JPH01135534A - Adsorbent of self-antibody and stripper - Google Patents

Abstract

PURPOSE:To selectively remove a self-antibody from body liquid by carrying aromatic sulfonate compd. shown in a specified formula on a water-insoluble porous carrier and forming an adsorbent of the self-antibody. CONSTITUTION:An adsorbent of a self-antibody is formed by fixing a compd. shown in the formula I [R shows aromatic ring, X shows the same or different monovalent cation in (n) pieces of SO3X, (n) shows an integer of 1-12] on a water-insoluble porous carrier. Exclusion limiting mol.wt. of globular protein of the water-insoluble porous carrier is preferably regulated to 0.4-20 million. A stripper of the self-antibody is constituted by providing filters 4, 5 capable of making fluid pass through and the components contained in this fluid but incapable of making the adsorbent 3 of the above-mentioned formula I pass through to the inside of a vessel 7 having an inflow port 1 and an outflow port 2 of the fluid and also introducing the adsorbent 3 into a column 6.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to an adsorbent for adsorbing and removing harmful components from body fluids and a removal device using the adsorbent. More specifically, the present invention relates to an adsorbent for removing harmful autoantibodies from body fluids and suppressing autoimmune diseases, and an autoantibody removal device using the adsorbent.

[Problems to be solved by conventional technology and invention]

Autoimmune diseases, as the name suggests, are diseases in which antibodies against the constituents of one's own tissues (hereinafter referred to as autoantibodies) appear, but in systemic lupus erythematosus (hereinafter referred to as SLE), a typical autoimmune disease, nuclear components of cells,
In particular, it is known that antibodies against deoxyribonucleic acid (hereinafter referred to as DNA) (hereinafter referred to as anti-DNA antibodies) appear in body fluids and are closely related to the disease state thereof.

Although the mechanism by which autoantibodies produced are involved in the onset of disease is not necessarily clear, there is a mechanism in which autoantibodies themselves damage cells, or autoantibodies combine with antigens to form immune complexes that are deposited in tissues. Mechanisms that cause tissue damage have been proposed. In the case of SIJ, the generated anti-DN
It is known that antibody A forms immune complexes with cell-derived DNA that has also leaked into the bloodstream and is deposited on blood vessel walls, renal glomerular basement membranes, etc., causing vasculitis and lupus nephritis, respectively. In fact, many cases of SLE result in death due to renal failure.

On the other hand, in addition to SLE, there are diseases accompanied by the production of autoantibodies. Typical diseases are rheumatoid arthritis (hereinafter referred to as RA) and polyarthritis nodosa (hereinafter referred to as PN), and the autoantibodies that appear in these diseases are antibodies called rheumatoid factor. It is known that this antibody forms an immune complex with immunoglobulin G (hereinafter referred to as IgG) in the blood and deposits on blood vessel walls, smooth layers, etc., thereby causing vasculitis and arthritis, respectively.

Since various symptoms are caused by the autoantibodies themselves or the immune complexes between the autoantibodies and the corresponding antigens produced in this way, controlling autoantibodies is extremely important for treatment.

Conventionally, steroids, immunosuppressants, immunomodulators, anti-inflammatory agents, and the like have been widely used for treatment for the purpose of suppressing the production of autoantibodies. Among these, steroids are the most commonly used, and short-term super-dose therapy called pulse therapy is also often performed. However, since steroids tend to cause side effects even when administered in small doses, it is obvious that short-term super-dose therapy of steroids tends to cause even greater side effects. In addition, these drugs are often used for a long period of time, in which case side effects are more likely to occur, and the dosage must be gradually increased due to drug resistance, so in some cases, these drugs may not be used. In many cases, it is impossible or does not have sufficient effect.

On the other hand, as an approach different from these drug treatments, attempts have been made to directly remove autoantibodies in body fluids by extracirculation. The simplest method is so-called plasma exchange therapy, in which a patient's plasma containing autoantibodies is exchanged with the plasma of a healthy person. This method has significantly reduced autoantibodies in the blood and improved symptoms.

However, this method not only requires a large amount of healthy plasma and is expensive, but also involves the risk of infection such as serum hepatitis during the therapeutic treatment, so it has not become widely used. - In plasma exchange therapy, all components in plasma are removed and replaced with healthy plasma, but in order to selectively remove autoantibodies, which are pathogenic substances, the pathogenic substances are removed depending on their molecular size. A plasma separation membrane method has been developed to separate

In this method, plasma is separated into a high molecular weight fraction and a low molecular weight fraction using a membrane, the high molecular weight fraction containing pathogenic substances is discarded, and the low molecular weight fraction containing the main protein albumin is discarded. However, since autoantibodies and albumin have similar molecular weights, their separation is not always sufficient, and when removing autoantibodies, a large amount of albumin is also removed, and all proteins with a molecular weight equal to or higher than that of the pathogenic substance are removed. There are disadvantages such as being removed.

Therefore, it has been desired to develop a means for removing autoantibodies, which are pathogenic substances, more selectively, with almost no loss of useful components in body fluids.

In view of these circumstances, the present inventors have conducted extensive research and have discovered an adsorbent that can selectively adsorb almost only autoantibodies without losing most of the active ingredients in body fluids, and have completed the present invention. It's arrived.

[Means for solving problems]

That is, the present invention is based on the general formula: %Formula%) (wherein, R is an aromatic ring, X is a monovalent cation that is the same or different in n 5osx, and n is 1 or more 12
An autoantibody adsorbent comprising a compound represented by the following integer) immobilized on a water-insoluble porous carrier, a container having an inlet and an outlet for a fluid, and a fluid contained in the fluid. However, the general formula: NH2-R(S03K) (where R%X
and n is the same as above) immobilized on a water-insoluble porous carrier, and which cannot pass an autoantibody adsorbent; The present invention relates to an autoantibody removal device comprising an adsorbent.

〔Example〕

General 2NH2-t used in the present invention? (SO3X) (
In the compound represented by the formula (where R, , refers to a ring that is susceptible to substitution reactions, satisfies Huckel's rule, and is polyvalent to two or more.

The compound having an aromatic ring may be a compound having one aromatic ring per molecule, or a compound having a plurality of aromatic rings arranged per molecule.

Representative examples of compounds having an aromatic ring used in the present invention include benzene, naphthalene, anthracene, naphthacene,
Examples include pentacene, anthraquinone, naphthoquinone, azulene, azulene and derivatives thereof. Further, the aromatic ring may be a heterocycle having a nitrogen, oxygen, or sulfur atom in the ring, and representative examples of compounds having such a heterocyclic aromatic ring include virol, pyridine, pyrimidine, purine, imidazole, indole,
Examples include, but are not limited to, furan, quinoline and their derivatives. Further, the hydrogen atom of the aromatic ring may be substituted with another atom and/or an atomic group.

Further, the number of SO3X groups bonded to the aromatic ring may be one or more, and if it is 12 or more, it will be unstable and the autoantibody adsorption ability will decrease, so the number is preferably 1 or more and 12 or less.

Monovalent cations include hydrogen atoms, cations of alkali metals such as sodium, potassium, lithium, rubidium, and cesium, and compounds such as ammonium, monoalkylammonium, dialkylammonium, trialkylammonium, and pyridinium. It is not limited to. In addition, these cations may be the same in n SO3X in a compound represented by general 2NH2-R(SOsX) (wherein RSX and n are the same as above),
They may all be different, or some may be the same and some may be different from others.

In this specification, body fluids include blood, plasma, serum, ascites, lymph, intraarticular fluid, and fractionated components obtained therefrom.
and other biologically derived humoral components.

The water-insoluble porous carrier used in the present invention preferably has large, continuous pores.

In other words, autoantibodies are composed of immunoglobulins such as l5rG and IgM (immunoglobulin N), and have a molecular weight of 16
Since the autoantibody is a large molecule with a molecular weight of 90,000 to 900,000, it is necessary for the autoantibody to easily penetrate into the water-insoluble porous carrier in order to efficiently adsorb it.

There are various methods for measuring pore diameter, and mercury intrusion method is the most commonly used, but it is difficult to apply to hydrophilic porous materials. Exclusion limit molecular weight is often used as an alternative guideline for pore diameter, and can be applied to both hydrophilic porous materials and hydrophobic porous materials. Exclusion limit molecular weight is the molecular weight that cannot enter the pores (excluded) in gel permeation chromatography, as stated in books (for example, Hiroyuki Hatano and Toshihiko Hana, Experimental High Performance Liquid Chromatography, Kagaku Doujin). The molecular weight of the substance with the smallest molecular weight.

It is known that the exclusion limit molecular weight varies depending on the target compound, and in general, it has been well investigated for globular proteins, dextran, polyethylene glycol, etc., but in the case of the carrier used in the present invention, the molecular weight that is most similar to autoantibodies is It is appropriate to use the values obtained using globular proteins that are thought to be present.

As a result of studies using various water-insoluble porous carriers with different exclusion limits, we found that the range of pore diameters suitable for adsorption of autoantibodies is as follows:
It has become clear that it is more than 10,000 and less than eooo million. That is, when a water-insoluble porous carrier having an exclusion limit molecular weight of less than 100,000 is used, the amount of autoantibody adsorbed is too small to be practical. In addition, when the exclusion limit molecular weight is 600,000 or more, the surface area is too small and the adsorption amount not only decreases noticeably, but also the adsorption of components other than the target autoantibody.
That is, non-specific adsorption increases and selectivity decreases significantly. Therefore, the exclusion limit molecular weight of the water-insoluble porous carrier used in the present invention is preferably 100,000 or more and 60,000,000 or less, and more preferably 400,000 or more and 20,000,000 or less from the viewpoint of a higher selective adsorption capacity.

Next, regarding the porous structure of the water-insoluble porous carrier, total porosity is preferable to surface porosity, and the pore volume is desirably 20% or more from the viewpoint of high adsorption capacity.

The shape of the water-insoluble porous carrier can be selected from any shape such as granules, spheres, fibers, membranes, and hollow fibers. When using a particulate water-insoluble porous carrier, if the particle size is less than 1, the pressure loss will be large.
If it exceeds oo or i, the adsorption capacity is small, so it is preferably at least 1 to 5,000.

The water-insoluble porous carrier used in the present invention may be either organic or inorganic, but it is preferably one that has little adsorption (so-called non-specific adsorption) of body fluid components other than the target autoantibody. It is preferable for the water-insoluble porous carrier to be hydrophilic rather than hydrophobic, since non-specific adsorption is less likely to occur if the carrier is hydrophilic.

Furthermore, it is advantageous if a vine functional group used for the immobilization reaction of the ligand is present on the surface of the carrier. Representative examples of these functional groups include a hydroxyl group, an amino group, an aldehyde group, a carboxyl group, a thiol group, a silanol group, an amide group, an epoxy group, a halogen group, a succinylimide group, an acid anhydride group, and the like.

Representative examples of water-insoluble porous carriers used in the present invention include:
Soft porous materials such as agarose, dextran, and polyacrylamide; inorganic porous materials such as porous glass and porous silica gel; synthetic polymers such as polymethyl methacrylate, polyvinyl alcohol, and styrene-divinylbenzene copolymer; and/or cellulose. Examples include, but are not limited to, porous polymer hard gels made from natural polymers such as .

When using the adsorbent of the present invention for extracorporeal circulation treatment, blood,
Due to the need to flow high viscosity fluids such as blood plasma at high speeds, it is preferred to use a rigid, water-insoluble porous carrier that has sufficient mechanical strength to avoid compaction. In other words, a hard porous body is one in which a water-insoluble porous carrier is uniformly packed into a cylindrical column and the relationship between pressure loss and flow rate is at least 0 when an aqueous fluid is passed through it, as shown in the reference example below.
A linear relationship up to -3kg/cJ.

As a method for immobilizing the compound represented by the general formula NH2-R(803X) (wherein R, Can be used. However, since the adsorbent of the present invention is used for extracorporeal circulation treatment, it is important for safety to suppress desorption and elution of the ligand as much as possible during sterilization or treatment. It is most preferable to do so.

The amount of said compound introduced per ml of adsorbent is
It is preferable that it is 0.1-1000μ-oR.

If it is less than 0.1 μmop, the adsorption capacity is insufficient and 1
If it exceeds 000 μraol, the amount of non-specific adsorption will be too large and it will be difficult to put it to practical use. More preferably, the amount of the compound introduced is 5 to 200 μraol.

There are various ways to use the adsorbent of the present invention for treatment.
Any method may be used, but the simplest method is to draw the patient's blood outside the body and store it in a blood bag, mix it with the adsorbent of the present invention to remove autoantibodies, and then pass it through a filter. There are methods to remove the adsorbent and return the blood to the patient. Although this method does not require complicated equipment, it has the drawbacks that the amount of treatment per treatment is small, the treatment takes time, and the operation is complicated.

Another example is one in which the adsorbent is packed into a column and installed in an extracorporeal circulation circuit to perform online adsorption and removal. That is, although the device of the present invention is a container having a fluid inlet and an outlet, the fluid and the components contained in the fluid can pass through, but the general 2NH2-R(SOs is the same as above),
Body fluid is passed through an autoantibody removal device consisting of a filter that cannot pass through the autoantibody adsorbent, which is immobilized on a water-insoluble porous carrier, and an autoantibody removal device that is filled in the container. This method is preferable because it is simple and requires a large amount of treatment at one time, and does not require a long time for treatment.

Further, processing methods include a method in which whole blood is directly perfused and a method in which plasma is separated from blood and then passed through a column.

The adsorbent and apparatus of the present invention can be used in either method, but as mentioned above, it is most suitable for on-line processing.

FIG. 2 shows a schematic cross-sectional view of an embodiment of the autoantibody removal device of the present invention. In FIG. 2, (1) and (2 are the respective fluid inlets and outlets, (3) is the adsorbent of the present invention, (
4) and (5) are filters or meshes through which the fluid and the components contained in the fluid can pass through, but the adsorbent of the present invention cannot pass through; (6) is the column 1. (7) is a container. Here, the filter (4) on the fluid inlet side may not be present.

Hereinafter, the present invention will be explained in more detail with reference to Examples.
The present invention is not limited to such embodiments.

Reference example Agarose gel was placed in a glass column (inner diameter 9 mm, column length 150 mm) equipped with a filter with a diameter of 15 ρ at both ends.
Biogel A5++ (Product name: Bio-Rad (Blo)
Toyopearl HW65 (product name, manufactured by Rado), a gel made of synthetic polymer (particle size: 50-100 mesh),
Toyo Soda Kogyo Co., Ltd., particle size: 50-100a+) and porous cellulose gel, Cellulofine GC-700 (trade name, Chisso, particle size: 45-100a+) were uniformly packed in the column using a Beristaltic bomb. Water was passed through the tube, and the relationship between flow rate and pressure loss ΔP was determined.

The results are shown in FIG. As is clear from the figure, agarose gel, which is a soft gel, becomes compacted when the flow rate exceeds a certain level, and the flow rate does not increase even if the pressure is increased, whereas hard gels such as Toyovar and Cellulofine undergo compression when the pressure increases. The flow rate increases approximately in proportion to.

Example 1 20 ml of porous cellulose gel CKA-3 (trade name, manufactured by Chisso ■, globular protein exclusion limit molecule ffi 50 million, particle size 45-105 Jl) was suspended in 20 ml of water, and this was mixed with 2M water. Sodium oxide aqueous solution 10ml
was added and heated to 40°C, then 4 m of epichlorohydrin was added.
After stirring at 40°C for further 2 hours, the mixture was washed with water to obtain a cellulose gel into which epoxy groups were introduced (hereinafter referred to as epoxidized gel).

Add 0.0 ml of sulfanilic acid to 5 ml of the resulting epoxidized gel.
A solution of 14 g dissolved in 7 ml of water and adjusted to pnto was added, and the mixture was shaken at 45° C. for 6 hours to obtain a cellulose gel on which sulfanilic acid was immobilized.

Example 2 Porous cellulose gel was prepared using CKA-22 (trade name, manufactured by Chisso Corporation, exclusion limit molecular weight for globular proteins of 20 million, particle size of 4).
5-105ρ, crosslinked gel), Cellulofine GCL-2
000 (trade name, manufactured by Chisso ■, exclusion limit molecular weight of globular protein 3 million, particle size 45-105 at, cross-linked gel),
Cellulofine GC-700 (trade name, manufactured by Chisso ■, exclusion limit molecule f1.4 million for globular proteins, particle size 45-105
Karasu), Cellulofine GC-200 Kai (trade name, manufactured by Chisso ■, exclusion limit molecular weight for globular proteins 120,000, particle size 45~
105 AlT11), (?le 07 Fin GCL-9
A cellulose gel with immobilized sulfanilic acid was obtained in the same manner as in Example 1, except that the gel was changed to 0 (trade name, manufactured by Chisso ■, exclusion limit molecular weight of globular protein 35,000, particle size 45-105 Jl).

Example 3 In Example 1, the epoxidized crosslinked agarose gel was replaced with epoxidized crosslinked agarose gel, Eboxy Activated Sepharose CL-6B (trade name, manufactured by Pharmacia Fine Chemicals, Globular Protein Exclusion Limit Molecule m4)
An agarose gel on which sulfanilic acid was immobilized was obtained in the same manner as in Example 1 except that a gel with a particle size of 45 to 1B5 was used.

Example 4 PP-)IG, a hydrophilic porous hard hydrogel mainly composed of polymethyl methacrylate (trade name, manufactured by Mitsubishi Kasei ■, exclusion limit molecular weight of globular protein 4 million, particle size 12)
A gel on which sulfanilic acid was immobilized was obtained in the same manner as in Example 1, except that sulfanilic acid (0,11111) was used.

Example 5 For each 0.1 ml of adsorbent obtained in Examples 1 to 4, 0.1
5M Tris-HCL buffer (PH7,6) 0.9 m
After adding 0.1 l of serum containing anti-dsDNA antibodies,
5ml was added and incubated at room temperature for 2 hours. After adsorption, the rgG concentration in the supernatant was measured by immunoturbidimetry, and the anti-dsDNA antibody titer was measured by enzyme-linked immunosorbent assay (ELISA).

To determine the anti-dsDNA antibody titer, drop a diluted sample onto a plate with DNA attached, perform an antigen-antibody reaction, drop a peroxidase-labeled anti-human immunoglobulin antibody, and perform an enzymatic color reaction using 5LT-210 (trade name, Lab Science). (manufactured by )). Table 1 shows the antibody titers in the supernatants after adsorption experiments using adsorbents in which sulfanilic acid is immobilized on various carriers, expressed as a relative antibody titer as a percentage of the antibody titer of the original serum.

From Table 1, the adsorbent with immobilized sulfanilic acid has anti-ds
It can be seen that DNA antibodies are adsorbed, but 1gG is hardly adsorbed.

Further, from the same table, it can be seen that the anti-dsDNA antibody binding ability of Cellulose GO-20h and Cellulose GCL-90, whose exclusion limit molecular weight is smaller than the anti-DNA antibody molecular weight of about 160,000, is slightly lower.

[Margin below]

Example 6 Using the adsorbents obtained in Examples 1 to 4, adsorption of rheumatoid factor was investigated in the same manner as in Example 5 using patient serum with high rheumatoid factor titer, and relative rheumatoid factor titer was determined. Ta. The rheumatoid factor titer was measured using the solid-phase enzyme antibody method (ELISA) in the same manner as in Example 5, except that rabbit IgG was used as the antigen and peroxidase-labeled anti-human IgM was used as the secondary antibody.
). The results are shown in Table 2.

From Table 2, it can be seen that all adsorbents on which sulfanilic acid is immobilized adsorb rheumatoid factors well.

Also, from the same table, cellulose GC-
It can be seen that the rheumatoid factor binding ability of cellulose GCL-90 is slightly reduced after 20 hours.

[Margin below]

Table 2 [Effects of the Invention] The adsorbent of the present invention and the removal device using the same are effective in selectively removing autoantibodies from body fluids.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the results of examining the relationship between flow rate and pressure drop using three types of gels, and FIG. 2 is a schematic cross-sectional view of one embodiment of the autoantibody removal device of the present invention. . (Main symbols in the drawing) (1); Inlet (J: Outlet (3): Adsorbent (4), (5): Filter (7): Container Pressure loss ΔP (kg/cm") >

Claims (1)

[Claims] 1 General formula: NH_2-R(SO_3X)_n (wherein, R is an aromatic ring, X is a monovalent cation that is the same or different in n SO_3X, and n is 1 or more 1
1. An autoantibody adsorbent comprising a compound represented by (representing an integer of 2 or less) immobilized on a water-insoluble porous carrier. 2 Claim 1: The water-insoluble porous carrier has an exclusion limit molecular weight of 400,000 to 20 million.
Adsorbent for autoantibodies described in Section 1. 3 A container with a fluid inlet and an outlet, through which the fluid and the components contained in the fluid can pass, but with the general formula: NH_2-R(SO_3X)_n (wherein R is an aromatic ring and X is an n number of Same or different monovalent cations in SO_3X, and n is 1 or more and 1
(representing an integer of 2 or less) immobilized on a water-insoluble porous carrier, the autoantibody adsorbent cannot pass through the filter, and the autoantibody adsorbent filled in the container Autoantibody removal device consisting of adsorbent.