JPH0424065B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an adsorbent for body fluid purification that selectively adsorbs and removes autoantibodies and/or immune complexes, etc., which are thought to be closely related to various diseases caused by biological immune function. As is well known, autoantibodies and/or immune complexes expressed in body fluids, such as blood, are associated with cancer, immunoproliferative syndromes, and autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus, as well as allergies and rejection during organ transplants. It is thought that there is a close relationship with the cause or progression of diseases and phenomena related to biological immune functions such as reactions. Therefore, by specifically adsorbing and removing the above-mentioned autoantibodies and/or immune complexes from body fluid components such as blood and plasma, it is possible to prevent the progression of the above-mentioned diseases, alleviate symptoms, and even cure them. It was hoped that this would be accelerated. The present inventors have developed a technology that selectively adsorbs autoantibodies and/or immune complexes with high efficiency, has little non-selective adsorption, is safe, can be easily sterilized, and can purify body fluids. Alternatively, as a result of intensive research aimed at providing adsorbents suitable for regeneration, we have developed various types of adsorption in which the carrier holds a substance with a special chemical structure that selectively interacts with the adsorbed substance. found the material and filed a patent application (Japanese Patent Application 1986-7152, 1984-18923,
56-76776, patent application No. 56-159444). The present invention relates to the previous invention, and was made as a result of a more detailed study of substances that selectively interact with adsorbed substances, and relates to improvements to the above-mentioned adsorbent. Desired properties of the adsorbent used for the purpose of the present invention are (1) to selectively and highly efficiently adsorb autoantibodies and immune complexes; (2) to adsorb substances other than the target substance; (3) it does not activate the coagulation and fibrinolytic system, (4) it can be sterilized, and (5) it has sufficient mechanical strength. The present inventors aim to provide an adsorbent for body fluid purification that can adsorb autoantibodies and immune complexes with even higher efficiency, that is, can be made into a compact adsorbent with a small priming volume. Then, I conducted further intensive research. When various compounds were immobilized on a carrier and the adsorption characteristics for autoantibodies and/or immune complexes were evaluated, it was found that the charge of the compound immobilized on the carrier and the number of carbon atoms in the backbone structure satisfied certain conditions. In other words, when the compound immobilized on the carrier has a negative charge and the number of carbon atoms in the backbone structure is relatively large, the adsorption capacity for autoantibodies and/or immune complexes is increased, and further, When the molecular weight of the compound immobilized on the carrier is relatively small, even if the compound detaches from the carrier and enters the living body, it is necessary to confirm that it does not have antigenicity before completing the present invention. I've reached it. That is, the present invention is directed to an organic low molecular weight compound having one substituent having a negative charge in one molecule and having 10 to 500 carbon atoms having an unsaturated bond in the skeleton structure, or Autoantibodies and/or antibodies characterized by having one substituent with a negative charge on the body and a low-molecular-weight organic compound having 20 to 500 carbon atoms in its backbone structure bound to an insoluble carrier. The negatively charged substituent which is the adsorbent of the composite is preferably a carboxyl group or a sulfonic acid group. The organic low-molecular compound portion referred to in the present invention is 1.
Has one substituent with a negative charge in the molecule and 10 or more carbon atoms with unsaturated bonds in the skeleton 500
or one molecule with one negatively charged substituent and 20 or more substituents in the skeletal structure.
It refers to something that has less than 500 carbons.
The molecular weight is preferably 10 ⁴ or less, preferably 10 ³ or less. Here, the negatively charged substituents are casboxyl groups (COO ^- , COOH,
COONa), sulfonic acid group (SO ₃ ^- , SO ₃ H, SO ₃
refers to characteristic groups that exhibit a negative charge in neutral electrolytes such as blood and body fluids, such as phosphoric acid groups (PO ₃ ^- , PO ₃ H, PO ₃ Na), etc. Examples of the organic low-molecular compound portion referred to in the present invention include cyclic compounds having one negatively charged substituent and aromatic properties such as naphthol-sulfonic acid and naphthylic acid, undecanoic acid, and leucyl- Examples include long-chain aliphatic compounds having one negatively charged substituent such as leucine. Any compound with aromatic properties can be usefully used, but benzene-based aromatic rings such as naphthalene and phenanthrene, oxygen-containing aromatic rings such as dibenzofuran and xanthene, and sulfur-containing aromatic rings such as thianthrene are useful. A method in which a negatively charged substituent is introduced gives good results. Among these, those in which a negatively charged substituent is introduced into the benzene aromatic ring give particularly good results. In the present invention, the number of carbon atoms in the skeleton structure of the organic low-molecular compound portion refers to substituents having a negative charge among the carbons contained in the organic low-molecular compound portion, that is,
Refers to all carbon atoms except those in carboxyl groups.
Here, the reason why the carbon of the carboxyl group was excluded is that the carboxyl group is hydrophilic and mainly exhibits only the effect of negative charge. The number of carbon atoms in substituents other than carboxyl groups, ie, carbons contained in alkoxyl groups, aldehyde groups, alkoxycarbonyl groups, etc., is counted. A hydrophobic interaction force (Van der Waals force) acts between the carbon contained in this organic low molecular compound portion and the autoantibody and/or immune complex. The organic low molecular compound portion must be exposed on the surface of the adsorbent, and is preferably scattered over a structure having as large a surface area as possible. The synergistic effect of the Coulombic force of the negatively charged substituents and the hydrophobic interaction force of the carbon skeleton makes it possible to improve the selective adsorption ability of autoantibodies and/or immune complexes. Negatively charged substituents increase the adsorption capacity of autoantibodies and/or immune complexes. Furthermore, as the number of carbon atoms in the skeleton structure of the organic low-molecular-weight compound increases, the interaction force with autoantibodies and/or immune complexes increases, and the adsorption capacity increases. In this case, the number of carbons and the strength of interaction with autoantibodies and/or immune complexes are somewhat different for unsaturated carbons and saturated carbons, with unsaturated carbons being better than saturated carbons. also exhibits an interaction force that is about twice as large. In the present invention, the number of carbon atoms in the skeleton structure of the organic low-molecular compound portion is required to be 10 or more if the carbon has an unsaturated bond, and 20 or more if not. This allows for very high adsorption of autoantibodies and/or immune complexes. Further, the upper limit of the number of carbon atoms is determined by the molecular weight, but the number of carbon atoms is preferably 500 or less, and preferably 50 or less. When the organic compound portion has a hydrophobic substituent such as chlorine or iodine, the adsorption characteristics of the target adsorbent substance are improved compared to those without a substituent. On the other hand, in the case of organic compound moieties that have many non-dissociable hydrophilic substituents, such as hydroxyl groups, thiol groups, and amide groups, the adsorption ability for the target adsorbent substance is lower than those without substituents. Undesirable. The number of non-dissociable hydrophilic substituents is preferably less than one per two carbon atoms in the skeleton structure of the organic compound portion, and desirably less than one per three carbon atoms. This is thought to be due to the fact that the hydrophobic interaction force exerted on the target adsorbent substance differs depending on the carbon bonding mode, the type of substituent, etc. The molecular weight of the organic compound portion bound to the insoluble carrier is preferably 10 ⁴ or less from the viewpoint of antigenicity even if the bond is broken and enters the living body. More preferably it is 10 ³ or less. The method for producing the adsorbent of the present invention will be described below, taking as an example a method for producing an adsorbent for affinity chromatography, in which a carrier is activated and a ligand is bound thereto. The carrier contains one negatively charged substituent in one molecule.
An organic low-molecular compound that has 10 to 500 carbon atoms with unsaturated bonds in its skeletal structure, or has one negatively charged substituent in one molecule and 20 carbon atoms in its skeletal structure. It is sufficient to bind organic low-molecular compounds having 500 carbon atoms or less, and both hydrophilic and hydrophobic carriers can be used; however, when using a hydrophobic carrier, non-selective adsorption of albumin to the carrier may occur. hydrophilic carriers give more favorable results. The shape of the insoluble carrier may be any known shape, such as particulate, fibrous, hollow fiber, or membrane. Preferably. The average particle size of spherical or particulate carrier is 25-2500μ
m can be used, but due to its specific surface area (adsorption ability as an adsorbent) and circulation of body fluids,
Particularly preferred is one with a diameter of 1500 μm. The specific surface area of the carrier is preferably 5 m ² /g or more, and 55
m ² /g or more is desirable. Particulate carriers that can be used include agarose-based, dextran-based, cellulose-based, polyacrylamide-based, glass-based, silica-based, and activated carbon carriers, but hydrophilic carriers having a gel structure give good results. Furthermore, known carriers commonly used for immobilized enzymes and affinity chromatography can be used without any particular limitations. Porous particles, especially porous polymers, can also be used as particulate carriers. The porous polymer particles used in the present invention are capable of immobilizing organic low-molecular compounds on their surfaces, and the exclusion limit molecular weight (protein) of the target adsorption substance of the present invention is 150,000 (IgG). More immune complexes especially IgM
In the case of immune complexes it reaches 10 million, so 15~
10 million is preferred. The most common exclusion limit molecular weight for purposes of this invention is 1 million to 5 million. Polymer compositions include polyamide, polyester, polyurethane, vinyl compound polymers, etc.
Although known polymers capable of forming a porous structure can be used, particularly preferred results are obtained using vinyl compound-based porous polymer particles made hydrophilic with a hydrophilic monomer. When using a fibrous carrier, the fiber diameter is
0.02 denier to 10 denier, more preferably
It is preferable to use one in the range of 0.1 denier to 5 denier. If the fiber diameter is too large, the amount and speed of adsorption of globulin-based compounds will be reduced; if it is too small, activation of the coagulation system, blood cell adhesion, and clogging are likely to occur. As the fibrous carrier that can be used, generally known fibers such as regenerated cellulose fibers, nylon, acrylic, and polyester can be used. Any known method such as covalent bonding, ionic bonding, physical adsorption, embedding, or precipitation insolubilization on the surface of a polymer can be used to bond a low-molecular-weight organic compound to an insoluble carrier. Therefore, it is preferable to use it by immobilizing it and making it insolubilized by covalent bonding. For this purpose, known methods for activating carriers and binding methods for ligands that are usually used in immobilized enzymes, affinity chromatography, and affinity chromatography can be used. Examples of activation methods include cyanogen halide method, epichlorohydrin method, bisepoxide method,
Examples include the halogenated triazine method, the bromoacetyl bromide method, the ethyl chloroformate method, and the 1,1'-carbonyldiimidazole method. The activation method of the present invention is not limited to the above examples as long as it can perform a substitution and/or addition reaction with a nucleophilic reactive group having active hydrogen such as an amino group, a hydroxyl group, a carboxyl group, or a thiol group of the ligand. However, in consideration of chemical stability, thermal stability, etc., a method using an epoxide is preferable, and an epichlorohydrin method is particularly recommended. There is no problem in binding two or more kinds of organic low molecular compounds referred to in the present invention to the carrier. Above, as a method for manufacturing the adsorbent of the present invention, the method of activating the carrier and then bonding the organic low molecular compound referred to in the present invention has been described in detail, but the present invention is not limited thereto. For example, in the present invention, a method of polymerizing (copolymerizing) using a polymerizable monomer or crosslinking agent having an organic low molecular compound moiety, or a method of polymerizing (copolymerizing) using a crosslinking agent or a polymerizable monomer having an organic low molecular compound moiety; The method used can also be used. Furthermore, after coating the insoluble substance with a polymer capable of binding organic low-molecular compounds,
A method of bonding an organic low-molecular compound or a method of coating an insoluble substance with a polymer having an organic low-molecular compound portion can also be used. At that time, the coat polymer can also be post-crosslinked if necessary. Alternatively, a method in which a carrier is bonded after activating the organic low-molecular compound can also be adopted. That is, the present invention is directed to an organic low molecular weight compound having one substituent having a negative charge in one molecule and having 10 to 500 carbon atoms having an unsaturated bond in the skeleton structure, or This effect is achieved by having an organic low-molecular compound part in the adsorbent that has one substituent with a negative charge and a skeleton structure of 20 to 500 carbon atoms. It doesn't depend on the method. The adsorbent of the present invention can be used by being filled and held in a container equipped with an inlet and outlet for body fluids. In the drawings, reference numeral 1 shows an example of an adsorption device using the autoantibody and/or immune complex adsorbent of the present invention. A cap 6 having a body fluid inlet 5 is screwed into the cylinder 2, and a cap 8 having a body fluid outlet 7 is screwed into the opening at the other end of the cylinder 2 through a packing 4' having a filter 3' on the inside. A container is formed, and an adsorbent is filled and held in the gap between the filters 3 and 3' to form an adsorbent layer 9. The adsorbent layer 9 may be filled with the adsorbent of the present invention alone, or may be mixed or laminated with other adsorbents. As other adsorbents, for example, adsorbents for other malignant substances (antigens) such as DNA, activated carbon having a wide range of adsorption capacity, etc. can be used. As a result, a wider range of clinical effects can be expected due to the synergistic effect of the adsorbent. The appropriate volume of the adsorbent layer 9 is about 50 to 400 ml when used for extracorporeal circulation. When the device of the present invention is used for extracorporeal circulation, there are roughly two methods as follows. First, blood taken from the body is separated into plasma components and blood cell components using a centrifuge or a model plasma separator, and the plasma components are passed through the device to be purified and then separated into blood cell components. One method is to return the blood to the body together with the blood, and the other method is to directly pass the blood taken out from the body through the device and purify it. In addition, regarding the passage speed of blood or plasma, since the adsorption efficiency of the adsorbent is extremely high, the particle size of the adsorbent can be made coarser, and the degree of packing can be lowered, so the shape of the adsorbent layer can be changed. Regardless, high passing speeds can be provided. Therefore, a large amount of body fluid can be treated. The method for passing body fluids may be either continuous or intermittent, depending on clinical needs or equipment conditions. As described above, the adsorbent of the present invention can adsorb and remove autoantibodies and/or immune complexes in body fluids at a high rate and selectively, and can easily and safely assemble a very compact adsorption device. used. The adsorbent of the present invention can be applied to general uses for purifying and regenerating body fluids such as autologous plasma and autologous blood, and is applicable to cancer, immunoproliferative syndrome, rheumatoid arthritis,
Collagen diseases such as systemic lupus erythematosus, autoimmune diseases such as severe joint asthenia, diseases and phenomena related to the body's immune function such as allergies, rejection reactions during organ transplants, kidney diseases such as nephritis, and liver diseases such as hepatitis. It can be effectively used for extracorporeal circulation treatment such as. Embodiments of the present invention will be explained in more detail with reference to Examples below. Example 1 CNBr, activated Cepharose 4B (Sweden,
An organic low-molecular compound having one negatively charged substituent and 10 carbon atoms with an unsaturated bond in its backbone structure is bonded to the compound (manufactured by Pharmacia) by a standard method to reduce excess activity. The groups were blocked with ethanolamine. The retained amount of various organic compounds is determined by converting the primary amino groups of the remaining organic compounds into 4-phenylspiro[furan-2(3H), 1'-phthalane]-
It was calculated by reaction bonding with 3,3'-dione ("Flurum" manufactured by Rochie). In the adsorption experiment, 3 volumes of ACD rheumatism patient plasma and 1 volume of adsorbent were mixed, incubated at 37°C for 3 hours, and then the supernatant plasma was evaluated. Evaluations were made for rheumatoid factor, immune complexes, and immunoglobulin G. Rheumatoid factor was measured using a latex agglutination test and a passive sensitized hemagglutination test. The latex agglutination test is a detection method that measures the property of latex particles agglutinating when patient plasma containing rheumatoid factor is applied to polystyrene latex particles adsorbed with human-gamma-globulin. A plasma dilution series is created and the rheumatoid factor concentration is evaluated at the plasma dilution rate at which latex particles no longer aggregate. Plasma containing a high concentration of rheumatoid factor has a high dilution factor, while plasma with a low concentration has a low dilution factor. The passive sensitization hemagglutination test involves adsorbing rabbit γ-globulin to sheep red blood cells.
The rest is the same as the latex agglutination test. Generally, passive sensitization hemagglutination tests are considered to have higher rheumatoid factor specificity than latex agglutination tests. A dilution series was prepared using glycine saline buffer, and the dilution ratio at which rheumatoid factor was negative in a latex agglutination test was determined. Latex agglutination test
This was done using a kit from the Japan Freeze Drying Research Institute. Similarly, passive sensitization hemagglutination test [RAHA test,
[manufactured by Fuji Organ Pharmaceutical Co., Ltd.] was used for evaluation. Immune complexes were prepared by polyethylene glycol precipitation. This method uses polyethylene glycol to precipitate and separate immune complexes into single cells.
The amount of immune complexes is determined by quantifying the amount of immunoglobulin using the radial immunodiffusion method. The operating method and conditions for this method are as follows. (1) Put 1.0ml of the sample into a test tube and add 8% PEG (polyethylene glycol, average molecular weight 6000-7500).
Add 1.0ml, stir, and leave at 4℃ for 60 minutes. (2) Centrifuge at 1000g at 4°C for 60 minutes, remove the supernatant, and redissolve the resulting precipitate in PBS (phosphate buffered saline) to make 1.0 ml. (3) Repeat steps (1) and (2) two more times to wash away any contaminating monomeric immunoglobulin. (4) Quantify immunoglobulin G in the PBS suspension of the finally obtained immune complex using the single radial immunodiffusion method. Immunoglobulin G was quantified using the single radial immunodeficiency method. The values of rheumatoid patient plasma used were 1280 for rheumatoid factor latex, 5120 for RAHA, 60 mg/d for immune complexes (measured for immunoglobulin G), and 1600 mg/d for immunoglobulin G. The results of the adsorption experiment are shown in Table 1. From Table 1, one molecule has one substituent with a negative charge, and
An adsorbent containing an organic low-molecular-weight compound with 10 carbon atoms with unsaturated bonds in its skeleton structure is used to treat rheumatoid factors,
Selective adsorption of immune complexes and immunoglobulin G
It can be seen that there is little non-selective adsorption of . In addition,
In Table 1, the number of carbon atoms in the skeleton structure also includes the number of carbon atoms in CNBr, which is the reagent used for activation.

[Table] Comparative Example 1 An adsorbent was prepared in the same manner as in Example 1 using an organic compound with no negative charge or an unsaturated bond and a carbon number of 9 or less, and an experiment was conducted in the same manner as in Example 1. . The results are shown in Table 2. From Table 2, it can be seen that those without negative charges have a lower ability to adsorb rheumatoid factors and immune complexes, and those with unsaturated bonds having 9 or less carbon atoms have a lower adsorption ability.

[Table] Example 2 Sepharose 4B (manufactured by Pharmacia, Sweden) was activated using 1,4-bis-(2,3-epoxypropoxy)-butane in a conventional manner, and then activated in a conventional manner. Therefore, an organic low-molecular compound having one negatively charged substituent and 20 carbon atoms was bonded to the adsorbent, and the excess active groups were blocked with ethanolamine to form an adsorbent. Furthermore, in the organic low molecular compound, carbon is 21
and 24 pieces were also used as adsorbents in the same manner as above. After determining the retention amount in the same manner as in Example 1, Example 1
Adsorption experiments were conducted in the same manner. The results of the adsorption experiment are shown in Table 3. From Table 3, it can be seen that the carbon 20
It can be seen that the adsorbent to which organic low molecular weight compounds having 1, 21 and 24 molecules are bound selectively adsorbs rheumatoid factors and immune complexes, and non-selective adsorption of immunoglobulin G is small. In Table 3, the number of carbon atoms is 1,4-bis-(2,
Contains the carbon contained in 3-epoxypropoxy)-butane.

[Table] Comparative Example 2 An experiment was conducted in the same manner as in Example 2 using an organic compound that had no negative charge or had 19 or less carbon atoms. The results are shown in Table 4. From Table 4, it can be seen that those without negative charge have a lower ability to adsorb rheumatoid factors and immune complexes, and those with 19 or less carbon atoms have a lower adsorption ability.

[Table] Example 3 Vinyl acetate 100g, triallylisocyanurate 52.0g (crosslinking degree X = 0.35), ethyl acetate 100g, heptane 100g, polyvinyl acetate (polymerization degree 500) 7.5g
and 3.8 g of 2,2'-azobisisobutyronitrile
A homogeneous mixed liquid consisting of polyvinyl alcohol 1
wt%, atrium dihydrogen phosphate 0.05 wt% and disodium hydrogen phosphate dodecahydrate
Add 400ml of water in which 1.5% by weight was dissolved into a flask, stir thoroughly, and then heat at 65℃ for 18 hours, then at 75℃.
Suspension polymerization was carried out by heating and stirring for 5 hours to obtain a granular copolymer. After washing with water and extraction with acetone, the copolymer was transesterified in a solution consisting of 46.5 g of caustic soda and 2 methanol at 40° C. for 18 hours. The average particle size of the obtained gel was 150 μm, and the vinyl alcohol unit (qOH) per unit weight was
The molecular weight was 10.0 meq/g, the specific surface area was 60 m ² /g, and the exclusion limit molecular weight by dextran was 6×10 ⁵ . Next, 10 g (dry weight) of the resulting gel was suspended in 120 ml of dimethyl sulfoxide, and this was mixed with 78.3 ml of epichlorohydrin and 10 ml of 30% sodium hydroxide.
was added, and the activation reaction was carried out with stirring at 30°C for 5 hours. After the reaction, the mixture was washed with dimethyl sulfoxide, water, and dehydrated under suction. This activated gel was then combined with 2.5 g of 3-amino-2-naphthylic acid.
0.1M sodium carbonate buffer (pH9.8) 160ml
suspended in it. The immobilization reaction was carried out at 50°C for 14 hours with stirring, and then 33 ml of a 60.6 mg/ml tris(hydroxyethyl) aminomethane solution was added.
A blocking reaction (to block remaining active groups) was further carried out at 50°C for 5 hours with stirring. Thereafter, it was thoroughly washed with water to obtain an adsorbent for body fluid purification. The amount of 3-amino-2-naphthylic acid immobilized on this adsorbent was 220 μmol/g (dry weight). The organic compound immobilized on this adsorbent has 10 carbon atoms with unsaturated bonds, 13 total carbon atoms, and 1 negative charge. This adsorbent was packed into a column with an inner diameter of 30 mm and a length of 70 mm, and 150 ml of ACD rheumatoid arthritis patient plasma was passed through the column at a flow rate of 5 ml/min. At this time, no decrease in the packing volume of the adsorbent in the column, no clogging, and no decrease in flow rate were observed, and the pressure loss across the column was 15 mmHg or less. Analysis of plasma proteins before and after passing through the column revealed that
Rheumatoid factor is 320 in latex before adsorption,
While it was 2560 for RAHA, both became negative after adsorption. Furthermore, the immune complex (measured by C/q solid-phase EIA) was 20 μg/ml before adsorption, but decreased to 3 μg/ml or less after adsorption. In contrast, immunoglobulin G is 1320mg/d and 1170mg/d.
It only decreased to mg/d.

[Brief explanation of drawings]

The drawing is a schematic diagram showing an example of an adsorption device in which a container is filled with the adsorbent of the present invention.

Claims (1)

[Claims] 1. One molecule with one negatively charged substituent and 10 carbon atoms with an unsaturated bond in the skeleton structure.
Organic low-molecular compound parts that have 500 or more carbon atoms, or organic low-molecular compound parts that have one substituent with a negative charge in one molecule and have 20 to 500 carbon atoms in the skeleton structure are insoluble. An autoantibody and/or immune complex adsorbent characterized by being bound to a carrier. 2. The autoantibody and/or immune complex adsorbent according to claim 1, wherein the negatively charged substituent is a carboxyl group or a sulfonic acid group.