JPS59186559A - Self-antibody and/or immunological composite adsorbing material - 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 expressed in body fluids, such as blood, and/-! or immune complexes are the cause of autoimmune diseases such as cancer, immunoproliferative syndromes, and rheumatoid arthritis; systemic lupus erythematosus, or diseases and phenomena related to the body's immune system, such as allergies and organ transplant rejection. Or, it is thought to have a close relationship with progression.

Therefore, by adsorbing and removing the above-mentioned autoantibodies and/or immune complexes from body fluid components such as blood and plasma, the progression of the above-mentioned diseases can be prevented, symptoms can be alleviated, and healing can be accelerated. That was expected.

The present inventors have discovered that autoantibodies and/or immune complexes can be selectively attached to rEl 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 creating an adsorbent material suitable for physical purification or regeneration, we have found that the carrier retains a substance with a special chemical structure that selectively interacts with the adsorbed substance. He discovered various adsorbents made of
23, Japanese Patent Application No. 56-76776, Japanese Patent Application No. 56-L-15
9444).

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: (2) adsorption of IL+ autoantibodies and immune complexes selectively and with high efficiency; (2) adsorption of substances other than the target substance; (31) It must be difficult to activate the coagulation and fibrinolytic system, (4) It must be sterilizable, and (5) It must have 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,
When certain conditions are met regarding the charge of the compound immobilized on the carrier and the number of carbon atoms in the skeleton structure, that is,
They found that 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 ability to adsorb autoantibodies and/or immune complexes is high. 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 is directed to an organic low molecular weight compound having one substituent having a negative charge in one molecule and having at least 10 carbon atoms having an unsaturated bond in the skeleton structure, or a part having a negative charge in one molecule. An adsorbent for autoantibodies and/or immune complexes, which is characterized by having one substituent having The charged substituent is preferably a carboxyl group or a sulfone group.

The organic low-molecular compound moiety referred to in the present invention is one that has one substituent group having a negative charge in one molecule and at least 10 carbons having an unsaturated bond in the skeleton structure, or one that has a negative charge in one molecule. It refers to a substance having a total of one charged substituent and at least 20 carbons in the skeleton structure, but the molecular weight is preferably 104 or less, and 10
It is desirable that it be 3 or less. Here, the substituent having a negative charge is a carboxyl group (COO-, C0OH, CO
ONa), sulfonic acid group (SOs, 5OsH, 5O
sNa ), phosphate group (pop.

Characteristic groups -tS that exhibit negative charge in neutral electrolytes such as blood and body fluids, such as POsH, PO5Na), etc.

Examples of all the organic low molecular weight compounds referred to in the present invention include naphthol-sulfonic acid, naphthylic acid, a cyclic compound having one substituent group having a negative charge and aromatic properties, undecanoic acid, and leucyl-leucine. Long-chain aliphatic compounds with one negatively charged substituent, such as 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 xanthine, 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-based aromatic ring give particularly good results. Among the carbons contained in the molecular compound part, it refers to all carbons except for substituents having a negative charge, that is, carbons of 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.

The hydrophobic interaction force (van der Waals force) lfi acts between the carbon contained in this specific low molecular compound portion and the autoantibody and/or the immune complex. They need to be exposed on the surface, and are preferably scattered over a structure with as large a surface area as possible.

The synergistic effect of the Coulomb 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. The negatively charged substituents increase the ability to adsorb 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 becomes less strong, 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 in the case of carbon having an unsaturated bond, and 20 or more in the case of carbon having an unsaturated bond.

This makes it possible to attach autoantibodies and/or immune complexes at a very high rate.Also, 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 50 or less. is desirable.

When the organic compound part 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 differs depending on the carbon bond type, the type of substituent, etc. Even if the molecular weight of the organic compound moiety bonded to the insoluble carrier Even if the bond is broken and enters the living body, a value of 104 or less is preferable in terms of antigenicity. It is preferably 103 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 to it. An organic low-molecular compound that has one substituent group with a negative charge in the molecule, and has an unsaturated bond in the skeleton structure and at least 10 carbon atoms, or has one substituent group with a negative charge in one molecule,
In addition, it is sufficient to bind an organic low-molecular compound having at least 20 carbons in the skeleton structure, and both hydrophilic and hydrophobic carriers can be used. However, when using a hydrophobic carrier, albumin may sometimes be added to the phase. Since non-selective adsorption occurs, hydrophilic carriers give better results. The shape of the insoluble carrier can be any known shape, such as particulate, hollow-fiber-like, hollow-fiber-like, or membrane-like. In view of the amount of organic low molecular weight compounds retained and ease of handling as an adsorbent, particulate or fibrous materials are preferred.

The average particle size of the spherical or particulate carrier is 25 to 2500 μm
However, from the viewpoint of specific surface area (adsorption capacity as an adsorbent) and number of particles, those with a diameter of 50 to 1500 μm are particularly preferable.

The specific surface area of the carrier is 5TL2/? or more is preferable, and 55
A value of 27 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. Generally, immobilized enzymes and known carriers used in affinity chromatography can be used without particular limitations.

Porous particles, especially porous polymers, can also be used as particulate carriers. The porous polymer particles used in the present invention are those that can immobilize organic low-molecular compounds on their surfaces, and the exclusion limit molecular weight (protein) is 150,000 (IgG) for the target adsorption substance of the present invention. In the case of immune complexes, particularly IgM immune complexes, the number reaches 10 million, so 15 to 10 million is preferable. The most general exclusion limit molecular weight for the purpose of the present invention is 1 million to 5 million. The polymer composition may be any known polymer that can eliminate porous structures, such as polyamide, polyester, polyurethane, or vinyl compound polymers. Polymers can be used, but in particular, vinyl compound-based porous polymer particles made hydrophilic with hydrophilic monomers give preferable results. When a fibrous carrier is used, the fiber diameter is 0.02 denier. It is preferably in the range of 10 to 10 denier, more preferably 0.1 denier to 5 tenier. If the fiber diameter is too large, the adsorption amount and rate of globulin compounds will be reduced; 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.

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. Therefore, commonly known methods for activating an immobilized enzyme, a carrier used in affinity chromatography, and a method for binding a ligand can be used.

Examples of activation methods include the rhodanide cyanide method, epichlorohydrin method, bisepoxide method, halogenated triazine method, bromoacetylbromide method, ethyl chloroformate method, and 1,1'-carbonyldiimidazole method. be able to. 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 amine group, hydroxyl group, carboxyl group, or thiol group of a ligand. However, in consideration of chemical stability, thermal stability, etc., the method using epoxide is preferable, and the epichlorohydrin method is particularly recommended. It is permissible to bond two or more types of organic low molecular weight 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 binding the organic low molecular compound referred to in the present invention after fully activating the carrier 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, a method of coating an insoluble substance with a polymer capable of binding an organic low molecular compound and then bonding the organic low molecular compound, or a method of coating an insoluble substance with a polymer having an organic low molecular compound moiety can also be used. At this time, the coat polymer can also be post-crosslinked if necessary. However, it is also possible to use a method in which a low-molecular-weight organic compound is activated and then bonded to a carrier.

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 at least 10 carbon atoms having an unsaturated bond in the skeleton structure, or a part having a negative charge in one molecule. This effect is achieved by having in the adsorbent an organic low-molecular compound moiety that has one substituent having , and has at least 20 carbon atoms in its skeleton structure, and it depends on the manufacturing method. isn't it.

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 Z is screwed into the opening at the other end of the cylinder 2 through a backing 4' having a filter 3' stretched inside. The adsorbent layer 9 is formed by filling and holding an adsorbent in the gap between the filters 3 and 6'.

In the adsorbent layer 9, the adsorbent of the present invention Q may be filled with light 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 capacities, 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 40.0 ml when used for extracorporeal circulation.

When the device of the present invention is used for extracorporeal circulation, there are two methods as follows: First, the liquid taken out from the body is separated into plasma components and blood cell components using a centrifugal separator or a model plasma separator, and then the plasma components are passed through the device. One method is to remove the blood from the body, purify it, and then return it to the body together with blood cell components.The other method is to directly pass the blood taken out from the body through the device and purify it.

As for the passage speed of blood or plasma, the adsorption capacity of the adsorbent is very high, so the particle size of the adsorbent can be made coarser, and the degree of light loading of the adsorbent can be lowered, so the adsorption rate of the adsorbent layer can be reduced. A high passing speed can be given regardless of the shape. 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 autoantibodies and/or immune complexes in body fluids, 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 applied to general uses for purifying and regenerating body fluids such as autologous plasma and autologous blood, and can be used to treat collagen diseases such as cancer, immunoproliferative syndrome, rheumatoid arthritis, systemic lupus erythematosus, and severe Effective for extracorporeal circulation treatment of autoimmune diseases such as ganasthenia, allergies, diseases and phenomena related to the body's immune function such as organ transplant rejection, kidney diseases such as renal moxibustion, and liver diseases such as hepatitis. Available.

Embodiments of the present invention will be explained in more detail with reference to Examples below.

Example 1 CNBr, activated Sepharose 4B (Sweden).

(manufactured by Pharmacia) by a normal method to bond an organic low molecular compound having one negatively charged substituent and at least 10 carbon atoms with unsaturated bonds in the backbone structure to remove excess active groups. was blocked with ethanolamine. The retained amount of various organic compounds is determined by converting the primary amine group of the remaining organic compound into 4-phenylspiro[furan-2(
31().

1'-phthalane) -3,3'-dione ("fluram■
The adsorption experiment calculated by reacting and bonding with “Roche Co., Ltd.”
Three volumes of ACD 01J equine patient plasma and one volume of adsorbent were mixed, incubated for 3 hours, and then the supernatant plasma was evaluated. The evaluation focused on rheumatoid factor, immune complexes, and immunoglobulin G.

Rheumatoid factor was measured using a latex agglutination test and a passive sensitization 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 γ-globulin. 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, while plasma with a low concentration has a low dilution factor.

The passive sensitization reagglutination test was performed using rabbit-γ on sheep red blood cells.
- Globulin is adsorbed, and the rest is the same as the latex agglutination test. Generally, the passive sensitization hemagglutination test is considered to be more specific for rheumatoid factor than the latex agglutination test.

A dilution series was prepared with 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 Japan Freeze Drying 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. In this method, the amount of immune complexes is determined by quantifying the amount of immunoglobulin using a single radial immunodiffusion method of the immune complexes precipitated and fractionated with polyethylene glycol. The operating method and conditions for this method are as follows.
), stir, and leave at 4C for 60 minutes.

+214C, 1000? After centrifuging for 60 minutes and removing the supernatant, the resulting precipitate was redissolved in PBS (IJ acid-washed physiological saline) and designated as '+' and 'omt'.

+31111 and +21 are repeated two more times to wash out contaminating monomeric immunoglobulin.

(4) The finally obtained immune complex suspension in PBS was subjected to single radial immunodiffusion method.
Quantitative determination of immunoglobulin G. Quantification of immunoglobulin G was determined by a single radial immunodeficiency method. The values of rheumatoid patient plasma used are 1280 for rheumatoid factor latex, 5120 for RAHA,
Immune complexes (measured in immunoglobulin G') 60~/d
t, immunoglobulin G was 16110 m9/dA.

The results of the adsorption experiment are shown in Table 1. According to Table 1, the adsorbent that has one substituent with a negative charge in one molecule and an organic low-molecular compound part that has at least 10 carbon atoms with unsaturated bonds in the skeleton structure is used for rheumatoid factors, immune complexes, etc. It can be seen that the whole body is selectively adsorbed, and non-selective adsorption of immunoglobulin G is small. In addition, in Table 1, the number of carbons in the skeleton structure also includes the number of carbons possessed by CNBr, which is the reagent used for activation.

Table 1 Comparative Example 1 The v! 1. The attachment was prepared and experimented in the same manner as in Example 1.

The results are shown in Table 2. From Table 2, those without negative charges are rheumatoid factor, immune complex rli! , 7f4 ability decreases, and those with unsaturated bonds having 9 or less carbon atoms have low adsorption ability.

Table 2 Example 2 Sepharose 4B (manufactured by Pharmacia, Sweden)
1.4-bis-(2,6-nipoxybropoquine)-
After activation using butane by a conventional method, an organic low-molecular compound having one negatively charged substituent and at least 20 carbon atoms is bonded by a conventional method, and excess activated groups are bonded. was blocked with ethanolamine and used as an adsorbent.

After determining the retention amount in the same manner as in Example 1, -
An adsorption experiment was conducted.

The results of the adsorption experiment are shown in Table 6. According to Table 3, the adsorbent, which is an organic low-molecular compound with one negatively charged substituent and at least 20 carbon atoms, is S-bonded and selectively targets rheumatoid factors and immune complexes. It can be seen that there is little non-selective adsorption of immunoglobulin G. Hikoo,
In Table 3, the number of carbon atoms is 1,4-bis-(
Carbon k of 2,6-epoxypropoquine)-butane
Including tr.

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 charges have a lower adsorption capacity for rheumatoid arthritis factor and immune complexes, and those with 19 or less carbon atoms have a lower adsorption capacity. Table 4 Example 6 e Vinyl acid 100f',) realyl isocyanurate s 2. Oy (crosslinking degree X-0,65), ethyl oxalate 1
00 S', heptane 100! i', a homogeneous mixture of 7.5 f of polyvinyl acetate (degree of polymerization 500) and 3.8 g of 2,2'-azobisisobutyronitrile, 1% by weight of polyvinyl alcohol, and sodium dihydrogen phosphate. 0.05% by weight of dihydrate and dihydrogen phosphate)
IJum-dihydrate 1.5 weight φ dissolved in water 400-
The mixture was placed in a flask and thoroughly stirred, followed by suspension polymerization by stirring under heat at 65 U for 18 hours and then at 75 C for 5 hours to obtain a granular copolymer.

After filtration, washing with water, and extraction with acetone, 1 ml was added at 4 DC in a solution consisting of 46°57 liters of soda and 2 t of methanol.
The transesterification reaction of the copolymer was carried out for 8 hours.

The average particle size of the obtained gel was 150 μm, and the vinyl alcohol unit per unit weight (q on) was 10.0 maq.
/g, the specific surface area was 60TrL2/7, and the exclusion limit molecular weight by dextran was 6 x 10'.

Next, the obtained gel 102 (dry weight) was suspended in dimethyl sulfoxide 120, to which was added 78.3 rnl of epichlorohydrin and 30% sodium hydroxide 10, and activated with stirring at 60C for 5 hours. The reaction was carried out. After the reaction, the mixture was washed with dimethyl sulfoxide, water, and dehydrated under suction. This activated gel was then converted into 5-amino-2-
0.1M sodium carbonate containing 2.51% naphthylic acid (
Tufa (pH 9,8) suspended in 160g, 50C
An immobilization reaction was carried out with stirring for 14 hours, and then 133ml of 60.6 mg/-Nodris (hydroxyethyl) aminomethane solution was added, and a blocking reaction (to block the remaining active groups) was carried out for 5 DC and 5 hours with stirring. . Thereafter, the adsorbent for body fluid purification was obtained by light washing.

The amount of 3-amino-2-naphthylic acid immobilized on this adsorbent is 220 μmot/? (dry weight).

The number of carbon atoms in the organic compound immobilized on this adsorbent having an unsaturated bond is 10, the total number of carbon atoms is 13, and the number of negative charges is 1.

This adsorbent was packed into a column with an inner diameter of 30 mm and a length of 70IIIO, and ACD rheumatoid patient plasma-257/
150 ml was flowed at a flow rate of vrin. At this time, no decrease in the packed volume of the adsorbent in the column, no clogging, and no decrease in the flow rate were observed, and the pressure loss before and after the column was 15 mmHg or less.

When all the plasma proteins were analyzed before and after passing through the column, rheumatoid factor was 620 for latex and 256 for RAHA before adsorption.
In contrast, both became negative after adsorption.

In addition, the immune complex (measured by C/q solid-phase EIA) was 20 μg/- before adsorption, but 6 μg/- after adsorption.
- immunoglobulin G decreased to below 1
620~/dA only decreased to 1170 mL;//at.

[Brief explanation of the drawing]

The drawing is a schematic diagram showing an example of an adsorption device in which all containers of adsorbent of the present invention are initially filled.

Claims (1)

[Scope of Claims] (111 having one substituent with a negative charge in the molecule and having at least 10 carbon atoms having an unsaturated bond in the skeleton structure)
an autoantibody characterized by having an organic low-molecular-weight compound moiety having an organic low-molecular compound moiety, or having one negatively charged substituent per molecule and an organic low-molecular-weight compound moiety having at least 20 carbon atoms in its skeleton structure. and/or immune complex adsorbents. (21) 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.