JP2011245267A - Sulfated polysaccharide immobilized porous polymeric base material and medical instrument - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a polymeric base material being a polymeric material for virus absorption for the purpose of removing viruses in the blood and having a function to selectively remove viruses without absorbing/removing blood components whose removal is not good, and to provide a medical instrument using the polymeric base material.SOLUTION: In the polymeric material for virus absorption, the surface of a porous polymeric support (C) is treated by graft polymerization reaction of a compound (A) having a hydroxyl group or amino group or a polymerized compound (B) having a functional group reactive to ammonia. Sulfated polysaccharide is combined with the compound (A) having the hydroxyl group or amino group or the polymerized compound (B) via covalent bond with ammonia, so that the polymeric material for virus absorption is immobilized on the porous high polymeric support (C).

Description

The present invention relates to a polymer substrate on which a sulfated polysaccharide obtained by treating the surface of a porous polymer support is immobilized. In addition, since the polymer substrate of the present invention has a function of adsorbing hepatitis virus or human immunodeficiency syndrome virus, the present invention relates to a medical device for removing hepatitis virus or human immunodeficiency syndrome virus using the polymer substrate. .

Hepatitis C is caused by chronic infection with hepatitis C virus (HCV), and a combination therapy of peginterferon and ribavirin is common as a therapeutic method using drugs. In patients with genotype 1b and a large amount of virus in the blood, the therapeutic result is about 50%, and since the rate of transition to cirrhosis and liver cancer is high, development of more effective treatment methods and drugs is desired. (Non-Patent Document 1). In general, treatment with drugs is known to have high therapeutic results when the amount of virus in the blood is low. When HCV in the blood is removed with a porous filter and combined with the drug, treatment is performed. There is a report that results are improved (Non-Patent Document 2). That is, it is presumed that the treatment results have been improved by reducing the amount of virus in the body.

However, the method of removing with the above filter is to remove the virus by once separating blood cells and plasma with a filter and then passing the plasma component through another filter. There is a need for a method of removing viruses from sucrose.

On the other hand, it is known that heparin is effective as a ligand that binds to HCV (Non-patent Document 3). Therefore, the base material on which heparin is immobilized may be able to remove HCV more easily, and is expected to provide an HCV removal module or the like with less burden on the patient.

Examples of the form of the substrate on which heparin is immobilized include beads and porous hollow fibers. The internal circulation type extracorporeal circulation module using a porous hollow fiber has less blood stagnation than the extracorporeal circulation module filled with the particulate heparin-immobilized base material, so it has the advantage of less thrombus formation due to its structure. Have When immobilizing heparin on the porous hollow fiber, the type of surface functional group and the immobilization density vary depending on the base material, and it is necessary to find an optimum method for each base material.

For example, in Patent Document 1, a blood inlet, an upstream blood circuit, a plasma separation unit, and a downstream blood circuit are connected in this order, and further, a plasma outlet of the plasma separation unit, an upstream plasma circuit, a plasma purification unit, and a downstream plasma The circuits are connected in this order, and the end of the downstream plasma circuit is a blood processing apparatus connected to blood plasma mixing means provided in the middle of the downstream blood circuit, and the blood plasma mixing means of the downstream blood circuit A device is described in which a blood cell treatment means comprising a water-insoluble carrier for removing at least viruses and virus-infected cells is provided on the downstream side, and the plasma purification means comprises a porous filtration membrane having a maximum pore diameter of 20 nm to 50 nm.

Patent Document 2 discloses an adsorbent excellent in safety and performance for adsorbing and removing harmful components in blood, and a compound having an anionic group and having a molecular weight of 1,000 to 1,000,000 is used as a water-insoluble carrier. An adsorbent for blood purification characterized in that it has an elution value of 0.001 or less and a thermal dissociation value of 0.01 or less and is covalently bound to 0.01 mg or more and 100 mg or less per mL of a water-insoluble carrier.

Patent Document 3 discloses an HIV and related substance removing material having a porous body and a polysulfate compound supported on the pore surfaces of the porous body, wherein the polysulfate compound is substantially A material for removing HIV and its related substances is described, characterized in that the sulfate groups are present in the form of potassium or sodium salts.

On the other hand, human immunodeficiency syndrome is caused by chronic infection with human immunodeficiency syndrome virus (HIV), and multidrug combination therapy is common as a therapeutic method using drugs. However, with the advent of drug-resistant viruses, the development of effective therapeutic methods and drugs that have never existed is desired. After removing or reducing HIV in blood, it is expected that the therapeutic effect can be enhanced by the combined use effect with the drug.
For example, Patent Document 4 discloses an HIV and related substance removing material having a porous body and a polysulfate compound supported on the pore surfaces of the porous body, the polysulfate compound being substantially In particular, a material for removing HIV and its related substances is described, wherein the sulfate group is present in the form of potassium salt or sodium salt.

JP 2005-230165 A JP-A-5-168707 Patent 3615785 Japanese Patent No. 3955379

Viral hepatitis-Advances in basic and clinical research-Japanese clinical volume 62 extra number 7 (2004) A. K. Fujiwara et al. Hepatol. Res. , 37, 701 (2007) Zahn, J. et al. P. Allain, J .; Gen. Virol. , 86, 677 (2005)

In the conventional techniques, most of the means for removing pathogenic bacteria such as viruses are filtration of blood containing pathogenic bacteria using a filtration membrane, and these means are the same for patients as hepatitis virus or human immunodeficiency syndrome virus. Since useful components of a size larger than about, such as lipids and immunoglobulins, are also removed together, the burden on the patient has been a big problem. Therefore, an object of the present invention is to adsorb and remove components in the blood that are adsorbed and removed from hepatitis virus or human immunodeficiency syndrome virus for the purpose of removing viruses in the blood, which are not preferable for removal. And providing a polymer substrate having a function capable of selectively removing hepatitis virus or human immunodeficiency syndrome virus, and a medical device using the same.

In order to solve the above problems, the present inventors selected a porous polymer support (D), a porous polymer support of sulfated polysaccharide having a function of adsorbing hepatitis virus or human immunodeficiency syndrome virus ( As a result of detailed studies on the immobilization method to D) and the virus adsorption ability, the present invention has been completed.

That is, the present invention is based on a graft polymerization reaction of a compound (A) having a hydroxyl group or an amino group, a compound (B) having at least two epoxy groups, or a polymerizable compound (C) having a functional group capable of reacting with ammonia. A polymer substrate for adsorbing hepatitis virus or human immunodeficiency syndrome virus, wherein the surface of the porous polymer support (D) is treated,
The sulfated polysaccharide is bonded to the polymerizable compound (C) through a covalent bond with a compound (A) having a hydroxyl group or an amino group, a compound (B) having at least two epoxy groups, or ammonia. The present invention relates to a polymer substrate for adsorbing hepatitis virus or human immunodeficiency syndrome virus, which is immobilized on a conductive polymer support (D).

According to the present invention, a polymer substrate having a function capable of selectively removing hepatitis virus or human immunodeficiency syndrome virus without adsorbing / removing the above-mentioned components in the blood that are not preferable to be removed, and the use thereof A medical device can be provided.

It is a schematic sectional drawing which shows an example of the medical device provided with the polymer base material of this invention.

That is, the present invention
1. Porous polymer support by graft polymerization reaction of compound (A) having hydroxyl group or amino group, compound (B) having at least two epoxy groups or polymerizable compound (C) having a functional group capable of reacting with ammonia A surface of the polymer substrate for adsorbing hepatitis virus or human immunodeficiency syndrome virus, wherein the surface of (D) is treated,
The sulfated polysaccharide is bonded to the polymerizable compound (C) through a covalent bond formed by a reaction with a compound (A) having a hydroxyl group or an amino group, a compound (B) having at least two epoxy groups, or ammonia. A polymer substrate for adsorbing hepatitis virus or human immunodeficiency syndrome virus, characterized by being immobilized on a porous polymer support (D),
2. 1. The porous polymer support (D) is a porous polymer support using a polyolefin as a substrate. A polymer substrate for adsorbing hepatitis virus or human immunodeficiency syndrome virus according to
3. 1. The porous polymer support is a hollow fiber. A polymer substrate for adsorbing hepatitis virus or human immunodeficiency syndrome virus according to
4). 2. The polyolefin is polyethylene. A polymer substrate for adsorbing hepatitis virus or human immunodeficiency syndrome virus according to
5). The compound (A) having a hydroxyl group or an amino group is a hydroxyalkylamine derivative in which the hydroxyl group or amino group may have a protecting group, or a polyvalent amine derivative having at least two amino groups having no protecting group. . ~ 4. A polymer substrate for adsorbing hepatitis virus or human immunodeficiency syndrome virus according to any one of
6). The compound (A) having a hydroxyl group or an amino group is selected from the group consisting of 2-aminoethanol, ethylenediamine, butylenediamine, hexamethylenediamine, 1,2-bis (2-aminoethoxy) ethane, polyethyleneimine, and polyallylamine. 4. Either A polymer substrate for adsorbing hepatitis virus or human immunodeficiency syndrome virus according to
7). The compound (B) having at least two epoxy groups is represented by the general formula (1)

（式中、ｍは１〜１００の整数、ｎは２〜４の整数を表す。）
で表される１．〜６．の何れかに記載の肝炎ウイルス又はヒト免疫不全症候群ウイルス吸着用高分子基材、
８．重合性化合物（Ｃ）が、（メタ）アクリル酸、グリシジル（メタ）アクリレート又は（メタ）アクリロイルオキシアルキルイソシアネートである１．〜７．の何れかに記載の肝炎ウイルス又はヒト免疫不全症候群ウイルス吸着用高分子基材、
９．硫酸化多糖が、ヘパリン、ヘパリン誘導体、デキストラン硫酸又はフコイダンである請求項１．〜８．の何れかに記載の肝炎ウイルス又はヒト免疫不全症候群ウイルス吸着用高分子基材、
１０．１．〜９．の何れかに記載の肝炎ウイルス又はヒト免疫不全症候群ウイルス吸着用高分子基材を備えた肝炎ウイルス又はヒト免疫不全症候群ウイルス除去器具、
１１．肝炎ウイルスがＢ型又はＣ型肝炎ウイルスである１０．に記載の肝炎ウイルス除去器具、
１２．８．〜１１．の何れかに記載の肝炎ウイルス又はヒト免疫不全症候群ウイルス除去器具を用いた肝炎ウイルス又はヒト免疫不全症候群ウイルスの除去方法、
に関する。

(In the formula, m represents an integer of 1 to 100, and n represents an integer of 2 to 4.)
1. ~ 6. A polymer substrate for adsorbing hepatitis virus or human immunodeficiency syndrome virus according to any one of
8). 1. The polymerizable compound (C) is (meth) acrylic acid, glycidyl (meth) acrylate or (meth) acryloyloxyalkyl isocyanate. ~ 7. A polymer substrate for adsorbing hepatitis virus or human immunodeficiency syndrome virus according to any one of
9. 2. The sulfated polysaccharide is heparin, heparin derivative, dextran sulfate or fucoidan. ~ 8. A polymer substrate for adsorbing hepatitis virus or human immunodeficiency syndrome virus according to any one of
10.1. ~ 9. A hepatitis virus or human immunodeficiency syndrome virus removal device comprising a polymer substrate for adsorbing hepatitis virus or human immunodeficiency syndrome virus according to any one of
11. 9. Hepatitis virus is hepatitis B or C Hepatitis virus removal instrument according to
12.8. ~ 11. A method for removing hepatitis virus or human immunodeficiency syndrome virus using the hepatitis virus or human immunodeficiency syndrome virus removal instrument according to any one of
About.

・ Compounds having a hydroxyl group or an amino group (A)
The compound (A) having a hydroxyl group or amino group of the present invention is a hydroxyalkylamine derivative in which the hydroxyl group or amino group may have a protecting group, or at least 2 in the molecule in which the amino group may have a protecting group. 2-aminoethanol, ethylenediamine, butylenediamine, hexamethylenediamine, 1,2-bis, which is an amine derivative having a specific number of amino groups, and the hydroxyl group or amino group may have a protective group. (2-aminoethoxy) ethane, polyethyleneimine, polyallylamine and the like can be mentioned.

This compound (A) may have a known and commonly used protective group used for a hydroxyl group or an amino group during the reaction with the polymerized compound (B). Examples of the protecting group used include, but are not limited to, a group protecting with a carbamate bond, a group protecting with an ester bond, a group protecting with an amide bond, or a group protecting with an ether bond.

Examples of the group protected by a carbamate bond include t-butyl carbamate (Boc group) and benzyl carbamate. Examples of the group protected by an ester bond include an acetyl group and a benzoyl group. Examples of the protecting group include an acetylamide group and a benzoylamide group, and examples of the group to be protected by an ether bond include a methoxymethyl ether group and a benzyl ether group. These groups can be appropriately selected depending on the type of compound (A) used, the type and conditions of the reaction performed with the polymerizable compound (C), and the like.

-Compound (B) having at least two epoxy groups
The compound (B) can be used without particular limitation as long as it is a compound having an epoxy group that can react with both the polymerizable compound (C) and the sulfated polysaccharide, but the reactivity with the reactive sulfated polysaccharide, General formula (1) for ease of handling

（式中、ｍは１〜１００の整数、ｎは２〜４の整数を表す。）
で表される化合物を挙げることができる。
より具体的には、多価アルコールとエピクロロヒドリンとの反応生成物を挙げることができ、用いることのできる多価アルコールとして、両末端に水酸基を持つ直鎖状アルキルジオールおよび、その縮合体、エチレングリコール、プロピレングリコール、ブタンジオール、ポリエチレングリコール、ポリプロピレングリコール、ポリブチレングリコールなどを挙げることができる。

(In the formula, m represents an integer of 1 to 100, and n represents an integer of 2 to 4.)
The compound represented by these can be mentioned.
More specifically, a reaction product of a polyhydric alcohol and epichlorohydrin can be mentioned. As the polyhydric alcohol that can be used, a linear alkyl diol having hydroxyl groups at both ends and a condensate thereof , Ethylene glycol, propylene glycol, butanediol, polyethylene glycol, polypropylene glycol, polybutylene glycol, and the like.

・ Polymerizable compound (C)
The polymerizable compound (C) used in the present invention has a group capable of reacting with the compound (A) having a hydroxyl group or an amino group, and is further ethylenic capable of undergoing a graft polymerization reaction on the porous polymer support (D). It can be used without limitation as long as it has an unsaturated group. However, in consideration of graft polymerization with the porous polymer support (D), it reacts with the compound (A) having a hydroxyl group or an amino group. A (meth) acrylic compound having a group to be obtained is preferred. As the (meth) acrylic compound, (meth) acrylic acid, glycidyl (meth) acrylate or (meth) acryloyloxyalkyl isocyanate is particularly preferable.

・ Porous polymer support (D)
The porous polymer support (D) used in the present invention is preferably a polymer compound having a methylene group in the main chain because radicals are generated by irradiation with ionizing radiation. As such a polymer compound, various compounds having high blood compatibility can be used. For example, olefin resin, styrene resin, sulfone resin, acrylic resin, urethane resin, ester Resin, ether resin or cellulose acetate, and more specifically, polyethylene terephthalate, ethylene vinyl alcohol copolymer, polymethyl methacrylate, polysulfone, polyether sulfone, polyacrylonitrile, polyethylene, polypropylene or poly-4-methyl. An example is pentene.

Other materials for the porous polymer support (D) include natural polymers such as cellulose and its derivatives, or polyolefin, polysulfone, polyethersulfone, polyacrylonitrile, polymethyl methacrylate, ethylene vinyl alcohol, polyamide, polyimide, Examples thereof include polymer materials such as polyvinylidene fluoride and a polymer alloy of polyethersulfone and polyarylate. Furthermore, what is surface-modified to the hydrophilic porous membrane by surface treatment is preferable.

The shape of the porous polymer support (D) is not particularly limited, and can be used in various forms such as hollow fibers, beads, and nonwoven fabrics. When applied to extracorporeal circulation, it can be used as beads or non-woven fabrics with a structure in which blood stays. It is desirable to use a hollow fiber.
Preferred examples of the porous polymer support (D) include hollow fibers having an average pore diameter in the range of 0.1 to 1 μm.

-Graft polymerization In the present invention, the porous polymer support (D) has an ethylenically unsaturated group of the polymerizable compound (C) supported by the radical generated by irradiating ionizing radiation to the porous polymer support (D). Graft polymerize on the body. Examples of the ionizing radiation source used in the graft polymerization include known and commonly used α-rays, β-rays, γ-rays, accelerated electron beams, X-rays and the like, and practically preferred are γ-rays and accelerated electron beams.

The graft polymerization method includes a simultaneous irradiation graft polymerization method in which the porous polymer support (D) and the polymerizable compound (C) are brought into contact with each other and irradiated with ionizing radiation, and the porous polymer support (D) is irradiated in advance. Any of the pre-irradiation graft polymerization methods for contacting with the polymerizable compound (C) can be used and can be selected according to the purpose.

In the graft polymerization method using ionizing radiation used in the present invention, the irradiation dose and the accelerating voltage differ depending on the porous polymer support (D). It is necessary to adjust appropriately considering the material, form, thickness, etc. of D). For example, when the irradiation amount is large, dielectric breakdown due to charging occurs, and when the irradiation amount is small, the polymerization reaction does not proceed. For this reason, in consideration of the material and form of the porous polymer support (D), the irradiation dose that does not cause dielectric breakdown due to charging and the polymerization reaction sufficiently proceeds may be appropriately adjusted. Further, the acceleration voltage is related to the permeability and varies depending on the thickness of the porous polymer support (D). In the case of a thin form such as a film, the acceleration voltage is generally small and may be selected according to the form of the porous polymer support.

For example, in the case where the porous polymer support (D) is a hollow fiber made of polyethylene and having a thickness of 10 (μm) to 100 (μm), it is preferably 10 (kGy) or more and 300 (kGy) or less. May be 90 (kGy) or less, and the acceleration voltage can be appropriately selected.

There is no particular limitation in the order of the contact step and the ionizing radiation irradiation step.
For example, the step of irradiating the porous polymer support (D) with ionizing radiation in this state after contacting the polymerizable compound (C) or the polymerizable composition containing the polymerizable compound (C) The porous polymer support (D) may be irradiated with ionizing radiation first, and then the porous polymer support (D) may be polymerized with the polymerizable compound (C) or the You may perform the process of making the polymeric composition containing a polymeric compound (C) contact in this order.

In the irradiation graft polymerization, radicals in the substrate after irradiation are quickly inactivated by temperature increase and contact with oxygen. Therefore, after irradiation, it is preferable to store it at a low temperature in a state where oxygen is sufficiently removed, and to quickly fix it. For the above reasons, the immobilization is preferably carried out under deoxygenation or in an inert gas.
The porous polymer support (D) after polymerization may be removed from the unreacted polymerizable compound by a method such as washing with a solvent.

There is no particular limitation on the bonding reaction between the compound (A) having a hydroxyl group or an amino group and the polymerizable compound (C), and the compound (A) having a hydroxyl group or an amino group and the polymerizable compound (C) are not covalently bonded. Anything can be obtained.

For example, when (meth) acrylic acid is used as the polymerizable compound, the compound (A) having an amino group that is a functional group capable of binding to the carboxyl group of (meth) acrylic acid is preferred.
In the case of this bonding mode, an amidation reaction between (meth) acrylic acid and a compound having an amino group is performed.
Amidation reaction methods include, for example, amidation with an active ester, amidation with a condensing agent, combinations thereof, mixed acid anhydride method, azide method, redox method, DPPA method, Woodward method, etc. The known and commonly used amidation reaction may be appropriately performed.

Examples of amidation with an active ester include NHS (N-hydroxysuccinimide), nitrophenol, pentafluorophenol, DMAP (4-dimethylaminopyridine), HOBT (1-hydroxybenzotriazole), and HOAT (hydroxyazabenzotriazole). And the like to form an active ester obtained by once condensing a group having a high leaving ability with a carboxy group, and reacting this with an amino group. Amidation with a condensing agent may be used alone or in combination with the active ester. As the condensing agent, EDC (1- (3-dimethylaminopropyl-3-ethyl-carbodiimide hydrochloride), HONB (endo-N-hydroxy-5-norbornene-2,3-dicarboxamide), DCC (dicyclohexylcarbodiimide) , BOP (benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate), HBTU (O-benzotriazol-1-yl-N, N, N ′, N′-tetramethyluronium hexafluorophosphate) , TBTU (O-benzotriazol-1-yl-N, N, N ′, N′-tetramethyluronium tetrafluoroborate), HOBt (1-hydroxybenzotriazole), HOOBt (3,4-dihydro-3- Hydroxy-4-oxo- , 2,3-benzotriazine), di-p-trioylcarbodiimide, DIC (diisopropylcarbodiimide), BDP (1-benzotriazole diethyl phosphate-1-cyclohexyl-3- (2-morpholinylethyl) carbodiimide), fluorine Cyanuric chloride, cyanuric chloride, TFFH (tetramethylfluoroformamidinium hexafluorophosphophosphate), DPPA (diphenylphosphoradidate), TSTU (O- (N-succinimidyl) -N, N, N ′, N′-tetramethyl Uronium tetrafluoroborate), HATU (N-[(dimethylamino) -1-H-1,2,3-triazolo [4,5,6] -pyridin-1-ylmethylene] -N-methylmethanaminium. Hexafluorophosphate / N-oxide) BOP-Cl (bis (2-oxo-3-oxazolidinyl) phosphine chloride), PyBOP ((1-H-1,2,3-benzotriazol-1-yloxy) -tris (pyrrolidino) phosphonium tetrafluorophosphate), BrOP (bromotris (dimethylamino) phosphonium hexafluorophosphate), DEPBT (3- (diethoxyphosphoryloxy) -1,2,3-benzotriazin-4 (3H) -one), PyBrOP (bromotris (pyrrolidino) phosphonium Hexafluorophosphate) and the like.

Among these, the method in which the carboxy group of (meth) acrylic acid is once NHS and then reacted with the amino group of compound (A) to be amidated is preferable.

As a solvent that can be used in these amidation methods, water and an organic solvent used for peptide synthesis can be used. For example, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), hexaphosphoroamide, dioxane, tetrahydrofuran ( THF), ethyl acetate, and the like, and mixed solvents and aqueous solutions containing these.

The proportion of the activated ester residue introduced into the carboxy group depends on the type of activator used and the amount of reagent used. In general, compared to the reaction in solution, the polymer obtained from the polymerizable compound is bonded, and the reactivity is lowered. Therefore, in order to increase the introduction amount, it is necessary to use a considerably excessive amount of the reaction reagent. It is thought that there is. Therefore, the ratio of the activator to the condensing agent reaction reagent with respect to the carboxy group of (meth) acrylic acid polymerized on the porous polymer support (D) cannot be defined unconditionally, but the active ester group is added to the carboxyl group. In order to introduce it in an equal amount, it is desirable to use about 1 to 10 in a molar ratio. Furthermore, by adjusting the excess amount of the reaction reagent, it is possible to arbitrarily adjust the introduction ratio of the active ester group in the range of 0.01 to 100%.

When glycidyl (meth) acrylate is used as the polymerizable compound (C), the hydroxyl group or amino group of the compound (A) having a hydroxyl group or amino group is reacted with the glycidyl group of the polymerizable compound (C). Can be combined. Examples of the reaction conditions include a solvent used in the reaction of a normal hydroxyl group or amino group and a glycidyl group, a reaction temperature, and a reaction time. As an example of preferable conditions, when the compound (A) having a hydroxyl group or an amino group is a solution, it may be used directly. When a solvent is used, the porous polymer support (D) is dissolved or dissolved. A solvent that does not swell, more preferably a solvent that can dissolve the polymer of the polymerizable compound (C) without dissolving and swelling the porous polymer support (D), and a compound having a hydroxyl group or an amino group ( The density | concentration of A) can be selected arbitrarily, Preferably, it can adjust and use for the density | concentration of weight concentration 0.1-50%.

The reaction temperature can be selected from the temperature at which the compound (A) having a hydroxyl group or amino group or the solvent does not vaporize, and is preferably 0 to 70 ° C, more preferably 20 to 60 ° C. The reaction time depends on the type and concentration of the compound (A) having a hydroxyl group or amino group, and the reaction temperature. The content of the compound (A) having a hydroxyl group or amino group in the reaction solution is determined by gas chromatography, liquid It is preferable to carry out until (A) is no longer consumed while monitoring by chromatography, titration or the like. In general, the reaction can be completed in 30 minutes to 12 hours, but is not limited thereto, and reaction conditions can be set by appropriately selecting.

When (meth) acryloyloxyalkyl isocyanate is used as the polymerizable compound (C), the hydroxyl group or amino group of the compound (A) having a hydroxyl group or amino group, and the isocyanate group possessed by the polymerizable compound (C) It can couple | bond by making it react. Examples of the reaction conditions include a solvent used in a reaction between a normal hydroxyl group or amino group and an isocyanate group, a reaction temperature, and a reaction time. As an example of preferable conditions, the solvent can be appropriately selected from solvents that do not dissolve and swell the porous polymer support (D), such as ethyl acetate and acetonitrile, and do not react with isocyanate. The reaction concentration can be arbitrarily selected, and specifically can be selected from 0.5 to 15% by weight concentration. The reaction temperature is preferably 0 to 50 ° C. because the reaction with isocyanate is an exothermic reaction. Although reaction time can mention 2 to 24 hours, it is not limited to these, It can select suitably and can set reaction conditions.

The kind of sulfated polysaccharide used in the present invention is not particularly limited as long as it is a sulfated polysaccharide having a function of adsorbing hepatitis virus or human immunodeficiency syndrome virus.
Examples of such sulfated polysaccharides include, for example, heparin, heparin derivatives, dextran sulfate, fucoidan, heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, gratan sulfate, heparitin sulfate, carrageenan, sulfated hyaluronic acid, xylan sulfate. , Caroline sulfate, cellulose sulfate, chitin sulfate, chitosan sulfate, pectin sulfate, inulin sulfate, alginate sulfate, glycogen sulfate, dextrin sulfate, laminari sulfate, rhamnan sulfate, curdlan sulfate, galactan sulfate, sulfated duran and their persulfates Derivatives such as isomers, desulfates, and degradation products can be exemplified.
Among these, preferred sulfated polysaccharides include heparin, heparin derivatives, dextran sulfate, and fucoidan.

As the heparin used in the present invention, generally known heparins can be used without limitation. Heparin exists widely in the small intestine, muscle, lungs, spleen, mast cells and the like, and is a kind of heparan sulfate that is chemically a glycosaminoglycan, β-D-glucuronic acid, or α-L-iduron. It is a polymer in which an acid and D-glucosamine are polymerized by 1,4-bonds, and has a feature that the degree of sulfation is particularly high compared to heparan sulfate.

Further, the average molecular weight of heparin is not particularly limited, but when the average molecular weight is large, the reactivity with the compound (A) having a hydroxyl group or an amino group is lowered, so that the efficiency of immobilizing heparin is considered to be poor. It is done. Therefore, the molecular weight of heparin is preferably about 500 to 500,000 daltons, more preferably 1,200 to 50,000 daltons, and even more preferably 5,000 to 20,000 daltons.

  The heparin derivative is a heparin derivative in which the primary or secondary hydroxyl group of the heparin is sulfate-esterified or a heparin derivative obtained by N-sulfate esterification of an acetyl group-eliminated N-acetyl group of the heparin. It has a special feature. The sulfuric esterification can be usually performed by a known method.

When synthesizing a heparin derivative in which a primary or secondary hydroxyl group of heparin is sulfated, for example, heparin amine salt is obtained by treating an alkali salt of heparin with an ion exchange resin (H ⁺ ) or the like and treating with an amine. Adjust. Thereafter, it can be treated with a sulfating agent to obtain the desired heparin derivative (A). As the sulfating agent, known and commonly used SO ₃ • pyridine is preferable.

Moreover, when synthesizing a heparin derivative obtained by N-sulfate esterification of an acetyl group-eliminated heparin N-acetyl group, for example, after deacetylating the heparin N-acetyl group with hydrazine or the like, a sulfating agent is obtained. To obtain the desired heparin derivative. As the sulfating agent, known and commonly used SO ₃ .NMe _{3 and the} like are preferable.

Moreover, a well-known and usual thing can be used for dextrin sulfuric acid or fucoidan.

The coupling | bonding of the sulfated polysaccharide of this invention and the compound (A) which has a hydroxyl group or an amino group can be performed by the said amidation reaction, for example.

Here, after subjecting the polymerizable compound (C) to a graft polymerization reaction, heparin may be immobilized after reacting with the hydroxyl group or amino group-containing compound (A) or ammonia, or the polymerizable compound (C ), A hydroxyl group- or amino group-containing compound (A) or ammonia, and then a graft polymerization reaction may be carried out to immobilize heparin.

The form of the medical device provided with the polymer substrate of the present invention is not particularly limited as long as it is a shape applicable to the above-mentioned use, and examples thereof include a hollow fiber module, a filtration column, and a filter. . In the hollow fiber module and the filtration column, the shape and material of the container are not particularly limited, but when applied to extracorporeal circulation of body fluid (blood), a cylindrical container having an internal volume of 10 to 400 mL and an outer diameter of about 2 to 10 cm It is preferable to use a cylindrical container having an internal volume of 20 to 200 mL and an outer diameter of about 2.5 to 4 cm.

As a method for using the medical device of the present invention, it is brought into contact with a liquid containing hepatitis virus or human immunodeficiency syndrome virus (for example, an aqueous solution containing hepatitis virus or human immunodeficiency syndrome virus, or a body fluid such as blood, plasma, serum). Any method may be used as long as hepatitis virus or human immunodeficiency syndrome virus in the solution can be adsorbed and removed. Examples of such methods include the following methods.
(1) A method of preparing a module having a polymer substrate of the present invention and passing a liquid containing hepatitis virus or human immunodeficiency syndrome virus through the module (2) Although the liquid can pass through the outlet, A method of passing a liquid containing a hepatitis virus or a human immunodeficiency syndrome virus through a column-like container in which a molecular substrate is not allowed to pass and a column-like container filled with the substrate is prepared. (3) The methods (1) and (2) are prepared by preparing a container, adding the liquid containing the hepatitis virus or human immunodeficiency syndrome virus and the polymer base material of the present invention to the container, and then collecting the supernatant. It is preferable in terms of simplicity, and it is possible to efficiently remove hepatitis virus or human immunodeficiency syndrome virus from a patient's body fluid or blood in-line by incorporating it in an extracorporeal circuit. It is. Among these, a more preferable method is the method (1).
In the methods (2) and (3), for example, when handling blood, it is necessary to separate only blood into blood cells and plasma to prevent coagulation, but in the method (1), Such a process is not required, the operation is the simplest, and the burden on the patient is small.

Evaluation of hepatitis virus adsorption ability The hepatitis virus targeted in the present invention is characterized by hepatitis B virus or hepatitis C virus. In this invention, in order to evaluate hepatitis virus adsorption ability, adsorption ability with respect to HCV E2 protein (His-tag) was evaluated. The E2 protein, together with the lipid membrane, constitutes the envelope of the hepatitis virus particle (envelope) and plays an important role in the entry of hepatitis virus. By evaluating the adsorption ability to the E2 protein, It is a protein that makes it possible to evaluate adsorption capacity.

As shown in the following examples, the polymer substrate of the present invention was confirmed to have the ability to adsorb HCV E2 protein, and it was also confirmed to have the ability to adsorb hepatitis virus.
-Evaluation of human immunodeficiency syndrome virus adsorption ability It evaluated by PCR method by the change in the number of viruses before and after treatment with a hepatitis virus or human immunodeficiency syndrome virus removal instrument.

The following examples illustrate the invention in more detail.

The following measurement items were performed by the methods shown below.
<Average pore diameter>
ASTM: Method described in F316-86 (half dry method)
Experimental conditions Liquid used: ethanol, pressurization speed: 0.01 atm / second Calculation formula Average pore diameter = 2860 × surface tension / half dry air pressure (Reference: JP 2001-157826 A)
<Porosity>
Calculation formula Porosity = (wet membrane weight−dry membrane weight) / membrane volume × 100

<Heparin immobilization amount>
The measurement was performed using a quantitative method using adsorption of a dye solution using toluidine blue. (Reference: P.K. Smith, Anal. Biochem., 109, 466 (1980))
Specifically, it was performed as follows.
A dye solution was prepared by dissolving 25 mg of toluidine blue in 500 mL with 0.01 M HCl containing 0.2% NaCl and making up the volume. 10 to 70 μg of heparin was added to 2.5 mL of the dye solution, 2.5 mL of 0.2% NaCl was added, 5 mL of hexane was further added, and the mixture was shaken vigorously for 30 seconds. The solution was separated by standing for a while, 1 mL of the aqueous layer was taken, diluted to 10 mL with methanol, and the absorbance was measured to prepare a calibration curve.
To about 10 cm ² of the hollow fiber, 2.5 mL of the dye solution was added and immersed for 10 minutes, and then diluted by adding 2.5 mL of 0.2% NaCl. 1 mL of the diluted solution was taken, diluted to 10 mL with methanol, the absorbance was measured, and the immobilized amount was calculated from the calibration curve.

<HCV-E2 adsorption amount>
The hollow fiber on which heparin was immobilized was cut into 5 cm ² , put into a microtube, 1% BSA / PBS solution was added as a blocking solution, and left overnight at 4 ° C. The blocking solution was removed, 500 μL of 2 to 4 μg / mL E2 solution (Abcam) was added, and the mixture was rotated at room temperature for 2 hours, and the amount of E2 before and after adsorption was measured by ELISA.

<Number of human immunodeficiency syndrome viruses>
PCR method (Roche, AMPLICOR HIV MONITOR Test ve
rsion 1.5 or COBAS TaqMan). Each laboratory strain (
AD8) PBS solution (number of viruses 3 × 10 ⁵ copies / mL) was added to 1 mL of 5 cm ^{2 of} the sulfated polysaccharide-immobilized hollow fiber prepared in each example, rotated, sampled in 2 hours, and the number of viruses Was measured. The results are shown in Table 2.

(Synthesis example) Preparation example of sulfated heparin derivative 1 g of heparin sodium salt was passed through 50 mL of an ion exchange resin (H ⁺ ), and the eluate was neutralized with a tributylamine-ethanol solution. Excess tributylamine was extracted and removed with Et ₂ O, and the remaining aqueous layer was lyophilized to prepare 1.7 g of heparin tributylammonium salt.
Next, a solution of SO ₃ · Py 2.8 g in anhydrous DMF in 100 mL was added dropwise to 60 mL of DMF in which n-tributylammonium salt of heparin prepared above (800 mg) was dissolved, and the mixture was reacted at 40 ° C. for 1 hour. The reaction solution was put into cold water, neutralized with 1M NaOH, desalted and purified by ultrafiltration, freeze-dried, and 470 mg of sulfated heparin derivative was obtained.

Example 1
A polyethylene hollow fiber bundle (average pore diameter 0.1 μm, porosity 70%, hollow fiber surface area 100 cm ² ) using an electron beam irradiation device “EC250 / 30 / 90L” manufactured by IELON BEAM Co., Ltd. to an oxygen concentration of 100 ppm or less. Nitrogen substitution was performed until it was, and an electron beam of 90 kGy was irradiated at an acceleration voltage of 200 kV. A hollow fiber bundle was put into a glass test tube, a septum was attached, and the atmosphere was purged with nitrogen under reduced pressure. Subsequently, a deoxygenated solution of glycidyl methacrylate (Tokyo Kasei) methanol solution 10 mg / mL was added to the test tube at 23 ° C. After 4 hours, the hollow fiber bundle was taken out from the test tube, washed several times, dried, then weighed, and the immobilization of glycidyl methacrylate was confirmed from the increase in weight. Polyethyleneimine (Wako Pure Chemical, molecular weight 10,000) 1g was melt | dissolved in methanol 15mL, the glycidyl methacrylate fixed hollow fiber was immersed, and reaction was performed at 40 degreeC for 16 hours. After removing the hollow fiber from the solution and washing, 30 mg of heparin (Aldrich), 12 mg of NHS (Wako Pure Chemical Industries), and 15 mg of EDC (Dojin Chemical) were dissolved in 15 mL of PBS and immersed in the solution. After soaking for 16 hours, the hollow fiber was taken out, washed, and then immersed in an Ac ₂ O / AcONa 1/2 v / v solution for 1 hour for acetylation treatment to obtain a heparin-immobilized hollow fiber. The amount of heparin immobilized was measured by a quantitative method using adsorption of a dye solution using the toluidine blue. The HCV E2 adsorption rate and the number of human immunodeficiency syndrome viruses were evaluated by the methods described above.

(Example 2)
In the same manner as in Example 1, glycidyl methacrylate was fixed, and instead of polyethyleneimine, 4 mL of 1,2-bis (2-aminoethoxy) ethane (Tokyo Kasei) was dissolved in 15 mL of methanol, and the glycidyl methacrylate-fixed hollow fiber was immersed therein. The reaction was carried out at 16 ° C. for 16 hours. Subsequently, heparin was immobilized in the same manner as in Example 1 to obtain a heparin-immobilized hollow fiber.

(Example 3)
A sulfated heparin derivative-immobilized hollow fiber was obtained in the same manner as in Example 1 except that the sulfated heparin derivative obtained in the above synthesis example was used instead of heparin.

(Example 4)
A dextran sulfate-immobilized hollow fiber was obtained in the same manner as in Example 1 except that dextran sulfate was used instead of heparin.

(Example 5)
A fucoidan-fixed hollow fiber was obtained in the same manner as in Example 1 except that fucoidan was used instead of heparin.

(Example 6)
In the same manner as in Example 1, hydroxyethyl methacrylate was immobilized on the hollow fiber. 1,4-butanediol diglycidyl ether 2.5 mL, NaBH ₄ 50 mg, 1M NaOH 2.5 mL, and H ₂ O 5 mL were mixed and reacted at 25 ° C. for 8 hours to obtain an epoxidized hollow fiber. After washing with water, a 100 mg / mL solution of heparin was prepared using a 0.1 M Na ₂ CO ₃ solution, and the epoxidized hollow fiber was immersed in this solution for 24 hours at 40 ° C. and reacted to obtain a heparin-immobilized hollow fiber. Obtained.

(Reference example)
<Preparation of human immunodeficiency syndrome virus>
-After transfection of HeLa cells with AD8, supernatant was collected and used 48 hours later.

(Comparative example)
Fixation of glycidyl methacrylate in the same manner as in Example 1 except that a hollow fiber bundle made of poly-4-methylpentene (average pore diameter 0.05 μm, porosity 30%, manufactured by DIC Corporation) was used instead of the polyethylene hollow fiber bundle. And treated with polyethyleneimine to immobilize and acetylate heparin.

Table 1 shows the HCV-E2 adsorption rate and the human immunodeficiency syndrome virus adsorption rate using the polymer base materials obtained in Examples 1 to 5 and Comparative Example.
In the table, a compound (A) having a hydroxyl group or an amino group, a compound (B) having at least two epoxy groups, or ammonia is referred to as a “linking compound”.

The polymer substrate of the present invention can be used as an instrument for removing hepatitis virus or human immunodeficiency syndrome virus.

1 outlet 2 inlet 3 sulfated polysaccharide 4 hollow fiber 5 container 6 partition

Claims (12)

Porous polymer support by graft polymerization reaction of compound (A) having hydroxyl group or amino group, compound (B) having at least two epoxy groups or polymerizable compound (C) having a functional group capable of reacting with ammonia A surface of the polymer substrate for adsorbing hepatitis virus or human immunodeficiency syndrome virus, wherein the surface of (D) is treated,
The sulfated polysaccharide is bonded to the polymerizable compound (C) through a covalent bond formed by a reaction with a compound (A) having a hydroxyl group or an amino group, a compound (B) having at least two epoxy groups, or ammonia. A polymer substrate for adsorbing hepatitis virus or human immunodeficiency syndrome virus, wherein the polymer substrate is immobilized on a porous polymer support (D). The polymer substrate for adsorbing hepatitis virus or human immunodeficiency syndrome virus according to claim 1, wherein the porous polymer support (D) is a porous polymer support using a polyolefin as a substrate. The polymer substrate for adsorbing hepatitis virus or human immunodeficiency syndrome virus according to claim 2, wherein the porous polymer support is a hollow fiber. The polymer substrate for adsorbing hepatitis virus or human immunodeficiency syndrome virus according to claim 3, wherein the polyolefin is polyethylene. The compound (A) having a hydroxyl group or an amino group is a hydroxyalkylamine derivative in which the hydroxyl group or amino group may have a protecting group, or a polyvalent amine derivative having at least two amino groups having no protecting group. Item 5. A polymer substrate for adsorbing hepatitis virus or human immunodeficiency syndrome virus according to any one of Items 1 to 4. The compound (A) having a hydroxyl group or an amino group is selected from the group consisting of 2-aminoethanol, ethylenediamine, butylenediamine, hexamethylenediamine, 1,2-bis (2-aminoethoxy) ethane, polyethyleneimine, and polyallylamine. The polymer substrate for adsorbing hepatitis virus or human immunodeficiency syndrome virus according to claim 5, which is any one of them. The compound (B) having at least two epoxy groups is represented by the general formula (1)

(In the formula, m represents an integer of 1 to 100, and n represents an integer of 2 to 4.)
The polymer base material for hepatitis virus or human immunodeficiency syndrome virus adsorption | suction in any one of Claims 1-6 represented by these. The polymerizable compound (C) is (meth) acrylic acid, glycidyl (meth) acrylate or (meth) acryloyloxyalkyl isocyanate, for adsorbing hepatitis virus or human immunodeficiency syndrome virus according to any one of claims 1 to 7. Polymer substrate. The polymer substrate for adsorbing hepatitis virus or human immunodeficiency syndrome virus according to any one of claims 1 to 8, wherein the sulfated polysaccharide is heparin, heparin derivative, dextran sulfate or fucoidan. A hepatitis virus or human immunodeficiency syndrome virus removing device comprising the polymer substrate for adsorbing hepatitis virus or human immunodeficiency syndrome virus according to any one of claims 1 to 9. The hepatitis virus removing instrument according to claim 10, wherein the hepatitis virus is a hepatitis B or C virus. The removal method of the hepatitis virus or human immunodeficiency syndrome virus using the hepatitis virus or human immunodeficiency syndrome virus removal instrument in any one of Claims 8-11.

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