JPH0257580B2 - - Google Patents

Description

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

Conventionally, many attempts have been made to immobilize physiologically active substances on carriers to make them insoluble, thereby stabilizing or sustaining their activity or making them easier to use.
In such attempts, it is important to use an appropriate carrier in order to effectively exert the various activities of the physiologically active substance. The functions required of such a carrier are that it has a high ability to immobilize physiologically active substances, that the immobilization reaction does not require high temperatures to the extent that the activity of the physiologically active substance is lost, and that It is possible to release biologically active substances in a sustained manner or to permanently retain them on a carrier depending on the purpose, and the carrier itself is stable in the place where it is used. Examples include the ability to obtain molded products of various shapes such as granules, and the ability to provide a strong coating layer that can be coated on various molded products. However, among the various carriers currently in use, although some of them retain each of the above-mentioned functions, they do not satisfactorily retain all of them. For example, conventionally known typical carriers such as Cephadex, glass beads, and activated carbon have only been available in fixed shapes. It was impossible to obtain such information, and there were limits to where it could be used. In addition, polymers having maleic anhydride groups, such as copolymers of aromatic vinyl monomers and maleic anhydride or copolymers of aliphatic vinyl ether and maleic anhydride, can be used as carriers for immobilizing physiologically active substances. Although examples in which they have been used are known, it is difficult to use them alone due to problems such as stringability, formability such as film forming properties, and water resistance, and no such examples have been found. In addition, filamentous materials made of the reaction product of the former copolymer and a crosslinking agent are known from U.S. Pat. No. 3,966,839, U.S. Pat. has poor ability to immobilize physiologically active substances. Furthermore, a molded article made of a reaction product of the latter copolymer and a crosslinking agent is known from U.S. Pat. No. 3,810,468;
The molded product is hard but brittle, making it impossible to use it as a column filler, filter, etc. Further, in this case, if the crosslinking reaction is carried out sufficiently to satisfy the above-mentioned functions, the functions will be drastically reduced. Homopolymers obtained from monomers having acidic groups other than maleic anhydride, such as carboxyl groups, sulfone groups, phosphonic groups, phosphine groups, phenolic hydroxyl groups, etc., and copolymers with other monomers copolymerizable with these monomers. There are many reports using these as carriers, but when these are used as carriers, high temperatures are required for the immobilization reaction (i.e., they do not fulfill the function), and when immobilized at low temperatures, the period of time they remain on the carrier is limited. The number of acidic groups in the polymer is small compared to the molecular weight (i.e., the function is not satisfied), and there are many things that cannot be molded or coated (i.e., the functionality is not satisfied). It cannot be used satisfactorily for reasons such as:

In view of these current circumstances, the present inventors have conducted intensive research in order to obtain a carrier that satisfies all of the functions as a carrier, and have found that (A) maleic anhydride and an aromatic vinyl monomer or The reaction product of an olefin monomer, (B) a copolymer of maleic anhydride and an aliphatic vinyl ether or aliphatic vinyl ester copolymerizable with it, and (C) a polyfunctional compound can be used as a carrier for immobilizing a physiologically active substance. The present invention has been developed based on the discovery that the method is superior.

That is, the present invention provides 0.5 to 99.5 parts by weight of (A) a copolymer of maleic anhydride and at least one monomer selected from the group consisting of styrene, ethylene, isobutylene, and propylene (hereinafter referred to as component (A)); , (B) a copolymer of maleic anhydride and at least one monomer selected from the group consisting of methyl vinyl ether, ethyl vinyl ether, butanediol vinyl ether, and vinyl acetate (hereinafter referred to as
(referred to as component (B)) 0.5 to 99.5 parts by weight, component (A) and (B)
1 to 200 parts by weight of (C) per 100 parts by weight of the total amount of ingredients
Component (A) is added to a compound containing two or more functional groups in one molecule that are reactive with a maleic anhydride group or a carboxyl group generated by hydrolysis of a maleic anhydride group (hereinafter referred to as component (C)). The gist of the present invention is a carrier for immobilizing a physiologically active substance, which is made of a reaction product obtained by reacting component (B) with component (B).

The carrier of the present invention is an excellent carrier that can satisfy all of the above-mentioned functions required for a carrier for immobilizing a physiologically active substance, but what is particularly noteworthy is that component (A)
The ability of the carrier of the present invention comprising a reaction product of component (B) and component (C) to immobilize a physiologically active substance is determined by the reaction product of component (A) and component (C); The fact is that the performance is far superior to that expected when the reaction product of the component and (C) component is mixed.

The term "physiologically active substance" as used in the present invention refers to a substance that has some pharmacological effect or physiological influence on living organisms, such as antigens and antibodies against them, hormones and receptors against them, enzymes and their substrates, inhibitors, and complements. Related substances such as enzymes, various blood proteins, drugs such as antibiotics and disinfectants,
Examples include antithrombotic agents. More specifically, for example, antigens extracted from the glomerular basement membrane of the kidney,
Antigens such as nuclear antigens and proteinaceous antigens in body fluids, B
Antibodies against hepatitis surfactant antigen ( _HBs antibody), l antibodies, antibodies against angiotensin,
Antibodies such as antibodies against cortisol, complements such as the g component of C complement, hormones, receptors for hormones, deoxyribonuclease, amylase, trypsin, chymotrypsin, aminoacylase, galactosidase, invertase, pectinase, L-asparaginase, glucose oxidase, Enzymes such as uriase and cellulase, substrates for enzymes, enzyme inhibitors such as aprotinin, blood proteins such as albumin, globulin, ceruloplasmin, transferrin, thyroglobulin, haptoglobin, hemopexin, conglutinin, amikacin, dipekacin,
Antibiotics such as kanamycin, streptomycin, gentamicin, fradiomycin, polymyxin, disinfectants such as acrinol, acrylfuran, benzalkonium chloride, benzethonium chloride, cyclohexidine, streptokinase, urokinase, plasmin, purinase, synthetic fibrinolytic activators, etc. Examples include fibrinolytic activators, anticoagulants such as heparin and coumarin derivatives, and antithrombotic agents such as platelet inhibitors such as dipyridamole, but in the present invention, various enzymes, antigens, antibodies, antibiotics, bactericidal agents, etc. is preferably used. In particular, _HBs antibodies are preferably used as antibodies, and _HBs antibodies in human blood, purified _HBs antigens or HBs antibodies recovered from antiserum obtained by immunizing humans or animals with vaccines, and _HBs antibodies by cell fusion can be used. Monoclonal _HBs antibodies, etc., obtained by this method are used.

As component (A) in the present invention, a copolymer of maleic anhydride and at least one monomer selected from the group consisting of styrene, ethylene, isobutylene, and propylene is used. Polymerization ratio is 1:1 to 1:5
and those having a molecular weight of 500 to 2,000,000 are preferably used.

In addition, as component (B) in the present invention, a copolymer of maleic anhydride and at least one monomer selected from the group consisting of methyl vinyl ether, ethyl vinyl ether, butanediol vinyl ether, and vinyl acetate is used. The copolymerization ratio of maleic acid and the above monomer is 1:1 or more
1:5 and a molecular weight of 500 to 2,000,000 is preferably used.

Furthermore, as component (C) in the present invention, a compound containing two or more functional groups in one molecule that is reactive with a maleic anhydride group or a carboxyl group generated by hydrolysis of a maleic anhydride group is used. used. Examples of such compounds include polyisocyanates such as hexamethylene diisocyanate, toluene diisocyanate, xylene diisocyanate, phenylene diisocyanate, isocyanate derivatives of aniline-formaldehyde, tetramethylene glycol, glycidyl ethers, etc. , polyeposides such as diglycidyl ether of diethylene glycol, polyvinyl alcohol, copolymers of polyvinyl alcohol with ethylene, propylene, vinyl chloride, allyl alcohol, N-vinylpyrrolidone, etc., methyl, ethyl, propyl, butyl, octyl of polyvinyl alcohol, Ether derivatives such as dodecyl and phenyl, formal of polyvinyl alcohol, acetal derivatives such as ethanal, butyral, and aminoacetal, ester derivatives of polyvinyl alcohol such as acetic acid, formic acid, butyric acid, caproic acid, lauric acid, stearic acid, and benzoic acid, ethylene Glycol, propylene glycol, butylene glycol, diethylene glycol, cyclohexanediol, pentaerythritol, glycerin,
Polyols such as 1,1,1-trimethylolpropane and their carboxymethylated products, polyether polyols such as polyethylene glycol, polypropylene glycol, polytetramethylene glycol and their carboxymethylated products, succinic acid, glutaric acid, adipic acid, sebacic acid,
Polyesters having hydroxyl groups at both ends obtained by condensing dicarboxylic acids such as isophthalic acid, phthalic acid, and terephthalic acid with glycols such as ethylene glycol, propylene glycol, butylene glycol, and their carboxymethylated products, starch, gelatin, and dextran. Examples include natural polymers such as and their carboxymethylated products, amino alcohols such as monoethanolamine and triethanolamine, and polyamines. Examples of polyamines include low-molecular polyamines such as ethylenediamine, trimethylenediamine, 1,2-diaminopropane, tetramethylenediamine, 1,3-diaminobutane, 2,3
-diaminobutane, pentamethylenediamine,
2,4-diaminopentane, hexamethylenediamine, octamethylenediamine, nonamethylenediamine, decamethylenediamine, undecamethylenediamine, dodecamethylenediamine, tridecamethylenediamine, octadecamethylenediamine, N,N-dimethylethylenediamine, N ,N
-diethyltrimethylenediamine, N,N-dimethyltrimethylenediamine, N,N-dibutyltrimethylenediamine, N,N,N'-triethylethylenediamine, N-methyltrimethylenediamine, N,N-dimethyl-p-phene Nylenediamine, N,N-dimethylhexamethylenediamine,
Diethylenetriamine, triethylenethitramine, tetraethylenepentamine, heptaethyleneoctamine, nonaethylenedecamine, 1,3-bis(2'-aminoethylamino)propane, bis(3-aminopropal)amine, 1,3 -bis(3'-aminopropylamino)propane, 1,2,
3-triaminopropane, tris(2-aminoethyl)amine, tetra(aminomethyl)methane,
Methyliminobispropylamine, methyliminobisethylamine, ethyliminobisethylamine, N-aminopropyl-2morpholine, N-aminopropyl-2-pipecoline, N-(2-hydroxyethyl)trimethylenediamine, xylylenediamine, nylenediamine, piperazine,
N-methylpiperazine, N-(2-aminoethyl)
Examples include ethanolamine, N-aminoethylpiperazine, N,N,N'-tetramethylethylenediamine, N,N,N',N'-tetramethyltetramethylenediamine, and polymeric polyamines such as (i) amines and alkylenes. Poly(allene polyamine) synthesized from dihalite or epichlorohydrin [encyclopidio of
Polymer Science and Technology,
(Encyclopedia of Polymer Science and
Technology) Volume 10, Page 616], (ii) Ethyleneimine,
Alkylene imine polymers obtained by ring-opening polymerization of alkylene imines such as propylene imine [Encyclopedia of Polymer Science and Technology, vol. 1, p. 734],
(iii) Others include polyvinylamine, polylysine, etc. Among these, polyvinyl alcohol and its derivatives, polyols, polyether polyols, natural polymers, and amino alcohols are preferably used, and particularly preferably polyvinyl alcohols are used. Alcohol, polyethylene glycol, triethylene glycol, and triethanolamine are used.

The reaction product constituting the carrier of the present invention is the above (A)
0.5 to 99.5 parts by weight of component, 0.5 to 99.5 parts by weight of component (B), and 1 part per 100 parts by weight of the total amount of component (A) and component (B).
It is a reaction product with ~200 parts by weight of component (C). (A)
The ratio of component (A) to the total amount of component and (B) is 0.5
If the amount is less than % by weight, the resulting reaction product water,
Too soluble in body fluids or blood;
On the other hand, if it exceeds 99.5% by weight, the ability to immobilize physiologically active substances will be significantly reduced, and moldability, film-forming properties, and threadability will deteriorate. It is necessary that the proportion of is 0.5 to 99.5% by weight, preferably 20 to 99.5% by weight, particularly preferably 30 to 95% by weight. If the ratio of component (C) to 100 parts by weight of the total amount of components (A) and (B) is less than 1 part by weight, depending on the type and ratio of components (A) and (B), The solubility of the reaction products produced may become too large;
If the amount exceeds 1 part by weight, the ability to immobilize physiologically active substances will be significantly reduced and moldability will also deteriorate.
The proportion of component (C) is 1 to 200 parts by weight, preferably 1 to 200 parts by weight.
It is necessary to use 150 parts by weight, particularly preferably 20 to 100 parts by weight.

The carrier of the present invention includes, for example, component (A), component (B), and (C)
It can be prepared by blending the ingredients in any manner and order, shaping the blend, and then subjecting it to heat treatment. The most preferred method for blending the three components is to dissolve the three components in water or an organic solvent such as acetone or alcohol. Heat treatment is usually performed at a temperature of 20 to 200℃ for 1 minute to 72
It will be done for about an hour. Molding can be easily carried out by known dry methods, wet methods, etc., and supports in the form of films, threads, nonwoven fabrics, pellets, granules, etc. can be obtained. The carrier of the present invention also includes one whose surface is coated with the above reaction product. In order to prepare such a carrier, for example, a molded article may be coated with a mixture of components (A), (B) and (C), and then heat treated.

As a method for immobilizing a physiologically active substance on the carrier of the present invention, for example, a method of immersing the carrier in a solution of a physiologically active substance, a method of spraying a solution of a physiologically active substance onto the carrier, etc. can be adopted. When the tube is tube-shaped, a method of circulating a physiologically active substance solution inside the tube is preferably adopted.

Immobilization as used in the present invention means insolubilizing a physiologically active substance by chemical bonding and making it remain on a carrier, but more specifically, it refers to both permanent immobilization and sustained release immobilization. means. Permanent immobilization means that the immobilized bioactive substance does not leave the carrier at the point of use, while sustained release immobilization means that it gradually separates from the carrier at the point of use. means.

The carrier of the present invention has the feature that permanent immobilization and sustained release immobilization can be carried out as appropriate depending on the purpose, and this is a great advantage as a carrier. For example, permanent fixation can be achieved by heat treating the carrier at a temperature of about 100 to 140°C for several hours and then fixing it at room temperature or below. Sustained release immobilization can be achieved by immobilization after immersion for a period of time.

The carrier of the present invention on which a physiologically active substance is immobilized has various uses. For example, it is used as a so-called immunoadsorbent, in which an antigen or antibody is immobilized and the corresponding antigen or antibody is removed. or by immobilizing drugs such as antibiotics or anticancer drugs so that their medicinal effects are exerted when they are used.
Antithrombotic agents can be immobilized and used to prevent blood coagulation when they are used. When used as an immunoadsorbent, it is often filled into a column and used as a filter for filtration. A material having a denier of 10 denier or less and a large surface area per mass is preferably used.

There are many possible uses for the immunoadsorbent in which antigens and antibodies as described above are immobilized on the carrier of the present invention, but in particular, the immunoadsorbent in which the _HBs antibody is immobilized is
The onset of hepatitis caused by blood transfusion has been known for a long time, and is highly effective in preventing hepatitis, and it is now well known that hepatitis is classified into type A, type B, and nonA-nonB types. Among these, the most notable is hepatitis B, which is caused by infection with the _hepatitis B virus. If used, this hepatitis B virus can be efficiently adsorbed. Therefore, if this filter is used to filter blood, the hepatitis B virus in the blood will be removed, and the onset of hepatitis due to blood circulation can be suppressed.

Hereinafter, the present invention will be explained in more detail with reference to Examples. Note that "parts" in the examples mean "parts by weight."

Examples 1 to 3 1 part of Gantrez AN169 (manufactured by GAF, copolymer of methyl vinyl ether and maleic anhydride),
1 part of SMA3000 (manufactured by ARCO Chemical, copolymer of styrene and maleic anhydride) and magrogol
400 (manufactured by Matsumoto Yushi Co., Ltd., polyethylene glycol) 1
1 part was dissolved in 7 parts of acetone, and a 10 denier thread was produced from this solution by dry spinning. The obtained yarn was heat treated at 140°C for 2 hours. This product was insoluble in hot water. Take 1 g of each of these threads and prepare three types of samples. Sample A is added to an aqueous solution of Hebusbrin (manufactured by Midori Juji Co., Ltd., dried anti-hepatitis B human immunoglobulin) [200 mg/50 ml], and sample B is added to an aqueous solution of dibekacin sulfate (PH). (adjusted to 8) [100
When sample C was immersed in urokinase aqueous solution [100 mg/50 ml] at 7°C for 24 hours, it was found that sample A contained 60 hebsbrin.
45 mg of dibekacin sulfate was immobilized on sample B, and 40 ft of urokinase was immobilized on sample C. The activity of the immobilized material remained unchanged after storage in air at 7° C. for 3 months.

Comparative Example 1 0.3 parts of Gantrez AN169 and 99.7 parts of SMA3000
and 50 parts of Magrogol 400 were dissolved in 350 parts of acetone, and spinning was attempted in the same manner as in Example 1 using this solution, but spinnability was poor and spinning was impossible.

Comparative Example 2 99.7 parts of Gantrez AN169 and 0.3 parts of SMA3000
and 50 parts of Magrogol 400 were dissolved in 350 parts of acetone and spun in the same manner as in Example 1 to obtain a thread of 11 denier. This yarn was heat treated at 140°C for 2 hours. This product was insoluble in cold water but soluble in hot water.

Example 4 1 part of Gantrez AN169, 1 part of SMA3000, and 1 part of Magrogol 400 were dissolved in 17 parts of acetone, and a 3-denier thread was produced from this solution by dry spinning. The obtained yarn was heat treated at 120°C for 3 hours. This product was insoluble in hot water. 1g of this thread was mixed with Hebusbrin aqueous solution [200mg/
50ml] at 7°C for 24 hours, 90mg of hebusbrin was immobilized.

When this thread was filled in a column with a capacity of 10 ml and plasma with an amount of _HBs antigen of 1: ^2.15 was filtered through this column, the amount of antigen in the filtered plasma was reduced to 1: ^2.2 . A large amount of HB _s antigen
It is clear that the _HBs antibody was adsorbed to the immobilized thread of this example.

Example 5 1 part of a copolymer of maleic anhydride and ethyl vinyl ether (copolymerization ratio 1:1, molecular weight 500,000) and a copolymer of maleic anhydride and ethylene (copolymerization ratio 1:1, molecular weight 500,000) 1.5 parts of triethylene glycol and 0.5 parts of triethylene glycol were dissolved in 7 parts of acetone, and a 10 denier thread was obtained in the same manner as in Example 1. The obtained thread was heat-treated at 120° C. for 3 hours, and then immobilized in the same manner as in Example 1, resulting in immobilization of 55 mg of hebusbulin, 50 mg of dibekacin sulfate, and 60 mg of urokinase. The activity of the immobilized substance remained unchanged even after 3 months.

Example 6 10 parts Gantrez AN139 and 90 parts Isoban
110 (manufactured by Kuraray, a copolymer of maleic anhydride and isobutylene) and 30 parts of polyvinyl alcohol (degree of polymerization 1700, completely saponified product) were dissolved in 2600 parts of water, and this aqueous solution was poured onto a polyester film. Rolled out, heat treated at 140℃ for 5 hours,
A 30μ film was made. The obtained film was cut into 5 cm squares and immobilized in the same manner as in Example 1, resulting in immobilization of 40 mg of hebusbulin, 35 mg of dibekacin sulfate, and 30 mg of urokinase. After the immobilized film was immersed in water at 25°C for 10 days, the activity remained unchanged from that at the beginning of immobilization.

Example 7 Two parts of Gantrez AN169 and two parts of SMA3000
A solution was prepared by dissolving 1.5 parts of Magrogol 400 in 100 parts of acetone, and a silicone catheter was immersed in this solution for 3 minutes at room temperature.
When taken out and heat treated at 120℃ for 2 hours,
A coating layer with a thickness of 10 μm was formed on the surface of the catheter. This coating layer was insoluble in hot water. Subsequently, this catheter was treated with Polymixi B aqueous solution [800000
unit/100ml] for 12 hours at 7℃
400,000 units of polymyxin B were immobilized. Even after the immobilized catheter was immersed in water at 37°C for 40 days, the pharmacological effects of polymyxin B were fully observed.

Example 8 Two parts of Gantrez AN169 and two parts of SMA3000
and 2 parts of Magrogol 400 were dissolved in 20 parts of acetone, this aqueous solution was cast onto a polyester film, and heat treated at 140°C for 3 hours to determine the thickness.
A 20μ film was made. This film was insoluble in hot water. 1 g of this film was mixed with γ-globulin (HUMAN COHN manufactured by Sigma Chemical Co., Ltd.).
FRACTION) aqueous solution [200mg/150ml] at 7℃
60 mg of γ-globulin was immobilized.

Comparative example 3 4 parts of Gantrez AN169 and 2 parts of tuna gor
400 was dissolved in 20 parts of acetone, and a 25 μm thick film was prepared in the same manner as in Example 8. 1 g of this film was immersed in a γ-globulin aqueous solution in the same manner as in Example 8. Although the film was slightly dissolved, the amount of γ-globulin immobilized per gram of film was 58 mg.

Comparative example 4 4 parts of SMA3000 and 2 parts of Tuna Gol 400 for 20
A film having a thickness of 10 μm was prepared in the same manner as in Example 8. This film was insoluble in hot water. When 1 g of this film was immersed in a γ-globulin aqueous solution in the same manner as in Example 8,
Only 4 mg of γ-globulin was immobilized on the film.

Claims (1)

[Claims] 1 (A) Copolymer of maleic anhydride and at least one monomer selected from the group consisting of styrene, ethylene, isobutylene and propylene 0.5~
99.5 parts by weight and (B) a copolymer of maleic anhydride and at least one monomer selected from the group consisting of methyl vinyl ether, ethyl vinyl ether, butanediol vinyl ether, and vinyl acetate.
0.5 to 99.5 parts by weight, and 1 to 200 parts by weight of (C) a maleic anhydride group or a carboxyl group generated by hydrolysis of a maleic anhydride group based on 100 parts by weight of the total amount of (A) and (B). A carrier for immobilizing a physiologically active substance, which is made of a reaction product obtained by reacting (A) and (B) with a compound containing two or more functional groups in one molecule with the reactivity of .