JPS59186558A - Adsorbing material of self-antibody and/or immunological 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, which are believed to be closely related to various diseases caused by the immune system of the body. 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 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 all of 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 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 holds a substance with a special chemical structure that selectively interacts with the adsorbed substance. He discovered an adsorbent and filed a patent application for it (Japanese Patent Application No. 7152/1983, Patent Application No. 189-23/1983).
, Japanese Patent Application No. 56-76776, Japanese 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) Adsorption of autoantibodies and immune complexes selectively and with high efficiency; (2) Target substances other than larvae? (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 aimed to provide an adsorbent that can adsorb autoantibodies and immune complexes with higher efficiency as an adsorbent for body fluid purification, that is, can be made into a compact inhaler with a small priming volume. With this goal in mind, I continued to do more research.

Various compounds are immobilized on a carrier to immobilize autoantibodies and/or
Or, when we evaluated the adsorption properties for immune complexes,
When the charge of the compound immobilized on the carrier and the number of carbon atoms in the skeleton structure meet certain conditions, that is,
We found that when the compound immobilized on the carrier has a large number of negative charges and the number of carbon atoms in the skeleton structure is relatively large, the ability to adsorb autoantibodies and/or immune complexes increases. The present invention was completed by confirming that if the molecular weight of the 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 has at least two substituents having all negative charges in one molecule, and at least 6 substituents in the skeleton structure.
It is an adsorbent for autoantibodies and/or immune complexes characterized by having an organic low-molecular-weight compound moiety having 5 carbon atoms, and the negatively charged substituent is a carboxyl group or/
and a sulfonic acid group are preferred.

In the present invention, the organic low-molecular compound moiety refers to a compound having two or more negatively charged substituents in one molecule and having six or more carbon atoms in its skeleton structure, but the molecular weight is It is preferably 104 or less, and desirably 103 or less. Here, the substituent having a negative charge is a carboxyl group (COO-).

C0OH,COONa),y71zphonic acid group (S
O3-, So, H.

So, Na), phosphate groups (PO3, PO3H, PO3
Refers to a characteristic group that exhibits a negative charge in neutral electrolyte solutions such as blood and body fluids, such as Na ).

Examples of the organic low-molecular compound portion referred to in the present invention include cyclic compounds having two or more negatively charged substituents and aromatic properties such as benzenedinulphonic acid and naphmelene dicarboxylic acid, and succinolytic navanine. Examples include long-chain aliphatic compounds having two or more negatively charged substituent groups, such as polyglutamic acid and polyaspartic acid. 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. 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 have two or more negatively charged substituents introduced into the aromatic ring. gives good results.

Among these, those in which two or more negatively charged substituents are 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 all carbons contained in the organic low-molecular-weight compound portion, excluding carbons of negatively charged substituents, that is, 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.

Due to the synergistic effect of the Coulomb force of the negatively charged substituents and the hydrophobic interaction force of the carbon skeleton, autoantibodies and /l;A can improve the selective adsorption of immune complexes. It becomes possible. The greater the number of negatively charged substituents, the greater the adsorption capacity for autoantibodies and/or immune complexes. When the organic low-molecular compound portion has one negatively charged substituent, more carbons are required in the skeleton structure of the organic compound portion. In the present invention, it is desirable that there be at least two negatively charged substituents.

In the present invention, the number of carbon atoms in the skeleton structure of the organic low-molecular-weight compound is 6 or more, but carbon atoms having unsaturated bonds are preferable in terms of fully improving the adsorption ability of autoantibodies and/or immune complexes. In the case of , the number is 6 or more, preferably 10 or more, and in the case of only carbon having a single bond, the number is preferably 10 or more, and preferably 15 or more. The upper limit is determined by the molecular weight, but the number of carbon atoms is 500.
The number is preferably 50 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 1 per 2 carbon atoms in the skeleton structure of the organic compound, and preferably 1 per 6 carbon atoms.
Less than one. This is thought to be due to the fact that the hydrophobic interaction force exerted on the -1 target adsorbent differs depending on the bonding mode of carbon, the type of substituent, etc.

The molecular weight of the organic compound moiety is preferably 104 or less from the viewpoint of antigenicity even if the bond is broken and enters the living body. It is preferably 10" or less.

Hereinafter, the method for producing the adsorbent ThN of the present invention will be explained, 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.

It is sufficient that the carrier has at least two substituent groups having a negative charge in one molecule and can bind an organic low-molecular compound having at least six carbon atoms in the skeleton structure, and a hydrophilic carrier,
Although any hydrophobic carrier can be used, a hydrophilic carrier is preferred since non-selective adsorption of albumin to the carrier sometimes occurs when hydrophobic carriers are used.

The shape of the insoluble carrier may be any known shape such as particulate, fibrous, hollow fiber, or membrane, but it must have at least two negatively charged substituents in one molecule and at least 6 carbons? Particulate or fibrous materials are preferred in terms of the amount of organic low-molecular compounds retained and ease of handling as an adsorbent.

Average particle size of spherical or particulate carrier: 25 to 2500 μm
However, from the viewpoint of its specific surface area (adsorption capacity as an adsorbent) and flow direction of body fluids, those with a diameter of 50 to 1500 μm are particularly preferable.

The specific surface area of the carrier is preferably 5 go/7 or more, and 55 go/2.
The above 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, 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 have at least two negatively charged substituents on their surface and are capable of immobilizing an organic low-molecular compound having at least 6 carbon atoms in the skeleton structure. As for the exclusion limit molecular weight (protein), the molecular weight of the object biting substance of the present invention is 150,000 (LgG).
In the case of the gM immune complex [FiIDOO reaches 10,000, so
15 to 10 million is preferable. The most common exclusion limit molecular weight for purposes of this invention is 1 million to 5 million.

As the polymer composition, known polymers having a porous structure such as polyamide, polyester, polyurethane, and vinyl compound polymers can be used, but in particular, vinyl made hydrophilic by a hydrophilic layer is used. Compound-based porous polymer particles give favorable results.

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 adsorption rate of globulin compounds will be reduced, and if it is too small, activation of the coagulation system, blood cell adhesion, and blemishes 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.

Methods for bonding an organic low-molecular compound having at least two negatively charged substituents and at least six carbon atoms in its skeleton structure to an insoluble carrier include covalent bonding, ionic bonding, physical adsorption, embedding, or polymerization. Any known method such as precipitation and insolubilization on the combined surface can be used, but in view of the elution properties of the bound substance, it is preferable to use it after fixation and insolubilization by covalent bonding. Therefore, it is possible to use conventional immobilized enzymes, known carrier activation methods and ligand binding methods used in affinity chromatography.
Ru.

Examples of activation methods include halogenated cyanide method, epichlorohydrin method, bis-e-boxide method, halogenated triazine method, bromoacetyl bromide method, ethyl chloroformate method, and 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 even if two or more types of organic low-molecular compounds referred to in the present invention are bonded to the carrier.

As described above, as a method for producing the adsorbent of the present invention, after activating the carrier, at least two substituent groups having negative charges in one molecule
Although the method of fully bonding an organic low-molecular compound having at least 6 carbon atoms in its skeleton structure has been described in detail, the present invention is not limited to l''IIC. For example, a method of polymerization (copolymerization) using a polymerizable monomer or a crosslinking agent having an organic low molecular compound moiety as referred to in the present invention, or a crosslinking agent having an organic low molecular compound moiety at the time of further post-crosslinking to the crosslinked polymer particles. A method using , etc. can also be used. Furthermore, it is also possible to use a method in which an insoluble substance is coated with a polymer capable of fully binding an organic low-molecular compound to fully bind the organic low-molecular compound, or a method in which an insoluble substance is coated with a polymer having an organic low-molecular compound portion. At this time, the coat polymer can also be post-crosslinked if necessary. Alternatively, a method of activating an organic low molecular compound and then bonding it to a carrier can also be adopted.

That is, the present invention has at least two negatively charged substituents in one molecule, and at least 6 substituents in the skeleton structure.
The effect is achieved by having the organic low-molecular-weight compounds that are fully bonded, and is not affected by 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 the drawings, reference numeral 1 shows an example of an adsorption device using the adsorbent for autoantibodies and/or immune complexes of the present invention, in which a backing 4 with a filter 3 lined inside is attached to an opening at one end of a cylinder 2. A cap 6 having a body fluid inlet 5 is screw-fitted through the cylinder 2, and a cap 8 having a body fluid outlet lower part is screw-fitted to the other end opening of the cylinder 2 through a backing 4' having a filter 3' stretched inside. A container is formed, and the gap between the filters 6 and 3' is completely filled with the adsorbent to form an adsorbent layer 91r.

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 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. When used for extracorporeal circulation, the appropriate volume of the adsorbent layer 9 is about 50 to 400 vessels.

When using the apparatus of the present invention for external circulation, there are two methods as shown below. One method is to use a total blood centrifuge or a model plasma separator to separate plasma components and blood cell components, which are taken out from the body, and then pass the plasma components through the device, purify them, and then separate the blood cells. One method is to return the blood to the body together with its components, and the other method is to pass blood taken from the body directly through a nuclear device for purification.

In addition, regarding the passage speed of blood or plasma, since the adsorption efficiency of the adsorbent is very high, the particle size of the adsorbent can be made coarser, and the degree of filling can be lowered, so the shape of the adsorbent layer can be changed. On the other hand, it is possible to provide a relatively high passing speed. 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 collagen diseases such as cancer, immunoproliferative syndrome, rheumatoid arthritis, and systemic lupus erythematosus. Effective for extracorporeal circulation treatment of autoimmune diseases such as myasthenia gravis, allergies, diseases and phenomena related to the body's immune function such as organ transplant rejection, or IPi-organs such as nephritis, and liver diseases such as hepatitis. Available for

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

Example 1 CNBr Activated Sepharose 4B (Sweden).

Pharmacia! l! First, an organic compound having two or more negative charges and six or more carbon atoms was fully bound by a conventional method, and blocked with an excess of the activated base ethanolamine. The retained amount of various compounds is determined by converting the primary amino group of the remaining organic compound into 4-phenylspiro[furan-2(3
H), 1'-phthalane]-3,3'-dione was calculated by reaction bonding with fluram (manufactured by Roche).

The adsorption test was conducted using 5 volumes of ACD rheumatism patient plasma and 1 adsorbent.
After thoroughly mixing the contents and incubating at 67°C for 6 hours,
The plasma on the upper floor was evaluated. Evaluation was conducted on 11IIIvi, rheumatoid factor, immune complex, 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 rich in rheumatoid factors is applied to polystyrene latex particles adsorbed with human γ-globulin. A dilution series is created and rheumatoid factor 4 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 mW becomes low.

Passive sensitization hemagglutination test was performed using rabbit-gamma on sheep red blood cells.
- All globulins are adsorbed, but the rest is the same as the latex agglutination test. Generally, the passive sensitization hemagglutination test is said to have higher specificity for rheumatoid factor than the latex agglutination test.

Dilution series with glycine saline buffer? The dilution ratio at which rheumatoid factor was negative in a latex agglutination test was determined. Latex agglutination tests were performed using kit i from Japan Freeze Drying Institute. Similarly, evaluation was carried out using the passive sensitization hemagglutination test CRAHA test (manufactured by Fuji Kinki Liang Co., Ltd.).

The immune complex was prepared by polyethylene glycol precipitation method K-r.
Knee h. In this method, the amount of immune complexes is determined by quantifying the amount of immunoglobulin by 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.

(1) Put sample 1.0- into a test tube, add 8% PEG (polyethylene glycol, average molecular weight 6000-7500)
) to 1. Om7! Add, stir and leave at 4℃ for 60 minutes.

(2) Centrifuge at 4°C, 1000f for 60 minutes and remove the supernatant, resulting in a precipitate of 1PBs (IJ acid buffered saline)
Re-dissolve in and set to 1.0-.

(31(1), (2+ 'repeat two more times,
Wash away contaminating monomeric immunoglobulins.

(4) The finally obtained immune complex suspension in PBS was subjected to single radial immunodiffusion method.
Quantify immunoglobulin G'z.

Immunoglobulin G levels were quantified using the single radial immunodeficiency method. The value of rheumatoid factor latex in IJ rheumatoid patient plasma is 12
80, RAHA 5120, immune complexes (immunoglobulin Gi measurement) 6 o mg/di −.

Immunoglobulin G was 1600 μ/a.

The results of the adsorption experiment are shown in Table 1. According to Table 1, adsorbents containing organic low-molecular compound moieties that have at least two negatively charged substituents in one molecule and that have at least six carbon atoms in their skeleton structure absorb rheumatoid factors and immune complexes. It can be seen that there is selective adsorption and less non-selective adsorption of immunoglobin G.

Note that 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 1 Comparative Example 1 An adsorbent was prepared in the same manner as in Example 1 using an organic compound having 1 or less negative charge or 5 or less carbon atoms, and the same experiment as in Example 1 was conducted. The results are shown in Table 2. This shows that the adsorption performance of rheumatoid factors and immune complexes deteriorates with organic compounds having one or less negative charges or five or less carbon atoms.

Table 2 Example 2 Vinyl acetate 1007,) realyl isocyanurate) 5
2. Or (crosslinking degree X=0.35), ethyl acetate i o
o l, heptane 10047', polyvinyl acetate (degree of polymerization 5 o o) 7. '5? and 2.2'-azobisisobutyronitrile 6.82% by weight of polyvinyl alcohol, 0.05% by weight of sodium dihydrogen phosphate dihydrate and 1% by weight of sodium dihydrogen phosphate dihydrogen phosphate. 40q of water in which 1.5% by weight of dihydrochloride was dissolved was placed in a flask, stirred thoroughly, and then heated at 65°C for 18 hours.
Further, suspension polymerization was carried out by heating and stirring at 75°C for 5 hours.
A granular copolymer was obtained.

After filtering, washing with water, and then extracting with acetone, add 46.
5f' and 2t methanol at 40°C.
The transesterification reaction of the copolymer was carried out for 18 hours.

The average particle size of the obtained gel was 150 μm.

Vinyl alcohol single Q (qOH) of p per unit weight is 1
0. Omeq/f, specific surface area was 6077Is15', and exclusion limit molecular weight by dextran was 6 x 10'.

Next, the obtained gel 102 (dry weight) was suspended in dimethyl sulfoxide 120, and 3 ml of epichlorohydrin 7B, 10 green onions of 30% sodium hydroxide were added thereto, and the mixture was stirred at 30°C for 5 hours to activate the gel. A chemical 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 7-amino-1,6
- Naphthalene-disulfonic acid 2. ! 1M'-i including 0.
1M Sodium Carbonate (1)H9,13)
160-. The immobilization reaction was carried out at 50°C for 14 hours with stirring, and then 33 parts of 6mg/mt of tris(hydroxyethyl)aminomethane was added, and the blocking reaction (residual activity) was further carried out at 50°C for 5 hours with stirring. Fund Block) was carried out. Thereafter, it was thoroughly washed with water to obtain an adsorbent for body fluid purification.

The amount of 7-amino-1,3-naphthalene-disulfonic acid immobilized on this adsorbent was 200 μmob/? (dry weight).

The number of carbon atoms in the organic compound immobilized on this sorbent is 13.
The number of negative charges is 2.

This adsorbent was packed into an IO column with an inner diameter of 50 mrm and a length of 70 ml, and 25 d/mL of ACD-added rheumatoid patient plasma was added.
1507 was flowed at the flow rate of B. At this time, there was no decrease in the packing volume of the adsorbent in the column, no clogging, and no decrease in the flow rate.
The pressure loss before and after Karajin was 1511+Hg or less.

When plasma proteins were analyzed before and after passing through the column, rheumatoid factor was 620 for latex and 25 for A (RA) before adsorption.
60, whereas both became negative after adsorption. -Also, the immune complex (measured by C/q solid-phase EIA) was 20μf'/me before adsorption, but it decreased to less than 6μ7/ml after adsorption. On the other hand, immunoglobulin G is 1620! rQ/dl is 1210~/dl
It only dropped to .

[Brief explanation of the drawing]

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 (2)

[Claims] (1) At least 2 substituents with negative charges in one molecule
1. An adsorbent for autoantibodies and/or immune complexes, characterized by having an organic low molecular weight compound moiety having at least 6 carbons and gold in its skeleton structure. (2) The negatively charged substituent is a carboxyl group or/
and a sulfonic acid group, the adsorbent for autoantibodies and/or immune complexes according to claim 1.