JPH0771632B2 - Adsorbent and removal device using the same - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to an adsorbent of a sulfatide-adhesive protein for adsorbing and removing or adsorbing and collecting a sulfatide-adhesive protein from a body fluid, and a removal device using the adsorbent.

[Problems to be Solved by Conventional Techniques and Inventions] Innumerable kinds of viruses, bacteria, fungi, parasites and other infectious foreign substances are present around us.
Both of them can cause diseases of the living body, and if they grow in the body indefinitely, they will eventually kill the living body. Therefore, the immune system exists in the living body as a function of protecting it. Antibody molecules play a major role in this immune system. In other words, foreign substances such as viruses and bacteria act as antigens, and when the immune system is stimulated, antibody molecules that selectively bind to the antigens are produced.

On the other hand, in autoimmune diseases, a humoral or cellular immune response occurs to the antigens of the cells and tissues of its own, rather than the external organisms, and antibodies against self (hereinafter referred to as autoantibodies) and antigens and antibodies are specific. A large amount of complexes (hereinafter referred to as immune complexes) that are formed by reacting with each other are produced. They are not processed by the physiological elimination mechanism and may be directly involved in the appearance of pathologies characteristic of autoimmune diseases by depositing in tissues such as liver, renal glomerulus, joint synovium, lungs, and blood vessel walls. Many.

Autoimmune hepatitis is one of autoimmune diseases and is characterized by positive sulfatide adhesion protein. Although the mechanism by which the produced sulfatide-adhesive proteins are involved in the onset of disease is not clear, it is believed that these sulfatide-adhesive proteins bind to hepatocytes and damage tissues.

Since the sulfatide-adhesive protein thus produced causes various symptoms, control of the sulfatide-adhesive protein is very important for the treatment of autoimmune hepatitis.

Steroids, immunosuppressants, immunomodulators and the like are used to suppress the production of sulfatide adhesive protein.
Of these, steroids are the most commonly used and effective. The initial dose is 30-60 mg /
The daily dose is maintained at 10 to 15 mg / day or 15 to 20 mg / every other day. As a guideline, the administration period is 6 months. However, since steroids are likely to cause side effects even when administered in small amounts, it is self-evident that long-term administration of steroids will cause more serious side effects. Further, if the dose is extremely reduced, there is a problem that the suppressed symptoms will recur.

On the other hand, as an approach different from these drug therapies, there is a method of directly removing the sulfatide-adhesive protein in the body fluid by extracorporeal circulation. The simplest method is so-called plasma exchange therapy, in which plasma of a patient containing sulfatide adhesion protein is replaced with plasma of a healthy person.

This method significantly reduces the sulfatide-adhesive protein in blood. However, this method requires a large amount of healthy plasma, is not only expensive, but also carries the risk of infection such as serum hepatitis during medical treatment.

In addition, plasmapheresis removes all components in plasma,
Although it is exchanged with healthy serum, there is a plasma separation membrane method which selectively separates and removes the sulfatide-adhesive protein, which is the causative agent, by molecular size. In this method, plasma separates plasma into a high molecular weight fraction and a low molecular weight fraction, discards the high molecular weight fraction containing the etiological agent, and removes the low molecular weight fraction containing the major protein albumin. Returned to the patient. However, the sulfatide-adhesive protein is mainly IgG (immunoglobulin G) having a molecular weight of about 160,000, and its separation from albumin (a molecular weight of about 60,000) is not always sufficient, and albumin is also removed when the sulfatide-adhesive protein is removed. It has a drawback that it is removed in a large amount and that all proteins having a molecular weight equal to or higher than that of the pathogenic substance are removed.

Therefore, it has been desired to develop a means for removing the sulfatide-adhesive protein more selectively so that useful components in the body fluid are hardly lost. Therefore, the present inventors have conducted extensive studies in view of such actual circumstances, and found an adsorbent capable of selectively adsorbing only sulfatide-adhesive protein with almost no adsorption of active ingredients in body fluids,
The present invention has been completed.

[Means for Solving the Problems] That is, the present invention provides an adsorbent of a sulfatide-adhesive protein consisting of a water-insoluble porous material having an anionic functional group, a container having a fluid inlet and a fluid outlet, The present invention relates to a device for removing sulfatide-adhesive protein, which comprises a filter that can pass a component contained in the fluid but cannot pass the adsorbent and the adsorbent filled in the container.

Example The adsorbent of the present invention comprises a water-insoluble porous material having an anionic functional group. The term "water-insoluble" refers to the property that the components of the porous body are not dissolved in water, and may be the property of swelling.

The water-insoluble porous body may be either organic or inorganic. In addition, hydrophilicity is preferable to hydrophobicity because adsorption of body fluid components other than the target sulfatide-adhesive protein (so-called nonspecific adsorption) is less.

Furthermore, it is convenient that the surface of the water-insoluble porous material has a functional group that can be used for the immobilization reaction of the ligand (compound having an anionic functional group). Representative examples of these functional groups include, for example, hydroxyl group, amino group, aldehyde group, carboxyl group, thiol group, silanol group, amide group, epoxy group, halogen group, succinylimide group, acid anhydride group and the like. It is not limited to these.

Typical examples of the water-insoluble porous material include, for example, soft porous materials such as agarose, dextran and polyacrylamide; inorganic porous materials such as porous glass and porous silica gel; polymethyl methacrylate, polyvinyl alcohol, styrene, divinyl. Examples thereof include, but are not limited to, a synthetic polymer such as benzene or a copolymer of monomers constituting these and / or a porous polymer hard gel made from a natural polymer such as cellulose as a raw material. Of these, natural polymers such as cellulose, which are hydrophilic and have sufficient mechanical strength, are preferable because they have good biocompatibility.

As the shape of the water-insoluble porous material, for example, an arbitrary shape such as a granular shape, a spherical shape, a fibrous shape, a film shape, or a hollow fiber shape can be selected according to the usage method of the adsorbent. Among them, when the water-insoluble porous material is used after being packed in a column, the granular or spherical shape is preferable because it forms a gap through which components other than the sulfatide-adhesive protein contained in the body fluid can sufficiently pass.

When a particulate water-insoluble porous material is used, its particle size is preferably 1 to 5000 μm. The particle size is 1 μm
If it is less than the above, the adsorption capacity becomes small, and if it exceeds 5000 μm, the adsorption capacity becomes small as well.

The pores of the water-insoluble porous body are preferably continuous pores having a large diameter in order to adsorb the sulfatide-adhesive protein. That is, since the sulfatide-adhesive protein is a large molecule having a molecular weight of at least 160,000, which is mainly composed of immunoglobulins such as IgG and IgM (immunoglobulin M), it may be a pore that can easily enter the porous body. is necessary.

By the way, there are various methods for measuring the pore diameter, and the mercury porosimetry method is most often used, but it is difficult to apply it in the case of a hydrophilic porous material. The exclusion limit molecular weight is often used as an alternative measure of the pore diameter, and can be applied to both hydrophilic porous bodies and hydrophobic porous bodies. Exclusion limit molecular weight cannot be penetrated into the pores in gel permeation chromatography as described in books such as Hiroyuki Hatano and Toshihiko Hanai, “Experimental High Performance Liquid Chromatography”, Kagaku Dojin. Excluded) The molecular weight of the molecule with the smallest molecular weight.

Regarding the exclusion limit molecular weight, generally, globular proteins, dextran, polyethylene glycol, etc. have been well investigated, but in the case of the adsorbent of the present invention, globular proteins that are considered to be most similar to sulfatide-adhesive proteins (viruses It is appropriate to use the value obtained using (including).

Therefore, as a result of examination using various water-insoluble porous materials having different exclusion limits using globular proteins, a range of pore diameters suitable for adsorption of sulfatide-adhesive proteins, that is, preferred water-insoluble porous materials used in the present invention It was revealed that the exclusion limit molecular weight range was 120,000 to 50,000,000. When the exclusion limit molecular weight is less than 120,000, the adsorbed amount of sulfatide-adhesive protein decreases, while as the exclusion limit molecular weight increases, the adsorbed amount of sulfatide-adhesive protein increases and eventually reaches 50 million. Beyond that, not only the surface area becomes smaller and the adsorption amount noticeably decreases, but also nonspecific adsorption increases and the selectivity remarkably decreases. Further, it is preferably 600,000 to 20,000,000 from the viewpoint that the selective adsorption capacity is larger.

Furthermore, for the porous structure of the water-insoluble multi-body, total porosity in which pores communicate with the interior of the porous body is preferable to surface porosity, and the pore volume has a large adsorption capacity.
20% or more is desirable.

When the adsorbent of the present invention is used for extracorporeal circulation treatment, it is necessary to flow a high-viscosity fluid such as blood or plasma at a high speed, so that the water-insoluble porous material does not cause compaction when it is, for example, a particle. It preferably has sufficient mechanical strength.
That is, as shown in the reference example below, the water-insoluble porous body is uniformly packed in a cylindrical column, and the relationship between the pressure loss and the flow rate when circulating an aqueous fluid is at least 0.3 kg / cm ^2.
Those having a linear relationship are preferable.

The water-insoluble porous body has an anionic functional group, and the ionic interaction between the anionic functional group and the sulfatide-adhesive protein causes the sulfatide-adhesive protein to be adsorbed.

As the anionic functional group, any functional group can be used as long as it is negatively charged near neutral pH. Representative examples thereof include, but are not limited to, a carboxyl group, a sulfonic acid group, a sulfone group, a sulfate ester group, a silanol group, a phosphate ester group, and a phenolic hydroxyl group. Of these, a sulfate ester group, a sulfonate group, a carboxyl group, and a phosphate ester group are preferable because they have a strong affinity for the sulfatide-adhesive protein.

There are various methods for introducing the anionic functional group into the water-insoluble porous material, and any method may be used, but typical examples include (1) anionic functional group or easily anionic functional group. A method of forming a water-insoluble porous body by polymerization using a compound having a functional group that can be converted into a group as a monomer or a crosslinking agent, (2) a method of fixing a compound having an anionic functional group to the water-insoluble porous body, (3) A method of directly fixing an anionic functional group to the water-insoluble porous body by reacting a compound having an anionic functional group with the water-insoluble porous body can be mentioned.

Of course, an anionic functional group-containing compound originally containing an anionic functional group such as glass, silica, or alumina may be used as the adsorbent.

The compound having an anionic functional group may be a compound having one anionic functional group per molecule, or may be a polyanion compound having a plurality of anionic functional groups. The polyanion compound is preferable because it has a large affinity for the sulfatide-adhesive protein and easily introduces many anionic functional groups into the unit amount of the water-insoluble porous material. Of these, compounds having a molecular weight of 1000 or more are preferable in terms of the amount of anionic functional groups having an affinity for sulfatide-adhesive proteins. The type of anionic functional group contained in the polyanion compound may be one type or two or more types.

Representative examples of the polyanion compound include, for example, polyacrylic acid, polyvinyl sulfuric acid, polyvinyl sulfonic acid, polyvinyl phosphoric acid, polystyrene sulfonic acid, polystyrene phosphoric acid, polyglutamic acid, polyaspartic acid, polymethacrylic acid, polyphosphoric acid, and styrene-malene. Examples include, but are not limited to, synthetic polyanion compounds such as acid copolymers, and anionic functional group-containing polysaccharides such as heparin, dextran sulfate and chondroitin sulfate.

Further, among the compounds having an anionic functional group, typical examples of the compound containing a sulfuric acid ester group include, for example, a sulfuric acid ester of a hydroxyl group-containing compound such as alcohol, saccharide, and glycol. Partially sulfated products, especially sulfated products of sugars, contain both a sulfate ester group and a functional group necessary for immobilization, and are preferable because both biocompatibility and activity are high. Furthermore, sulfated polysaccharides are easily prepared. It is particularly preferable because it can be fixed to a water-insoluble porous body.

Furthermore, the type of the compound having an anionic functional group fixed to the water-insoluble porous body may be one type or two or more types.

Typical examples of the monomer or cross-linking agent having an anionic functional group or a functional group that can be easily converted into an anionic functional group used in the method (1) above include acrylic acid and its ester, methacrylic acid and its ester,
Examples thereof include styrene sulfonic acid, but are not limited thereto.

Examples of the method (2) include a method by physical adsorption, a method by ionic bond, a method by covalent bond,
Although any method may be used, it is important that a compound having an anionic functional group is not eliminated for the storage stability and stability of the adsorbent, and thus a covalent bond method capable of firmly fixing is desirable. .

When the compound having an anionic functional group is immobilized on the water-insoluble porous body by a covalent bond, the compound having an anionic functional group preferably has a functional group other than the anionic functional group that can be used for immobilization. Representative examples of functional groups that can be used for immobilization include amino groups, amide groups, carboxyl groups, acid anhydride groups, succinylimide groups, hydroxyl groups, thiol groups, aldehyde groups, halogen groups, epoxy groups, silanol groups, and the like. However, it is not limited to these.

The compound having an anionic functional group having a functional group capable of covalent bonding is not particularly limited, but typical examples thereof include sulfanilic acid, hydrogen sulfate, 2-aminoethyl, terephthalic acid, phosphorylethanolamine and the like. To be

Next, as a typical example of the method (3), there is a reaction of introducing a hydroxyl group-containing sulfate ester group, for example. In this case, the sulfate ester group can be directly introduced by reacting the hydroxyl group-containing water-insoluble porous material with a reagent such as chlorosulfonic acid or concentrated sulfuric acid.

As described above, the adsorbent of the present invention selectively and efficiently adsorbs the sulfatide-adhesive protein, and thus the body fluid, that is, blood, plasma, serum, ascites, lymph, synovial fluid, and fractions obtained from them. It is useful for the removal of sulfatide-adhesive protein from components and other liquid components of biological origin.

The sulfatide-adhesive protein adsorbent of the present invention can be used for treatment by various methods.

The simplest method is to extract the patient's blood out of the body, store it in a blood bag, and mix it with the adsorbent of the present invention to remove the sulfatide-adhesive protein, and then remove the adsorbent through a filter to remove blood. There is a way to return it to the patient. This method does not require a complicated device, but has the drawbacks that the amount of treatment performed once is small, the treatment requires time, and the operation becomes complicated.

On the other hand, a method of filling the adsorbent in a column, incorporating it in an extracorporeal circulation circuit, and adsorbing and removing it online is preferable because a large amount of body fluid can be treated in a short time.

Next, a device for removing sulfatide-adhesive protein of the present invention using the adsorbent, which can be incorporated into an extracorporeal circulation circuit, is shown in FIG. explain.

In FIG. 1, (1) is a fluid inlet, (2) is a fluid outlet, (3) is a sulfatide-adhesive protein adsorbent of the present invention, (4) and (5) are fluids and fluids. A filter that can pass the contained components but cannot pass the adsorbent, (6) is a column, and (7) is a container.

The shape and material of the container are not particularly limited, but preferred specific examples are, for example, a volume of about 150 to 400 ml and a diameter of 4
An example is a cylindrical container of about 10 cm.

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Reference example A glass gel column (inner diameter 9 mm, column length 150 mm) equipped with filters with a diameter of 15 μm on both ends, agarose gel (Bio-Rad A5m (trade name) manufactured by Bio-Rad, particle size 50-100 mesh), gel composed of synthetic polymer (Toyopearl HW65 (trade name) manufactured by Toyo Soda Kogyo Co., Ltd., particle size 50-100μ
m) and a porous cellulose gel (Cellulofine GC700 (trade name) manufactured by Chisso Corp., particle size 45 to 100 μm) are uniformly filled, and water is circulated in the column by a peristaltic pump to flow and pressure. The relationship with the loss ΔP was obtained. The results are shown in FIG.

As is clear from Fig. 2, agarose gel, which is a soft gel, consolidates above a certain flow rate and does not increase even if the pressure is increased, whereas hard gels such as Toyopearl and Cellulofine do not The flow rate increased almost in proportion to the increase.

Production Example 1 Porous cellulose gel CK gel A manufactured by Chisso Corporation
-3 (trade name), globular protein exclusion limit 50 million, particle size 45
~ 105μm) Add 60ml water and 80ml 2M NaOH to 100ml 45
The mixture was stirred at 0 ° C for 1 hour. After stirring, 25 ml of epichlorohydrin was further added and stirred at 45 ° C. for 2 hours. After completion of the reaction, the gel was separated by filtration and washed with water to obtain a cellulose gel having an epoxy group introduced (hereinafter referred to as epoxidized gel).

Example 1 To 10 ml of the epoxidized gel obtained in Preparation Example 1 was added 0.14 g of sulfanilic acid dissolved in 7.5 ml of water, and 2 M of
After adjusting the pH of the solution to 10 by adding NaOH, the solution was left at 45 ° C. for 20 hours. After the reaction was completed, the gel was separated by filtration and washed with water to obtain a cellulose gel having sulfanilic acid immobilized thereon. The amount of anionic functional group introduced by the immobilized sulfanilic acid was 10 μmol per 1 ml of the adsorbent.

After thoroughly washing the obtained adsorbent with physiological saline, 1.
0 ml was taken by the examiner and 1.0 ml of autoimmune hepatitis patient serum was added and incubated at room temperature for 2 hours. After completion of this adsorption operation, centrifugation was performed to sediment the gel, and the level of sulfatide-adhesive protein in the collected supernatant was measured by the solid-phase enzyme antibody method (ELIS
It was measured according to A).

The sulfatide-adhesive protein level is obtained by adding a diluted sample to a microplate to which sulfatide has been adhered, performing an antigen-antibody reaction, dropping a peroxidase-labeled anti-human immunoglobulin antibody, and performing an enzyme color reaction by LabScience Co., Ltd. It was measured with SLT-210 (trade name). The results are shown in Table 1 as a relative level of the sulfatide-adhesive protein level in the supernatant of the supernatant after the adsorption experiment with respect to the level of the raw serum as the sulfatide-adhesive protein level.

Example 2 Phosphorylethanolamine instead of sulfanilic acid
A cellulose gel on which phosphorylethanolamine was immobilized was obtained in the same manner as in Example 1 except that 0.11 g was used. The amount of anionic functional groups introduced by the immobilized phorphorylethanolamine was 100 μmo from 1 ml of the adsorbent.
It was l.

The performance of the obtained adsorbent was evaluated in the same manner as in Example 1. The results are shown in Table 1.

Example 3 10 ml of the epoxidized gel obtained in Production Example 1 had a molecular weight of about 5,000,
5 g of sodium dextran sulfate having a sulfur content of 18% and 8 ml of water were added, 2 M NaOH was further added to adjust the pH of the solution to 10, and the mixture was allowed to stand at 45 ° C. for 17 hours. After completion of the reaction, the gel was separated by filtration, washed with water, added with 0.5% monoethanolamine aqueous solution and allowed to stand at room temperature for 20 hours to seal unreacted epoxy groups. After the reaction was completed, the gel was separated by filtration and washed with water to obtain a cellulose gel on which dextran sulfate was fixed. The amount of anionic functional group introduced by the immobilized dextran sulfate was 29 μmol per 1 ml of the adsorbent.

The performance of the obtained adsorbent was evaluated in the same manner as in Example 1. The results are shown in Table 1.

Examples 4 to 8 Porous cellulose gel was changed to CK-225 (trade name, exclusion limit of globular protein, molecular weight 20 million, particle size 45 to 105 μm, crosslinked gel), Cellulofine GCL-2000m (trade name, exclusion limit of globular protein) Molecular weight 3 million, particle size 45-105μm, cross-linked gel), Cellulofine GC-700m (trade name, exclusion limit molecular weight of spherical protein 400,000, particle size 45-105μm), Cellulofine GC-200m (trade name, spherical protein) Exclusion limit of molecular weight 12
10,000, particle size 45-105μm, Cellulofine GC-90 (trade name, exclusion limit molecular weight of globular protein 35,000, particle size 45-105μ)
m) (all manufactured by Nitrogen Co., Ltd.), except that the dextran sulfate sodium-immobilized cellulose gel was obtained in the same manner as in Production Example 1 and Example 3. The amount of anionic functional groups introduced by the immobilized dextran sulfate was 16, 18, 24, 30, and 37 μmol per 1 ml of the adsorbent, respectively.

The performance of the obtained adsorbent was evaluated in the same manner as in Example 1. The results are shown in Table 2.

Production Example 2 Ethylenediamine was added to 40 ml of the epoxidized gel obtained in Production Example 1.
What melt | dissolved 0.5 g in 30 ml was added, and it left at 45 degreeC for 20 hours. After completion of the reaction, the gel is separated by filtration and washed with water to give an amino group-introduced cellulose gel (hereinafter referred to as aminated gel).
I got it.

Example 9 To 10 ml of the aminated gel obtained in Production Example 2 was added 5 g of dextran sodium sulfate used in Example 3 and 8 ml of water, 0.5 ml of 2M NaOH was further added, and the mixture was left at 45 ° C. for 1 hour. NaBH ₄ 0.1g was added to this and it was left to stand at room temperature for 24 hours. After the reaction was completed, the gel was separated by filtration and washed with water to obtain a cellulose gel having dextran sodium sulfate immobilized. The amount of anionic functional group introduced by the immobilized dextran sulfate was 39 μmol per 1 ml of the adsorbent.

The performance of the obtained adsorbent was evaluated in the same manner as in Example 1. The results are shown in Table 1.

Example 10 10 ml of CK gel A-3 was washed with water and suction-filtered, 6 ml of dimethyl sulfoxide, 2.6 ml of 2N-NaOH and 1.5 ml of epichlorohydrin were added, and the mixture was stirred at 40 ° C. for 2 hours. After the reaction, the gel was separated by filtration and washed with water to obtain a cellulose gel having an epoxy group introduced therein.

6 ml of concentrated aqueous ammonia was added thereto, and the mixture was reacted at 40 ° C. for 2 hours to obtain an aminated cellulose gel.

A solution of 0.2 g of sodium polyacrylate having a molecular weight of 190,000 to 500,000 dissolved in 10 ml of water and adjusted to pH 4.5 was added to 5 ml of this gel, and 200 mg of 1-ethyl-3- (dimethylaminopropyl) carbodiimide was further added to pH 4 Add while keeping at 0.5
It was agitated at 4 ° C for 24 hours. After the reaction was completed, the gel was separated by filtration and washed with water to obtain a poly (acrylic acid) -introduced cellulose gel. The amount of anionic functional group introduced by the immobilized polyacrylic acid was 14 μmol per 1 ml of the adsorbent.

The performance of the obtained adsorbent was evaluated in the same manner as in Example 1. The results are shown in Table 1.

It can be seen from Table 1 that the adsorbent having dextran sulfate or polyacrylic acid immobilized thereon has a particularly excellent adsorbability for sulfatide-adhesive proteins.

From Table 2, it can be seen that the adsorbent using the porous body having the exclusion limit molecular weight of 40 to 20 million has a particularly excellent ability to adsorb the sulfatide-adhesive protein.

[Advantages of the Invention] The adsorbent of the present invention can selectively adsorb and remove sulfatide-adhesive proteins from body fluids, and a removal device using the adsorbent can be incorporated into an extracorporeal circulation circuit to allow sulfatide-adhesive proteins in a fluid to be online. The effect that protein can be removed efficiently is exhibited.

[Brief description of drawings]

FIG. 1 is a schematic cross-sectional view of an embodiment of the removing apparatus of the present invention, and FIG. 2 is a graph showing the results of investigating the relationship between flow velocity and pressure loss using three types of gels. (Main symbols in the drawing) (1): Inlet (2): Outlet (3) Adsorbent (4), (5): Filter (7): Container

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location C07K 17/14 8318-4H

Claims (6)

[Claims] 1. An adsorbent for a sulfatide-adhesive protein, which comprises a water-insoluble porous material having an anionic functional group. 2. The adsorbent according to claim 1, wherein a compound having an anionic functional group is immobilized on a water-insoluble porous body. 3. The adsorbent according to claim 1, wherein the water-insoluble porous body comprises a compound having a hydroxyl group. 4. The adsorbent according to claim 1, wherein the anionic functional group is at least one selected from the group consisting of a sulfuric acid ester group, a sulfonic acid group, a carboxyl group and a phosphoric acid ester group. 5. The adsorbent according to claim 2, wherein the compound having an anionic functional group is a polyanionic compound having a plurality of anionic functional groups in one molecule. 6. A container having a fluid inlet and a fluid outlet,
A device for removing sulfatide-adhesive protein comprising a filter and an adsorbent filled in the container, which is capable of passing a fluid and components contained in the fluid but not the adsorbent according to claim 1.