JP3176753B2 - Adsorbent for blood processing - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an adsorbent for removing unwanted substances from whole blood or plasma.

[0002]

2. Description of the Related Art As a method of removing unnecessary substances present in blood, there is known a method of purifying blood by contacting the blood with an adsorbent having a functional group capable of binding to the unnecessary substance on a porous carrier. Have been. In such an adsorbent for treating blood, as a functional group having a binding property with an unnecessary substance, and / or
Alternatively, in order to increase the wettability of blood, a group having a negative charge is often introduced on the surface for hydrophilicity. However, when blood comes into contact with the surface of a material having a large amount of negative charge such as glass, activation of blood coagulation factor XII occurs, and kallikrein is generated from prekallikrein by the activated blood coagulation factor XII. It is known that high-molecular-weight kinino gel is limitedly decomposed to produce kinin (blood kinin: Bradykinin). It is also known that this kinin is one of the causative substances of anaphylactic reactions such as lowering of blood pressure, hot flush, conjunctival hyperemia, smooth muscle contraction, and pain, that is, one of anaphylatoxins. However, on the other hand, quantitative information on the production of kinin and the negative charge on the surface of the material has not been sufficiently obtained, and in particular, the optimal negative charge on the surface of clinically usable blood adsorbent materials has not been studied. Not done. Further, there is no clinically known quantitative relationship between the anaphylactic reaction symptoms and the amount of kinin.

[0003]

SUMMARY OF THE INVENTION It is an object of the present invention to provide an adsorbent for blood treatment which has excellent blood wettability and does not cause an increase in kinin during the treatment of blood or plasma. Specific uses of the adsorbent include, for example, cholesterol, autoantibodies, immune complexes, bilirubin, hydrophobic amino acids, plasma proteins such as drugs, lipids, amino acids, nucleic acids, adsorption removal of components in plasma such as low molecular weight compounds, Collection and separation.

That is, according to the present invention, the exclusion limit molecular weight is 5
The water-insoluble carrier surface consisting of a polymer is 0,000 or more 10,000,000 A adsorbents for blood processing comprising a functional group capable of binding with unwanted material, Table
Negatively charged surface and acid / base titration and / or dye
Total charge specified by surface charge measurement method by adsorption
An adsorbent for treating blood, wherein the amount is −30 μeq / g or more and −0.01 μeq / g or less . Further, in the present invention, the adsorbent comprises a water-insoluble carrier and a ligand containing a functional group capable of binding to an unnecessary substance immobilized on the water-insoluble carrier, and the ligand is chain-like, and a part of the ligand is immobilized on the carrier. The adsorbent according to claim 1, wherein the other portion is in a state where it can move flexibly. In the present invention, the term “total charge amount” means a charge amount after both charges are canceled out when a positive charge amount is represented by plus (+) and a negative charge amount is represented by minus (−), that is, from the positive charge amount to the negative charge amount. Charge minus amount
Refers to the amount . Therefore, the expression that the total charge amount is −30 μeq / g or more means that the absolute amount of the negative charge is 30 μeq / g or less .

[0005] The anticoagulant for blood, which exhibits antithrombin activity such as heparin, and forms complexes with divalent metal ions such as citric acid and salts thereof and ethylenediaminetetraacetic acid and salts thereof. That show anticoagulant activity by Nafamostat Mesilate
te) and the like. In our study, nafamostat mesilate inhibits kallikrein activity and has a suppressive effect on kinin production, whereas quinine exhibits anticoagulant activity by forming a complex with a divalent metal ion. Inhibits the activity of decomposing enzymes (kininase) and suppresses kinin decomposition. For this reason, any anticoagulant can be used, but when an anticoagulant that exhibits anticoagulant activity by forming a complex with a divalent metal ion is used, the negative charge on the surface is 30 μeq / The kinin rise was more problematic with filter materials above g, especially above 50 μeq / g. Furthermore, captopril (Captopril), enalapril (En)
When angiotensin converting enzyme inhibitors such as arapril) were present in plasma, the degradation of kinin was inhibited, and kinin elevation was again high. Therefore, an anticoagulant such as citric acid is preferable for the evaluation of the kinin-forming property of the filtration material,
Practically, nafamostat mesilate is preferable in terms of safety because it can suppress the kinin elevation. The total charge on the surface is-
According to an in vitro blood test, a blood filtration material having a large negative charge of 30 μeq / g or less, particularly −50 μeq / g or less, is brought into contact with blood containing about 0.1 to 20% by weight of citric acid and salts thereof in a flask. It was found that the kinin concentration in plasma increased to a high concentration of 4000 pg / ml or more. Further, in practical use, the blood filtration material increases the kinin concentration in plasma to 4000 pg / ml or more,
In addition, it was found that when plasma having a kinin concentration of 4000 pg / ml or more entered the body, anaphylactic symptoms such as flushing of the face and lowering of blood pressure were exhibited. Therefore, in order to perform safe blood purification without causing side effects, the kinin concentration must be 4000 p.
It is necessary that the adsorbent does not increase to g / ml or more. The present inventors have paid attention to the relationship between the kinin increasing property of the adsorbent for blood treatment and the negative charge on the surface of the adsorbent material, and when they studied, there is a clear positive correlation between the two, and the negative charge on the surface of the adsorbent material. It was found that by reducing the amount of kinin, the increase in kinin caused by the adsorbent can be prevented. As a result of our research, the total charge on the surface of the adsorbent was -30.
Above μeq / g, there was little activation of blood coagulation factor XII in the plasma, and therefore the increase in kinin concentration was modest, and in vitro blood tests showed no increase above 4000 pg / ml. The lower the surface negative charge, the more preferable. The more preferable range of the total charge on the surface of the adsorbent is −25 μeq / g or more. More preferably -2
0 μeq / g or more. The smaller the amount of negative charge on the surface of the adsorbent, the better, and there is no particular lower limit. But,
On the other hand, it is preferable that the surface has a negative charge because of the wettability and blood compatibility of the blood when the adsorbent is used, and further, the non-specific adsorption of the plasma protein is low, and 0.01 μeq /
g or more is preferable. It is more preferably at least 0.1 μeq / g, most preferably at least 1 μeq / g. Although the charge amount on the surface of a substance is generally expressed as the charge density per surface area, in our studies, even if the charge density is low, the larger the surface area, the more opportunities for kinin to be generated, Therefore, it was not preferable to simply express the charge density per surface area.

The surface as referred to in the present invention refers to the entire surface of the adsorbent to which blood can come into contact, and does not include the inside of the adsorbent which cannot contact blood. Even if the surface has been subjected to any surface treatment, it refers to the entire surface that can be contacted by blood, including the treatment. The surface charge refers to a charge existing on the surface and in the vicinity of the surface, which can exert an electrostatic action on blood components. The charge that exists between the surface and a depth of 10 ° from the surface is to be quantitatively indicated. In addition, the surface charge amount referred to in the present invention is the surface of the water-insoluble carrier surface that can be contacted with a body fluid such as blood, and the total charge resulting from the cancellation of the negative charge amount and the positive charge amount depending on the ligand molecule. Say the amount. As the adsorbent in the present invention, there are a substance consisting essentially of a water-insoluble porous carrier and a substance consisting of a water-insoluble porous carrier having a ligand immobilized thereon. In the former, the carrier itself has a functional group having specific or selective affinity for the substance to be adsorbed on the surface of the carrier, and in the latter, the carrier itself is specific or selective for the substance to be adsorbed. It does not have a functional group having a high affinity and the ligand formed on the surface has the function. When a coating layer is provided on a porous carrier for the purpose of increasing the affinity of blood or the like, the coating layer is included in the carrier according to the present invention. The negative charge and the positive charge are groups that show negative or positive at neutral pH (pH 7.0), respectively, and the amount of the group means a group that is in a dissociated state at neutral pH.

The term "negative charge" as used in the present invention refers to a neutral charge such as a carboxyl group, a phosphate group, a phosphite group, a sulfonic acid group, a sulfate ester group, a sulfite group, a hyposulfite group, a sulfide group, a phenol group, a hydroxyl group and the like. Refers to an acidic group that is negative at a pH of. Among them, carboxyl groups, sulfonic acid groups and sulfate groups are particularly important because of their high charge strength. This negative charge is derived from the negative group originally possessed by the material constituting the adsorbent of the present invention, and is hydrolyzed by heat, chemicals such as oxides, acids, and alkaline solutions, and radiation during the process of producing the adsorbent. And a compound having a negative charge introduced by a method such as covalent bonding, grafting, physical adsorption, ionic bonding, or embedding. Further, it includes those introduced as a result of graft polymerization of a monomer having a negative group by radiation grafting or plasma grafting, or negative charges newly generated in the monomer or carrier when a monomer having no negative group is graft-polymerized. Thus, consequently all negative charges present on the surface of the adsorbent material when the adsorbent is used are included.

The positive group referred to in the present invention includes amines, amine derivatives, and the like, and includes tertiary and quaternary amino groups. As a preferred example, pK
b is 2.0 or more, and more preferably, pKb is 3.0 or more. Further, since heparin is usually used as an anticoagulant in blood or plasma, it is more desirable that heparin is less adsorbed. From this point, a more preferable example is expressed by the following equation (A).

[0009]

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There are no particular restrictions on the substituents R1, R2 and R3, and any substituent can be provided, but any of the substituents (for example, R1) is shared with any main chain on the adsorbent material. They are connected by coupling. For example, it may be a hydrocarbon substituent such as hydrogen, a methyl group, an ethyl group, a propyl group, a phenyl group, a benzyl group, or a substituent containing a heteronuclear atom such as methylol or ethylol. Furthermore, R1 and R2 (or R3)
And may constitute a main chain. R2 and R3 may be two or more and cyclic, and examples thereof include pyridine, imidazole, piperidine, pyrrole, and pyrimidine. When an example of a positive (functional) group is exemplified by a monomer name, allylamine, diallylamine,
N, N-dimethylallylamine, N, N-diallylpiperazine, N, N-diallylaniline, N, N-diallylmelamine, aminostyrene, N, N-dimethylaminostyrene, N, N-diethylaminostyrene, vinylpentylamine, Vinylphenethylamine, N, N-dimethylvinylphenethylamine, N, N-diethylvinylphenethylamine, N-propylvinylphenethylamine, vinylpyridine, 2-methyl-5-vinylpyridine, 2-ethyl-5-vinylpyridine, 2-vinylquinoline , 2-vinylimidazole, 4-vinylimidazole, vinylpyrazoline, vinylpyrazine, 4-vinylpyrimidine, vinylamine, vinylcarbazole, ethyleneimine, N-phenylethyleneimine, N, N-diethyl-N-vinylphen Ethylamine, diethylaminoethyl methacrylate, dimethylaminoethyl methacrylate, and monomers such as diethylaminoethyl styrene, oligomers or polymers to these either polymerized units may be mentioned. Further, they may be quaternary ammonium groups. Among these, especially R2, R3
Is preferably hydrogen (H) or an alkyl chain having 1 to 12 carbon atoms. Further, R2 and R3 are more preferably hydrogen (H) or an alkyl chain having 1 to 6 carbon atoms. Specific examples include diethylaminoethylstyrene, N, N-diethyl-N-vinylphenethylamine,
More preferred examples include diethylaminoethyl methacrylate, dimethylaminoethyl methacrylate, diethylaminoethyl acrylate, and dimethylaminoethyl acrylate. Further, these polymerized units and other polymerizable monomers may be copolymerized with, for example, hydroxystyrene, hydroxymethylstyrene, vinyl alcohol, 2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylate, segmented polyurethane, segmented polyester, and the like. It may be united. In particular, it is preferable that the polymer has a hydroxyl group from the viewpoint of improving blood compatibility. There is no particular limitation on the mode of bonding of the hydroxyl group in the polymer. In particular, a copolymer with a polymer unit having a hydroxyl group containing 1 to 50% by weight of a polymer unit having a positive group represented by the formula (A) is preferable.

The amount of charge on the surface of the adsorbent refers to the amount of negative groups only when the surface has only negative groups or negative groups and non-ionic groups and no positive groups. When negative and positive coexist on the surface, in the blood around pH 5 to 9 when the adsorbent is used, the total charge obtained by subtracting the negative charge from the coexisting positive charge is -30 μeq / g or more. I just want it.

The method for measuring the total charge of the adsorbent in the present invention is as follows. Calculate 5 ml of the adsorbent under the conditions used. The adsorbent is transferred to a glass filter, and 500 ml of distilled water for injection is slowly poured from above and washed. The adsorbent is transferred to a beaker, 2N hydrochloric acid is added, and the mixture is stirred for 30 minutes using a stirrer. 30 minutes later, the adsorbent in the beaker was transferred onto a glass filter, and 2 L of distilled water for injection was used.
Wash with. At this time, it is confirmed that the pH of the filtrate is 4 or more and 7 or less. If the pH of the filtrate is 4 or less, the washing is repeated with distilled water for injection. PH 7
In the above case, 2N hydrochloric acid is added again, the mixture is stirred for 30 minutes, and the washing is repeated. After checking the pH, transfer the adsorbent to a beaker and add 4%
Add 50 ml of an aqueous sodium chloride solution and stir for 1 hour using a stirrer. The adsorbent is filtered using a glass filter, 20 ml of the filtrate is precisely weighed, and titrated with a 0.01 N aqueous sodium hydroxide solution. From the results of this titration, the negative charge is determined. Method for Measuring Charge of Adsorbent by Titration (Positive Charge) Among the above measurement methods, measurement is performed by replacing hydrochloric acid and aqueous sodium hydroxide solution, and performing the same operation.
First, titration is performed using sodium hydroxide and, at the time of titration, using hydrochloric acid. From this, the amount of positive charge is determined. 5 ml of the adsorbent is weighed under the conditions of use. The adsorbent is transferred to a glass filter, and 500 ml of distilled water for injection is slowly poured from above and washed. Transfer the adsorbent to a beaker,
Add 2N sodium hydroxide and stir with a stirrer for 30 minutes. After 30 minutes, the adsorbent in the beaker is transferred onto a glass filter and washed with 2 L of distilled water for injection. At this time, it is confirmed that the pH of the filtrate is 7 or more and 10 or less. If the pH of the filtrate is 7 or less, an aqueous sodium hydroxide solution is added again, and the mixture is stirred for 30 minutes and the washing is repeated. When the pH is 10 or more, washing is repeated with distilled water for injection. After checking the pH, the adsorbent is transferred to a beaker, 50 ml of a 4% aqueous sodium chloride solution is added, and the mixture is stirred for 1 hour using a stirrer. The adsorbent was filtered using a glass filter, 20 ml of the filtrate was precisely weighed,
Titrate with 1N hydrochloric acid. From the result of this titration, the amount of positive charge is determined. Method for measuring the amount of charge of the adsorbent by titration (system in which positive and negative charges coexist) The measurement method is the same as the method for measuring the negative charge. However,
According to the study of the present inventors, in the coexisting system, the positive charge and the negative charge are ionic-bonded in the system, and the remaining negative charge, that is, the negative charge as the difference obtained by subtracting the positive charge from the negative charge. The amount of charge can be determined.

A simplified method for measuring the amount of negative charge in the presence of an excess of positive charge This method is a simple method for easily determining the presence or absence of negative charge on a filter material, in which negative charge and positive charge coexist and the amount of positive charge is high. Method. A dye having a negative charge is adsorbed on the negative functional group, and the negative charge is determined based on the amount of adsorption. An example is shown below. The filter material is weighed in an amount of 0.01 g in a glass container capable of being sealed. Into this, 10 ml of 0.013% by weight aqueous solution of safranin O was added,
After sealing, shake at 30 ° C. for 48 hours. After 48 hours,
Only the supernatant is taken out and the absorbance is measured at a wavelength of 515 nm. Further, the same operation is performed without adding a filtering material, and the absorbance at the time of non-adsorption is measured. A calibration curve is prepared in advance by the negative charge measurement method using the material whose negative charge is known, and the presence or absence of negative charge and the negative charge thereof are determined from the amount of safranin O adsorbed on the filtration material.

In the present invention, the total charge of the adsorbent is -30 μm.
The means to be on the eq / g or more there is the following method. (1) A method for preventing hydrolysis and thermal decomposition during the production of an adsorbent. (2) A method of reducing the coexistence amount of oxygen during radiation, electron beam, plasma irradiation, or the like, to thereby prevent generation of peroxide (peroxide). (3) A method of scientifically blocking a negative group, such as by covalently bonding a negative group to a non-negative compound. (4) A method of coating a negative group by a physical method such as coating a non-negative compound. (5) A method of chemically or physically introducing a positive group to neutralize electrostatically. Either method can be used. A more preferable method differs depending on the type and use of the material, and cannot be definitely determined. However, in general, a method of forming a coating layer of a compound having a neutral or positive hydrophilic group or a polymer is easiest and practically preferable.

In the porous material used as the adsorbent, there are many fine pores into which blood coagulation factor XII and the like cannot enter.
There are very many surfaces that are not involved in raising or activating bradykinin. For this reason, blood coagulation factor XII (molecular weight of about 10
000), kallikrein (molecular weight about 100,00
0), high molecular weight kininogen (molecular weight about 70,000)
Although it is important to define the amount of negative (functional) groups on the surface to which the triple complex can bind, i.e., the molecular weight and the size of the pores into which 300,000 compounds can enter, Quantitation was difficult.

In the present invention, it is necessary to express the weight of the adsorbent by the weight in the closest state when the adsorbent is used.
Since the adsorbent is porous and has a very large surface area to which the substance to be adsorbed can contact and has a high expansion coefficient, the surface area per unit weight is large between the dry state and the used state (ie, swollen state). In contrast, the negative charge density, most important in terms of bradykinin elevation, also varies significantly. In our studies, the negative charge per weight during use was most closely related to bradykinin elevation. The use state as used herein refers to a state in which the adsorbent swells in a solution similar to the pH and osmotic pressure at the time of use. Specifically, pH 6.5-
7.5 in a physiological saline solution having an osmotic pressure of about 280 mOsm. The weight at the time of use of the adsorbent according to the present invention refers to the weight in a so-called bed state including the solution present in the space between the adsorbents when the adsorbent is densely filled in a certain container and swollen. In the case of an adsorbent in the form of beads, chips, or the like, it may be considered that the volume is equal to the bed volume. Specifically, for example, after swelling, the solution was filled in a container having an inlet and an outlet for the solution and having a function of preventing the adsorbent from leaking, such as a mesh that does not allow the adsorbent to pass through the outlet portion, and the solution was suction-filtered from the outlet. Thereafter, it can be determined by measuring the volume of the adsorbent. In the case of a fibrous, hollow fiber, fibrous, or the like, the length can be measured in a similar manner after a short cut. According to the study of the present inventors, it has been found that an increase in bradykinin can be suppressed by setting the surface negative charge absolute amount to 50 μeq or less per unit weight of the adsorbent. Furthermore, it was found that by setting the absolute amount of surface negative charge to 30 μeq / g or less, clinical shock symptoms caused by an increase in bradykinin can be substantially eliminated.

According to the study of the present inventors, when the sum of the negative charge on the surface of the water-insoluble carrier and the negative charge in the ligand compound exceeds 50 μeq / g, the increase in bradykinin remarkably depends on the negative charge. Become. On the other hand, when the total negative charge was 50 μeq / g or less, the bradykinin-elevating property was greatly reduced, and particularly when it was 30 μeq / g or less, the bradykinin elevation, which was particularly problematic, did not occur.

In the case of an adsorbent in which a ligand is held on the surface of a water-insoluble carrier, the increase in bradykinin greatly differs depending on the electrostatic characteristics of the ligand and the electrostatic characteristics of the surface of the water-insoluble carrier. When a ligand mainly having a negative group (negative ligand) electrostatically binds to a substance to be adsorbed by the negative group, the higher the amount of the ligand or the negative charge density in the ligand, the more the adsorbent can be bound per unit capacity. The amount of the substance to be adsorbed, that is, the adsorption capacity is increased. On the other hand, however, the increase in bradykinin increases and problems arise. In our studies was found to be kept low elevated bradykinin by the total charge amount -30μeq / g or more on the adsorbent surface, thus, the support surface is substantially electrically neutral In this case, the type and fixed amount of the ligand may be controlled so that the negative charge of the ligand is 30 μeq / g. When high capacity of the adsorbent is expected, a group having a positive charge to offset a part of the negative charge of the ligand is provided on the surface of the carrier while the absolute amount of the negative charge derived from the ligand exceeds 30 μeq / g. Preferably, it is introduced. In addition, in the case of an adsorbent having a negative ligand on the surface, the amount of negative groups derived from the ligand on the surface of the adsorbent and the amount of positive groups on the carrier surface were offset to ensure sufficient adsorption capacity. Is preferably 10 μeq / g or more.

According to the study of the present inventors, it was found that the amount of bradykinin caused by the negative group increased less when the blood contacted with the flexible negative surface than with the rigid negative surface. Therefore, the negative ligand is preferably present in a more flexible state on the water-insoluble carrier. For this reason, it is preferred that the high molecular weight ligand has a chain-like molecular structure and extends in a whisker-like manner from the surface of the water-insoluble carrier. Further, it is preferable that the negative ligand has this structure in order to extend its effect farther than the surface of the carrier and to facilitate multipoint bonding with the substance to be adsorbed.

Negative Group Density in Negative Ligand Molecules The higher the density of negative groups present in a negative ligand molecule, the higher the binding to the substance to be adsorbed and the better the adsorbing ability. It was found that if the density was too high, the increase in bradykinin was more likely to occur.
From the viewpoint of the binding to the substance to be adsorbed, the density of the negative group is preferably such that at least one negative group exists per 600 molecular weight of the ligand, more preferably one or more per 300 molecular weight, and further preferably 200 per 200 molecular weight. Is one or more. Further, from the viewpoint that it does not easily cause an increase in bradykinin, the number is preferably 1 or less per 80 molecular weight of the ligand, more preferably 1 or less per 120 molecular weight, and further preferably 1 or less per 150 molecular weight. Specific Examples of Ligands Negative ligands include peptides, sugars, and synthetic organic compounds. More specifically, examples of the negative ligand include those having a sulfate group, a carboxyl group, a phosphate group, or the like in its molecule, and nucleic acids such as adenine, thymine, guanine, cytosine, and uracil and derivatives thereof, or glutamic acid and the like. Amino acids of monoaminodicarboxylic acid such as aspartic acid and derivatives thereof, and compounds and oligomers thereof, polymers thereof, or heparin, dextran sulfate, chondroitin sulfate, chondroitin polysulfate, heparan sulfate, keratan sulfate, heparitin sulfate, xylan sulfate, Karonine sulfate, cellulose sulfate, chitin sulfate, chitosan sulfate, pectin sulfate, inulin sulfate, alginate sulfate, glycogen sulfate, polylactose sulfate, carrageenan sulfate, starch sulfate, polyglucose sulfate, lamiraline sulfate, galactan sulfate, Sulfated polysaccharides such as bansulfate and mepesulfate, phosphotungstic acid, polysulfated anethole, polyvinyl alcohol sulfate, polyphosphoric acid, polyacrylic acid, 2-sulfoethyl methacrylate, crotonic acid, vinylsulfonic acid, allylsulfonic acid, meta Ril sulfonic acid and the like. Among these, synthetic compounds are preferred because they are less likely to exhibit immunogenicity when the ligand is released, and are less likely to cause other physiological reactions. Further, since the adsorption amount of divalent metal ions in plasma is relatively small, a sulfate group is preferred. Are preferred. A preferred example is dextran sulfate.

In the case where the positive group is insolubilized on the water-insoluble carrier in the adsorbent on which the negative ligand is immobilized, the positive group is preferably in the following immobilized state. The positive group may be insolubilized on the carrier surface in the form of a monomer, or may be in the form of a polymer. It is not preferable because it acts obstructively to the function of the ligand. As the method for insolubilizing the positive group, the method used for immobilizing the ligand can be similarly used. However, it is not preferable because it is fixed in a whisker-like manner by the grafting method. Usually, it is preferable to insolubilize a monomer having a positive group by a covalent bond or coat a polymer containing a monomer having a positive group as a component. For example, a method of providing a coating layer is used. When a coating layer is provided, the negative ligand may be insolubilized on the carrier surface or on the coating layer, but is more preferably insolubilized on the coating layer. Most preferably, a method of covalently bonding a monomer having a positive group and a molecular weight of 1,000 or less to the surface of the carrier.

The positive group introduced into the carrier of the adsorbent having the negative ligand does not necessarily need to be present on the side chain of the polymer chain constituting the carrier, and may be one that forms the main chain. A method of insolubilizing a negative functional group in a carrier, such as a method of introducing a positive group into a carrier as a side chain or a method of physically bonding a positive group to a carrier surface without a covalent bond when the polymer is a polymer. Can be selected, and these methods are easier to manufacture than introducing them into the main chain. As an example of a method for obtaining a positive group on a carrier or its coating layer surface, a cyanogen halide method,
Epichlorohydrin method, bisepoxide method, bromoacetyl bromide method and the like are known. Specific examples include an amino group, a carboxyl group, a hydroxyl group, a thiol group, an acid anhydride group, a succinylimide group, a chlorine group, an aldehyde group, an epoxy group, and a tresyl group. Among them, an epoxy group derived by the epichlorohydrin method is particularly preferable because of its stability during heat sterilization.

When a ligand mainly having a positive group (positive ligand) electrostatically binds to a substance to be adsorbed by the positive group, a negative group should be retained on the surface of the carrier in order to enhance blood compatibility of the carrier. Is more preferred. The positive ligand is preferably a flagellated high molecular substance on the carrier surface, while the negative group on the carrier surface has a molecular weight of 1,000.
It is preferable that it has the following low molecular weight or is in a coated state and is not flagella-like. As described above, the rise of bradykinin is more likely to occur on a rigid surface than on a flexible surface. Therefore, in the case of an adsorbent having a positive ligand, the amount of negative groups on the surface of the carrier strongly affects the increase in bradykinin. Therefore, in the case of an adsorbent having a compound having a positive functional group as a ligand and having a negative group on the surface of the carrier, the absolute amount of negative charge remaining as a result of canceling the amount of positive charge derived from the ligand and the amount of positive charge on the surface of the carrier is 30 μeq.
/ G or less, the increase in bradykinin can be suppressed for the time being, but the absolute negative charge on the carrier surface itself is 30 μep / g.
The following can prevent a significant rise in bradykinin,
Particularly preferred.

The positive ligands include allylamine, diallylamine, N, N-dimethylallylamine, N, N-
Diallyl piperazine, N, N-diallyl aniline, N,
N-diallylmelamine, aminostyrene, N, N-dimethylaminostyrene, N, N-diethylaminostyrene, vinylpentylamine, vinylphenethylamine,
N, N-dimethylvinylphenethylamine, N, N-diethylvinylphenethylamine, N-propylvinylphenethylamine, vinylpyridine, 2-methyl-5-vinylpyridine, 2-ethyl-5-vinylpyridine, 2-
Vinylquinoline, 2-vinylimidazole, 4-vinylimidazole, vinylpyrazoline, vinylpyrazine,
-Vinylpyrimidine, vinylamine, vinylcarbazole, ethyleneimine, N-phenylethylenimine,
Polymers containing monomers such as N, N-diethyl-N-vinylphenethylamine, diethylaminoethyl methacrylate, dimethylaminoethyl methacrylate, and diethylaminoethylstyrene can be preferably used. More preferred specific examples of the positive ligand include diethylaminoethylstyrene, N, N-diethyl-N-vinylphenethylamine, diethylaminoethyl methacrylate, and dimethylaminoethyl methacrylate. Further, these polymerized units and other polymerizable monomers such as hydroxystyrene, hydroxymethylstyrene, vinyl alcohol,
It may be a copolymer with 2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylate, segmented polyurethane, segmented polyester, or the like.
In particular, it is preferable that the polymer has a hydroxyl group from the viewpoint of improving blood compatibility. There is no particular limitation on the mode of bonding of the hydroxyl group in the polymer. In particular, 1 to 50% by weight of a polymer unit having a positive functional group represented by the formula (A)
It is preferably a copolymer with a hydroxyl-containing polymer unit.

It is particularly preferable that the positive ligand and the positive ligand are both macromolecules and have at least two or more negative or positive groups in the molecule. If the number of negative (or positive) groups per ligand molecule is small, it will electrostatically bind to the oppositely charged groups on the carrier surface,
The binding property with the substance to be adsorbed as a ligand cannot be sufficiently exhibited. If the number of groups per ligand molecule is large, even if some groups are electrostatically bonded to the oppositely charged groups on the surface of the carrier, the remaining groups can be extended away from the carrier, which is preferable. Furthermore, there is a problem that a ligand is partially decomposed by a sterilization operation in a manufacturing process or by pH, oxygen, ultraviolet light or the like during long-term storage, and this decomposed product elutes into a living body to cause a physiological reaction.
If the carrier has a charge opposite to the ligand, the groups in the degraded ligand and the groups on the surface of the carrier are electrostatically bonded to each other, thereby substantially eliminating elution of the ligand.

The molecular weight of the positive ligand and the negative ligand of the present invention is preferably 1,000 or more and 1,000,000 or less. When the molecular weight is 1,000 or less, the number of functional groups in the molecule is small, and the effect of the above-mentioned polymer is low. On the other hand, when the molecular weight exceeds 1,000,000, when the ligand at the time of production is immobilized on a water-insoluble carrier, The viscosity of the solution increases, making it difficult to maintain uniform conditions. In addition, the risk when the ligand is eluted by mistake becomes particularly large, which is not preferable. In particular, when the substance to be adsorbed is a high molecular weight substance such as a protein, it is more preferable that the ligand has a molecular weight of 5,000 or more because the substance can be bonded at more multipoints and firmly bonded. Further, it is preferably 500,000 or less due to the upper limit of the molecular weight. More preferably, the molecular weight of the positive ligand is 200,000 or less.

When the ligand has a negative group and a positive group, the total charge after these are offset at neutral pH is the charge of the ligand. Examples of the ligand having a negative group and a positive group include a polymer of a positive group-containing compound and a negative group-containing compound, an amino acid and a derivative thereof, or a compound containing the same. is there. Of these, amino groups or derivatives thereof, or compounds containing these, are more preferably monomers, oligomers, polymers, and the like, and most preferably monomers or oligomers. Although any number of molecular weights of these ligands can be used, it is preferable that the molecular weight be 200,000 or less from the viewpoint of the effect on the living body when the ligand is released. In particular, amino acids or derivatives thereof, or compounds containing these, have a risk of showing immunogenicity. Therefore, the amount is more preferably 30,000 or less. Further, it is preferably 5,000 or less, most preferably 1,000 or less. When the ligand has no particular group, the total charge amount on the water-insoluble carrier is important, and it is also desirable that the total charge amount is -30 μeq / g or more.

Ligand insolubilization method As a method for immobilizing the ligand on the surface of the water-insoluble carrier, any known method such as covalent bond, ionic bond, physical adsorption, embedding, or precipitation insolubilization can be used. From the viewpoint of elution properties, it is preferable that they are fixed and insolubilized by covalent bonds. When the molecular weight of the ligand is 1,000 or more and one or more negative groups such as a carboxyl group, a sulfate group, and a sulfate group are present per 300 ligand molecular weights, ionic bonds can be formed by multipoint bonding. Since it can be firmly bonded, it can be preferably used. The method of covalently binding a ligand includes a method for activating a known water-insoluble carrier, a method for immobilizing a ligand, and a method for immobilizing a ligand, which are usually used for immobilizing an enzyme or for immobilizing a ligand substance by affinity chromatography or the like. A graft polymerization means having a ligand as a branch can be used. Examples of the activation method include a cyanogen halide method, an epichlorohydrin method, a bisepoxide method, a halogenated triazine method, a bromoacetyl bromide method, an ethyl chloroformate method, a 1,1′-carbonyldiimidazole method, a tresyl chloride method, Acetamide method and the like can be mentioned. The activation method of the present invention may be any method as long as it can perform a substitution and / or addition reaction with a nucleophilic reactive group having an activated hydrogen such as an amino group, a hydroxyl group, a carboxyl group, or a thiol group of a ligand. Absent. However, in terms of chemical stability and thermal stability, a particularly preferable example is a method using an epoxide, and the epichlorohydrin method can be particularly recommended.
Examples of the graft polymerization method include a chain transfer method, an emulsion polymerization method, a graft polymerization method using various initiators such as a cerium salt, a persulfate-lithium halide, and a hydrogen peroxide-metal salt, a perester group, and a mercapto group. , A graft polymerization method using a compound having a functional group such as a diazo group, a graft polymerization method using air or ozone oxidation, a radiation grafting method, a plasma polymerization method, and the like. Among them, a polymerizable monomer having a negative functional group in a carrier having a reducing group such as a hydroxyl group, a thiol group, an aldehyde, and an amine using a cerium salt, an iron salt, or the like as an initiator, or in radiation or plasma such as γ-rays. Can be preferably used, and a method using radiation or plasma is particularly preferable.

The ligand to be immobilized is 1 m of the water-insoluble carrier.
The suitable amount is 0.01 mg or more and 100 mg or less per liter. From the viewpoint of the adsorption performance of the target substance as an adsorbent and the cost of the ligand used, the fixed amount is 1 ml of the water-insoluble carrier.
In particular, 0.1 mg or more and 10 mg or less are particularly preferable.

The binding strength between the ligand and the water-insoluble carrier is as follows:
The number of covalent bonds per one molecule of the high molecular compound of the ligand varies depending on the number of covalent bonds. In addition, there are also physically adsorbed ligands, which generally have lower bonding strength than covalent bonds, and these cannot be measured individually. However, the ratio of the high molecular compound released by a good solvent of the high molecular compound, for example, a physiological saline solution or a 40% ethanol solution, to the total high molecular compound fixed on the water-insoluble carrier is determined by an elution value, phosphoric acid, carbonic acid, citric acid, or the like. 121 with a neutral aqueous solution containing an acid or a salt thereof
Of the polymer compound released after 1 hour of treatment at a temperature of
When the ratio to the total amount of the high molecular compound fixed on the water-insoluble carrier is defined as a thermal dissociation value, the elution value or the thermal dissociation value can be used as an index of the physical adsorption amount and the strength of the covalent bond. Any of the adsorbents can bind the ligand with a fixed strength of an elution value of 0.05 or less and a thermal dissociation value of 0.01 or less. Further, binding strengths with an elution value of 0.01 or less and a thermal dissociation value of 0.001 or less are also possible. In fact, the present invention provides an elution value of 0.0
It was possible to make the thermal dissociation value 0.001 or less and 0.0001 or less.

Specific Examples of Water-Insoluble Carrier As the carrier of the adsorbent of the present invention, either an inorganic compound or an organic compound can be used. Examples include amylna, silica gel, activated carbon, glass, apatite, natural or synthetic organic compounds, and the like. Any of these may be used, but an organic polymer is preferable because it has a small amount of eluted substances with respect to warm water and can easily and precisely control the pores of the porous body. Examples of such are polypropylene,
Polymers and copolymers of vinyl compounds such as polystyrene, polymethacrylate ester, polyacrylate ester, polyacrylic acid, and polyvinyl alcohol; polyamide compounds such as nylon 6 or 66; polyester compounds such as polyethylene terephthalate; Examples include plant-derived polysaccharide compounds. In the examples, polymers and copolymers of vinyl compounds are more preferably used because of easiness of polymerization and easy introduction of negative and positive groups. An example of this is
Styrene, methylstyrene, diphenylethylene, ethylethylene, dimethylstyrene, vinylnaphthalene, vinylphenanthrene, vinylmesitylene, 3,4,6-
Hydrocarbon compounds such as trimethylstyrene and 1-vinyl-2-ethylacetylene: chlorostyrene, methoxystyrene, bromostyrene, cyanostyrene, fluorostyrene, dichlorostyrene, N, N-dimethylaminostyrene, nitrostyrene, chloromethyl Styrene derivatives such as styrene, trifluorostyrene, trifluoromethylstyrene and aminostyrene: acrylonitrile, α-
Acrylonitrile derivatives such as acetoxyacrylonitrile: acrylic acid, methacrylic acid: acrylic acid esters such as methyl acrylate, lauryl acrylate, chloromethyl acrylate, ethyl acetoxy acrylate: cyclohexyl methacrylate, dimethylaminoethyl methacrylate, glycidyl methacrylate, methacrylic acid Methacrylates such as tetrahydrofurfuryl acid and hydroxyethyl methacrylate: diethyl maleate, diethyl fumarate: vinyl ketones such as methyl vinyl ketone and ethyl isopropenyl ketone, vinylidene compounds such as vinylidene chloride, vinylidene bromide and vinylidene cyanide: Acrylamide, methacrylamide, N-butoxymethylacrylamide, N-phenylacrylamide, diacetone acrylamide, N Acrylamide derivatives such as N-dimethylaminoethyl acrylamide: fatty acid vinyl derivatives such as vinyl acetate, vinyl butyrate, and vinyl caprate: thiofatty acid derivatives such as phenyl thiomethacrylate, methyl thioacrylate, and vinyl thioacetate: further N-vinyl succinimide , N-vinylpyrrolidone, N-vinylphthalimide, N-vinylcarbazolevinylfuran, 2-vinylbendfuran, vinylthiophene, vinylimidazole, methylvinylimidazole, vinylpyrazole,
Vinyl oxazolindone, vinyl thiazole, vinyl tetrazole, vinyl pyridine, methyl vinyl pyridine,
There are heterocyclic vinyl compounds such as 2,4-dimethyl-6-vinyltriazine and vinylquinoline. Examples of the crosslinkable polymerizable monomer that is a constituent unit of the porous support include divinylbenzene, divinyltoluene, divinylxylene, divinylnaphthalene, divinylethylbenzene, divinylphenanthrene, trivinylbenzene, divinyldiphenyl, divinylphenylether, and divinyldiphenylsulfur. FID, dipinyldiphenylamine, dipinylsulfone, divinylketone, dipinylfuran, divinylpyridine, dipyrylquinoline, di (vinylpyridinoethyl) ethylenediamine, diallyl phthalate, diallyl maleate, diallyl fumarate, diallyl succinate, diallyl carbonate, oxalic acid Diallyl, diallyl adipate, diallyl sebacate, diallyl tartrate, diallylamine, triallylamine, triallyl phosphate, triaryl tricarballylate , Triallyl aconitate, triallyl citrate, N, N-ethylenediacrylamide, N, N-ethylenedimethacrylamide, N, N-methylenedimethacrylamide, ethylene glycol methacrylate, triethylene glycol dimethacrylate, polyethylene glycol dimethacrylate, Trimethylolpropane trimethacrylate, pentaerythritol tetramethacrylate, 1,3-butylene glycol diacrylate,
1,6-hexanediol diacrylate, trimethylpropane triacrylate, pentaerythritol tetraacrylate, triallyl isocyanurate,
2,5-triacryloylhexahydro-1,3,5-
Triazine, diarylmelamine and the like. Among them, methyl methacrylate, polyvinyl alcohol,
A rigid polymer composed of a synthetic polymer containing one or more of styrene, divinylbenzene, vinyl acetate, mataleic anhydride, polyamide, and the like, and / or a natural polymer such as cellulose as a raw material. Gels are preferred.

Physical Properties of Water-Insoluble Carrier (1) Pore Size When the pore size of the water-insoluble carrier is represented by the exclusion limit molecular weight, a large effective surface area to which the substance to be adsorbed easily enters and which can actually bind to the substance to be adsorbed is secured. 10,000
Must be 0 or more. On the other hand, if the pores are too large, the substance to be adsorbed easily enters the pores, but the effective surface area decreases, and the strength of the water-insoluble carrier decreases, which is not preferable. Therefore, it is preferably not more than 10,000,000. More preferably, it is 100,000 or more, and 5,000,000 or less. When the pore size is measured by the mercury intrusion method, the average pore size is preferably 50A or more, more preferably 2A, for the same reason.
00A or more, more preferably 300A or more. The upper limit is 5,000 A or less, and more preferably, 3,
000A or less. Since the measurement principle is different between the mercury intrusion method and the exclusion limit, it is more important to satisfy the pore size determined at the exclusion limit molecular weight closer to practical conditions.

Physical Properties of Water-Insoluble Carrier (2) Pore Size Distribution It is preferable that the above-mentioned pore distribution is as uniform as possible and concentrated on the target pore size. Specifically, 10
It is preferable that the volume of the pores having a size of 0 A to 5,000 A is 40% or more of the total volume. Further, it is preferable that the content is 60% or more within the above range. On the other hand, the size of the target substance is not completely uniform, and the substance to be adsorbed bonded near the entrance of the pore does not block the entrance of the pore and is effectively used for adsorption to the inside of the pore. Is also not preferred. When the average pore diameter is D,
The amount of pores in the range of 0.5D to 2.0D is preferably 70% or less by volume ratio, more preferably 50% or less.
Further, the porosity is preferably at least 30%, more preferably at least 50%, in order to secure many pores. If the porosity is too high, the strength as a carrier is reduced. Therefore, the porosity is preferably 95% or less, more preferably 90% or less. The pores referred to here are preferably values in a state as close as possible to the use of the adsorbent. In the case where the surface has a coating layer, the pore values refer to values after the coating treatment. Also,
If the shape changes due to the drying process at the time of measurement by the mercury intrusion method, the change in particle diameter is measured, and the surface area is corrected by the square of the change rate of the particle diameter, and the pore volume is corrected by multiplying the cube of the particle diameter by the cube of the particle diameter. I decided that. That is, when the particle diameter is 1 / X times, the surface area is 1 / X ² times and the pore volume is 1 / X ³ times. Similarly, the pores are converted based on the change rate of the particle diameter. Make corrections based on apparent specific gravity and expansion rate as necessary.

Physical Properties of Water-Insoluble Carrier (3) Specific Surface Area The surface area of the porous body can be measured by physical adsorption, heat of immersion, permeation, chemical adsorption, mercury intrusion, etc. The adsorption method (BET method) is generally and often used. The specific surface area of the water-insoluble carrier of the present invention is preferably 5 m ² / g or more per dry weight, more preferably 10 m ² / g or more, and further preferably 20 m ² / g or more. Most preferably, it is 30 m ² / g or more. In the mercury intrusion method, the specific surface area for each pore size is determined. Here, since the measurement by the mercury intrusion method is accurately required, the ratio of the surface area at each pore size to the layer surface area of the pores having a pore size of 60A to 80,000A is 100A The ratio of the surface area of not less than 5,000 A and not more than 5,000 A is preferably 30% or more. It is more preferably at least 50%.

Physical properties of the water-insoluble carrier (4) Specific shape As the shape, any of spherical, granular, fibrous, hollow fiber, flat membrane, etc. can be effectively used. Granular is most preferably used. Spherical or granular average particle size is 10μm to 10,000μ
m is easy to use, but 25 μm to 1,000 μm
Is more preferable, and more preferably 50 μm to 6 μm.
00 μm.

Physical Properties of Water-Insoluble Carrier (5) Others The preferable specific gravity of the water-insoluble carrier is 0.5 or more and 2.0 or less. It is more preferably 0.7 or more.
5 or less. The ratio of the volume at the time of swelling to the volume including the internal volume of the pores at the time of drying, that is, the swelling ratio is 1 or more and 30 or less. If it exceeds 30, the strength of the carrier decreases, which is not preferable in practical use. It is more preferably 20 or less, and further preferably 10 or less. The water-insoluble carrier is used after being filled in a container, but is preferably rigid because it hardly deforms when passing through a body fluid such as blood or plasma and does not cause an increase in pressure. When the degree of rigidity is exemplified by a specific numerical value, a container having a diameter of 10 mm and a length of 50 mm is filled, and the pressure difference between the inlet and the outlet is 20.
A preferable example is that the rate of decrease in volume when passing water at 0 mmHg at night is 10% or less.

Surface State of Water-Insoluble Carrier The water-insoluble carrier is provided with a coating layer made of a polymer for controlling the adhesion of platelets which is hydrophilic on the surface of the porous support in order to improve the affinity with blood. It may be held. The original purpose of the hydrophilic coating layer is to increase blood compatibility, and it is necessary to show the degree of blood compatibility, but it is difficult to obtain blood stably and there are differences between blood. , A common stable evaluation is difficult. The degree of hydrophilicity can be expressed simply. If the preferred range of the degree of hydrophilicity is intentionally shown, the contact angle of air bubbles on the surface of a sheet or film in water at 25 ° C. is 20 ° or more.

Coating Layer of Water-Insoluble Carrier The hydrophilic coating layer is preferably a polymer to prevent peeling during use. Specific examples of the hydrophilic coating layer include 2-hydroxyethyl acrylate, 2-hydroxypropyl acrylate, 2-hydroxyethyl methacrylate and 2
-Hydroxypropyl methacrylate, 2-hydroxybutyl methacrylate, diethylaminoethyl methacrylate, dimethylaminoethyl methacrylate, methoxydiethylene glycol methacrylate, methoxytriethylene glycol methacrylate, glycerol monomethacrylate, acrylic acid or methacrylic acid and derivatives thereof, methoxytriethylene glycol, etc. Methoxypolyethylene glycols, styrene derivatives such as diethylaminoethylstyrene, hydroxystyrene, hydroxymethylstyrene, monomers having a vinyl group such as vinylamine and vinyl alcohol, segmented polyurethane, segmented polyester, mono (2
A polymer, a block copolymer, a graft copolymer, or a polyethylene oxide chain containing at least one monomer such as -methacryloyloxyethyl) acid phosphate and mono (2-acryloyloxyethyl) acid phosphate; And a graft copolymer such as another polymerizable monomer. In particular, the polymer preferably has a hydroxy group. There is no particular limitation on the mode of bonding of the hydroxyl group in the polymer. The polymer may be a homopolymer of the above polymerized unit, or may be a copolymer of two or more. Further, it may be a copolymer containing a polymer unit having a hydroxyl group and containing 0.1 to 20% by weight of a polymer unit having a positive group.
These polymers are not particularly concerned with polymerization forms such as linear polymers, graft polymers and crosslinked polymers. The coating layer of the polymer may be obtained by any of a grafting method, a precipitation method, a coating method, a covalent bonding method using a functional group on the surface of the porous body, and the like. Among them, the coating method is particularly preferable in practical use because the production operation is easy.

The amount of the coating layer on the water-insoluble carrier The amount of the coating layer thus obtained, that is, the coating layer is expressed by the weight of the coating layer per dry weight of the adsorbent in order to exert the effect as the coating layer. At least 1 μg / g, 100
mg / g or less, more preferably 10 μg / g or more and 10 mg / g or less.

Shape of Adsorber The adsorbent of the present invention has a mesh for preventing leakage of the adsorbent in a container provided with a body fluid outlet, and the adsorbent is filled and held between the meshes. It is generally used. The present adsorbent may be filled alone, mixed with another adsorbent, or laminated. The capacity of the adsorber is
When used for external circulation, about 10 ml to about 1000 ml is appropriate.

Sterilization Method It is preferable that the present adsorber is sterilized before use. Sterilization methods such as a method using a gas such as ethylene oxide gas, a method using heating, and a method using radiation or ultraviolet light can be used. preferable.

Filling liquid at the time of sterilization Although the adsorbent can be supplied in a dry state, it is necessary to eliminate the operation of wetting during use and to eliminate the operation of drying the adsorbent in the final step of production. From the viewpoint of the stability of the ligand, the container is preferably filled in a wet state. It is preferable that the container is filled with an aqueous solution together with the adsorbent, since the generation of bubbles during use can be prevented. This aqueous solution is used to bring osmotic pressure closer to physiological conditions.
It is preferable that the solution contains a salt, and it is more preferable that the solution is a buffer solution because the pH can be controlled. As the buffer, an acid or a base generally used can be used, but the pH is preferably 9 or less, and more preferably 5 or more. Further, a reducing compound such as pyrosulfite may coexist.

Clinical Implementation Method This adsorber perfuses blood taken from the body or blood filled in a container such as a blood bag, or separates plasma components from blood by a known method, and then perfuses only plasma. Can be used. Bradykinin is gradually degraded by kininolytic enzymes such as angiotensin converting enzyme in plasma. If plasma is injected into the body after a certain time after the treatment with the adsorber, the increase in bradykinin does not pose a problem. The adsorbent of the present invention exerts a high effect when the plasma treated by the present adsorber is quickly injected into the body. Specifically, when plasma is used for infusion within 1 hour after flowing out of the adsorber, the effect is exhibited. 30 more
A higher effect is obtained when the injection is performed within 10 minutes, and it is particularly preferable when the composition is used for the purpose of injection within 10 minutes. Most preferably, it is used for extracorporeal circulation. The extracorporeal circulation can be specifically carried out by the following method. The blood taken out from the body is separated into blood plasma components and blood cell components using centrifugation or a membrane-type plasma separator, and the blood plasma components are passed through an adsorber, purified, and returned to the body together with the blood cell components. Can be implemented. In addition, blood taken from the body can be directly passed through the adsorber for purification. Among them, the plasma purification method in which the plasma component is passed through the adsorber for purification is more preferable because the flow resistance in the adsorber is small and the total adsorption capacity can be increased. The plasma perfusion method can be performed without using a pump by utilizing a natural head or blood pressure, but a method using a pump is preferable because the perfusion rate can be freely set and the control is easy. Also, as a method of passing bodily fluids,
It may be passed continuously or intermittently according to the necessity in use or the state of the equipment of the equipment. At this time, the longer the plasma stays in the adsorber, the more bradykinin is activated. Therefore, when the residence time of the plasma in the adsorber is 10 seconds or more, the present invention is preferably used. The time is more preferably 1 minute or more, and still more preferably 10 minutes or more. Also, if plasma stays in the adsorber for a long time, there is a risk of causing non-specific adsorption of plasma components and plasma coagulation, and therefore, the residence time is preferably 30 minutes or less.

Uses of adsorbents Uses of adsorbers filled with negative ligand adsorbents include:
Low-density and / or very low-density lipoproteins, sulfatide-adhering proteins, activated complement components, amyloid protein A, immune complexes, autoantibodies such as anti-DNA antibodies and rheumatoid factors, and / or immunity producing the autoantibodies B cells, immunoglobulin light chain, blood coagulation factor VIII, blood coagulation factor IX,
β ₂ microglobulin, and the like. Among them, the use as a low-density and / or extremely high-density lipoprotein adsorbent using a polymer compound having a negative group is highly clinically useful.

The adsorber packed with the positive ligand adsorbent is used for amyloid P protein, transthyretin, immunosuppressive acidic protein, β-amyloid substance, serum amyloid A protein, serum amyloid A precursor protein, bilirubin, uric acid , Bile acids, albumin binding compounds, β
_2- microglobulin, fatty acids and the like. Among them, a plasma protein having an isoelectric point of 4.5 or less is preferable, and in particular, amyloid P protein, transthyretin serum amyloid A protein, and serum amyloid A precursor protein can be mentioned. Further, as an adsorber filled with an adsorbent holding a ligand having a positive group and a negative group or a ligand having no functional group on the surface, an anti-acetylcholine substance antibody, an anti-RNA antibody, an anti-blood group substance, an anti-platelet Autoantibodies such as antibodies, antiviral antigen antibodies, blood group substances, hormones, interleukins, cancer cell necrosis factor, transforming growth factor, cytokines such as platelet growth factor, fibroblast growth factor, or prostaglandins, etc. is there.

Example 1 A polyvinyl alcohol gel having an average particle size of 100 μm, a vinyl alcohol unit (qOH) per unit weight of 6.0 meq / g, and an exclusion limit molecular weight of 5 × 10 ⁶ was used as a water-insoluble carrier. The average pore size and pore volume were measured using a mercury porosimeter (Micromeritix, manufactured by Shimadzu Corporation).
The pore size was 9320). The measurement results were obtained using a pore plot system (manufactured by Shimadzu Corporation, 9320-PC2).
(V1.0). The average pore size is 1,120 °, the pore volume of 100 ° to 5,000 ° is 92.5% of the total pore volume, and 0.5 D when the average pore size is D
The pore volume of 2.0 D or less is 64.1% of the total pore volume.
Met. The specific surface area measured by a nitrogen adsorption method (BET method) was 180.6 m ² / g. Next, a 100 g dry weight polyvinyl alcohol gel was epoxy activated using epichlorohydrin. The epoxy equivalent at this time was 116.6 μeq per 1 ml of the adsorbent. Next, 100 ml of the epoxy-activated polyvinyl alcohol gel was mixed with 12.8 ml (8 v / v%) of dimethylacetamide containing 1.6 g (1 w / v%) of dextran sulfate having a molecular weight of 500,000 and a sulfur content of 18.2 wt%, Amine 1.7 ml, 0.1 M sodium hydroxide aqueous solution 22
The suspension was suspended in 8 ml of a mixture and reacted at 50 ° C. for 16 hours to obtain an adsorbent having dimethylamino groups and dextran sulfate fixed on the surface. When the surface negative charge of the obtained adsorbent was determined by a neutral salt decomposition method, 3.0 μeq /
g. In order to roughly confirm this value, the total amount of each of the sulfate groups and dimethylamino groups on the surface of the adsorbent was determined as follows. The amount of sulfate groups fixed on the adsorbent was determined as follows. Dextran sulfate was hydrolyzed by adding 10 volumes of 1N hydrochloric acid to 1 volume of the adsorbent sufficiently washed with physiological saline and reacting at 121 ° C. for 1 hour. The filtrate was collected, and the amount of sugar was determined using an enzyme used for a glucose test and a coloring reagent. The amount of sulfate group was determined from the measured amount of sugar and the sulfur content. The amount of sulfate groups in the obtained adsorbent is 9
It was 8.4 μeq / ml. The molecular weights of dextran sulfate and dimethylamine differ greatly, and the presence or absence of dextran sulfate is not considered to have a significant effect on the fixation reaction of dimethylamine.Therefore, polyvinyl alcohol gel was similarly reacted without adding dextran sulfate. The amount of dimethylamino groups on the surface of the carrier obtained as described above was determined by a neutral salt decomposition method to obtain the amount of positive groups on the surface of the adsorbent. The amount of positive groups on the surface of the obtained adsorbent was 96.2 μeq / g. As described above, the amount of surface negative charge obtained by separately obtaining and canceling the amount of the negative group and the positive group was 2.2 μeq / g. When the amount of kinin produced was measured using this adsorbent in the same manner as for a nonwoven fabric, it was 227 pg / ml, and there was almost no increase in kinin. Next, the adsorbent was filled in a container having an inlet and an outlet for plasma and fitted with a mesh for preventing leakage of the adsorbent at each of the outlet and inlet to obtain an adsorber. The filling amount of the adsorbent in the adsorber was 1 ml. 8 ml of healthy human plasma obtained by collecting blood under 2,000 IU / ml of heparin was perfused into the adsorber at 0.06 ml / min, and plasma flowing out of the adsorber was collected in a fraction size of 1 ml. The collected plasma immediately stopped the production and decomposition of kinin in the same manner as in the batch method. The maximum kinin concentration during the 8 ml perfusion was taken as the bradykinin value in the column flow. The bradykinin value of the present adsorber was 126 pg / ml.

Example 2 10 ml of the polyvinyl alcohol gel used in Example 1
With 2-sulfoethyl methacrylate (sodium salt)
It was immersed in 20 ml of a 10% aqueous ethanol solution containing 2 w / v%, and subjected to a graft reaction under irradiation of 25.0 kGy of γ-ray to obtain an adsorbent. The amount of negative charge on the surface of the obtained adsorbent obtained by the neutral salt decomposition method was 12.3 μeq / g. The kinin production of this adsorbent is 120.3 pg / ml
Met. The bradykinin value measured in the same manner as in Example 1 was 45.6 pg / ml.

Example 3 The polyvinyl alcohol gel before the introduction of the epoxy resin used in Example 1 had a negative charge of 1.2 μeq / g on the surface. The kinin production of this gel was 277 pg / ml, and almost no increase was observed. The bradykinin value measured in the same manner as in Example 1 was 87 pg / ml.

Example 4 Using the adsorbent of Example 1, the elution value and the thermal dissociation value were measured. The elution value was determined by using an adsorbent previously washed with distilled water having a volume 50 times the volume of the adsorbent at room temperature, and applying a 10% volume 0.8% aqueous sodium chloride solution at 37 ° C. to a linear velocity of 1.0 ml / min · cm ^2. Flowed away. After collecting all of this solution and concentrating,
Detection was performed by a refraction method using high performance liquid and chromatography. From the calibration curve measured in advance, the amount of dextran sulfate eluted in a 0.8% aqueous sodium chloride solution was determined. The ratio of the amount of dextran sulfate eluted to the amount of dextran sulfate fixed to the adsorbent was defined as the elution value. The thermal dissociation value was measured using an adsorbent previously washed at room temperature with 5 volumes of distilled water. PH with sodium bicarbonate
After heating at 121 ° C. for 1 hour in twice the amount of sodium pyrosulfite solution adjusted to 7.2, the amount of eluted dextran sulfate was determined in the same manner as the measurement of the elution value. The ratio of the amount of dextran sulfate eluted to the amount of dextran sulfate immobilized on the adsorbent was defined as the thermal dissociation value. Elution of dextran sulfate from the adsorbent was not detected and the adsorbent 100 ml
0.015 mg or less and an elution value of 5 × 10 ⁻⁵
It was below. The thermal dissociation amount was below the detection limit, 0.01 mg per 100 ml of the adsorbent, and the thermal dissociation value was 3.
It was 1 × 10 ⁻⁵ or less.

Example 5 A polyvinyl alcohol gel was obtained in the same manner as in Example 1. The average pore size of this polyvinyl alcohol gel was 2.2.
The pore volume of 100 ° to 5,000 ° is 88.6% of the total pore volume. When the average pore size is D, the pore volume of 0.5D to 2.0D is less than the total pore volume. 6
2.3%. The specific surface area measured by a nitrogen adsorption method (BET method) was 64.9 m ² / g. Next, a 100 g dry weight polyvinyl alcohol gel was epoxy activated using epichlorohydrin. At this time, the epoxy equivalent was 97.8 μeq per 1 ml of the adsorbent. Next, 10 ml of the epoxy activated polyvinyl alcohol gel was
The suspension was suspended in 20 ml of a 0.1 molar sodium hydroxide aqueous solution containing 0.125 w / v% of taurine, and reacted at 50 ° C. for 16 hours to obtain an adsorbent. The negative charge on the surface of the obtained adsorbent was 24.0 μeq / g. The kinin production of this adsorbent was 285 pg / ml, and there was no significant increase. The bradykinin value measured in the same manner as in Example 1 was 171 pg / ml.

Example 6 A dextran sulfate fixed adsorbent was obtained in the same manner as in Example 1 except that the dextran sulfate concentration was changed to 5 w / v%. The surface charge of this adsorbent determined by the neutral salt decomposition method was 33.
It was 2 μeq / ml. The amount of sulfate groups obtained in the same manner as in the example was 127.9 μeq / g, and the amount of surface charge obtained by individually obtaining the amounts of negative groups and positive groups was 29.5 μe.
q / g. The kinin production of this adsorbent is 680p
g / ml, with no significant increase. Example 1
The bradykinin value measured in the same manner as described above was 499 pg / m
l.

Example 7 10 ml of the epoxy-activated polyvinyl alcohol gel obtained in Example 1 was mixed with 0.1 ml of 25 mM / L aspartic acid.
Suspend in 20 ml of 2 molar carbonate buffer (pH 9.6)
The reaction was carried out at 0 ° C. for 16 hours to obtain an adsorbent. The negative charge on the surface of the obtained adsorbent was 21.5 μeq / g. The bradykinin value of this adsorbent measured in the same manner as in Example 1 was 123 pg / ml. The amount of kinin produced was 2,030 pg / ml.

Example 8 An adsorbent was obtained in the same manner as in Example 7 except that aspartic acid was 30 mM / L. The negative charge on the surface of the obtained adsorbent was 25.0 μeq / g. The bradykinin value of this adsorbent measured in the same manner as in Example 1 was 226 pg / m
l. The amount of kinin produced was 515 pg / ml.

Example 9 A dextran sulfate fixed adsorbent was obtained in the same manner as in Example 1 except that the dextran sulfate concentration was changed to 0.1 w / v%. The surface charge of this adsorbent determined by the neutral salt decomposition method was 2 μeq / ml or less. The amount of sulfate groups obtained in the same manner as in Example 1 was 77.4 μeq / g, and the amount of surface charge obtained by individually obtaining the amounts of negative groups and positive groups was larger for positive charges than for 18 groups. 0.8 μeq / g. In addition, when the charge amount of the adsorbent is measured under the condition of measuring the positive charge amount by the neutral salt decomposition method, the positive charge amount is larger, and the amount is 1
The measured value was 7.1 μeq / g. The kinin production of this adsorbent was 88 pg / ml, and there was no significant increase.
The bradykinin value measured in the same manner as in Example 1 was 7
It was 1 pg / ml.

Comparative Example 1 In Example 1, the amount of dextran sulfate added was 11.
An adsorbent was obtained in the same manner except that 2 g (7 w / v%) was used. The negative charge on the surface of the obtained adsorbent is 50.4 μe.
q / g. When the amount of kinin produced was measured using this adsorbent in the same manner as for a nonwoven fabric, it was found to be 7,230 pg / m
1 and there was a significant increase in kinin. Example 1
The bradykinin value measured in the same manner as in
g / ml.

Comparative Example 2 The same operation as in Example 2 was carried out except that the amount of 2-sulfoethyl methacrylate (sodium acid) was changed to 10 w / v%. The negative charge on the surface of the obtained adsorbent is 63.2 μeq.
/ G. The kinin production of this adsorbent is 4,810
pg / ml. The bradykinin value measured in the same manner as in Example 1 was 3,500 pg / ml.

Comparative Example 3 An adsorbent was obtained in the same manner as in Example 5 except that the concentration of taurine was changed to 1.5 w / v%. The negative charge on the surface of the obtained adsorbent was 79.9 μeq / g. The amount of kinin produced by this adsorbent was 5.219 pg / ml, showing a remarkable increase. The bradykinin value measured in the same manner as in Example 1 was 8,943 pg / ml.

Comparative Example 4 An adsorbent was obtained in the same manner as in Example 7 except that aspartic acid was changed to 50 mM / L. The negative charge on the surface of the obtained adsorbent was 37.8 μeq / g. The bradykinin value of this adsorbent measured in the same manner as in Example 1 was 3,920 pg
/ Ml. The kinin production was 9,750 pg.
/ Ml.

Comparative Example 5 An adsorbent was obtained in the same manner as in Example 9 except that aspartic acid was changed to 60 mM / L. The negative charge on the surface of the obtained adsorbent was 41.6 μeq / g. The bradykinin value of this adsorbent measured in the same manner as in Example 1 was 12,100 p
g / ml. The kinin production was 11,900.
pg / ml.

──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-4-7024 (JP, A) JP-A-62-244442 (JP, A) JP-A-60-114340 (JP, A) JP-A 60-114 90038 (JP, A) JP-A-60-87854 (JP, A) JP-A-59-200655 (JP, A) JP-A-2-265566 (JP, A) JP-A-6-7429 (JP, A) JP-A-6-7431 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) A61K 35/14 A61M 1/00-1/34

Claims (2)

(57) [Claims] (1) an exclusion limit molecular weight of 50,000 or more;
The surface of the water-insoluble carrier composed of a polymer having a molecular weight of not more than 0,000,000 has a functional group capable of binding to an unnecessary substance.
An adsorbent for blood treatment , having a negative charge on the surface,
And surface charge measurement by acid / base titration and / or dye adsorption
-30μeq / g total charge specified by
As described above, the adsorbent for blood treatment is −0.01 μeq / g or less . 2. An adsorbent comprising a water-insoluble carrier and a ligand containing a functional group capable of binding to an unnecessary substance immobilized on the water-insoluble carrier, wherein the ligand is in a chain form, and a part of the ligand is immobilized on the carrier. 2. The adsorbent according to claim 1, wherein the other portion is in a state where it can move flexibly.