JPS59204601A - Manufacture of molded article having physiological activity - Google Patents

Abstract

PURPOSE:To obtain a catalyst for chemical reactions, adsorbent for separation and purification, material for clinical tests, medical materials, etc., by immobilizing a physiologically active substance on the surface of a molded article consisting of a copolymer of an epoxy-group containing monomer and a specific monomer, e.g. (chlorinated) olefin, etc. CONSTITUTION:A physiologically active substance, e.g. an enzyme, coenzyme, enzyme inhibitor, hormone, antibiotic substance, germicide, anticancer agent, physiologically active substance for immunological reaction, etc. is immobilized on the surface of a molded article consisting of a copolymer of (A) an epoxy- group containing monomer and (B) a monomer selected from the group of a (chlorinated) olefin, e.g. ethylene or vinyl chloride, a vinyl ester, e.g. vinyl acetate, and a diene, e.g. butadiene, preferably a terpolymer of 60-99wt% ethylene, 1-40wt% vinyl acetate and 0.5-40wt% glycidyl (meth)acrylate, through covalent and ionic bonds.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing a bioactive molded article.

Enzymes, coenzymes, enzyme inhibitors, hormones, antibacterial agents.

Physiologically active substances such as antigens and antibodies are immobilized on various polymeric substances, and the obtained physiologically active substance-immobilized polymeric substances can be used as specific adsorbents for separation and purification of chemical reaction catalysts, materials for clinical testing, and medical treatment. It is used as a raw material. For example, Japanese Patent Publication No. 56-223588 describes that a polysaccharide gel such as agarose or cellulose is reacted with epichlorohydrin, and then physiologically active substances such as enzymes and enzyme inhibitors are immobilized via epoxy groups. There is. Epoxy groups are preferred functional groups because they can react with physiologically active substances such as enzymes under mild conditions, but this polysaccharide
Since it is gel-like, it is difficult to handle and is not suitable for industrial use as a specific adsorbent for separation and purification of chemical reaction catalyst 1. Furthermore, when actually used as clinical test materials or medical materials, it is desired to impart physiological activity to molded articles such as fibers and tubes depending on the purpose of use.

The present inventors have been concerned with these circumstances and have investigated various methods for imparting physiological activity to molded bodies made of polymers having epoxy groups. , tlylated olefins, vinyl esters.

We discovered that bioactive substances can be added to molded bodies made of copolymers with monomers selected from dienes.

This has led to the present invention.

That is, the present invention fixes a physiologically active substance on the surface of a molded product made of a copolymer of a monomer having an epoxy group and a II-mer selected from the group consisting of olefins, chlorinated olefins, vinyl esters, and dienes. This is a method for producing a bioactive molded article, characterized by:

Examples of the epoxy group-containing monomer used in the present invention include glycidyl acrylate and glycidyl nucleate. itaconate monoglycidyl ester, itaconate diglycidyl ester, butenetricarboxylic acid monoglycidyl ester, butenetricarboxylic acid triglycidyl ester, butenetricarboxylic acid triglycidyl ester,
Unsaturated glycidyl esters such as P-styrene carboxylic acid glycidyl ester, allyl glycidyl ether, 2
- Glycidyl ethers such as methylallyl glycidyl ether, styrene-P-glycidyl ether, and P-glycidylstyrene, 3.4-epoxy-1-butene, 3,
4-epoxy-3-methyl-1-butene, 3.4-epoxy-3-methyl-1-butene, 3.4-epoxy-1
Examples include epoxy alkenes such as -pentyne, 3,4-epoxy-3-methyl-1-pene, thea, 5,6-epoxy-1-hexene, and vinylcyclohexene monoxide.

The olefin to be copolymerized with the above monomer having an epoxy group is a hydrocarbon having one double bond, such as ethylene and propylene.

α-olefins such as 1-butene, 1-pentene, 1-hexene, 1-heptene, 1-octene, 1-nonene, 1-decene, 4-methylpentene, 3-methylpentene, isobutylene, 2-methyl-1 -butene, vinylidene type olefins such as 2-methyl-1-pentene, vinyl Schiff 1-cohexane.

Examples include norbornene and any alicyclic olefins.

Examples of chlorinated olefins include mono-logenated ethylenes such as vinyl chloride, vinyl bromide, and vinyl fluoride; di-logenated ethylenes such as vinylidene chloride, 1,2-dichloroethylene, and vinylidene bromide; trichlorethylene; Examples thereof include trihalogenated ethylene such as trifluoroethylene, tetra-halogenated ethylene such as tetrachlorethylene and tetrafluoroethylene, and allyl halides such as allyl chloride and allyl bromide.

Examples of vinyl esters 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.

Examples of the diene include conjugated dienes such as butadiene, isoprene, chloroprene 1,3-pentadiene, 1
, 4-hexadiene, 1.5-hexadiene, 1.6-
Examples include non-conjugated dienes such as heptadiene.

Copolymerization of a monomer having an epoxy group with a monomer selected from the group consisting of olefins, chlorinated olefins, vinyl esters, and dienes is described, for example, in Murahashi, Imoto, and Tani (eds.), Synthetic Polymer IJ (Hatsusan Shoten).

Copolymers preferably used in the present invention can be carried out by known methods such as those described in "Synthetic Polymer ■" (Hatsusan Shoten, 1971) edited by Murahashi, Iki, and Tani. is selected from the group consisting of 0.1 to 95% by weight, particularly 0.5 to 70% by weight of one or more monomers having an epoxy group, and olefins, chlorinated olefins, vinyl esters, and dienes. One or more monomers5~
It is a copolymer of 99.9% by weight, particularly 30 to 99.5% by weight. Monomer with epoxy content is 0
．． If the amount is less than 1% by weight, the amount of physiologically active substance immobilized will be small, making it difficult to impart sufficient physiological activity for practical purposes.On the other hand, if the monomer having an epoxy group exceeds 95% by weight, This is not preferred because the mechanical properties of the molded product obtained from the copolymer are impaired.

Among these copolymers, the copolymers that have good moldability and impart high physiological activity and are particularly preferably used in the present invention contain 60 to 99% by market weight of ethylene and 1 to 1% of vinyl acetate.
It is a terpolymer copolymerized with 40% by weight of glycidyl methacrylate or 0.5 to 40% by weight of glycidyl acrylate.

The copolymers thus obtained can be used, for example, as beads, films, plates, monofilaments, nonwoven fabrics, sponges, textiles, and laminated materials, depending on the purpose.

It can be formed into various shapes such as short fibers, tubes, and hollow fibers. For molding processing, as necessary.

Plasticizers, modifiers, stabilizers, oil agents, etc. may be added, or various polymers may be blended.

The physiologically active substances used in the present invention include, for example, enzymes,
Coenzymes, enzyme inhibitors, proenzymes, hormones, antibiotics,
Substances that have an important effect on the physiological functions of animals and plants, such as bactericidal agents, anticancer agents, and immune response physiologically active substances.

An example of an enzyme is alcohol dehydrogenase.

Lactate dehydrogenase, glucose-6-phosphate dehydrogenase,
Glucose oxidase, luciferase.

Redox enzymes such as L-amino acid oxidase, catalase, tyrosinase, and peroxidase.

Transferases such as hexokinase, pyruvate dehydratase, carbamate kinase, acetate kinase, ribonuclease, lipase, acetylcholinesterase, steroid esterase, amylase, cellulase, dextranase, invertase, pepsin,
Renin, trypsin, chymotrypsin, papain, ficin, thrombin, kallikrein, streptokinase.

Urokinase, plasmin, briclase. Examples include hydrolytic enzymes such as asparakinase, urease, penicillin amidase, and avilase; glueases such as pyruvate decarboxylase, alpartase, and threonine deaminase; isomerases such as glucose isomerase; and glue gauzes such as tyrosyl-TRNA synthetase and acetyl-CoA synthetase.

Examples of coenzymes include pyridoxal phosphate and nicotine adenine dinucleotide.

Examples of enzyme inhibitors include ohomucoids.

Kunitz soybean tripcin inhibitor, aprotinin.

Antitropin ■, α2-macroglobulin, α1-antitrypsin, α1-antiplasmin.

Examples include plasminogen antiactivator and heparin.

An example of a proenzyme is plasminogen.

Examples include prothrombin and blood coagulation factor X.

Examples of hormones include cortisone, testosterone, estrone, estradiol, progesterone,
Examples include insulin, tumastatin, and gonadotropins.

An example of an antibiotic is cloxacillin.

Penicillins such as cycloxacillin, flucloxacillin, ampicillin, hetacillin, talampicillin, cyclacillin, amoxicillin, bibmecillinum, piperagiline, cephalolidine, zephaloglycine, cephalexin, cefazolin.

Cephalosporin drugs such as cephapirin, cefrazine, cefrazole, cefoxitin, and cefatridine 1. Streptomycin, kanamycin.

Fradiomycin, paromomycin, gentamicin,
Aminoglycosides such as bekanamycin, lipostamycin, dibekacin, amikacin, tobramycin, spectinomycin, tetracyclines such as oxytetracycline, tetracycline, demethylchlortetracycline, meccycline, doxycycline, minocycline, erythromycin, kitasamycin,
Macrolides such as oleandmycin, spiramycin, josamycin, and midecamycin, lincomycins such as lincomycin and talindamycin, and antigram-positive bacteria such as micamicin, gramicidin S, and gramicidin.

Colistin, polymyxins such as polymyxin B, biomycin, cabreomycin, enbiomycin,
Antimycobacteria such as cycloserine, polyene macrolides such as amphotericin B, pimaricin, and rifampicin.

Examples include pyrrolnidoline, mitomycin C, actinomycin, pleomycin, daunorubicin, doxorubicin, and neocarzinostatin.

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, and iodine complexes such as povidone rot. Examples include amphoteric surfactants such as alkyldiaminoethylglycine hydrochloride.

Examples of anticancer agents include nitrogen mustard, nitromine, chloramdacil 5-cyclophosphamide, melphalan, and uracil mustard.

Mannomustine, dopane, BCNtl, triethylenemelamine, thio-TEPA, Aza-TEP
^, alkylating agents such as trenimon, inprocuone, busulfan, dimethylmyleran, pivosulfan, etogluside, epoxypropicine, epoxypiperazine, hex-methylmelamine, dibromomannitol, pivobroman, folic acid, aminopritene.

Methotrexate, guanine, 8-azaganine.

5-fluorouracil, cytarabine, azaserine,
Antimetabolites such as diazomycin, actinomycin D
, antibiotics such as sacromycin, mitomycin C, daunomycin, pleomycin, chromomycin, cardinophilin, synthetic agents such as 5-HP, IQ-1, thioteba, cyclophosphamide, xorubicin, daunorubicin.

Examples include plant components such as neocarcinostane, Hg-hematoporphyrin, Co-protoporphyrin, stilbestrol, hydrociurea, procarbazine, methylglyoxalubisuguanylhydrazone, and L-asparaginase.

Immunoreactive substances refer to substances capable of forming immunological bonds, such as antigens and antibodies. An antigen is a substance that can induce an antigen-antibody reaction, and generally includes peptides, proteins, polysaccharides, glucoproteins, steroids, and the like. An antibody is a protein that is produced in a living body upon stimulation by an antigen and specifically binds to the antigen, and its chemical substance is an immunoglobulin. Such immunoreactive substances include, for example, filamentous fungi, yeasts, protozoa.

Virus-like microorganisms, their immunologically active components 1
Antibodies, serum components, toxins, hormones, enzymes, alkaloids, and cells isolated from humans and animals.

Examples include 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 body by covalent bonding, the physiologically active substance is immobilized with water, methanol, ethanol, or propatool.

Dissolved or dispersed in a mixture of water and a water-miscible solvent such as acetone, tetrahydrofuran, dioxane, dimethylsulfoxide, dimethylformamide,
The surface of the molded product is preferably coated with the obtained liquid to a concentration of -20 to 100
°C, particularly preferably 0 to 80 °C, preferably 5
By treating for 100 minutes to 100 hours, preferably 10 minutes to 80 hours, the epoxy group on the surface of the molded article and the physiologically active substance can be covalently bonded. At that time, pll is preferably 2 to 12. In order to particularly preferably adjust the value to 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.

If necessary, a spacer can be inserted between the surface of the molded body and the physiologically active substance for the purpose of increasing the physiological activity of the immobilized physiologically active substance by reducing steric hindrance. The spacer is preferably a compound having at least two groups containing active hydrogen, such as a carboxyl group or an amine group, which can react with both the epoxy group on the surface of the molded article and the physiologically active substance. Examples of the spacer include adipic acid, polyethylene glycol having carboxyl groups at both ends, alginic acid, carboxymethyl cellulose,
Polyacrylic acid, polymethacrylic acid.

Maleic acid-methyl vinyl ether copolymer.

Polycarboxylic acids such as maleic acid-styrene copolymer, maleic acid-isobutylene copolymer, maleic acid-ethylene copolymer, and polyglutamic acid.

Hexamethylenediamine, polyethyleneimine.

Polylysine, chitosan, polyethylene glycol with amino groups at both ends, polyvinylamine.

Examples include polyamines such as aminoacetalized polyvinyl alcohol. The reaction between the surface of the molded article and the spacer, Δ, 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″-dicyclohexylcarposiimide, 1-cyclohexyl-3
-(2-morpholinoethyl)-2-carposiimido-metho-P-toluenesulfonate, 1-ethyl-3-(3
-dimethylaminopropyl)carposiimide hydrochloride, dehydration condensation agents such as N-cyclohexyl-5-phenylisoxazolium-3'-sulfonate, or glutaraldehyde, dialdehyde starch.

Cyanuric chloride, maleic anhydride-methyl vinyl ether, maleic anhydride styrene copolymer, maleic anhydride
This can be carried out by a conventional method using a coupling agent such as isobutylene copolymer or maleic anhydride styrene copolymer. Alternatively, 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.

To immobilize a physiologically active substance on the surface of a copolymer by ionic bonding, for example, an amine group is added to the surface of the molded product.

An ion exchange group such as a carboxyl group or a sulfonate group may be introduced, and then a physiologically active substance may be ionically bonded to this ion exchange group. Polycamponic acid, polyamine, ε-aminocaproic acid, and glutamic acid were used as spacers to introduce amine groups and carboxyl groups onto the surface of bioforms.

This can be carried out by reacting an aminocarboxylic acid such as lysine. A sulfonate group can be introduced onto the surface of the molded article by reacting it 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.

A molded article having a physiologically active substance immobilized on its surface in this manner can be used, for example, as a specific adsorbent for separation and purification of a chemical reaction catalyst, 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 is used for the production of L-DOPA, and a molded product with immobilized amylase or cellulase is used for the production of glucose.

The molded product with immobilized aspartase is used for the production of L-aspartic acid, the molded product with immobilized acetate kinase or carbamate kinase used for the regeneration of ATP, and the molded product with immobilized glycose isomerase used for the production of fructose. Can be used.

Moreover, a molded body on which a physiologically active substance is immobilized can be used as a specific adsorbent for separation and purification. For example, molded bodies with immobilized enzymes are used for the separation and purification of coenzymes and enzyme inhibitors, molded bodies with immobilized coenzymes or enzyme inhibitors are used for the separation and purification of enzymes, and molded bodies with immobilized hormones. is used for the separation and purification of hormone receptors, the molded body with immobilized antigens is used for the separation and purification of antibodies, and the molded body with immobilized antibodies is used for the separation and purification of antibodies. It can be used for lt purification. In addition, these specific adsorbents are used as clinical test materials for enzyme immunoassay and radioimmunoassay.
Thyroid stimulating hormone, thyroid hormone.

It can be used for the quantification of insulin, sterodione, h-/Lzmon, human placental gonadotropin, angiotensin, α-fetoprotein, ferritin, lIn5 antigen, etc. And 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 cokinase, streptokinase, brinolase, and plasun, and blood coagulation system inhibitors such as heparin and Annato1 compin are used as antithrombotic molded body materials for vascular indwelling catheters. , bypass tubes, drain catheters, etc. A molded article on which an antibacterial substance such as an antiseptic substance or a bactericidal agent is immobilized can be used as an antibacterial material for a urinary catheter or the like. The molded article on which the anticancer agent is immobilized can be used as a local therapeutic agent for cancer.

Table 1 Application of specific adsorbent to treatment 1 (Continued) L -□□□-□-
−〜−−−−−−−−−−L−−1−−−−−−−
------1 The molded product obtained by the present invention has various shapes depending on the purpose of use, and has the feature that the surface of the molded product endowed with physiological activity has high activity. It has a wide range of uses, including specific adsorbents for separation and purification of reaction catalysts, materials for clinical testing, and medical materials.

The present invention will be explained in more detail below by giving examples.

Example 1 7 parts by weight of glycidyl methacrylate and 84 parts by weight of ethylene were prepared by the method of Example 3 of JP-A-48-11388.
A terpolymer was obtained in which parts by weight and 9 parts by weight of vinyl acetate were copolymerized. By pressing this copolymer, a FILNO having a thickness of 0.3+n was created.

This film was cut into a circular shape with a diameter of 5 mm, and the obtained film pieces were added to a urokinase phosphate buffer solution (urokinase 2.400 units/m' + phosphorus fVI buffer concentration 1/IOM, pH 7.0). Soak at 7°C for 4
After reacting for 8 hours, it was thoroughly washed with physiological saline solution.

Fibrinolytic activity was measured by preparing a fibrin plate according to the revised and expanded 28th edition of ``Clinical Testing Method Summary'' edited by Kanai and JV (Metal Publishing), Vl-105, and placing a piece of film on top of the fibrin plate to observe fibrin dissolution. As a result, after 24 hours, fibrin was found to have melted over a diameter of 15 mm around the film piece on which urokinase was immobilized12. It was immersed in a 1% aqueous solution of the copolymer and heated at 50°C for 2 hours.
After reacting for 4 hours, it was thoroughly washed with water. The film piece into which carboxyl groups were introduced in this way was treated with an aqueous solution of dibekacin sulfate [50 μ (potency) / 100 mj! ) to,
pH to 0. 9 using an aqueous sodium hydroxide solution of IN
The mixture was immersed for 2 hours at 7°C while adjusting the temperature to obtain a film piece on which dihecasin sulfate was immobilized.

This film was cut into a circular shape with a diameter of 5 fl, and Bacillus 5ubtilis ATC
When the antibacterial activity was measured by the cylindrical plate method using C6633 as a test bacterium, inhibitory particles were observed around the film piece after 16 hours at 37°C.

Example 3 The terpolymer obtained in Example 1 was melt-spun.

A fiber with a thickness of 5.2 d was obtained. 1 g of the obtained fiber was
Hepatitis human immunoglobulin (HBs antibody) 1% phosphate buffer (1/IOM, pH 8,0) 10 ml
After being immersed in water at 7°C for 24 hours, it was thoroughly washed with physiological saline solution.

The thus obtained anti-hepatitis B human immunoglobulin-immobilized fiber was packed into a 10 ml column, and when plasma with an 11Bs antigen titer of 1:2 was filtered through this column, the antigen amount in the plasma after filtration was decreased by 1:2. In addition, the measurement of llBs antigen titer is as follows.

The test was carried out in accordance with the revised and expanded 28th edition of "Clinical Testing Law Proposal" edited by Kanai and JV (Metal Publishing) XX-40.

Example 4 The terpolymer obtained in Example 1 was melt-molded.

A tube having an outer diameter of 2.0+n+n and an inner diameter of 1.5 cm was obtained.

In the resulting tube, add urokinase phosphate buffer (2,400 units of urokinase in 1 m & phosphate buffer concentration 1
/ 10 M, pH 9,0), reacted at 7°C for 24 hours, and then thoroughly washed with physiological saline.

When the tube in which urokinase was immobilized was cut into slices and placed on a fibrin plate in the same manner as in Example 1, 24
After some time, it was observed that the fibrin membrane around the ring section had melted.

Patent applicant: Unitika Co., Ltd.

Claims (1)

[Claims] (11) A bioactive material on the surface of a copolymer of a monomer having an epoxy group and an olefin, a monomer selected from the group consisting of chlorinated olefins, vinyl esters, and dienes. A method for producing a physiologically active molded article, which comprises immobilizing a substance.