) heparin soda solution (commercially available heparin soda 150 I u /m9), closed at both ends, and left at 60° C. for 24 hours.

Next, 500 ml of 25% sodium chloride was prepared, connected to a pump, and circulated through the tube at a rate of 100 ml/min.

After circulating for 10 hours, it was thoroughly washed with water, filled with physiological saline, and stored at 4°C.

This tube was cut into a length of about 30 cm, filled with blood that had just been collected from the jugular vein of a beagle dog using the double syringe method, and both ends were closed with hemostatic forceps and placed in a constant temperature bath at 37°C.

After 60 minutes have elapsed, at predetermined intervals, use forceps to grasp a new position approximately 2 crrL from the end of the tube with upper surface forceps, and cut the blood contained within the length of approximately 2 crrL from the tube. The sample was taken out and dropped into physiological saline, and the presence or absence of coagulation was visually determined.

Regarding the blood sampling interval, a preliminary test was performed at first every 1 to 2 hours, and the next day, using blood from the same dog, the clotting time was accurately determined to have an error of about ±10 minutes.

(The experiment was conducted on 3 dogs and the average value was used.) Table 4 shows the above samples, as well as those with only hydroxyl groups introduced.
The blood coagulation time measured for soft PVC tubes (artificial kidney circuit, commercially available, inner diameter 4.5 mm, outer diameter 7 mm), silicone tubes (commercial medical products, inner diameter 2.0 mm, outer diameter 4.0 mm), etc. It is shown in Table 4.

Claims (1)

[Claims] 1. Consisting of a conjugated diene compound and an aromatic vinyl compound,
1, in which glycidyl acrylate or glycidyl methacrylate is necessarily added as one component to a block copolymer in which the double bond portion is hydroxylated in the presence of a polymerization initiator.
1. A method for producing an anti-blood coagulant block copolymer, which comprises graft-polymerizing more than one component of a vinyl monomer and binding heparin to the graft polymer. 2. The manufacturing method according to claim 1, wherein the conjugated diene compound is butadiene or isoprene, and the aromatic vinyl compound is styrene. 3. The manufacturing method according to claim 1, wherein the block copolymer is a molded product. 4. The manufacturing method according to claim 3, wherein the molded body is in the form of a film. 5. The manufacturing method according to claim 3, wherein the molded body is tubular. 6. The manufacturing method according to claim 1, wherein the polymerization initiator is hydrogen peroxide-ferrous sulfate or ceric ammonium nitrate. 7 Contains one or more of the following vinyl monomers: methyl acrylate, N-vinylpyrrolidone, acrylamide, acrylic acid or its salt, 2-hydroxyethyl methacrylate, acrylonitrile, sodium parastyrene sulfonate, sodium allylsulfonate, or vinyl acetate. A manufacturing method according to claim 1.