JPS59186560A - Adsorbing material of self-antibody immnological composite - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention is an adsorption system for body fluid purification that selectively adsorbs and removes autoantibodies and/or immune complexes that are thought to be closely related to various diseases caused by biological immune function. Regarding materials.

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 organ transplants. It is thought to be closely related to the cause or progression of diseases and phenomena related to the body's immune function, such as rejection 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 all symptoms, and even hasten healing. That was expected.

The present inventors have discovered that autoantibodies and/or immune complexes can be selectively adsorbed with high efficiency, non-selective adsorption is low, safety is achieved, and sterilization can be easily performed.
As a result of intensive research aimed at providing adsorbents suitable for body fluid purification or regeneration, we have developed a variety of materials in which the carrier retains all substances with special chemical structures that selectively interact with the adsorbed substance. discovered an adsorbent and filed a patent application (Japanese Patent Application No. 7152/1983, Patent Application No. 18923/1983,
Patent application 1986-76776, patent application 1987-L59444)
.

The present invention relates to the previous invention, and was made as a result of detailed study on 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) Adsorption of autoantibodies and immune complexes selectively and with high efficiency; (2) Substances other than the target substance (3j) does not fully activate the coagulation and fibrinolytic system, (4) can be sterilized, and (5) has sufficient mechanical strength.

The present inventors aim to provide an adsorbent that can adsorb autoantibodies and immune complexes with even higher efficiency as an adsorbent for body fluid purification, that is, can be made into a compact adsorbent with a small priming volume. Then, I conducted further intensive research.

Various compounds are immobilized on a carrier to immobilize autoantibodies and/or
Or, when we evaluated the adsorption properties for immune complexes,
We discovered that autoantibodies and/or immune complexes can be well adsorbed by immobilizing two types of low-molecular-weight compounds, a negatively charged low-molecular compound and an organic low-molecular compound with at least 6 carbons, on a carrier. The present invention was completed by confirming that when the molecular weight of the immobilized compound is relatively small, even if the compound detaches from the carrier and 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 fact that it is an adsorbent for autoantibodies and/or immune complexes, which is characterized by the fact that it is an adsorbent for autoantibodies and/or immune complexes. The molar ratio to the binding compound is 2
A coefficient of 0 or more gives preferable results.

In the present invention, it has a negative charge and has 5 carbon atoms in its skeleton structure.
Low-molecular compounds with carbon atoms or less are those that exhibit a negative charge in neutral electrolyte solutions such as blood or mosquito fluid, such as carboxyl groups, sulfonic acid groups, phosphoric acid groups, etc., and have 5 or fewer carbon atoms in their skeletal structure. The molecular weight is 104 or less, preferably 103 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 skeleton structure is
The number of carbon atoms in the skeleton structure is 6 or more, and the molecular weight is 1.
04 or less, preferably 1.03 or less. Among these, cyclic compounds with aromatic properties give good results.

Any compound with 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 preferred. Aromatic rings such as sulfur-containing aromatic rings, nitrogen-containing 6-membered rings such as phenanthrene, 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 a low molecular compound and an organic low molecular compound as used in the present invention refers to all carbons contained in the low molecular compound and organic low molecular compound, excluding carbons of carboxyl groups.

Here, the carbon of the carboxyl group was removed because 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.

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 low-molecular compounds that have a negative charge and have a skeleton structure of 5 or less carbon atoms in the total bonded compounds is 20% in molar ratio.
to 95%, preferably more than 50%, more preferably more than 60%.

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 case of carbon with bond 6
The number is preferably 10 or more, and in the case of only single bonds, the number is preferably 10 or more, and 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 500 or less carbon atoms.

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 preferably less than one per three carbon atoms. This is thought to be due to the fact that the aqueous interaction force exerted on the target adsorbent differs depending on the bonding mode of carbon, the type of substituent, etc.

The molecular weight of the organic low-molecular-weight compound bound to the insoluble carrier is preferably j04 or less in terms of antigenicity, even if the compound is released from the bond and enters the living body. It is preferably 108 or less.

Hereinafter, the entire method for manufacturing the adsorbent of the present invention will be explained in detail, starting with the method for manufacturing the adsorbent for 7-finity chromatography, in which the carrier is activated and a ligand is bound. .

The carrier may be a low-molecular compound having a negative charge and 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 hydrophobic carriers are used, non-selective adsorption of albumin to the carrier sometimes occurs, so hydrophilic carriers give preferable 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 the spherical or particulate carrier is 25-25-00μ
m can be used, but depending on its specific surface area (adsorption capacity as an adsorbent) and body fluid flow path, it is 50 to 1500 μm.
Particularly preferred are those.

The specific surface area of the carrier is preferably 5 go/g or more, and 55 go/g or more is heavy.

Particulate carriers that can be used include agarose-based, Dastran-based, cellulose-based, polyacrylamide-based, glass-based, silica-based, activated carbon-based carriers, etc., but hydrophilic carriers having a gel structure give good results. Furthermore, immobilized enzymes and known carriers commonly used in 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. All compounds can be immobilized, and the exclusion limit molecular weight (protein) is
The molecular weight of the target adsorbent of the present invention is from 150,000 (IgG) to 1,000 in the case of immune complexes, especially IgM immune complexes.
15 to 10 million is preferable. The most general exclusion limit molecular weight for purposes of this invention is io.

~5 million.

Polymer compositions include polyamides, polyesters, polyurethanes, vinyl compound polymers, etc. with porous structures.
Although any conventional polymer can be used, particularly preferred results are obtained from vinyl compound-based porous polymer particles made hydrophilic with hydrophilic monomers.

When using a fibrous carrier, the fiber diameter is preferably in the range of 0.02 denier to 10 denier, more preferably 0.1 denier to 5 denier. If the fiber diameter is too large, the adsorption amount and rate of globulin compounds will be reduced, and if it is too small, activation of the coagulation system, blood cell adhesion, and clogging may occur. As the fibrous carrier that can be used, generally known fibers such as regenerated cellulose fibers, nylon, acrylic, and polyester can be used.

Low-molecular-weight compounds that have a negative charge and have 5 or less carbon atoms in their skeleton structure, and organic low-molecular-weight compounds that have at least 6 carbon atoms in their skeletal structure. Methods for bonding to total insoluble carriers 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 from the viewpoint of the elubility of the bound substance, it is preferable to use it after fixation and insolubilization by covalent bonding. Therefore, it is possible to use commonly known methods for activating immobilized enzymes, carriers used in affinity chromatography, and binding methods for ligands.

Examples of activation methods include cyanogen halide method, epichlorohydrin method, bisepoxide method, halogenated triazine method, bromoacetyl bromide method, ethyl chloroformer method, 1.1'-carbonyldiimidazole method, etc. I can give it to you. The activation method of the present invention is not limited to the above-mentioned 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 a 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 weight 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 of bonding the organic low-molecular-weight 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); The method used can also be used. Furthermore, a method of coating an insoluble substance with a polymer capable of bonding the compound of the present invention and then fully 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 may be post-crosslinked if necessary.

1. It is also possible to adopt a method of activating the compound referred to in the invention and then binding it to a carrier.

In other words, non-invention is 1. This effect is due to the fact that a low molecular compound with a negative charge and 5 or less carbon atoms in the skeleton structure, and an organic low molecular compound with at least 6 carbon atoms in the O. skeleton structure are bonded to the adsorbent. It demonstrates the
It does not depend on the manufacturing method.

The uninvented adsorbent can be used by filling and holding it in a container equipped with all the inlets and outlets for body fluids.

In FIG. 1, reference numeral 1 shows an example of the N suction device using the adsorbent for autoantibodies and immune complexes of the present invention. A cap 6 having a body fluid inlet 5 is fitted with a screw through a backing 4 covered with a filter 3, and a filter 6 is inserted inside the opening at the other end of the cylinder 2.
A cap 8 having a body fluid lead-out lower part is screwed through a gold-plated backing 4 to form a container, and an adsorbent is filled and held in the gap between the filters 3 and 6' to form an adsorbent layer 9. That's what happens.

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, activated carbon or the like can be used, which has a wide adsorption capacity and adsorption capacity for other malignant substances (antigens) such as DNA. As a result, a wider range of clinical effects can be expected due to the reciprocal effect of the adsorbent. The appropriate volume of the adsorbent layer 9 is about 50 to 400 m1 when used for extracorporeal circulation.

When the fMk device of the present invention is used in extracorporeal circulation, there is the following two-part method. First, blood taken from the body is separated into plasma components and precursor components using a centrifuge or a model plasma separator, and the plasma components are passed through the device and purified, followed by blood cell components. One method is to return the blood to the body along with the blood, and the other method is to purify the blood taken from the body by passing it directly through a nuclear device.

In addition, regarding the passage speed of blood or plasma, the adsorption efficiency of the adsorbent is very high, so the particle size of the adsorbent can be adjusted to the full size, and the degree of packing can be lowered, so the shape of the adsorbent layer can be changed. It is possible to give the passage speed without any damage. 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 the status of the equipment.

As described above, the adsorbent of the present invention selectively adsorbs and removes autoantibodies and immune complexes in body fluids at a high rate, allows for the construction of a very compact adsorption device, and allows for simple and safe use. used.

The adsorbent of the present invention can be used for general purposes such as total purification and regeneration of body fluids such as autologous plasma and autologous blood, and can be used to treat agglutination of cancer, immunoproliferative disorders, rheumatoid arthritis, systemic lupus erythematosus, etc. Extracorporeal circulation treatment for diseases and phenomena related to the immune system of the body, such as autoimmune diseases such as myasthenia gravis, allergies, rejection reactions during organ transplants, kidney diseases such as nephritis, and liver diseases such as hepatitis. It can be used effectively.

Hereinafter, embodiments of the present invention will be explained in more detail with reference to Examples.

Example 1 Vinyl acetate 100fi',) realyl isocyanurate 52. OS' (crosslinking degree X-0,65), ethyl acetate 1
007, heptane 1007, polyvinyl acetate (polymerization degree 5
00) 7.5 A homogeneous mixed liquid consisting of 3.87 S' and 2,2'-azobisisobutyronitrile, 1% by weight of polyvinyl alcohol, 5% by weight of sodium dihydrogen phosphate dihydrate J0, and phosphoric acid Put 400 ml of water in which 1.5% by weight of disodium hydrogen-dihydrate was dissolved into a flask, stir thoroughly, then heat and stir at 65°C for 18 hours, then at 75°C for 5 hours to perform @minami polymerization. , a granular copolymer was obtained.

After filtering, washing with water, and then extracting with acetone, add 46.
1 at 40°C in a solution of 57 and 2t methanol.
The transesterification reaction of the copolymer was carried out for 8 hours.

The average particle size of the resulting gel was 150 μm, vinyl alcohol units per unit weight (QOH).

meq/9, specific surface area is 60 @”/f, exclusion limit molecular weight by dextran id 6 x 105
Met.

By the epichlorohydrin method using this Kel as a carrier,
A negatively charged low-molecular compound and a compound having six or more carbon atoms were combined, and excess active groups were blocked with ethanolamine.

The ratio of low-molecular-weight compounds with negative electricity 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 (all comparative examples where only one of them is immobilized). ). The total binding amount was adjusted to around 50 μmol/ml gel.

The table below shows the external addition of the reagents used in this example.

In the adsorption experiment, 3 volumes of ACD rheumatism patient plasma and 1 adsorbent were used.
After mixing and incubating at 37°C for 5 hours,
The supernatant plasma was evaluated. Evaluation: rheumatoid factor, immunity = b
This was done for immunoglobulin G.

Rheumatoid factor was measured using a latex agglutination test and a passive sensitization hemagglutination test. The latex agglutination test is a method for detecting the property that latex particles agglutinate when polystyrene latex particles completely adsorbed with human γ-globulin are exposed to whole patient plasma containing rheumatoid factor. A 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 to become negative.
On the contrary, plasma with low concentration becomes low.

Passive sensitization hemagglutination test was performed using rabbit-gamma on sheep red blood cells.
- Globulin is adsorbed, and the rest is the same as the latex agglutination test. In general, the receptor sensitized hemagglutination test has better rheumatoid factor specificity than the latex agglutination test.

A dilution series was prepared with a mild glycine saline + 5 IJ solution, and a dilution ratio at which rheumatoid factor was negative in a latex agglutination test was determined. The latex agglutination test was performed using a kit from Japan Freeze Drying Research Institute. Similarly, passive sensitization dish ball longitudinal collection test [RAHA test, manufactured by Tomi±iMki Pharmaceutical Co., Ltd.]
It was evaluated.

Immune complexes were prepared by polyethylene glycol precipitation. This method involves precipitating and fractionating the immunoconjugate with polyethylene glycol in a single radial immunofluorescence system.
The amount of complete throw complex is determined by quantifying the amount of immunoglobulin using the diffusion method. The operating method and liquid properties of this method are as follows.

(1) Specimen 1. Place 81PEG (polyethylene glycol, average molecular weight 6000-7500) in an Odi test tube.
Add 1.0 ml of f, stir, and leave at 4°C for 60 minutes.

(2) Centrifuge at 1000g at 4°C for 60 minutes, remove the supernatant, and add the resulting precipitate to PBS (IJ acid buffered saline).
Redissolve in solution to make 1.0rnl.

(3) (1), (2) k Repeat two more times to completely wash away any contaminating monomeric immunoglobulin.

(・1) Immunoglobulin G2 is quantified using the single radial immunodiffusion method in the entire PBS suspension of the finally obtained immune complex.

Immunoglobulin G was quantified using the single radial immunodeficiency method.

The values of the rheumatoid patient's plasma used are 320 for rheumatoid factor latex, 1280 for RAHA, and 50/dl for immune complex (measurement of immunoglobulin G).

Immunoglobulin G was 1600119/dl'.

The results of the adsorption experiment are shown in Figures 2 to 5, in which the broken lines indicate the values of patient plasma.

From these figures, we can see that adsorption in which an insoluble carrier has a negative charge and is bound to a low-molecular compound with 5 or less carbon atoms in the skeleton structure, and an organic low-molecular compound with at least 6 carbon atoms in the skeleton structure is bonded to the insoluble carrier. It can be seen that the material selectively adsorbs rheumatoid factor and immune complexes.

In particular, an adsorbent in which the molar ratio of a low-molecular compound having a negative charge and having 5 or less carbon atoms in the skeleton structure to the total bonded compounds bonded to an insoluble carrier is 20 to 80%. It can be seen that it gives 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 the tip embodiment.

Claims (2)

[Claims] (1) A low-molecular compound containing 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. adsorbent for autoantibodies and/or immune complexes. (2) The claim has a power purchaser bonded to an insoluble carrier, and the molar ratio of a low-molecular compound whose skeleton structure has 5 or less carbon atoms to the total bonded compounds is 20 or more. The autoantibody described in item 1 and/-! or the adsorbed side of the immune complex.