JPS5932145B2 - Method for imparting anticoagulant properties to acrylonitrile polymers - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for imparting anticoagulant properties to an acrylonitrile polymer by heparinizing it.

One of the main problems with polymeric materials currently used in the medical field is their anti-coagulant properties, and as a way to improve this anti-coagulant property, it has been widely attempted to immobilize heparin on the materials. There is. For example, G. Schumer et al.
For Artificial Internal Organs, Vol. 22, p. 654 (1976) proposes heparin immobilization by so-called covalent bonding, in which heparin is immobilized by treating a cellulose membrane with bromine cyanide. However, in this method, the amount of heparin immobilized is small and the anticoagulant properties are insufficient, and there are also safety problems because highly toxic substances such as bromine cyanide are used. On the other hand, acrylonitrile (hereinafter abbreviated as AN)-based polymers are attracting attention as materials for artificial kidneys. Compared to copper ammonia rayon, which has traditionally been used as a material for artificial kidneys, this AN
This type of polymer is characterized by excellent ultrafiltration rate, permeability to medium-molecular-weight solutes, and strength, and is expected to be used in fields other than artificial kidneys in the future. When using this AN-based polymer as a material, various methods have been attempted to improve anti-blood coagulability. For example, JP-A-54-344
According to Publication No. 16, an attempt is made to impart anti-blood coagulability to the material by copolymerizing AN with a copolymerizable monomer having a sulfonic acid group or a salt thereof. However, simply introducing a sulfonic acid or a sulfonate does not necessarily provide sufficient anticoagulant properties. The present inventors have conducted extensive research aimed at developing a combination of heparin, a good natural anticoagulant, and an AN-based polymer. Good medical materials can be obtained by graft polymerizing glycidyl acrylate (hereinafter abbreviated as GA) or glycidyl methacrylate (hereinafter abbreviated as GMA) in the presence of a polymerization initiator such as, and reacting the reaction product with heparin. I have already proposed it (patent application 1982-
51602).

Furthermore, as shown in Japanese Patent Application No. 51,601/1984, if GA or GMA is reacted with heparin, the epoxy groups of heparin and GA or GMA can be easily bonded, and a heparin derivative having a double bond can be obtained. It's solved. Furthermore,
Vinyl monomers other than GA or GMA alone are A
It is difficult to graft polymerize to N-based polymers, but GA or GM
It has now been discovered that when mixed with A into the reaction system, it easily graft-copolymerizes with AN-based polymers. For these reasons, A
It has been found that a material with extremely high anticoagulant properties can be obtained by bonding heparin to an N-based polymer, and the present invention has been completed. That is, the present invention can be obtained by reacting (a) an acrylonitrile-based polymer and (b) a monomer having a polymerizable double bond and an epoxy group in one molecule, such as GMA or GA, with heparin. a heparin derivative having a polymerizable double bond; (c) at least one vinyl monomer selected from GA and GMA; (d), (c)
) with at least one other vinyl monomer other than ) in an aqueous solvent in the presence of a polymerization initiator. It provides: The AN-based polymer used in the present invention has an AN content of 30% by weight.
It is sufficient if the amount is more than 80%, but in order to express the characteristics of the AN-based polymer, it is desirable to contain 80% or more of AN.

The copolymer components contained in the AN-based polymer are not particularly limited in type, but include acrylic acid, methyl acrylate,
Acrylamide, itaconic acid, vinyl chloride, sulfuric acid,
Vinylidene chloride, methyl methacrylate, sodium allylsulfonate, etc. can be used. The AN polymer can be produced by a commonly used industrial method, such as aqueous suspension polymerization or solution polymerization. Heparinization rate indicating the amount of heparin bound in the present invention (the definition thereof is shown in Example 1).

) varies depending on the reaction conditions, the shape of the reaction target, etc.
For example, a film having a thickness of 50 μm and solidified by being poured into water from a nitric acid solution has a heparinization rate of approximately 2% or more. GA or GMA used in the present invention is AN
It is used for grafting to the system polymer, and although any may be used, it is preferable to use GMA, which is easy to handle industrially.

The heparin derivatives used in the present invention are, for example,
5-51601, a product obtained by reacting GA or GMA with heparin in the presence of a radical polymerization inhibitor in an aqueous solvent can be used.

The physical, chemical and biochemical properties of this substance are almost the same as heparin. The polymerizable vinyl monomer as component (d) used in the present invention can be a copolymer of GMA or GA grafted onto an AN polymer;
It is thought to connect the double bond between A and the heparin derivative,
Ceric ammonium nitrate in aqueous solvent, 2.2'
- Anything that polymerizes in the presence of a polymerization initiator such as azobis(2-amidinopropane) hydrochloride, hydrogen peroxide monosulfate, etc. is suitable, and it is desirable that it be hydrophilic. Examples of such vinyl monomers include acrylamide, acrylic acid, 2-hydroxyethyl methacrylate, acrylonitrile, N-vinylpyrrolidone, sodium p-styrenesulfonate, vinyl acetate, sodium allylsulfonate, methyl acrylate, and methacrylate. There are methyl acids, etc. Water is preferable as the aqueous solvent used in the graft copolymerization reaction of the present invention, and when the monomer is insufficiently dissolved, an organic solvent can be added to the water to the extent that the heparin derivative is dissolved.

For example, an organic solvent can be added up to a ratio of acetone to water of about 1:2 (volume ratio) and a ratio of formamide to water of about 1:9 (volume ratio). In addition, hydrophilic solvents such as N-N-dimethylacetamide, N-N-dimethylformamide, methyl ethyl ketone, and dioxane can be added. By using these mixed solvents, it is possible to graft copolymerize even non-hydrophilic monomers such as styrene in small amounts. Examples of the polymerization initiator used in the graft copolymerization of the present invention include a cerium salt-based initiator represented by ceric ammonium nitrate, and a monometallic hydrogen peroxide salt represented by ferrous hydrogen peroxide monosulfate. System initiator, 2.2'-
Water-soluble azo compounds such as azobis(2-amidinopropane) hydrochloride, potassium persulfate, monoacidic sodium sulfite, and the like, which are typical redox initiators, can be used.
Preferred polymerization initiators are the former three, particularly ceric ammonium nitrate, hydrogen peroxide monosulfate ferrous, and 2,2'-azobis(2-amidinopropane) hydrochloride. The AN polymer of the present invention is preferably shaped into a flat membrane, hollow membrane, fiber, or the like.

The reason for this is that in the case of unmolded products, the reaction solvent is extremely limited, and it is extremely difficult to find a solvent that can dissolve heparin derivatives.Also, even if it is sufficient to fix heparin only on the surface, It is extremely uneconomical, as it is fixed to the extent of In a preferred embodiment of the present invention, two or more vinyl monomers and GA or GMA, which necessarily contain either GA or GMA in the aqueous solvent,
Add an appropriate amount of MA-bound heparin derivative to the extent that it dissolves, add an AN-based polymer molded product, such as a flat membrane or fibrous material, add a polymerization initiator and a small amount of nitric acid as necessary, and completely dissolve. React with nitrogen substitution. The charging ratio in the reaction of the AN polymer, GMA (or GA), polymerized vinyl monomer, and heparin derivative is as follows, based on 1 part by weight of the solvent at around 20°C. The lower limit of the amount of the AN-based polymer is not particularly limited and may be arbitrary, but the upper limit can be up to about 4 parts by weight based on experience.

The amount of GMA (or GA) is 0.0001 to 0.012
A range of parts by weight is preferred, with the lower limit determined by reactivity and the upper limit determined by solubility.

The amount of polymerized vinyl monomer other than GMA (or GA) is generally added in a larger amount than GMA or GA, but is usually about 0.0005 to 6.0 parts by weight.

However, the upper limit may be determined primarily from the solubility of the polymerizable vinyl monomer in the solvent. The amount of heparin derivative is 0.001 to 0.5 parts by weight, preferably 0.00
It is carried out in a range of 5 to 0.3 parts by weight. The lower limit is determined by the reaction rate and reaction amount, and the upper limit is determined by the melting range or viscosity. The amount of the polymerization initiator is 0.01 to 50 mm0 per 11 of the solvent in any case, such as hydrogen peroxide, ceric ammonium nitrate, 2,2'-azobis(2-amidinopropane) hydrochloride, etc. , preferably 0.05
It is about ~5mm0'.

The amount of ferrous sulfate and nitric acid added to hydrogen peroxide and cerium salt, respectively, is preferably in the range of Z to 5 times the amount of each main polymerization initiator. The aqueous solvent should be prepared in advance from O to 90°C, preferably from O to 6°C.
Keep it at 0℃.

If the AN-based polymer is a hollow system and a heparin derivative is bonded to its inner surface, a module with a form similar to a completely sealable hollow fiber artificial kidney is made, and heparin is added to this from a similarly sealable flask. The reaction is carried out by introducing and circulating an aqueous solution containing a derivative, a vinyl monomer polymerization initiator, and a small amount of nitric acid. Of course, the reaction starts after the entire system including the module, flask, and reaction solution is completely purged with nitrogen. The temperature is within the above range, and the reaction time varies depending on the temperature, initiator, monomer concentration, monomer type, amount of GA or GMA bound to the heparin derivative, etc., but is in the range of about 10 minutes to 10 hours. It is. The amount of the reacted heparin derivative and bonded monomer can be accurately estimated from the difference in weight of the AN polymer film before and after the reaction, and by estimating the amount of heparin using a nephelometric method or sulfur combustion method for the sulfur content of heparin. I can do it. To routinely observe heparin binding, it is sufficient to stain with Azure A and observe metachromaticity to deep blue. In the case of acrylonitrile-based polymer flat membranes, the effectiveness of bound heparin can be determined by measuring the clotting time with Lindformcer using canine whole blood.

This measurement method is called the Lindholm test, and its outline is as follows. That is, a heparin-bound film with a dry weight of about 1 g was added to 500 d of 25% saline solution.
After 0 hours of soaking, this operation was repeated twice, replacing the saline solution. This removes ionic bonds and similar easily dissociated heparin. Wash this film with physiological saline, place it on a glass plate, and place it 20mm in the center from the top.
A Lindform cell is assembled by pressing a 5 mm thick piece of silicone with a hole of φ with a skate, and then pressing and tightening it with a glass plate having a hole of the same diameter from above.
After draining the first 0.5 ml from the dog's jugular vein, collect about 5 ml of whole blood and immediately spread 0.5 ml evenly on a film, every 5 minutes for the first 20 minutes.
After 20 minutes, tilt the tube at 450 degrees every 2 minutes to measure the time until the blood coagulates and stops moving. Each of the three dogs was measured once, and the blood coagulation time of the three dogs was expressed as an average value. The anticoagulant AN-based polymer obtained by the present invention may contain a small amount of initiator and vinyl monomer as impurities, but these can be relatively easily removed by washing with water, and the AN-based Vinyl monomers that are graft-polymerized to polymers are highly practical because they can be relatively inexpensive.

In addition, compared to the method of introducing an anticoagulant component into the main chain of an AN-based polymer, it is possible to immobilize heparin, a naturally good anticoagulant substance, in large quantities very efficiently, resulting in better anticoagulant properties. Shows blood clotting properties. If appropriate reaction conditions are selected, this AN-based polymer can be used as a flat membrane or hollow fiber for dialysis or filtration, and if made in the form of fibers, it can be used as a surgical suture. Available for use. Next, the present invention will be explained in more detail with reference to Examples.

Example 1 Heparin sodium (Hani Chemicals, 1501U/η)
Dissolve 10 grams in distilled water to make a 10% by weight solution.

This was added with 0.1 P-methoxyphenol as a polymerization inhibitor.
% of GMA was added thereto, and the mixture was heated to 50° C. with stirring and allowed to react for 16 hours. After the third reaction, the reaction solution was concentrated using a rotary evaporator and poured into 11 methanol with stirring to obtain a white powder precipitate. Collect the obtained precipitate, thoroughly wash it with methanol, then dissolve it in a small amount of water again, perform the same operation, collect the precipitate, and reach a constant weight in a vacuum dryer.
It was dried to 4. The resulting material has approximately the same physical properties as the starting material heparin, being a white, hygroscopic powder, readily soluble in water, and insoluble in organic solvents such as methanol, acetone, etc. When this product was dissolved in water and measured by gel perfusion chromatography (GPC),
A chromatogram almost completely unchanged from that of the starting heparin was obtained, indicating that the double bond of GMA bound to heparin was not polymerized. It was also confirmed from the infrared spectrum and proton NMR spectrum that GMA and heparin were bound together. The amount of bound GMA determined from the infrared spectrum is approximately 0.2m per gram of heparin.
It was m01. This heparin derivative was designated as [f] and was charged into a 500 ml four-necked separable flask with the following formulation. AN-based polymer film q-[MeH-gami] 0.32 g GMA (Tokyo Kasei Grade 1) was distilled under low pressure at 50°C, and other chemicals were purchased (Wako Pure Chemical Industries) as they were. Using.

AN polymer film is produced by suspending monomers consisting of AN/methyl acrylate/sodium methallylsulfonate (93/6/1 by weight ratio) in an aqueous system using a polymerization initiator based on potassium persulfate and sodium monosulfite. After polymerization in a liquid, washing with water and drying, it was dissolved in 70% nitric acid, spread uniformly on a glass plate with an applicator, solidified in water, washed with water, and dried under vacuum. Immediately after preparing the above mixture, repeat the operation of reducing the pressure to expel air and feeding nitrogen from another port three times, and finally, while continuously feeding nitrogen at a rate of about 30 (ml/min), pour the solution magnetically. The reaction proceeded with stirring using a stirrer.

The temperature of the flask was controlled in advance at 30.0°C, and after 1 hour, the AN polymer film was taken out.
The reaction was stopped by immersing it in a large amount of water. Furthermore, after washing this film with a large amount of water, it was compared with an unreacted film by the grafting rate, heparinization rate, Lindholm test, etc. The grafting rate and heparinization rate are calculated using the following formula (1)
According to the definition in (2). Amount of heparin bound by reaction (estimated from the amount of sulfate radicals obtained by removing the sulfate radicals of the melon reaction product by acid hydrolysis, adding a gelatin solution of barium chloride to this, and measuring by turbidimetry) Next, we will show the results of the measurements. From these results, it is clear that 1.7 x 10-5 (g/g) of heparin is bound to this film, which significantly prolongs the blood coagulation time. I understand.

When the water permeation rate of this film was measured at a pressure difference of 50 m7ILHg, it was found that the unreacted film had a water permeation rate of 130 (ml/Hg) at 37°C.
rm2, mmHg), compared to 93.6 (ml/ml/
Hr, I, 1 LmHg), the performance as a membrane did not deteriorate much, and it was found that it has great potential as a membrane for dialysis and filtration. Example 2 5 grams of heparin sodium was dissolved in distilled water to make a 5% solution, to which 2 ml of GA containing 0.5% of P-methoxyphenol was added and reacted at 40° C. for 24 hours.

The reaction product was recovered and purified in the same manner as for heparin derivative [1] in Example 1. This product was confirmed from an infrared spectrum to be a heparin derivative bound to GA having a double bond. This product is used as a heparin derivative []
500 in a constant temperature bath controlled at 40℃ with the following composition.
14 m was placed in a flask. After replacing the film with nitrogen, the reaction was started, and the film was allowed to react for 30 minutes at 40° C. After thorough washing with water, the following tests were conducted in comparison with an unreacted film.

The above results show that the method of the present invention was able to obtain a film in which heparin was sufficiently bound and had high anticoagulant properties.

Example 3 Using heparin derivative [1] in the AN polymer, polymerization initiator, polymerizable vinyl monomer, and solvent shown in Table 1 below, the reaction was carried out in the same manner as in Example 1, and the grafting was carried out in the same manner. % measurement, heparinization rate measurement, Lindholm test, etc.

Claims (1)

[Scope of Claims] 1 (a) an acrylonitrile polymer; (b) a heparin derivative obtained by bonding glycidyl acrylate or glycidyl methacrylate to heparin;
c) At least one vinyl monomer selected from glycidyl acrylate and glycidyl methacrylate, and at least one other vinyl monomer other than the vinyl monomers (d) and (c) are combined with a polymerization initiator in an aqueous solvent. 1. A method for imparting anticoagulant properties to an acrylonitrile polymer, the method comprising reacting the same in the presence of an acrylonitrile polymer. 2. Claim 1, wherein the acrylonitrile-based polymer contains 80% by weight or more of acrylonitrile.
Manufacturing method described in section. 3. The manufacturing method according to claim 1, wherein the acrylonitrile-based polymer is an acrylonitrile polymer. 4 The polymerization initiator is hydrogen peroxide-F^2_e+ system, ceric ammonium nitrate or 2,2'-azobis(2
-amidinopropane) hydrochloride. 5. The method according to claim 1, wherein the aqueous solvent is water. 6. The manufacturing method according to claim 1, wherein the reaction temperature is above the freezing point and below 60°C.