JPS6361025B2 - - 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 it would speed up the process. The present inventors have developed a technology that selectively adsorbs autoantibodies and/or immune complexes, etc. with high efficiency, has little non-selective adsorption, is safe, can be easily sterilized, and can be used for body fluids. As a result of intensive research aimed at providing adsorbents suitable for purification or regeneration, we have developed various types of adsorbents in which the carrier holds a substance with a special chemical structure that selectively interacts with the adsorbed substance. He discovered an adsorbent and filed a patent application (Japanese Patent Application No. 7152, No. 56-1892, No. 18923, No.
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. Desirable properties of the adsorbent used for the purpose of the present invention are (1) to selectively adsorb autoantibodies and immune complexes with high efficiency; (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 the carrier and their adsorption properties for autoantibodies and/or immune complexes were evaluated, two types of antibodies were found: low-molecular-weight compounds with negative charges and organic low-molecular-weight compounds with at least 6 carbons. They found that autoantibodies and/or immune complexes can be well adsorbed by immobilizing the carrier, and furthermore, when the molecular weight of the compound immobilized on the carrier is relatively small, even if the compound is detached from the carrier, We have completed the present invention by confirming that even if it enters the living body, it does not have antigenicity. That is, the present invention provides an insoluble carrier having a negative charge and bonded to a low-molecular compound having 5 or less carbon atoms in its skeletal structure and an organic low-molecular compound having at least 6 carbon atoms in its skeletal structure. It is an adsorbent for autoantibodies and/or immune complexes, which is characterized by the total bonding of a low molecular weight compound having a negative charge and having a skeleton structure of 5 or less carbon atoms bound to an insoluble carrier. The molar ratio to the compound is 20
% or more gives preferable results. In the present invention, low-molecular compounds that have a negative charge and have 5 or less carbon atoms in their skeleton structure include blood, carboxyl groups, sulfonic acid groups, phosphoric acid groups, etc.
Refers to a substance that exhibits a negative charge in a neutral electrolyte such as a body fluid, has a skeleton structure of 5 or less carbon atoms, and has a molecular weight of 10 ⁴ or less, preferably 10 ³ or less. Examples include aliphatic amino acids such as glycine, alanine, and aspartic acid, fatty acids such as γ-amino-n-butyric acid and ε-amino-caproic acid, sulfamic acid, and taurine carbamyl phosphate.
An organic low-molecular compound having at least 6 carbons in its skeletal structure refers to an organic compound having 6 or more carbon atoms in its skeletal structure and a molecular weight of 10 ⁴ or less, preferably 10 ³ or less. Among these, cyclic compounds with aromatic properties give good results. Any compound having aromatic properties can be usefully used, but benzene-based aromatic rings such as benzene, naphthalene, and phenanthrene, oxygen-containing aromatic rings such as dibenzofuran, fromene, and benzofuran, and thianaphthene and thianthrene are useful. Aromatic rings such as sulfur-containing aromatic rings, nitrogen-containing 6-membered rings such as phenanthridine, quinoline, and acridine, and nitrogen-containing 5-membered rings such as indole and carbazole give good results. Among these, benzene-based aromatic rings give particularly good results. The number of carbon atoms in the skeleton structure of the low-molecular compound and organic low-molecular compound referred to in the present invention is, among the carbons contained in the low-molecular compound and organic low-molecular compound,
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 the organic low-molecular compound and the autoantibody and/or immune complex. The proportion of the low-molecular compound having a negative charge and having 5 or less carbon atoms in the skeleton structure in the total bonded compounds is 20 to 95%, preferably more than 50%, and more preferably 60%. The range exceeds. In the present invention, the number of carbon atoms in the skeleton structure of the organic low molecular compound bound to the insoluble carrier is 6 or more, but unsaturated In the case of carbon having a bond, 6 or more, preferably
The number is 10 or more, and in the case of only carbon having a single bond, the number is preferably 10 or more, and the number is preferably 15 or more. Further, the upper limit 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 low-molecular compound is an organic compound having 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, organic compounds with many non-dissociable hydrophilic substituents such as hydroxyl groups, thiol groups, and amide groups are preferable because their adsorption to the target adsorbent is lower than those without substituents. do not have. The number of non-dissociable hydrophilic substituents is preferably less than one per two carbon atoms in the skeleton structure of the organic compound, and desirably less than one per three carbon atoms. This is thought to be due to the fact that the hydroplastic interaction force exerted on the target adsorbent substance differs depending on the bonding mode of carbon, the type of substituent, etc. The molecular weight of the organic low-molecular compound bound to the insoluble carrier is preferably 10 ⁴ or less from the viewpoint of antigenicity, even if the compound is released from the bond 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 may be a hydrophobic carrier as long as it has a negative charge and can bind a low-molecular compound having 5 or less carbon atoms in its skeleton structure, or an organic low-molecular compound having at least 6 carbon atoms in its skeleton structure. However, when a hydrophobic carrier is used, non-selective adsorption of albumin to the carrier sometimes occurs, so a hydrophilic carrier gives more favorable results. The shape of the insoluble carrier can be any known shape such as particulate, fibrous, hollow fiber, or membrane. Particulate or fibrous materials are preferable in terms of the amount of compounds and organic low-molecular compounds having at least 6 carbon atoms in their skeletal structure that can be retained and the ease of handling as an adsorbent. The average particle size of spherical or particulate carrier is 25-2500μ
50~m can be used, but due to its specific surface area (adsorption ability as an adsorbent) and circulation with 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-based 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 low-molecular compounds that have negative charges on their surfaces and have 5 or less carbon atoms in their skeleton structure, and organic low-molecular compounds that have at least 6 carbon atoms in their skeleton structure. It is capable of immobilizing compounds, and the exclusion limit molecular weight (protein) of the target adsorbent of the present invention is 150,000 (IgG), and in the case of immune complexes, especially IgM immune complexes, it reaches 10 million. , 15 to 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, and acrylic polyester can be used. Methods for bonding low-molecular compounds that have a negative charge and have 5 or less carbon atoms in the skeleton structure, and organic low-molecular compounds that have at least 6 carbon atoms in the skeleton structure to an insoluble carrier include covalent bonding and ionic bonding. Any known method can be used, such as physical adsorption, embedding, or precipitation and insolubilization on the surface of a polymer, but in view of the elution properties of the bound substance, it is preferable to fix and insolubilize the binder 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. As described above, as a method for producing the adsorbent of the present invention, after activating the carrier, a low molecular compound having a negative charge and having 5 or less carbon atoms in the skeleton structure, and a low molecular compound having at least 6 carbon atoms in the skeleton structure. Although the method for bonding organic low-molecular compounds has been described in detail, the present invention is not limited thereto. for example,
A method in which the compound referred to in the present invention is bonded to a polymerizable monomer or a crosslinking agent and then polymerized (copolymerized); a method in which a crosslinking agent to which the compound referred to in the present invention is bonded is used at the time of further post-crosslinking to the crosslinked polymer particles, etc. can also be used. Furthermore, a method of coating an insoluble substance with a polymer that can bind the compound of the present invention and then bonding the compound of the present invention, or a method of bonding the compound of the present invention to a polymer and then coating the insoluble substance may also be used. I can do it. At this time, the coat polymer can also be post-crosslinked if necessary. It is also possible to adopt a method in which the compound referred to in the present invention is activated and then bound to a carrier. In other words, the present invention is characterized by the fact that a low molecular weight compound having a negative charge and having 5 or less carbon atoms in its skeleton structure, and an organic low molecular compound having at least 6 carbon atoms in its skeleton structure are bonded to an adsorbent. , which exhibits its effects and is not dependent on the manufacturing 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 FIG. 1, reference numeral 1 shows an example of an adsorption device using the adsorbent for autoantibodies and immune complexes of the present invention, in which a packing 4 with a filter 3 stretched inside is attached to an opening at one end of a cylinder 2. 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' 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 selectively adsorbs and removes autologous antibodies and immune complexes in body fluids at a high rate, allows for the construction of a very compact adsorption device, and is easy and safe to use. . 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 myasthenia gravis, allergies, diseases and phenomena related to the body's immune function such as rejection 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 Vinyl acetate 100g, triallylisocyanurate 52.0g (crosslinking degree X = 0.35), ethyl acetate 100g,
100g heptane, polyvinyl acetate (degree of polymerization 500)
A homogeneous mixture of 7.5 g and 3.8 g of 2,2'-azobisisobutyronitrile, 1% by weight of polyvinyl alcohol, 0.05% by weight of sodium dihydrogen phosphate dihydrate, and disodium hydrogen phosphate dodecahydrate. Add 400ml of water in which 1.5% by weight of the compound was dissolved into a flask, stir thoroughly, and then heat at 65℃ for 18 hours.
Suspension polymerization was carried out by heating and stirring at 75° C. for 5 hours to obtain a granular copolymer. After washing with water and then extracting with acetone, add 46.5 g of caustic soda and 2 methanol.
The transesterification reaction of the copolymer was carried out at 40°C for 18 hours in a solution consisting of: The average particle size of the obtained gel was 150 μm, and the vinyl alcohol unit (qOH) per unit weight was 10.0 μm.
eq/g, specific surface area was 60 m ² /g, and exclusion limit molecular weight by dextran was 6×10 ⁵ . Using this gel as a carrier, a negatively charged low-molecular compound and an organic compound having 6 or more carbon atoms were bound by the epichlorohydrin method, and excess active groups were blocked with ethanolamine.
The ratio of low-molecular-weight compounds with negative charges to the total bound compounds was adjusted by changing the ratio of reagents added during the binding reaction, and adsorbents with various ratios were obtained (comparative examples are those in which only one of them is immobilized). ). In addition, the total binding amount was adjusted to around 50 μmol/ml gel. The reagents used in this example are listed in the table below.

[Table] 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 tendency of latex particles to agglutinate when patient plasma containing rheumatoid factor is applied to polystyrene latex particles adsorbed with human-gamma-globulin. Create a plasma dilution series and
The rheumatoid factor concentration is evaluated based on the plasma dilution ratio 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, the passive sensitization hemagglutination test is considered to have higher rheumatoid factor specificity than the latex agglutination test. 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 tests were performed using a kit from the Japan Freeze Drying Research Institute. Similarly, evaluation was performed using a passive sensitization hemagglutination test (RAHA test, manufactured by Fuji Organ Pharmaceutical Co., Ltd.). 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 4°C, 1000g for 60 minutes, remove the supernatant, and redissolve the resulting precipitate in PBS (phosphate buffered saline) to make 1.0ml. (3) Repeat steps (1) and (2) two more times to wash away contaminating monomeric immunoglobulins. (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 the rheumatoid patient's plasma used were 320 for rheumatoid factor latex, 1280 for RAHA, 50 mg/dl for immune complexes (measured for immunoglobulin G), and 1600 mg/dl for immunoglobulin G. The results of the adsorption experiments are shown in FIGS. 2 to 5.
In the figure, the broken line indicates the value of patient plasma. From these figures, the insoluble carrier has a negative charge,
and an adsorbent in which a low-molecular compound with a skeleton structure of 5 or less carbon atoms and an organic low-molecular compound with a skeleton structure of at least 6 carbon atoms are bonded,
It can be seen that rheumatoid factor and immune complexes are selectively adsorbed. In addition, the molar ratio of a low molecular compound having a negative charge and having 5 or less carbon atoms in its skeleton structure to the total bonded compound is 20 to 80%, which is bonded to an insoluble carrier.
It can be seen that the adsorbent material gives particularly good results.

[Brief explanation of drawings]

FIG. 1 is a schematic diagram showing an example of an adsorption device in which a container is filled with the adsorbent of the present invention, and FIGS. 2 to 5 are graphs showing the results of Examples.

Claims (1)

[Scope of Claims] 1. A low-molecular compound having a negative charge and having a skeleton structure of 5 or less carbon atoms and an organic low-molecular compound having at least 6 carbon atoms in a skeleton structure are bonded to an insoluble carrier. An adsorbent for autoantibodies and/or immune complexes. 2 has a negative charge attached to an insoluble carrier;
and/or the autoantibody and/or the autoantibody according to claim 1, wherein the molar ratio of the low molecular weight compound having a skeleton structure with 5 or less carbon atoms to the total bound compounds is 20% or more.
or adsorbents for immune complexes.