JPS60224637A - Carrier for immobilizing physiologically active substance - Google Patents

Abstract

PURPOSE:The titled carrier, obtained by treating a structure, having a shape of beads, powder, etc., and made from cellulose as a material with a solution of an acid anhydride based high polymer, and capable of immobilizing easily a large amount of a physiologically active substance. CONSTITUTION:A carrier for immobilizing a physiologically active substance obtained by treating a structure, having a shape of beads, powder, flake, tube, sponge, sheet, film or membrane, and made from cellulose as a material with a solution of an acid anhydride based high polymer, e.g. polymaleic anhydride. The above-mentioned carrier is treated with the acid anhydride based high polymer, and therefore capable of easily immobilizing the physiologically active substance, e.g. enzyme or coenzyme, with either one of covalent bond or ionic bond and introducing reactive functional groups in larger amounts than with a low-molecular compound. Thus, a large amount of the physiologically active substance can be immobilized. The carrier and the same containing the immobilized physiologically active substance find various uses, e.g. chemical reaction catalysts, specific adsorbents for separation and purification, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a carrier used for immobilizing physiologically active substances.

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

Physiologically active substances such as antigens and antibodies are made of polyacrylamide.
Those immobilized on carriers such as cellulose, agarose, and glass are used as specific adsorbents for separation and purification of chemical reaction catalysts, materials for clinical tests, and medical materials.

enzyme,? ! Chemical reaction catalysts are bioactive substances such as enzymes, hormones, antibacterial agents, antigens, and antibodies immobilized on carriers such as polyacrylamide and glass.

It is used in adsorbents, clinical test materials, medical materials, etc. Cellulose is a preferable material as a single substance because it is non-toxic to living organisms and relatively inexpensive.

Because it only has a hydroxyl group with low reactivity in its molecule.

As it is, it is impossible to react with a physiologically active substance under mild conditions, and it cannot be used as a carrier for immobilizing a physiologically active substance. Therefore, research has been conducted to activate cellulose by introducing highly reactive functional groups. For example, JP-A-52-5393 discloses that carboxymethylated cellulose molded articles are suitable as carriers for immobilizing physiologically active substances such as urokinase. However, this substance physically or ionically adsorbs physiologically active substances.

If it is necessary to immobilize by covalent bond.

It is necessary to use a dehydration condensation agent such as dicyclohexylcarposiimide, and the immobilization operation and washing after immobilization are troublesome. Furthermore, since this is carboxymethylation using a low-molecular compound, the degree of substitution of hydroxyl groups to carboxymethyl groups is theoretically at most 1, and usually about 0.25 is considered to be the highest. Therefore, a sufficient amount of immobilization cannot be expected.

Furthermore, in JP-A No. 56-64788, the present applicant previously reported that a polymer having acid anhydride groups was reacted with a polyol on the cellulose surface to remove unreacted acid anhydride groups on the cellulose surface. It is proposed that scales with a film formed thereon can be suitably used as a carrier for enzyme immobilization. This material has the advantage that enzymes and the like can be immobilized by covalent bonds without using a dehydration condensation agent, and is a better carrier than the aforementioned carboxymethylated cellulose. However, even with this product, in addition to the polymer having acid anhydride groups, it is necessary to use a polyol that can react with the polymer, and a common solvent for both must be selected. 3. Tomo 30-5
0℃1 Normally, a high temperature exceeding 10℃ is required, and the acid anhydride group that should normally be used for bonding with enzymes etc. is consumed in reaction with polyol and is reduced, so enzymes etc. It has drawbacks such as a decrease in the amount of binding.

In light of this current state of affairs, it is possible to easily immobilize physiologically active substances in large quantities by covalent bonding, ionic bonding, or adsorption as needed without using other reagents. With the aim of obtaining a carrier for immobilizing physiologically active substances that can be produced, as a result of further intensive research, it was discovered that the objective could be achieved by treating cellulose with an acid anhydride-based polymer solution 1. The present invention. has been reached.

That is, the present invention relates to beads, powders, flakes, tubes, sponges, and sheets made of cellulose.

This is a carrier for immobilizing physiologically active substances, which is made by treating a structure in the form of a film, membrane, etc. with an acid anhydride-based polymer solution.

In the present invention, cellulose means that glucose is β-1,
4,-Glucoside-linked polyBs.

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 immunoreactive substances.

An example of an enzyme is alcohol dehydrogenase.

Lactate dehydrogenase, glucose-6-phosphorus MFm hydrogenase, 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.

Hydrolytic enzymes such as urokinase, plasmin, purinolase, asparakinase, urease, penicillin amidase, avirase, lyases such as pyruvate decarboxylase, albaltase, threonine deaminase, isomerases such as glucose isomerase, thi l: + sil - T RNA synthetase, acetyl- CoA
Examples include glue gauze such as synthetase.

Examples of the coenzyme include pyridoxa-luphosphoric acid and nicotine adenine dinucleotide.

Enzyme inhibitors include, for example, the ohomucoid Kunit
z Soybean trypsin inhibitor, aprotinin 1 antitropin ■, α2-macroglobulin, α1-antitrypsin, αtsuta-antiplasmin.

Examples include plasminogen antiactivator and heparin.

An example of a proenzyme is plasminogen.

Examples include fibrinogen, prothrombin, and blood coagulation box factor X.

Examples of hormones include cortisone, testolone,
Examples include estrone, estradiol 9, progesterone, insulin, tumastatin, and gonadotropins.

An example of an antibiotic is cloxacillin.

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

Cephalosporins such as cephapirin, cefrazine, cefrazole, cefoxitin, and cefatridine; aminoglycosides such as streptomycin, kanamycin, fradiomycin, paromomycin, gentamicin, hekanamycin, ribostamycin, dibekacin, amikacin, tobramycin, and specdinomycin; Tetracyclines such as tetracycline, tetracytalline, demethylchlortetracycline, methacycline, doxycycline, methacycline, erythromycin, kitasamycin 5 oleandomycin,
Macrolides such as spiramycin, josamycin, and midecamycin; lincomycins such as lincomycin and talindamycin; antigram-positive bacteria such as micamycin, gramicidin S, and gramicidin; and polymyxins such as colistin and polymyxin B.

Antimycobacteria such as biomycin, cabreomycin, enviomycin, and cycloserine; polyene macrolides such as amphotericin B and pimaricin; rifampicin, virolnidoline, mitomycin C, and acunanomycin. Examples include pleomycin, daunorubicin, doxorubicin, and neocarzinostatin.

Examples of bactericidal agents include pigment preparations such as acrinol and acrylflavin, furan preparations such as nitrofurazone, quaternary ammonium salts such as benzalkonium chloride and benzethonium chloride, guanidine salts such as chlorhexidine, and iodine preparations such as povidone-iodine. complexes, amphoteric surfactants such as alkyldiaminoethylglycine hydrochloride, and the like.

Examples of anticancer drugs include 2) r+gen mustard, nitromine, chlorambucil, cyclophosphamide,
Melhuaran, uracil mustard.

Mantuno, Sten, Dovan, BCNU, triethylenemelamine, thio-TEPA, ^za-TEP^, trenimon, inprocuone, busulfan, dimethylmyleran, piposulfan, ethoglucide, epoxypropidine, uboxypiperazine, hexamethylmelamine,
Dibromomannitol, and alkylating agents such as pobromane, 3-folic acid, aminopriten methotrexate, guanine, and 8-azaganine.

6-mercaptopurine, azathioprine, uracil, 5
- Metabolic inhibitors such as fluorouracil, cytarabine, azaserine, diazomycin, anti-11F [, 5-HP, IQ-1] such as actinomycin D, sacromycin, mitomycin C, daunomycin, pleomycin, chromomycin, cardinophilin Synthetic agents such as thioteva, cyclophosphamide, doquinrubicin, daunorubicin.

Examples include plant components such as Neocarcinoscum, Hg-hemadoporphyrin, Co-protoporphyrin, stilbestrol, hydroxyurea, procarbazine, methylglyoxalubisuguanylhydrazone 9 L-asparaginase, and the like.

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.

Generally, they are peptides, proteins, polymas, glucoprotein 5 steroids, etc. 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, yeast,
Microorganisms such as protozoa and viruses and their immunologically active components.

Examples include antibodies isolated from humans and animals, serum components, toxins, hormones, enzymes, alkaloids, cell and tissue extracts, blood cells, and lectins.

A substance that can support the above-mentioned physiologically active substances through an immobilization process as described below is called a physiologically active substance immobilization carrier. It becomes physiologically active when exposed to water.

Examples of acid anhydride polymers in the present invention include polycarboxylic anhydrides such as polymaleic anhydride, polyitaconic anhydride, polyacrylic anhydride, and polymethacrylic anhydride, and these polycarboxylic anhydrides. A copolymer having as a unit.

Maleic anhydride/methyl vinyl ether copolymer.

Maleic anhydride/ethyl vinyl ether copolymer.

Copolymers of maleic anhydride and aliphatic vinyl ethers such as maleic anhydride/butanediol divinyl ether copolymers, maleic anhydride/ethylene copolymers, maleic anhydride/propylene copolymers, maleic anhydride/isobutylene copolymers Copolymers of maleic anhydride and olefin monomers such as polymers, copolymers of maleic anhydride and aromatic vinyl heptamers such as maleic anhydride/styrene,
Examples include copolymers of maleic anhydride and aliphatic vinyl esters, such as maleic anhydride/vinyl acetate copolymers, and in the present invention, one or a combination of these may be used.

In the present invention, the treatment of a cellulose-based structure with an acid anhydride polymer solution can be carried out using two acid anhydride polymers such as methanol, ethanol, propatool, isopropanol, butanol, tetrahydrofuran, acetic acid, etc. Ethyl, methyl acetate, benzaldehyde, formaldehyde, acetone, cyclohexane, methyl ethyl ketone, mesityl acetate, diacetone alcohol, 2-pyrrolidone, N-methyl-2-pyrrolidone, N-vinyl-2-
Bisridone, butyrolactone, acetic acid, dibutylformamide, pyridine, etc. or a mixed solvent thereof preferably contains 0.1 to 30% by weight1, particularly preferably 0.5 to 30% by weight.
The structure is dissolved in a solution having a concentration of 10% by weight, preferably about 0.01 to 10% by weight, particularly preferably 0.05 to 2% by weight, of sulfuric acid, hydrochloric acid, etc. as a catalyst. 0-150°C, preferably about 0-100°C, particularly preferably about 20-80°C, for about 10 minutes to 72 hours, preferably 30 minutes to 36 hours, or about 30 minutes if treated at high temperature. After treatment for ~10 hours, wash thoroughly and, if necessary, preferably at about 40-150°C, particularly preferably at 8°C.
This is carried out by heat treatment at 0 to 120°C, preferably for about 1 to 48 hours, particularly preferably for about 6 to 24 hours. Treatment methods include method 8, for example, immersion method, and method 1 for spraying.
A known method such as a coating method can be employed.

Among the carriers for immobilizing physiologically active substances of the present invention prepared in this manner, the acid anhydride group content per 1 g of carrier is 0.0002 to 20 as carboxyl group content.
Milliequivalents, even 0.002 to 10 milliequivalents, especially 0
．． 02 to 5 milliequivalents are preferred.

Fl! Anhydride group content is 0.0 as carboxyl group amount
If the amount is less than 0.002 milliequivalents, the ability to immobilize physiologically active substances tends to be poor, while if it exceeds 20 milliequivalents, the strength of the structure tends to be weakened or when used as a carrier, acid Anhydrous polymers tend to elute. In the present invention, the acid anhydride group content is determined as the amount of carboxyl groups by a neutralization titration method after hydrolyzing the acid anhydride groups of the carrier to form carboxyl groups.

Since the carrier for immobilizing a physiologically active substance of the present invention is treated with an acid anhydride-based polymer, it has many advantages such as 5 or less. In other words, physiologically active substances can be easily immobilized by either covalent bonds or ionic bonds, and it is possible to introduce a larger amount of reactive functional groups than by introducing reactive functional groups using low-molecular compounds. It is possible to immobilize a large amount of physiologically active substances, and the acid anhydride polymer itself acts as a so-called spacer during immobilization, making it easy for the physiologically active substances to react with the carrier.

As a result, a large amount of physiologically active substances can be immobilized and acid anhydride groups can be used for immobilization reactions without waste.

In the adsorption or covalent immobilization of the physiologically active substance on the carrier of the present invention, the physiologically active substance can be mixed with water or water such as methanol, ethanol, propatool, acetone, tetrahydrofuran, dioxane, dimethylsulfoxide, dimethylformamide, etc. The carrier of the present invention is dissolved or dispersed in a mixed solution of a solvent and water, and the resulting solution contains the carrier of the present invention at a temperature of preferably -20 to 100°C and (preferably 0 to 100°C).
This can be carried out by treating at 80°C, preferably for 5 minutes to 100 hours, particularly preferably for 10 minutes to 80 hours. At that time, the pH is preferably 2 to 12. In order to particularly preferably adjust the value to 4 to 10, a 1ffi solution such as phosphate or acetate may be used, or hydrochloric acid, sodium hydroxide, etc. may be added. In addition, the physiologically active substance is adsorbed onto the carrier of the present invention or immobilized by ionic bonding, after the carrier is once treated with water, preferably for about 5 minutes to 96 hours, particularly preferably for about 2 to 48 hours, and then as described above. This can be done by treating with a physiologically active substance as shown below.

The carrier of the present invention with a physiologically active substance immobilized thereon can be used, for example, as a specific adsorbent for separation and purification of chemical reaction catalysts, materials for clinical testing, medical materials, etc. .

That is, a biologically active substance on which an enzyme is immobilized can be used as a chemical reaction catalyst. For example, tyrosinase is immobilized for the production of L-〇OP^.
Those with immobilized amylase and cellulase are used for the production of glucose, and those with immobilized aspartase are used for L.
- For the production of aspartic acid, those with immobilized acetate kinase or carbamate kinase can be used for the regeneration of TP, and those with immobilized glycose isomerase can be used for the production of fructose.

Furthermore, the carrier of the present invention on which a physiologically active substance is immobilized can be used as a specific adsorbent for MIi production. For example, those with immobilized enzymes are used for the separation and purification of coenzymes and enzyme inhibitors, those with immobilized coenzymes or enzyme inhibitors are used for the separation and purification of enzymes, and those with immobilized hormones are used for hormone receptors. Those with immobilized antigens can be used for separation and purification of antibodies, and those with immobilized antibodies can be used for separation and purification of antigens. In addition, their specific adsorbents are 1 thyroid stimulating hormone and thyroid hormone as clinical test materials for enzyme, immunoassay and radioimmunoassay.

Insulin, steroid hormone, human placental gonadotropin, angiotensin, α-fetop1 protein 5
It can be used for quantifying ferritin, HBsjA source, etc. and as shown in Table 1.

Various diseases can be treated by removing various resident substances from body fluids using these specific adsorbents.

Furthermore, the carrier of the present invention on which a physiologically active substance is immobilized can be used as a medical material.

For example, urokinase, streptokinase.

Immobilized fibrinolytic active enzymes such as brinolase and plasun, and blood coagulation system inhibitors such as heparin and antithrombin can be used as antithrombotic molding materials for vascular indwelling catheters, bypass tubes, drain catheters, etc. can. Materials with immobilized antibacterial substances such as antibiotics and bactericides can be used as antibacterial materials in urinary catheters and the like. Those with immobilized anticancer drugs.

It can be used as a topical therapy for cancer.

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 8g of cellulophane GH-25-m (cellulose gel, manufactured by Chisso Corporation) beads were mixed with 4% by weight of Gant.
rez AN 169 (manufactured by GAF, maleic anhydride/methyl vinyl ether copolymer) acetone solution 100
ml and treated at 55° C. for 24 hours. After treatment, perform acetone cleaning and hot water cleaning to remove attached Gan.
After thoroughly rinsing off frez AN 169, l]O
A carrier for immobilizing a physiologically active substance was obtained by vacuum drying for 10 hours. The content of acid anhydride groups per gram of this carrier was determined to be about 0.48 milliequivalents as the amount of carboxyl groups by neutralization titration.

After this, 1g of anti-hepatitis B human immunoglobulin (HBs)
antibody) in 1% by weight phosphoric acid mild iH solution (1/'10M.

After being immersed in 10+wl (pH 8,0) at 7°C for 24 hours, it was thoroughly washed with a physiological saline solution.

200 mg of anti-hepatitis B HBs antibody-immobilized Cellulofine thus obtained was packed into a 1 ml column, and the plasma 21 with an HBs antigen titer of 1:26 was passed through the column.
When filtered, the amount of plasma antigen after filtration was reduced to 1:22. Following this column, 4 times (ie 2nd to 5th time) Blood with HBs antigen titer 1:28! +12111
When filtered, the amount of antigen after filtration was 1:2'' for the second and third times, and 1:23 for the fourth and fifth times.
The antigen titer was measured outside the laboratory according to the revised and expanded 28th edition of "Clinical Test Overview" edited by Zenben (Kanehara Publishing) XX-40.

Example 2 Gant
Treatment with rez solution and treatment with HBs antibody solution were performed. When 20 g of the HBs-immobilized cellulose powder obtained in this manner was packed into a column with a capacity of 11, and 2 Ill of plasma containing 28 HBs antigens was filtered through the column, the amount of plasma antigen after filtration was 1:22. Diminished.

When 2 ml of plasma with an HBs antigen titer of 1:2s was filtered through this column four times (i.e., 2nd to 5th times), the amount of antigen after filtration was 1:22° in the 2nd time, and 1:22° in the 3rd to 5th time.
The time was 1:23.

Comparative Example 1 Cellulofine G) I-25 similar to that used in Example 1
- m methanol (17.3% by weight) containing 3g of sodium hydroxide - water (13.7% by weight) -2-
164g of mixed solvent consisting of propatool (69% by weight)
After being alkalized by immersion in water at 20°C for 35 minutes.

4.2 g of monochloroacetic acid was added to this reaction system.

Carboxymethyl groups were introduced by treatment at 70° C. for 3 hours. After the reaction, the reaction system was cooled and treated with hydrochloric acid, and the carboxymethyl group-introduced cellulophane beads were washed repeatedly with water until the pH reached 3 or higher, and then dried and the carboxymethylated carrier for immobilizing a physiologically active substance was added. Obtained.

When 200 mg of this product was packed into a 111 ml column and 2 ml of plasma containing HBs antigen + i11 + 1:28 was filtered through this column, the amount of plasma antigen after filtration was 1:
It decreased to 24. Using this column, repeat 4 times.
When 12 ml of blood of llBs antigen (i [111B) was filtered, the amount of antigen after filtration was 1:2' for the second and third times.

The 4th and S5 times were 1:25.

Example 3 10g of cell 1-course film was mixed with 10ffi amount % SM
＾3000(8RCO/Chemical Comp
immersed in 100 ml of methyl ethyl ketone solution of maleic anhydride/styrene copolymer (manufactured by Any Co., Ltd.) at 60°C for 30 minutes.
It was treated for 6 hours. After treatment.

After thoroughly washing away the attached SMA 3000 by washing with methyl ethyl ketone and acetone,
The carrier was vacuum-dried at ℃ for 10 hours to obtain a carrier for immobilizing a physiologically active substance. The acid anhydride group content per gram of this carrier was about 0.04 milliequivalent in terms of carboxyl group content.

Example 4 SMA 3000 (8 RCO/Chemical
A carrier for immobilizing a physiologically active substance was prepared in the same manner as in Example 3, except that a methyl ethyl ketone solution of maleic anhydride/ethylene copolymer was used in place of the methyl ethyl ketone solution of maleic anhydride/methyl vinyl ether copolymer manufactured by Obtained.

The acid anhydride group content of this carrier was about 0.03 milliequivalent as carboxyl group per gram of carrier.

Example 5 5g of cellulose sponge was added to 2fii% (7) Gantr
ez^N 169 (manufactured by GAF, maleic anhydride/methyl vinyl ether copolymer) immersed in 100 ml of acetone solution.

After being treated at 50°C for 24 hours, it was washed with acetone and hot water, and vacuum dried at 110°C for 8 hours to obtain a carrier for immobilizing a physiologically active substance. The acid anhydride group content of this carrier was 0.38 milliequivalent as carboxyl group per gram of carrier. Add this to urokinase phosphate buffer (p
H7,0,]/IOM, 2000unit10w1)
After being immersed in 100 ml for 24 hours, it was thoroughly washed with physiological saline solution. Fibrinolytic activity was measured outside the laboratory.

More revisions to “Clinical Testing Method Overview” edited by Zenben? A fibrin plate was prepared according to i28 edition (Kanehara Publishing) -105.

A piece of fabric (circular with a diameter of lCo1) was placed on top of it, and the dissolution of fibrin was observed. As a result, after 24 hours, it was observed that fibrin had dissolved over a distance of 3.3 cm around the fabric piece on which urokinase was immobilized.

Patent applicant: Unitika Co., Ltd.

Claims (1)

[Claims] +11 Beads, powder, and flakes made of cellulose. A carrier for immobilizing physiologically active substances, which is made by treating a structure in the form of a tube, sponge, sheet film, film, etc. with an acid anhydride polymer solution.