JPS61206457A - Material and apparatus for adsorbing malignant substance - 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 (Industrial Application Field) The present invention removes fibrinogen, immunoglobulin, rheumatoid factor, autoantibodies such as anti-acetylcholine receptor antibodies, and immune complexes from blood, plasma, and other body fluids. The present invention relates to an adsorbent and an adsorption device using the same.

In particular, the present invention relates to an adsorbent that is excellent in removing fibrinogen and immunoglobulin from body fluids, and an adsorption device using the same.

The present invention selectively adsorbs and removes fibrinogen, immunoglobulin, autoantibodies, and immune complexes present in the body fluids of patients with collagen diseases such as rheumatoid arthritis and myasthenia gravis. The purpose of the present invention is to provide a simple adsorbent and an adsorption device that purify and regenerate the disease, prevent the progression of the disease, alleviate symptoms, and hasten healing.

(Conventional technology) Conventionally, rheumatoid arthritis, systemic lupus erythematosus (S
Collagen diseases such as LE), myasthenia gravis, systemic scleroderma, dermatomyositis, and periarteritis nodosa are considered intractable diseases, and there are no effective treatments.

In recent years, these collagen diseases have been understood as autoimmune diseases or immune complex diseases, and the presence of various autoantibodies is thought to have an important relationship with the occurrence of various symptoms, as well as vasculitis and renal lesions. The mechanism by which antigen-antibody immune complexes are deposited in tissues has been emphasized as the cause of the disease.

In order to respond to the demand for selective adsorption and removal of malignant substances such as autoantibodies and immune complexes, the present inventors have conducted extensive research and found that a special carrier that has a chemical selective interaction with the adsorbed substance has been developed. He discovered various adsorbents that hold substances through chemical bonds and applied for a patent earlier (Japanese Unexamined Patent Publication No. 57-122).
875, Japanese Patent Publication No. 57-192560, Japanese Patent Publication No. 58-
61752, JP-A-57-134164).

(Problems to be Solved by the Invention) On the other hand, with the progress of research on the clinical pathology of collagen diseases, it is becoming more and more important to not only remove autoantibodies and immune complexes from the body fluids of patients, but also to eliminate the predisposition to highly viscous body fluids. There is an increasing demand for the removal of fibrinogen and immunolobulin.

(Means for Solving the Problems) The present invention was developed as a result of intensive studies on compounds that selectively interact with fibrinogen and immunoglobulin, which cannot be expected to be sufficiently removed using the conventional techniques described above. , has been reached.

That is, the present inventors have studied in more detail the interaction between the adsorbent and the adsorbed substances, such as autoantibodies and immune complexes, in the prior art as described above. It was discovered that an adsorbent having at least one type of low molecular weight compound having a heterocyclic aromatic skeleton (hereinafter referred to as an organic ligand) exhibits excellent adsorption ability for fibrinogen and immunoglobulin, and the present invention was completed. .

The present invention provides an adsorbent for malignant substances in body fluids, which is characterized by having at least one low molecular weight compound having a nitrogen-containing heterocyclic aromatic skeleton on its surface, and a fluid containing one or more of the adsorbents. This is an adsorption device characterized by being housed in a container having an inlet and outlet.

The adsorbent of the present invention is suitable for diseases in which fibrinogen and/or immunoglobulin in body fluids act as exacerbating factors of collagen disease, and is suitable for diseases in which fibrinogen and/or immunoglobulin in body fluids acts as an exacerbating factor of collagen disease. Corrective effects can be expected.

Specifically, rheumatoid arthritis, myasthenia gravis, SLE
, mixed combined fibrosis (MCTD), progressive systemic sclerosis, Siegren's syndrome, dermatomyositis (polymyositis), silicosis, amyloidosis, Hashimoto's disease, chronic hepatitis, diabetes, bronchiectasis, etc. be.

The aromatic skeleton of the low-molecular-weight compound of the present invention plays an important role in inducing interaction with the hydrophobic part of the adsorbed substance, or with the skeleton part having pi electrons, and its structure is based on Hieckel's ( 4n+2) It follows the law of π electrons.

Furthermore, the fact that the organic ligand is a nitrogen-containing heterocyclic compound is an important factor in expressing cationic charge interaction with the adsorbed substance. Therefore, when the organic ligand is a compound having a nitrogen-containing heterocyclic aromatic skeleton, it is possible to form strong bonds with the adsorbed substance through hydrophobic interactions, pi-electron interactions, and charge interactions. can. Specifically, examples of organic ligands include five-membered cyclic compounds that have virole, pyrazole, imidazole, triazole, and tetrazole skeletons, and six-membered cyclic compounds that have pyridine, pyridazine, pyrimidine, pyrazine, and triazine skeletons. It is.

Moreover, among these, compounds having a pyrazole ring are more likely to exhibit cationic properties and give preferable results for the purpose of the present invention.

As organic ligand, pyrazole, mono-2°G.

Trimethylpyrazole, phenylpyrazole, oxypyrazole, alkoxypyrazonyl, aminopyrazole, acetylmethylpyrazole, aminoindazole,
Mention may be made of aminopyrazolopyrimidines and their derivatives. By having at least one of these on the surface of the adsorbent, the effects of the present invention can be easily obtained.

In the present invention, a low molecular weight compound is a derivative or low polymer of a compound having the above-mentioned skeleton, and has a molecular weight of 1
It is a substance with a molecular weight of 1,000 or less, more preferably a substance with a molecular weight of 1,000 or less. This results in protein A (molecular weight 42,000
) handling during immobilization compared to natural polymers such as
It can also be easily stored after fixation. Furthermore, even if the substance is eluted from the carrier, a substance with a molecular weight of 10,000 or less has negligible antigenicity to living organisms, is safe, and can be easily sterilized.

As the carrier used in the present invention, any carrier can be used as long as it is insoluble in water, but it is preferable to use a wood-philic carrier in order to reduce non-specific adsorption. The shape of the insoluble carrier may be particulate, fibrous, hollow fiber, membrane, etc.; Preferable carriers are those in the form of solid or fibrous materials.

When a fibrous carrier is used, the fiber diameter thereof 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 autoantibodies will be reduced, and if it is too small, activation of the coagulation system, blood cell adhesion, and clogging are likely to occur. As a fibrous carrier,
Known fibers such as regenerated cellulose fibers, nylon, acrylic, and polyester can be used.

The average particle diameter of the particulate carrier is preferably in the range of 25μ to 2500μ, more preferably in the range of 150μ to 1500μ. Average particle size is JIS-Z-
After classification in running water using a sieve specified in 8801, the intermediate value between the upper limit particle size and the lower limit particle size of each class is taken as the particle size of each class, and the average particle size is calculated as the weight average. Further, the particle shape is preferably spherical, but is not particularly limited. If the average particle size exceeds 2500μ, the adsorption amount and adsorption rate of malignant substances will decrease, and if it is less than 25μ, pressure drop will be large and clogging will easily occur when used for extracorporeal circulation, and activation of the coagulation system and blood cell adhesion are likely to occur. . Particulate carriers that can be used include agarose-based, cross-linked dextran-based,
Supports such as cellulose, crosslinked polyacrylamide, porous silica beads, activated carbon, and porous glass can be used. Furthermore, immobilized enzymes and known carriers commonly used in affinity chromatography can be used without any particular limitations. Particularly preferably, a hard gel with good shape stability is used as the carrier.

In the present invention, a hard gel refers to a gel whose physical property values maintain a certain value or more when an external force is applied. Specifically, when gel is filled into a container with a diameter of 10+am+ and a length of 50Il111 and water is passed through it, the pressure difference between the inlet and outlet of the container is 2.
00mm) The volume reduction rate of the gel is 10% or less in the Ig state.

Moreover, porous synthetic polymer particles can be used as the particulate carrier used in the present invention. The particles are capable of fixing the substance used in the present invention described above on their surfaces, and have an average pore diameter of 300 to 9,000 pores, more preferably 1,000 to 6,000 pores. As the polymer composition of the particles, polyamide-based, polyester-based, polyurethane-based, or vinyl compound polymers can be used, but particularly hydrophilic vinyl compound-based porous synthetic polymer particles give preferable results.

The porous structure preferably has an average pore size in the range of 300 to 9000 pores, but if the average pore size is too small, the amount of adsorption will be small, and if it is too large, the strength of the polymer particles will decrease. Measurement of the average pore size, which is impractical due to the reduction in surface area, can be performed with a mercury intrusion porosimeter. In this method, mercury is injected into a porous material, and the amount of pores is determined from the amount of mercury that has entered, and the pore diameter is determined from the pressure required for injecting, and pores of 40 Å or more can be measured. The average pore size is the pore size r.

When the cumulative pore volume measured with a porosimeter is V,
dv/dj! Let it be the value of r when the value of ogr is maximum.

Among porous polymers, crosslinked copolymers with hydroxyl groups have the following properties: ■ have little interaction with solutes such as proteins in plasma, minimizing nonspecific adsorption, and ■ complement system and coagulation in plasma. Due to the characteristics such as small interaction with the system,
It can be preferably used. In particular, a crosslinked copolymer containing vinyl alcohol units as a main component gives good results in terms of the above characteristics.

Due to its hydrophilic nature, the carrier made of a crosslinked copolymer mainly composed of vinyl alcohol units has a small interaction with solutes such as proteins in plasma, and reduces nonspecific adsorption to a minimum. It also has extremely excellent properties such as not interacting with the complement system and coagulation system in plasma. In terms of physical properties, it has heat resistance, enables heat sterilization, and has excellent physical and mechanical strength and hardness, which are characteristics of synthetic polymers. When used as a carrier for adsorbent for whole blood, it also has extremely excellent properties such as having little interaction with blood cell components and minimizing thrombus formation, non-specific adhesion of blood cell components, residual blood, etc. .

The higher the density of hydroxyl groups in the crosslinked copolymer of the present invention, the higher its hydrophilicity, which is advantageous for minimizing interaction with blood components, and when activated with an activation reagent. The active group density can also be maintained at a high level, which is preferable, but on the other hand, the physical and mechanical strength decreases. therefore,
The hydroxyl group density is preferably 5 to 17 s+eq/g, more preferably 6 to 15 g*eq/g.

The hydroxyl group density can be determined by reacting the carrier with acetic anhydride in a pyridine solvent and measuring the amount of acetic anhydride consumed by the reaction with the hydroxyl group or the change in weight of the carrier. The hydroxyl group density when 1 g of dry carrier reacts with 1a+moffi of acetic anhydride is 1 meq/g.

A crosslinked polymer having vinyl alcohol units as a main component can be synthesized by introducing a hydroxyl group through a chemical reaction of a polymerized polymer of a monomer having a hydroxyl group, or by introducing a hydroxyl group using a crosslinking agent having a hydroxyl group. Examples of polymerization methods include:
A radical polymerization method can be used. The crosslinking agent may be introduced by copolymerization during polymerization, or by chemical reaction between polymers (between polymers or between polymer and crosslinking agent), or both may be used in combination.

For example, it can be produced by copolymerizing a vinyl monomer and a vinyl or allyl crosslinking agent. Examples of the vinyl monomer in this case include carboxylic acid vinyl esters such as vinyl acetate and vinyl propionate, vinyl ethers such as methyl vinyl ether and ethyl vinyl ether, and vinylene carbonate II.

Examples of crosslinking agents include allyl compounds such as triallyl isocyanurate and triallyl cyanurate, and di(meth)acrylates such as ethylene glycol dimethacrylate and diethylene glycol dimethacrylate. , polyvinyl ethers such as butanediol divinyl ether, diethylene glycol divinyl ether, and tetravinylglyoxal, polyallyl ethers such as diarylidene pentaerythritol and tetraallyloxyethane,
Glycidyl acrylates such as glycidyl methacrylate can be used. Furthermore, copolymerized comonomers with other comonomers can also be used, if necessary.

In the case of vinyl copolymers, triallyl isocyanurate crosslinking of polyvinyl alcohol obtained by copolymerizing a vinyl ester of carboxylic acid and a vinyl compound (allyl compound) containing an isocyanurate ring and hydrolyzing the copolymer. The body provides a particularly good support in terms of strength, hardness, chemical stability and thermal stability.

Although the case of a vinyl copolymer has been exemplified above, the present invention
It is not limited to this.

As mentioned above, the shape of the crosslinked copolymer having hydroxyl groups can be effectively used, such as spherical, granular, filamentous, hollow fiber, and flat membrane. spherical or granular shapes are particularly preferably used from the viewpoint of flow of body fluids during extracorporeal circulation. Therefore, as a method for synthesizing the carrier, the known suspension polymerization method is particularly effectively used.

In the present invention, a crosslinked copolymer having a specific surface area of at least 5rd/g is used.

The specific surface area is expressed as the surface area occupied by nitrogen gas adsorbed per unit weight of the dry crosslinked copolymer. That is, the specific surface area indicates how effectively the substance constituting the crosslinked copolymer per unit weight forms the surface in a dry state.

Generally, a crosslinked copolymer swells in a medium that is compatible with the crosslinked copolymer and shrinks when dried.

In the case of a soft gel in which the pores filled with the medium during swelling are maintained only by a network of crosslinks, the network collapses when drying, and the pores almost disappear. The specific surface area in this case generally shows a low value of 1 rrr/g or less because it is a value only for the outside of the particle. Since agarose conventionally known for use in affinity chromatography is a soft gel, its bores tend to disappear when it dries. Therefore, sterilization cannot be performed easily, and furthermore, since it has a soft mesh that is easily crushed, even when packed in a column and used for extracorporeal circulation, body fluids cannot be passed through it at a high flow rate for a long period of time.

On the other hand, in the case of a hard gel (crosslinked copolymer) that has a solid structure and can withstand freeze-drying and heat sterilization, the bores will shrink somewhat when dried, but will maintain most of their swollen state. That is, it had permanent bores, and the specific surface area showed a value higher than that of soft gel, and showed a value of at least 5 rrr/g or more.

The specific surface area of the present invention was determined by the most common BET method using nitrogen gas. In addition, the sample used for specific surface area measurement must be sufficiently dry, but since the crosslinked copolymer of the present invention is difficult to dry, it is necessary to It was dried under reduced pressure at 60° C. or lower and used for measurement.

Water retention capacity (hereinafter referred to as W) of the crosslinked copolymer used in the present invention.

) is suitably in the range of 0.5 to 16 g/g, preferably in the range of 1.0 to 15.0 g/g.

WR is when the crosslinked copolymer is equilibrated with physiological saline,
The amount of physiological saline that can be contained within the particles is expressed as a value per dry weight of the crosslinked copolymer. That is, W.R.
is a guideline for the weight within the crosslinked copolymer. If the WR becomes thick, the weight of the part that forms the skeleton per unit volume of the crosslinked copolymer in water, that is, the weight of the crosslinked copolymer itself, decreases relatively, and therefore, the crosslinking becomes difficult in physiological saline and even body fluids. The mechanical strength of the copolymer decreases.

Furthermore, as the W becomes smaller, the weight per unit weight (or unit volume) effective for adsorption decreases, resulting in a decrease in adsorption capacity. Therefore, it is preferable for the carrier of this purpose that Wl is within an appropriate range.

WR is the weight (Wt) of the crosslinked copolymer that has been sufficiently dried in advance
After measuring, the crosslinked copolymer that has been sufficiently equilibrated with physiological saline is centrifuged to remove the physiological saline adhering to the surface of the crosslinked copolymer, and its weight (Wt) is measured. It can be determined by the formula.

The exclusion limit molecular weight (protein) of the carrier is preferably 15 to 10 million, since the molecular weight of the target adsorbent of the present invention is 150,000 (IgG) and reaches 10 million in the case of immune complexes, particularly IgM immune complexes. . The most common exclusion limit molecular weight for purposes of this invention is 1 million to 5 million.

All known methods such as covalent bonding, ionic bonding, physical adsorption, embedding, or precipitation and insolubilization on the polymer surface can be used to bond the above-mentioned low molecular weight substances to the carrier. fixed by covalent bond,
It is preferable to use it after insolubilization. Therefore, known methods for activating immobilized enzymes and carriers commonly used in affinity chromatography can be used.

Examples of activation methods include halogenated cyanide method, epichlorohydrin method, bisepoxide method, halogenated triazine method, bromoacetyl bromide method, ethyl chloroformate method, 1,1'-carbonyldiimidazole method, etc. can. 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 bond. do not have.

In addition, if necessary, between the carrier and the low molecular weight substance,
Molecules (spacers) of arbitrary length can also be introduced and used. For example, the hydroxyl group of the carrier and the isocyanate group on one side of hexamethylene diisocyanate are reacted and bonded, and the remaining isocyanate group is reacted with the amino group, hydroxyl group, thiol group, carboxyl group, etc. of the bond.
Each combination can be carried out. As for the spacer length, a range from no spacer to 20 atoms in the spacer gives particularly preferable results.

In the present invention, the amount of ligand bound to the carrier is 1 g of carrier (
dry weight) per O,lμw+offi or 1000
μl1OIl range, more preferably 0.5μt
The range is from mol to 100μIIIol.

A hydroxyl group, an amino group, a carboxyl group, a phosphoric acid group, etc. can be used as the portion of the ligand (low molecular weight substance) to be bound to the carrier.

The method for activating a carrier and binding a ligand has been described above in detail as a method for manufacturing the adsorbent of the present invention.
The present invention is not limited to this. For example, a method in which a ligand is bound to a polymerizable monomer or a crosslinking agent and then polymerized (copolymerized), a method in which a crosslinking agent bound to a ligand is used during post-crosslinking, etc. can also be used. Furthermore, we will discuss how to bind a ligand after coating an insoluble substance with a polymer capable of binding a ligand, and how to bind a ligand to an insoluble substance after binding a ligand to a polymer.
A method of coating an insoluble substance can also be used. At this time, the coat polymer can also be post-crosslinked if necessary. Alternatively, a method can be adopted in which the ligand is activated and then bound to the carrier.

That is, the present invention exhibits its effects as long as the ligand of the present invention is present on the surface of the adsorbent, and it goes without saying that it is not dependent on the manufacturing method.

The mechanism of action of this adsorbent is thought to be similar to that of an antigen-antibody reaction, but sufficient adsorption and removal ability can be achieved by a low-molecular-weight substance without using biological polymers or similar synthetic polymers, which are the original antigens. It is surprising that such substances exist.Furthermore, low molecular weight substances have the advantage of being easily metabolized in the body.

In the adsorbent of the present invention, the adsorbent described above is filled in a container having a fluid inlet/outlet, so that body fluids can pass through while coming into contact with the adsorbent.

A concrete example of this is shown in FIGS. 1 and 2. Figure 1 shows an adsorber (1) with a backing (4) with filters (3) and (3'') placed inside the openings at both ends of a cylinder (2).

Body fluid outlet (5) through (4'), cap (6) with (5'), (6”) screwed (7),
(7"), and the gaps between the filters at both ends are filled with adsorbent (8).

As the adsorbent, the adsorbent of the present invention may be filled alone, or may be mixed or laminated with other adsorbents.The volume of the adsorbent layer is 50 to 400 when used for extracorporeal circulation.
■About 1 is appropriate.

When using the adsorber of the present invention in extracorporeal circulation, there are two methods as shown below by Ohji. First, blood taken from the body is separated into plasma components and blood cell components using a centrifuge or a cavity-type plasma separator, and then the plasma components are passed through the adsorber of the present invention for purification. There is a method in which the blood is then returned to the body together with blood cell components, and the other method is to directly pass the blood taken out from the body through the adsorption device of the present invention 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 packing can be lowered, so the shape of the adsorbent layer can be changed. However, high passing speeds can be achieved. Therefore, a large amount of body fluid can be treated.

The method for passing body fluids may be either continuous or intermittent, depending on clinical needs or the installation status of the equipment.

(Effects of the Invention) As described above, the adsorbent and adsorption device of the present invention are excellent in removing fibrinogen and immunoglobulin from body fluids, and also remove autoantibodies such as rheumatoid factor and immune complexes at a high rate. It specifically adsorbs and removes, is extremely compact, and is simple and safe. It also has the effect that sterilization operations can be carried out easily and reliably.

In particular, when a crosslinked copolymer containing vinyl alcohol units as a main component is used as a carrier, it has extremely excellent properties such as low nonspecific adsorption of plasma proteins and low interaction with the complement system and coagulation system. In addition, it has excellent physical and mechanical strength, and there is extremely little chipping or flaking due to adsorbent preparation or handling.

Also, since it is hard, body fluids can flow through it at a high flow rate. Furthermore, since it has heat resistance, it also has the effect that ordinary sterilization methods (ethylene oxide gas sterilization, heat sterilization such as high-pressure steam, γ-ray sterilization, etc.) can be carried out easily and reliably. Furthermore, when used as an adsorbent for whole blood, since the interaction with blood cell components is small, it has the advantage of minimizing thrombus formation, non-specific adhesion of blood cell components, residual blood, etc.

The present invention is applicable to the general method of purifying and regenerating body fluids such as autologous plasma, and is effective for safe and reliable treatment of diseases related to the immune function of the body, particularly for the treatment of collagen diseases such as rheumatoid arthritis. It is.

In addition, the adsorbent of the present invention can be used not only as a therapeutic device by filling it into a device, but also as an adsorbent for separating and purifying autoantibodies and immune complexes such as fibrinogen, immunoglobulin, and rheumatoid factor, and for measuring these. It can also be used extremely effectively as a base material.

(Example) Hereinafter, embodiments of the present invention will be described in more detail with reference to Examples.

Example 1 Sepharose CL-4B (manufactured by Pharmacia, Sweden) 20mj! After washing with dimethyl sulfoxide 80n11, dimethyl sulfoxide 20o + 15 epichlorohydrin 20mj! suspended in a solution consisting of 30
% sodium hydroxide was added thereto, and the mixture was stirred at 30°C for 5 hours to obtain an embossed activated carrier.

Using the epoxy bonded gel, 2-aminobi-lysine (manufactured by Aldrich) was bonded to create an adsorbent. 2
- Prepare a solution of aminopyridine dissolved in 5% potassium hydroxide solution to a concentration of 100 μviol/ml, and add 20 mj of the epoxy bonded gel! Add to the ratio of 40-i,
The reaction was carried out at 50°C for 20 hours. .

Excess active groups were blocked with 0.15 oil/II glycine. The amount of 2-aminopyridine immobilized was calculated from the 285 ns absorption of 2-aminopyridine remaining in the reaction solution. The amount of 2-aminopyridine bound to the adsorbent was 55 μmol/ra1.

After thoroughly washing the adsorbent with physiological saline, it was dehydrated and used in the experiment.

In the adsorption experiment, 3 volumes of ACDCD medium patient plasma and 1 adsorbent were used.
After mixing and incubating at 37°C for 3 hours,
The supernatant plasma was evaluated. Evaluation was performed on fibrinogen and immunoglobulin G1 rheumatoid factor albumin.

Fibrinogen, immunoglobulin G, and albumin were quantified using the single radial immunodeficiency method. Measurement of rheumatoid factor was performed by passive sensitization hemagglutination test.

Passive sensitization hemagglutination test uses rabbit-T on sheep red blood cells.
- Globulin is adsorbed, and the detection method is to measure the property of passively sensitized blood cells aggregating when exposed to patient plasma containing rheumatoid factors. The rheumatoid factor concentration is evaluated based on the plasma dilution ratio at which sensitized blood cells 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 rheumatoid factor was measured using a passive sensitized hemagglutination test (RAHA test, manufactured by Fuji Organ Pharmaceutical Co., Ltd.).

The rheumatism patient plasma used had fibrinogen of 350 g/dll and immunoglobulin G of 1250 g/dll.
g/dlt, albumin is 2.9sg/dJI,
Rheumatoid factor RAHA was 1280.

The results of the adsorption experiment are shown in Table 1. 2-aminopyridine was shown to have high adsorption of fibrinogen and immunoglobulin G, and furthermore, rheumatoid factor was also highly reduced. On the other hand, the decrease in albumin was small, indicating that the selective adsorption of malignant substances was good.

Comparative Example 1 The same adsorption characteristics as in Example 1 were evaluated using Sepharose CL-4B (manufactured by Swedish Pharmacia). The results are shown in Table 1.

Example 2 All experiments were carried out under the same conditions as in Example 1, except that 4-aminopyrazolo(3,4,d)pyrimidine (hereinafter referred to as APP) was used as the binding ligand. The results are shown in Table 1.

Example 3 All experiments were carried out under the same conditions as in Example 1, except that aminopyrazine was used as the binding ligand. The results are shown in Table 1.

Example 4 All experiments were carried out under the same conditions as in Example 1, except that 3-amino-1,2,4-triazine was used as the binding ligand.

The results are shown in Table 1.

Example 5 All experiments were carried out under the same conditions as in Example 1, except that histamine was used as the binding ligand. The results are shown in Table 1.

Example 6 All experiments were carried out under the same conditions as in Example 1, except that 5-aminotetrazole was used as the binding ligand.

The results are shown in Table 1.

Example 7 All experiments were carried out under the same conditions as in Example 1, except that aminoindazole was used as the binding ligand. The results are shown in Table 1.

Example 8 0.5 denier benbelub long fiber [manufactured by Asahi Kasei Kogyo ■]
activated with epichlorohydrin in a conventional manner,
It was fixed using APP. The adsorbent Ig and patient plasma 10
+++ 1 was incubated at 37°C for 3 hours. All other conditions were the same as in Example 1. The results are shown in Table 1.

Comparative Example 2 0.5 denier Benbelb long fiber [manufactured by Asahi Kasei Kogyo ■]
The adsorption properties were evaluated in the same manner as in Example 8.

The results are shown in Table 1.

Example 9 The vinyl compound-based porous synthetic polymer was a ternary copolymer of hydroxyl ethyl methacrylate-ethylene glycol dimethacrylate-glycidyl methacrylate, with an average particle size of 100 μm and an average pore size of 1500 μm.
Epoxy density 1moj! APP was immobilized on % of the polymer. Unreacted epoxide was blocked using glycine.

Use this as an adsorbent for 10sj! An adsorption experiment was conducted using the experimental system shown in FIG.

That is, 30 g of the same patient's plasma (10) is placed in the container (9).
+4, pumped out at a flow rate of 1111 per minute by a pump (11), sent to a malignant substance adsorption device (1), passed through a drip chamber (14) and a sampling port (12), and returned to a container (9). A tube (13) was installed.

After circulating the blood for 3 hours using the above device, the blood was sampled, and fibrinogen, immunoglobulin G1 albumin, and rheumatoid factor in the plasma were measured in the same manner as in Example 1. The results are shown in Table 1.

Comparative Example 3 A similar evaluation was performed using the untreated vinyl compound-based porous synthetic polymer of Example 9. The results are shown in Table 1.

Example 10 Vinyl acetate 100g, ) realyl isocyanurate 24
．． 1g (X -0,20), ethyl acetate 124g,
124g heptane, polyvinyl acetate (degree of polymerization 500)
A homogeneous liquid mixture consisting of 3.1 g and 3.1 g of 2.2'-azobisisobutyronitrile, 1% by weight of polyvinyl alcohol, and 0.05% of sodium dihydrogen phosphate dihydrate.
Weight % and disodium hydrogen phosphate dodecane hydrate 1.5
400+ aj of water dissolved in weight%! After stirring thoroughly, the mixture was heated at 6.5°C for 18 hours and then at 75°C.
The suspension was polymerized by heating and stirring for 5 hours to obtain a granular copolymer. After filtration, washing with water, and extraction with acetone, the copolymer was transesterified in a solution consisting of 46.5 g of caustic soda and 2N methanol at 40° C. for 18 hours. The average particle size of the obtained particles was 150 μm.

When the hydroxyl group density (qOH) was determined using the above method, it was found to be 13
meq/g.

This gel was packed into a stainless steel column with an inner diameter of 7.5 mm and a length of 25 cm, and aqueous solutions of dextran and polyethylene glycol with various molecular weights, albumin,
When a phosphate buffered salt solution of immunoglobulin G1 and immunoglobulin M1β-riboprotein was measured, each sample was eluted in descending order of molecular weight. The exclusion limit molecular weight for dextran was approximately 3 x 10', and the exclusion limit molecular weight for protein was approximately 18 x 10'. Furthermore, when solutions of human γ-globulin and human albumin were passed through an aqueous solution containing 0.3M sodium chloride and 0.1M sodium phosphate as a solvent, almost 100% recovery rate was obtained, and gelation was achieved. The nonspecific adsorption of was very low. All sample measurements were performed at a flow rate of 1+++β/sin.

The gel 50 was then transesterified and thoroughly washed with water.
Suspend cc in 200ml of water, add 3g of cyanogen bromide and stir. p using a 2N aqueous sodium hydroxide solution.
The reaction was carried out for 8 minutes while keeping H at 10 to 11. After the reaction was completed, it was immediately filtered through a glass filter and then washed with water 21 to obtain an activated gel. APP was bound to the activated gel by a conventional method, and excess active groups were blocked with glycine.

After treatment with 6N hydrochloric acid at room temperature for 24 hours, the ligand was released, and the retained amount calculated from the absorption at 26 nm was 115 μmo.
It was l/g. After thoroughly washing the adsorbent with physiological saline, it was dehydrated and used in the experiment.

The adsorption experiment was conducted by mixing 3 volumes of systemic lupus erythematosus patient plasma and 1 volume of adsorbent and incubating the mixture at 37°C for 3 hours. In addition, unactivated Sepharose 4B was used as a control.

Evaluation was performed on fibrinogen, immunoglobulin G, albumin, and immune complexes.

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.

(1) Specimen 1. Onj! into a test tube, add 8% PEG (
Polyethylene glycol, average molecular weight 6000-750
0) to 1.0mj? Add, stir and leave at 4°C for 60 minutes.

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

131+11. (Repeat step 21 two more times to wash away contaminating monomeric immunoglobulin.

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

The value of the patient's plasma used was 270m of fibrinogen.
g/di, immunoglobulin G is 1530 mg/d.
1, albumin was 3.2 g/dl, and immune complexes (measured by immunoglobulin G) were 230 mg/dl.

The results are shown in Table 2.

From Table 2, it is clear that APP bound to the crosslinked copolymer mainly composed of vinyl alcohol units adsorbs fibrinogen, immunoglobulin G, and immune complexes selectively and at a high rate.

Table 2 Example 11 The adsorbent of Example 10 was placed in a container as shown in FIG. 1 to prepare an adsorbent. Two adsorbers were made, one of which was autoclaved. Adsorption experiments were conducted for each adsorber using the experimental system shown in FIG.

That is, myasthenia gravis patient serum (10) is placed in a container (9).
20 mji, and pump (11) pumps 0.5 m/min.
A tube (13) was arranged so that the sample was pumped out at a flow rate of J, sent to the adsorber (1), passed through the drip camper (14) and the sampling port (12), and returned to the container (9).

After circulating the serum for 1 hour using the above device, the serum was sampled and protein components in the serum were measured. Anti-acetylcholine receptor antibody was tested by radioassay using a conventional method.

The results are shown in Table 3, showing an excellent removal rate of immunoglobulin G by APP and a high removal rate of anti-acetylcholine receptor antibodies.

Table 3

[Brief explanation of drawings]

FIG. 1 is a cross-sectional view showing an example of the adsorption device of the present invention, and FIG. 2 is an explanatory diagram of a model experiment in the example. 1.-adsorption remover, 2-cylindrical, 3.3'-filter,
4.4''--backing, 5,5''--integrated liquid inlet/outlet, 6.6'-m-cap, 7.7'--screw, 8-
・Adsorbent, 9-container, 10-...-plasma, 11-pump,
12-sampling port, 13-tube, lt-drip chamber. Figure 1

Claims (2)

[Claims] (1) An adsorbent for malignant substances in body fluids, which has at least one low molecular weight compound having a nitrogen-containing heterocyclic aromatic skeleton on its surface. (2) Adsorption characterized by containing one or more types of adsorbent having at least one type of low molecular weight compound having a nitrogen-containing heterocyclic aromatic skeleton on the surface in a container having a fluid inlet/outlet. Device.