JPH0261266B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing a bioactive molded article. Physiologically active substances such as enzymes, coenzymes, enzyme inhibitors, hormones, antibacterial agents, antigens, and antibodies are immobilized on various polymeric substances, and the obtained physiologically active substance-immobilized polymeric substances can be used as chemical reaction catalysts and separations. It is used as a specific adsorbent for purification, clinical testing material, medical material, etc. The present inventors first reacted a polymer having an acid anhydride group with a polyol on a solid surface having various shapes such as a film, a tube, and a fiber.
Forming a film containing unreacted acid anhydride groups on the solid surface, and then bonding the unreacted acid anhydride groups on the film with an enzyme to impart enzyme activity to the solid surface. (Japanese Patent Application Laid-Open No. 56-64788). However, a film having acid anhydride groups can be formed by contacting a solid surface with a solution prepared by dissolving this invention's acid anhydride group-containing polymer and polyol in dioxane, tetrahydrofuran, acetone, methyl ethyl ketone, etc., and then heating the solid surface. Therefore, the process of obtaining a molded article having an acid anhydride group introduced onto its surface has the following drawbacks. (1) Harmful and flammable organic solvents must be used. (2) Two steps are required: forming a film on the solid surface and reacting the acid anhydride group with the polyol. In view of the above problems, the present inventors have developed a method for introducing acid anhydride groups onto the surface of a molded product without using an organic solvent, and a method for introducing acid anhydride groups onto the surface of various molded products in one step. As a result of various studies, we have arrived at the present invention. That is, the present invention is a copolymer obtained by melt-molding a monomer having an acid anhydride group and a monomer selected from the group consisting of olefin, chlorinated olefin, vinyl ester, vinyl ether, and diene. This is a method for producing a bioactive molded body, which is characterized by immobilizing a physiologically active substance on the surface of the molded body. Examples of the monomer having an acid anhydride group in the present invention include acrylic anhydride, methacrylic anhydride, itaconic anhydride, maleic anhydride, citraconic anhydride, dimethyl maleic anhydride, β-unsaturated acid anhydride, 4-methylcyclohex-4-ene-1,2-dicarboxylic anhydride, cyclohex-4-ene-1,2-dicarboxylic anhydride (tetrahydrophthalic anhydride), 1,
2,3,4,5,8,9,10-octahydronaphthalene-2,3-dicarboxylic anhydride, bicyclo[2,2,2]oct-5-ene-2,3-dicarboxylic anhydride, Bicyclo[2,2,1]oct-7-ene-2,3,5,6-tetracarboxylic acid-2,3,4,6-dianhydride, endo-bicyclo[2,2,1]-1 , 2,3,4,7,7-hexachloro-2-heptene-5,6-dicarboxylic anhydride (chlordic anhydride), bicyclo[2,2,1]
hept-5-ene-2,3-dicarboxylic anhydride, endo-bicyclo[2,2,1]-5-heptene-2,3-dicarboxylic anhydride (hymicic anhydride), 5-oxabicyclo[2, 2,1] unsaturated condensed ring carboxylic acid anhydrides such as hept-5-ene-2,3-dicarboxylic anhydride. The olefin to be copolymerized with the monomer having an acid anhydride group is a hydrocarbon having one double bond, such as ethylene, propylene, 1-
Butene, 1-pentene, 1-hexene, 1-pentene, 1-octene, 1-nonene, 1-decene,
α of 4-methylpentene, 3-methylpentene, etc.
-olefin, isobutylene, 2-methyl-1-
Examples include vinylidene type olefins such as butene and 2-methyl-1-pentene, and alicyclic olefins such as vinylcyclohexane and norbornene. Examples of chlorinated olefins include monohalogenated ethylenes such as vinyl chloride, vinyl bromide, and vinyl fluoride, dihalogenated ethylenes such as vinylidene chloride, 1,2-dichloroethylene, and vinylidene bromide, trichloroethylene, and trifluoroethylene. Examples include trihalogenated ethylene such as, tetrahalogenated ethylene such as tetrachlorethylene and tetrafulethylene, and allyl halides such as allyl chloride and allyl bromide. Examples of vinyl ester include vinyl acetate,
Examples include aliphatic vinyl carboxylates such as vinyl butyrate, vinyl caproate, vinyl laurate, and vinyl stearate; and vinyl aromatic carboxylates such as vinyl benzoate and vinyl 1-naphthoate. Vinyl ether in the present invention includes methyl vinyl ether, methyl vinyl ether, n-
Thropyl vinyl ether, isopropyl vinyl ether, t-butyl vinyl ether, isoamyl vinyl ether, n-hexyl vinyl ether,
Alkyl vinyl ethers such as n-octyl vinyl ether and 2-ethylhexyl vinyl ether;
Cyclohexyl vinyl ether, decahydro-2
- alicyclic vinyl ethers such as naphthyl vinyl ether and menthyl vinyl ether, phenyl vinyl ether, o, m or p-chlorophenyl vinyl ether, o, p or m-oxyquenyl vinyl ether, o, m or p-methyl phenyl vinyl ether, Examples include aryl vinyl ethers such as α- or β-naphthyl vinyl ether. Examples of the diene include conjugated dienes such as butadiene, isoprene, chloroprene and 1,3-pentadiene, and non-conjugated dienes such as 1,4-hexadiene, 1,5-hexadiene and 1,6-heptadiene. Copolymerization of a monomer having an epoxy group with a monomer selected from the group consisting of olefins, chlorinated olefins, vinyl esters, vinyl ethers, and dienes can be carried out, for example, in "Synthetic Polymers," edited by Murahashi, Imoto, and Tani. (Asakura Shoten, 1970) Murahashi, Imoto, Tani eds. "Synthetic Polymers" (Asakura Shoten, 1971) It can be carried out by a known method, and the method preferably used in the present invention The polymer contains one or more monomers having an acid anhydride group at 0.1
~60% by weight, especially olefin with 0.5-40% by weight
40 to 99.9% by weight, especially 60 to 99.9% by weight of one or more monomers selected from the group consisting of chlorinated olefins, vinyl esters, vinyl ethers, and dienes
99.5% by weight. A copolymer of a monomer having an acid anhydride group and a monomer selected from olefin, chlorinated olefin, vinyl ester, vinyl ether, and diene is
First, one or more monomers selected from olefins, chlorinated olefins, vinyl esters, vinyl ethers, and dienes are polymerized, and the resulting polymer contains monomers having one or more acid anhydride groups. It can also be produced by graft copolymerization in the presence of a radical initiator. The copolymerization ratio in this case is also preferably within the same range as above. This graph copolymerization is described, for example, in Japanese Patent Publication No. 52-39636,
This can be carried out by the known method described in Japanese Patent Publication No. 53-1319 and the like. A copolymer with an acid anhydride group
If the amount is less than 0.1% by weight, the amount of physiologically active substances immobilized will decrease, making it difficult to impart sufficient biological activity for practical purposes.On the other hand, if the amount of monomers having acid anhydride groups exceeds 60% by weight, This is not preferable because it not only makes melt molding difficult, but also impairs the mechanical properties of the molded product obtained from the copolymer. If the molecular weight of the copolymer is small, the mechanical properties of the molded article will be poor, so the molecular weight of the copolymer is preferably at least 5,000, particularly preferably 10,000 or more. The copolymer thus obtained can be used as beads, films, plates, monofilaments, nonwoven fabrics, sponges, woven fabrics, knitted fabrics, etc. depending on the purpose.
It can be formed into various shapes such as short fibers, tubes, and hollow fibers. During the molding process, plasticizers, modifiers, stabilizers, oil agents, etc. may be added as necessary, or various polymers may be blended. Physiologically active substances used in the present invention include enzymes, coenzymes, enzyme inhibitors, proenzymes, hormones, antibiotics, bactericides, anticancer agents, immune response physiologically active substances, etc. that have important effects on the physiological functions of animals and plants. A substance that gives Examples of enzymes include alcohol dehydrogenase,
Oxidoreductases such as lactate dehydrogenase, glycose-6-phosphate dehydrogenase, glycose oxidase, luciferase, L-amino acid oxidase, catalase, tyrosinase, peroxidase, hexokinase, pyruvate dehydrogenase, carbamate kinase, acetate kinase, Transferases such as ribonuclease, lipase, acetylcholinesterase, steroid esterase, amylase, cellulase, dextranase, invertase, pepsin, renin, trypsin, chymotrypsin, papain, huicin, thrombin, kallikrein, striptokinase,
Hydrolytic enzymes such as urokinase, plasmin, brinolase, asparakinase, urease, penicillin amidase, apyrase, lyases such as pyruvate decarboxylase, alpartase, threonine deaminase, isomerases such as glucose isomerase, ligases such as tyrosyl-TRNA synthetase, acetyl-CoA synthetase, etc. can be given. Examples of the coenzyme include pyridoxal phosphate and nicotine adenine dinucleotide. Examples of enzyme inhibitors include ohomucoids,
Kunitz soybean trypsin inhibitor, aprotinin,
antithrobin, α ₂ -acroglobulin, α ₁ -
Examples include antitrypsin, α ₁ -ancplasmin, plasminogen antiactivator, heparin, and the like. Examples of proenzymes include plasminogen,
Examples include prothrombin and blood coagulation factor. Examples of hormones include cortisone, testosterone, estrone, estradiol, progesterone, insulin, somastatin, and gonadotropin. Examples of antibiotics include penicillins such as cloxacillin, cycloxacillin, flucloxacillin, ampicillin, hetacillin, talampicillin, cyclacillin, amoxicillin, pipmecillinum, and piperacillin, cephalozin, zephaloglycine, cephalexin, cefazolin, cefapirin, cefrazine, and cestesol. , cephalosporins such as cefoxitin and cephatridine, aminoglycosides such as streptomycin, kanamycin, frangiomycin, paramomycin, gentamicin, bekanamycin, lipostamycin, dibekacin, amikacin, tobramycin, and spectinomycin, oxytetracycline, tetracycline, dimethyl Tetracyclines such as chlortetracycline, methacycline, doxycycline, minocycline, macrolides such as erythromycin, kitasamycin, oleandomycin, spiramycin, diyosamycin, midecamycin, lincomycins such as lincomycin, clindamycin, micamycin, gramicidin S , antigram-positive bacteria such as gramicidin, polymyxins such as colistin and polymyxin B, antimycobacteria such as biomycin, capreomycin, enviomycin, and cycloserine, polyene macrolides such as amphotericin B and pimaricin, Examples include rifampicin, pyrrolnitrin, mitomycin C, actinomycin, bleomycin, daunorubicin, doxorubicin, neocarzinostatin, and the like. Examples of disinfectants include pigment preparations such as acrinol and acrylflavin, furan preparations such as nitrofurazone, quaternary ammonium salts such as benzalkonium chloride and benzethonium chloride, guanidium salts such as chlorhexidine, iodine complexes such as povidone-iodine, Examples include amphoteric surfactants such as alkyldiaminoethylglycine hydrochloride. Examples of anticancer drugs include nitrogen mustard, nitramine, chlorambucil, cyclophosphamide, melphalan, uracil mustard, mannomustine, dopan, BCNU, triethylenemelamine, thio-TEPA, and Aza-
Alkylating agents such as TEPA, trenimon, improquion, busulfan, dimethylmilleran, piposulfan, ethoglucide, epoxypropidine, epoxypiperazine, hexamethylmelamine, dibromomannitol, pipobroman, folic acid,
Aminopriten, methotrexate, guanine,
Metabolic inhibitors such as 8-azaganine, 6-mercaptopurine, azathioprine, uracil, 5-fluorouracil, cytarabine, azaserine, diazomycin, actinomycin D, sacromycin,
Antibiotics such as mitomycin C, daunomycin, bleomycin, chromomycin, cardinophilin, synthetic agents such as 5-HP, IQ-1, plant ingredients such as thioteba, cyclophosphamide, doxorubicin, daunorubicin, neocarcinostane,
Examples include Hg-hematoporphyrin, Co-protoporphyrin, stilbestrol, hydroxyurea, procarbazine, methylglyoxal-bis-guanylhydrazone, and L-asparaginase. Immunoreactive substances refer to substances capable of generating immunological binding, such as antigens and antibodies. An antigen is a substance that can induce an antigen-antibody reaction, and is generally a peptide, protein, polysaccharide, glycoprotein, steroid, etc. Antibodies are proteins produced in vivo by stimulation of antigens that specifically bind to antigens, and their chemical substance is immunoglobulin. Such immunoreactive substances include, for example, microorganisms such as filamentous fungi, yeasts, protozoa, and viruses, their immunologically active components, antibodies isolated from humans and animals, serum components,
Examples include toxins, hormones, enzymes, alkaloids, cell and tissue extracts, blood cells, and lectins. The above-mentioned physiologically active substances can be immobilized on the surface of the molded article through covalent bonds or ionic bonds. When a physiologically active substance is immobilized on the surface of a molded product by covalent bonding, the physiologically active substance is mixed with water or, if necessary, water such as methanol, ethanol, propanol, acetone, tetrahydrofuran, dioxane dimethyl sulfoxide, dimethylformamide, etc. It is dissolved or dispersed in a mixture of a miscible solvent and water, and the molded surface is heated with the resulting liquid preferably at -20 to 100°C, particularly preferably at 0 to 80°C, preferably for 5 minutes to By treating for 100 hours, particularly preferably from 10 minutes to 80 hours, the acid anhydride group on the surface of the molded article and the physiologically active substance can be covalently bonded. At that time, in order to adjust the pH to preferably 2 to 12, particularly preferably 4 to 10,
A buffer such as phosphate or acetate may be used, or hydrochloric acid, sodium hydroxide, etc. may be added. When covalently immobilizing physiologically active substances, by reducing steric hindrance as necessary,
In order to increase the physiological activity of the immobilized physiologically active substance, a spacer can be inserted between the surface of the molded body and the physiologically active substance. The spacer is preferably a compound having at least two groups containing active hydrogen, such as amino groups, which can react with both the acid anhydride group on the surface of the molded article and the physiologically active substance. Examples of the spacer include hexamethylene diamine, polyethyleneimine, polylysine, chitosan, polyamines such as polyethylene glycol having amino groups at both ends, polyvinylamine, and aminoacetalized polyvinyl alcohol. The reaction between the surface of the molded article and the spacer can be carried out under the same conditions as those for directly reacting the surface of the molded article with the physiologically active substance. The reaction between the spacer and the physiologically active substance is N,N'-dicyclohexylcarbodiimide, 1-cyclohexyl-3-(2
-morpholinoethyl) 2-carbodiimide-meth-P-toluenesulfonate, 1-ethyl-3-
Dehydration condensation agents such as (3-dimethylaminopropyl)carbodiimide hydrochloride, N-cyclohexyl-5-phenyl isoxazolium-3'-sulfonate, glutaraldehyde, dialdehyde starch, cyanuric chloride, maleic anhydride This can be carried out by a conventional method using a coupling agent such as acid-methyl vinyl ether, maleic anhydride-styrene copolymer, maleic anhydride-isobutylene copolymer, maleic anhydride-ethylene copolymer, or the like. It is also possible to first react the coupling agent and the physiologically active substance and then covalently bond the physiologically active substance to the surface of the molded article via the coupling agent. In order to immobilize a physiologically active substance on the surface of a copolymer by ionic bonding, an ion exchange group such as an amino group, a carboxyl group, or a sulfonate group is introduced onto the surface of the molded product, and then the physiologically active substance is immobilized on the ion exchange group. All you have to do is make an ionic bond between them. Amino groups can be introduced onto the surface of the bioform by reacting a polyamine used as a spacer. In order to introduce carboxyl groups onto the surface of the molded product, the surface of the molded product is preferably heated to 10 to 120°C.
Particularly preferably at 20-100℃, preferably for 10 minutes-10
This can be carried out by hydrolyzing for a period of time, particularly preferably from 30 minutes to 8 hours. Introducing a sulfonate group onto the surface of the molded article can be carried out, for example, by reacting with an aminosulfonic acid such as taurine. The immobilization of the physiologically active substance on the surface of the molded body by ionic bonding can be carried out under the same conditions as the conditions for immobilization by covalent bonding described above. In this way, a molded article with a physiologically active substance immobilized on its surface can be used, for example, as a chemical reaction catalyst, a specific adsorbent for separation and purification, a material for clinical testing, a medical material, and the like. That is, a molded article on which an enzyme is immobilized as a physiologically active substance can be used as a chemical reaction catalyst. For example, a molded product with immobilized tyrosinase can be used for the production of L-DOPA, a molded product with immobilized amylase or cellulase can be used for the production of glycose,
The molded product with immobilized aspartase is used for the production of L-aspartic acid, and the molded product with immobilized acetate kinase or carbamate kinase is used for the production of ATP.
The molded body on which glycose isomerase is immobilized can be used for the production of fructose. In addition, molded bodies with immobilized physiologically active substances,
It can be used as a specific adsorbent for separation and purification. For example, a molded product with immobilized enzymes can be used for the separation and purification of coenzymes and enzyme inhibitors, a molded product with immobilized coenzymes or enzyme inhibitors can be used for the separation and purification of enzymes, and a molded product with immobilized hormones can be used for the separation and purification of enzymes. can be used for the separation and purification of hormone receptors, the molded body with immobilized antigens can be used for the separation and purification of antibodies, and the molded body with immobilized antibodies can be used for the separation and purification of antigens. In addition, these specific adsorbents are used as clinical test materials for enzyme immunoassays and radioimmunoassays such as thyroid stimulating hormone, thyroid hormone, insulin, steroid hormones, human placental gonadotropin, angiotensin, α-fetoprotein, ferritin, It can be used for quantifying HBs antigen, etc. As shown in Table 1, various diseases can be treated by removing various harmful substances from body fluids using these specific adsorbents. Furthermore, molded bodies with immobilized physiologically active substances are used as medical materials. For example, molded bodies immobilized with fibrinolytic active enzymes such as urokinase, streptokinase, purinolase, and plasun, and blood coagulation system inhibitors such as heparin and antithrombin can be used as antithrombotic molded body materials for vascular indwelling catheters, bypass tubes, etc. It can be used for drain catheters, etc. A molded article on which antibacterial substances such as antibiotics and bactericides are immobilized can be used as an antibacterial material for urinary catheters and the like. The molded article on which the anticancer agent is immobilized can be used as a local therapeutic agent for cancer. As described above, according to the present invention, a molded article having an acid anhydride on the surface can be obtained in one step without using an organic solvent, and therefore various molded articles having molding activity are industrially advantageous. can be manufactured. Hereinafter, the present invention will be specifically explained with reference to Examples.

【table】

【table】

[Table] Note that "parts" in the examples mean "parts by weight." Example 1 2 parts of di-tert-butyl peroxide was added to a mixture of 1000 parts of ethylene-vinyl acetate copolymer (80% by weight of ethylene, 20% by weight of vinyl acetate) and 50 parts of hymic anhydride, and the mixture was mixed using a Henschel mixer. and mixed at room temperature. The obtained mixture was supplied to an extruder and extruded at 180°C to obtain an ethylene-vinyl acetate copolymer grafted with hymic anhydride and to prepare a tube having an inner diameter of 1.5 mm and an outer diameter of 2.0 mm. The obtained tube was immersed in urokinase phosphate buffer (urokinase 2400 units/ml, phosphate buffer concentration 1/10M, PH7.0) and incubated at 7°C.
After reacting for 48 hours, it was thoroughly washed with physiological saline solution. To measure fibrinolytic activity, prepare a fibrin plate according to Kanai and Kanai (eds.), ``Clinical Test Methods Recommendations,'' Revised and Expanded 28th Edition (Kanehara Publishing) VI-105, and place a tube (2 mm in length) on top of the fibrin plate. Dissolution was observed. As a result, after 24 hours, a 16 mm diameter area was formed around the tube piece on which urokinase was immobilized.
Fibrin was observed to be dissolved. Example 2 1 part of benzoyl peroxide was added to a mixture of 1000 parts of polypropylene and 30 parts of tetrahydrophthalic anhydride, and the mixture was mixed at room temperature using a Henschel mixer. The resulting mixture was fed to an extruder and extruded at 200°C to obtain polypropylene grafted with tetrahydrophthalic anhydride and fibers with a thickness of 6.5 d. 1 g of the obtained fiber was immersed in 10 ml of anti-hepatitis B human immunoglobulin (HBs antibody) 1% phosphate buffer (1/10M, PH8.0) at 7°C for 24 hours, and then soaked in physiological saline solution. Washed well. The thus obtained anti-hepatitis B human immunoglobulin-immobilized fiber was packed into a column with a capacity of 10 ml, and when plasma with an HBs antigen titer of <sup>1:28</sup> was filtered through this column, the amount of antigen in the plasma after filtration was is 1:
2 decreased to <sup>2</sup> . The HBs antigen titer was measured in accordance with Kanai and Kanai (eds.), ``Clinical Test Methods Recommendation,'' Revised and Expanded 28th Edition (Kanehara Publishing)-40. Example 3 Ethylene, vinyl acetate and maleic anhydride were polymerized in an autoclave using benzoyl peroxide as a catalyst to produce 75% by weight of ethylene and 17% of vinyl acetate.
%, and a terpolymer consisting of 8% by weight of maleic anhydride was obtained. This copolymer was extruded at 200°C to create a film with a thickness of 0.3 mm. This film was hydrolyzed at 70°C for 5 hours to convert acid anhydride groups to carboxyl groups. This film piece was added to dibekacin sulfate aqueous solution [50 mg (potency)/100 ml] at PH
was immersed at 7° C. for 2 hours while adjusting the temperature to 9 using a 0.1N aqueous sodium hydroxide solution to obtain a film piece on which dibekacin sulfate was fixed. When this film was cut into circular pieces with a diameter of 5 mm and the antibacterial activity was measured using the cylindrical plate method using Bacillus subtilis ATCC 6633 as a test bacterium, an inhibition circle was observed around the film piece after 16 hours at 37°C. Admitted.

Claims (1)

[Claims] 1 A monomer having an acid anhydride group, an olefin,
A physiologically active substance characterized by melt-molding a copolymer with a monomer selected from the group consisting of chlorinated olefins, vinyl esters, vinyl ethers, and dienes, and immobilizing a physiologically active substance on the surface of the obtained molded product. A method for producing an active molded body.