JPS6353971B2 - - Google Patents

Description

Detailed Description of the Invention The present invention is an adsorption system for body fluid purification treatment that specifically adsorbs and removes autoantibodies and/or immune complexes that are thought to be closely related to various diseases caused by the immune system of the body. Regarding materials. As is well known, autoantibodies and/or immune complexes expressed in the blood are associated with autoimmune diseases such as cancer, immunoproliferative syndromes, rheumatoid arthritis, and systemic lupus erythematosus, allergies, and rejection during organ transplants. It is thought that there is a close relationship with the causes or progression of diseases and phenomena related to the body's immune function, such as: Therefore, by specifically adsorbing and removing the above-mentioned autoantibodies and/or immune complexes from body fluid components such as blood and plasma, it is possible to prevent the progression of the above-mentioned diseases, alleviate symptoms, and even cure them. It was hoped that this would be accelerated. Conventionally, adsorbents that can be used for this purpose include adsorbents in which protein A is immobilized on an insoluble carrier, and porous acrylic ester resins (e.g.
Cation exchangers such as XAD-7 (manufactured by Rohm and Haas) or carboxymethyl cellulose have been proposed. However, although protein A is immobilized on an insoluble carrier and has the ability to specifically adsorb autoantibodies and/or immune complexes, it is difficult to secure raw materials because it is a physiologically active protein derived from Staphylococcus aureus. The disadvantage is that the product costs are high, and since the activity is unstable, it is easily deactivated due to handling during immobilization, storage after immobilization, etc. Furthermore, it is difficult to use when in contact with body fluids. In this case, there is a risk of harmful effects caused by elution of Protein A, and in addition, there is a problem in that it is difficult to sterilize while suppressing deactivation. In addition, acrylic ester-based porous resins and carboxymethyl cellulose have the disadvantages of low adsorption capacity and low adsorption specificity.Furthermore, they also adsorb albumin in body fluids, resulting in abnormal osmotic pressure. It was impossible to use it as a safe treatment device. In view of the problems of the adsorbent for autoantibodies and/or immune complexes based on the prior art as described above, an object of the present invention is to provide a material that can be generally used, and which is highly active and capable of absorbing autoantibodies and/or immune complexes. To provide an adsorbent that specifically adsorbs, maintains stable activity, is stable, and can be easily sterilized, and is suitable for body fluid purification or regeneration, and an adsorption device using the same. It is something. As a result of intensive research in line with the above objectives, the present inventors bound various compounds to insoluble carriers and evaluated their binding activity to autoantibodies and immune complexes.It was truly surprising to find that the insoluble carriers The bound hydrophobic compound hardly adsorbs albumin, and highly and specifically binds autoantibodies,
It was discovered that immune complexes can be adsorbed, and the present invention was completed. That is, the present invention provides that a hydrophobic compound having a solubility in physiological saline at 25° C. of 100 mmol/dl or less and/or an oligomer containing the hydrophobic compound with a molecular weight of 1000 or less can be used as an insoluble carrier. The present invention relates to an adsorbent for body fluid purification treatment of autoantibodies and/or immune complexes, characterized in that 0.1 to 30 mg of autoantibodies and/or immune complexes are bound per ml. The adsorbed substances targeted by the present invention are autoantibodies and/or immune complexes, but to explain in more detail, rheumatoid factors, anti-nuclear antibodies, anti-DNA antibodies,
anti-lymphocyte antibody, anti-erythrocyte antibody, anti-platelet antibody,
acetylcholine receptor antibody, serum demyelinating antibody,
Autoantibodies such as anti-thyroglobulin antibodies, anti-microsomal antibodies, and anti-colon antibodies, immunoglobulin derivatives such as reduction products of autoantibodies and chemical modification products, antigens and antigens between autoantibodies or between autoantibodies and other substances, etc. complexes with similar substances, etc. Among these, autoantibodies and immune complexes, which are closely related to the cause and progression of autoimmune diseases, are particularly preferred as adsorbent substances targeted by the present invention. The hydrophobic compound used in the present invention refers to a compound having a solubility in physiological saline of 100 mmol/dl or less (at 25°C), more preferably 30 mmol/dl or less. A compound having a solubility in physiological saline of more than 100 mmol/dl becomes too hydrophilic and has a reduced affinity for autoantibodies and/or immune complexes, resulting in an extremely reduced adsorption capacity. Furthermore, an affinity for albumin, which is more hydrophilic, is generated, and albumin is also non-specifically adsorbed, which is undesirable. Among the hydrophobic compounds, compounds having at least one aromatic ring give particularly favorable results. Aromatic ring means a cyclic compound having aromatic properties, and any of them can be usefully used, but examples include benzene-based aromatic rings such as benzene, naphthalene, and phenanthrene, pyridine, quinoline, acridine, isoquinoline, and phenanthridine. nitrogen-containing 6-membered rings such as indole, carbazole, isoindole, indolizine, porphyrin, 2,3,2',
Nitrogen-containing 5-membered rings such as 3',-pyrrolopyrrole, pyridazine, pyrimidine, sym-triazine, sym-tetrazine, quinazoline, 1,2-naphthyridine,
Polyvalent nitrogen-containing 6-membered rings such as pteridine and phenazine,
Polyvalent nitrogen-containing 5-membered rings such as pyrazole, iminazole, 1,2,4-triazole, 1,2,3-triazole, tetrazole, benziminazole, imidazole, purine, norharman ring, perimidine ring, benzofuran, isobenzofuran, Oxygen-containing aromatic rings such as dibendofuran, sulfur-containing aromatic rings such as bendothiophene, thienothiophene, thiepin, oxazole, isoxazole, 1,
Aromatic rings such as oxygen-containing heteroaromatic rings such as 2,5-oxadiazole and benzoxazole, sulfur-containing heteroaromatic rings such as thiazole, isothiazole, 1,3,4-thiadiazole and benzothiazole, and derivatives thereof. A hydrophobic low-molecular organic compound having at least one of these gives preferable results. Among them, compounds containing an indole ring such as tryptamine give particularly favorable results. This can be interpreted as a result of the hydrophobicity and molecular rigidity of the compound effectively acting on the binding of the compound to the autoantibody and/or immune complex. In addition, as a result of intensive research in search of inexpensive hydrophobic compounds that can be used more safely and practically, the present inventors have found that hydrophobic amino acids and their derivatives can be used in autoantibodies and/or It was discovered that immune complexes can be adsorbed and removed. What are hydrophobic amino acids and their derivatives?
Tanfor d. Nozaki (J.Am.Chem.Soc., 184 4240
(1962), J.Biol.Chem. 246 2211 (1971)) [Tanford, Nozaki (Journal of American
Chemical Society, 184 , 4240 (1962), Journal of Biological Chemistry
246, 2211 (1971)], amino acids and their derivatives with a solubility in physiological saline of 100 mmol/mol or more are 1500 cal/mol or more.
means a compound below dl. For example, lysine, valine, leucine, tyrosine, phenylalanine,
These include isoleucine, tryptophan and its derivatives. Among these hydrophobic amino acids and their derivatives, tryptophan and its derivatives give particularly good results. Furthermore, the amino acid can be used without any particular limitation on the 1 and d conformations. Among hydrophobic compounds, non-dissociable, zwitterionic, and cationic compounds adsorb autoantibodies and/or immune complexes more selectively when bound to insoluble carriers than anionic compounds. preferable. Anionic compounds have an affinity for albumin and adsorption of autoantibodies and/or immune complexes, contrary to what might be expected since albumin molecules are more electronegative than autoantibodies and immune complexes. quantity has decreased. For example, phenylalanine methyl ester, phenylalanine, and tyrosine do not differ greatly in their hydrophobicity, but when their amino groups were covalently bonded to an insoluble carrier and their autoantibody adsorption ability was evaluated, phenylalanine methyl ester > Phenylalanine and tyrosine, and an example of this can be seen in the fact that the adsorption ability of phenylalanine and tyrosine decreases. Furthermore, non-aromatic ring compounds also showed similar results. In particular, a more hydrophilic compound having a sulfonic acid group as an anionic group had more affinity for albumin than autoantibodies and/or immune complexes, and acted as an albumin adsorbent. This is presumed to be due to the interaction between the charge of the compound, albumin, and the charge of the microenvironment within the molecule. In view of the above experimental results, it is believed that the mechanism of action of the present invention is based on the hydrophobic interaction force (Van der Waals force) between the hydrophobic compound bound to the insoluble carrier and the autoantibody and/or immune complex. In addition, it is presumed that the interaction force between charges (Coulomb force) as a long-distance force acts secondarily. The oligomer containing the hydrophobic compound of the present invention has a molecular weight of 1000 or less. This makes it easier to handle during immobilization and to store after immobilization compared to natural polymers such as protein A (molecular weight 42,000). Furthermore, even when the substance is eluted from the insoluble carrier, oligomers with a molecular weight of 10,000 or less are safe and have negligible antigenicity to living organisms, and can be easily sterilized. The oligomer can be obtained by copolymerizing the hydrophobic compound monomer alone or with other compounds. As the hydrophobic compound monomer, for example, a vinyl derivative of a compound containing an indole ring such as tryptamine, or a hydrophobic amino acid such as tryptophan can be used. The insoluble carrier used in the present invention can be either a hydrophilic carrier or a hydrophobic carrier, but when a hydrophobic carrier is used, nonspecific adsorption of albumin to the carrier sometimes occurs, so a hydrophilic carrier is preferable. gives the desired result. The shape of the insoluble carrier may be any known shape such as particulate, fibrous, hollow fiber, or membrane, but from the viewpoint of the amount of hydrophobic compound retained and ease of handling as an adsorbent, particulate and fibrous carriers are preferred. Preferably. The particulate carrier has an average particle size of 150μ to 2500μ.
It is preferable that it is in the range of . Average particle size is JIS-
After classifying in running water using a sieve specified in Z-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. In addition, the particle shape is preferably spherical, but
It is not particularly limited. Average particle size is 2500μ
If it is more than 150μ, the adsorption amount and rate of autoantibodies and/or immune complexes will decrease, and if it is less than 150μ, activation of the coagulation system and blood cell adhesion are likely to occur. Particulate carriers that can be used include agarose-based, dextran-based, cellulose-based, polyacrylamide-based, glass-based, and activated carbon-based carriers, but hydrophilic carriers having a gel structure give good results. Furthermore, known carriers commonly used for immobilized enzymes and affinity chromatography can be used without any particular limitations. Porous particles, in particular porous polymers, can also be used as particulate carriers. The porous polymer particles used in the present invention are capable of immobilizing a hydrophobic compound on their surfaces, and have an average pore diameter in the range of 300 Å to 9000 Å, more preferably 1000 Å to 6000 Å. For the polymer composition, known polymers that can have a porous structure, such as polyamides, polyesters, polyurethanes, and vinyl compound polymers, can be used, but in particular, porous vinyl compounds made hydrophilic with hydrophilic monomers can be used. Polymerized particles give favorable results. The porous structure has an average particle size of 300 Å to 9000 Å
However, if the average pore size is too small, the amount of autoantibodies and/or immune complexes adsorbed is small; if it is too large, the strength of the polymer particles decreases, and Not practical due to reduced surface area. The average pore diameter was measured using a mercury intrusion porosimeter. In this method, mercury is injected into a porous material, and the pore volume is determined from the amount of mercury infiltrated, and the pore diameter is determined from the pressure required for intrusion, and pores of 40 Å or more can be measured. What is the hole of the present invention?
Defined as communicating pores from the surface with a pore diameter of 40 Å or more.
The average pore diameter is dv/d log r, where r is the pore diameter and V is the cumulative pore volume measured with a porosimeter.
The value of r when the value of is the maximum. When using a fibrous carrier, the fiber shape is
0.02 denier to 10 denier, more preferably
Something in the range of 0.1 denier to 5 denier is best. If the fiber diameter is too large, the adsorption amount and adsorption rate of autoantibodies and/or immune complexes will be reduced, and if it is too small, activation of the coagulation system, blood cell adhesion, and clogging are likely to occur. As the fibrous carrier that can be used, generally known fibers such as regenerated cellulose fibers, nylon, acrylic, and polyester can be used. All known methods can be used to immobilize the hydrophobic compound on the surface of the insoluble carrier, such as covalent bonding, ionic bonding, physical adsorption, embedding, or precipitation insolubilization on the polymer surface. Considering its properties, it is preferable to use it after it is fixed and insolubilized by covalent bonding. Therefore, it is possible to use commonly known methods for activating immobilized enzymes, insoluble carriers used in affinity chromatography, and methods for binding with hydrophobic compounds. Furthermore, if necessary, a molecule (spacer) of any length may be introduced between the insoluble carrier and the hydrophobic compound. For example, by reacting and bonding the hydroxyl group of agarose and the isocyanate group on one side of hexamethylene diisocyanate, the remaining isocyanate group and the amino group of the hydrophobic compound,
It can be carried out by reacting and bonding hydroxyl groups, sulfhydryl groups, carboxyl groups, etc. As for the spacer length, a range from no spacer to 20 atoms in the spacer gives particularly preferable results. The amount of hydrophobic compound bound to the insoluble carrier in the present invention ranges from 0.1 mg to 30 mg per ml of the insoluble carrier.
mg range. More preferably 0.5 mg to 15
mg range. If the retained amount is less than 0.1 mg/ml, the adsorbed amount of autoantibodies and/or immune complexes will be extremely reduced, and if it exceeds 30 mg/ml, the adsorption specificity will be decreased, which is not preferable. The autoantibody and/or immune complex adsorption device used in the present invention is one in which the autoantibody and/or immune complex adsorbent described above is filled and held in a container equipped with a body fluid inlet/outlet. It is. In FIG. 1, reference numeral 1 indicates an example of an adsorption device for autoantibodies and/or immune complexes used in the present invention, in which a packing 4 with a filter 3 stretched inside is attached to an opening at one end of a cylinder 2. A cap 6 having a body fluid inlet 5 is screwed into the cylinder 2, and a cap 8 having a body fluid outlet 7 is screwed into the opening at the other end of the cylinder 2 through a packing 4' having a filter 3' inside. A container is formed, and an adsorbent is filled and held between the filters 3 and 3' to form an adsorbent layer 9. The adsorbent layer 9 may be filled with the adsorbent of the present invention alone, or may be mixed or laminated with other adsorbents. As other adsorbents, for example, adsorbents for other malignant substances (antigens) such as DNA, activated carbon having a wide range of adsorption capacity, etc. can be used. As a result, a wide range of clinical effects can be expected due to the synergistic effect of the adsorbent. The appropriate volume of the adsorbent layer 9 is about 50 to 400 ml when used for extracorporeal circulation. When using the device of the present invention for extracorporeal circulation, there are roughly two methods as follows. One method is to separate blood taken from the body into plasma components and blood cell components using a centrifuge or membrane plasma separator, and then pass the plasma components through the device to purify them and separate them into blood cells. One method is to return the blood to the body together with its components, and the other method is to directly pass blood taken from the body through the device for purification. In addition, regarding the passage speed of blood or plasma, since the adsorption efficiency of the adsorbent is extremely 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. Regardless, high passing speeds can be provided. Therefore, a large amount of body fluid can be treated. The method for passing body fluids may be continuous or intermittent, depending on clinical needs or equipment conditions. As described above, the adsorbent and adsorption device of the present invention adsorb and remove autoantibodies and/or immune complexes in body fluids at a high rate and specifically, are extremely compact, and are simple and safe. . It also has the advantage that sterilization operations can be carried out easily and reliably. The present invention is applicable to the general use of purifying and regenerating body fluids such as autologous plasma, and is suitable for safe and reliable treatment of diseases related to the body's immune function, especially autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. Effective in treating diseases. 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 immunoglobulins, autoantibodies, immunoglobulin derivatives, and immunoglobulin complexes, and as a substrate for measuring these. It can also be used extremely effectively as a material. Hereinafter, embodiments of the present invention will be explained in more detail with reference to Examples. Reference example 1 CNBr activated Sepharose 4B (Sweden,
Hydrophobic compounds containing various aromatic rings and hydrophobic amino acids or derivatives thereof were bonded to a compound (manufactured by Pharmacia Co., Ltd., average particle size: 260 μm) by a conventional method, and excess active groups were blocked with ethanolamine. The amount retained is determined by converting the primary amino group of the remaining hydrophobic compound into 4-phenylspiro[furan-2
475-
It was calculated by measuring with 490 nm fluorescence (excitation wave 390 nm) and subtracting the amount of physical adsorption, which was separately tested with unactivated Cepharose 4B. The adsorbent after ethanolamine blocking is
After repeated washing with PH4-0.1M acetic acid buffer and PH8.5-0.1M boric acid buffer, the sample was washed with physiological saline, drained, and used for experiments. In the adsorption experiment, 1 volume of human plasma and 1 volume of adsorbent were mixed and incubated at 37°C for 3 hours. Measure the amount of globulin and albumin after adsorption using an A/G test kit [A/GB test Wako, manufactured by Wako Pure Chemical Industries, Ltd.]
Measured at We also conducted adsorption experiments using unactivated Cefarrows and used them as a controller. The results are shown in Table-1. It is clear from Table 1 that hydrophobic compounds containing at least one aromatic ring specifically and highly adsorb globulin. [Table] Furthermore, using 1 ml of the adsorbent (tryptamine/cephalose 4B) after the adsorption experiment, adsorbed globulins were quantified by immunoglobulin class.
After washing the adsorbent 5 times with ice-cold PBS and dehydrating it,
ml ice-cold 0.1M glycine-hydrochloride buffer (PH2.5)
The adsorbed globulin was eluted. Single radial immunodeficiency (SRID) eluent
Quantification of each globulin class was carried out. SRID is
Plates manufactured by Medical and Biological Research Institute Co., Ltd. were used. The detected amounts for each globulin class were 6 mg for immunoglobulin G, 0.6 mg for immunoglobulin M, and 1.4 mg for immunoglobulin A. This shows that the adsorbent of the present invention adsorbs immunoglobulin with high activity regardless of its class. Reference Example 2 An adsorption experiment was conducted in the same manner as Reference Example 1 except that cyclohexylamine and isoleucine methyl ester were used as hydrophobic compounds.
Good results were obtained: 12%, albumin adsorption rate 3%, globulin adsorption rate 25% with isoleucine methyl ester, and albumin adsorption rate 6%. Reference Example 3 An experiment similar to Reference Example 1 was conducted using a hydrophilic compound instead of a hydrophobic compound. Display the results -
Shown in 2. It is clear from this that hydrophilic compounds are significantly inferior in globulin adsorption ability and adsorption specificity. [Table] Example 1 An experiment similar to Reference Example 1 was conducted except that 4-aminodiphenylamine was used as the hydrophobic compound and human rheumatoid arthritis patient disease was used instead of human plasma. The ability to adsorb and remove rheumatoid factor (autoantibody) and immune complexes, which are malignant substances in rheumatoid arthritis, was measured. Rheumatoid factor was measured by Terrax agglutination test and passive sensitized hemagglutination test. The latex agglutination test is a detection method that measures the tendency of latex particles to agglutinate when patient plasma containing rheumatoid factor is applied to polystyrene latex particles adsorbed with human-gamma-globulin. A plasma dilution series is created and the rheumatoid factor concentration is evaluated at the plasma dilution rate at which latex particles no longer aggregate. Plasma containing a high concentration of rheumatoid factor has a high dilution factor, while plasma with a low concentration has a low dilution factor. The passive sensitization hemagglutination test involves adsorbing rabbit γ-globulin to sheep red blood cells.
The rest is the same as the latex agglutination test. Generally, the passive sensitization hemagglutination test is considered to have higher rheumatoid factor specificity than the latex agglutination test. A dilution series was prepared using glycine saline buffer, and the dilution ratio at which rheumatoid factor was negative in a latex agglutination test was determined. Latex agglutination test
This was done using a kit from the Japan Freeze Drying Research Institute. Similarly, passive sensitization hemagglutination test [RAHA test,
[manufactured by Fuji Organ Pharmaceutical Co., Ltd.] was used for evaluation. In addition, immune complexes were measured using polyethylene glycol and complement hemolysis. This method uses polyethylene glycol to precipitate and separate immune complexes.
The amount of immune complexes is evaluated by reacting with complement in the serum of a healthy human, and measuring the amount of remaining complement by the amount of hemolysis of red blood cells bound to antibodies.
The operating method and conditions for this method are as follows. (1) Pour 0.3ml of sample into a separation tube. 0.2M EDTA50μ
Add and stir. Boric acid buffer (PBS)
Add 50μ and stir. Add 0.1ml of 12.5% PEG (polyethylene glycol) (Mw7500) and stir.
Leave at 4℃ for 90 minutes. (2) Centrifuge at 4°C, 1700g for 10 minutes, and wash the resulting precipitate with 1.0ml of 2.5% PEG. Centrifuge at 1700g for 15 minutes and drain the supernatant. (3) Add 30μ of GVB ⁺⁺ (gelatin veronal buffer containing divalent cations) at 37°C to dissolve the precipitate. Pool healthy human serum 10μ as complement source
Add. React the immune complex and complement at 37°C for 30 min. (4) Add 1.0 ml of 1.5×10 ⁸ /ml EA (antibody-sensitized red blood cells) and soak at 37°C for 60 minutes to promote hemolytic reaction due to residual complement. (5) After the reaction, add 6.5ml of saline at 4℃ and centrifuge.
Measure the absorbance (OD ₄₁₄ ) of the supernatant. (6) Calculate the inhibition rate of hemolysis relative to the control (healthy serum) and use the unit as PEG-cc%. [Rejection rate = Control - Specimen absorbance / Control absorbance x 100] [Removal rate = Untreated - Treated plasma inhibition rate / Untreated plasma inhibition rate x 100] The EA is for complement value measurement manufactured by Japan Lyophilization Institute. Sensitized red blood cells (KW) were used. Further, globulin was measured by the method used in Reference Example 1. Unactivated Cephalose was used as a control in the same manner as in Reference Example 1, and the results are shown in Table 3 in terms of removal rate when the control is set as 0. From Table 3, it is clear that 4-aminodiphenylamine, which is a hydrophobic compound, specifically adsorbs and removes globulin, rheumatoid factor, and immune complexes at a high rate. [Table] Example 2 The same procedure as in Example 1 was carried out except that tryptophan, tryptophan methyl ester, tryptamine, and l-phenylalanine methyl ester were used instead of 4-aminodiphenylamine. The results are shown in Table-4. [Table] From the above table, it is clear that compounds containing aromatic rings specifically and highly adsorb globulin, and also adsorb rheumatoid factors and immune complexes at an even higher rate and specifically. . Comparative Example 1 Using Protein A-Sepharose CL-4B (manufactured by Pharmacia, Sweden), Example 1
conducted a similar experiment. Globulin was quantified using a high performance liquid chromatograph (2 columns 3000SW, manufactured by Toyo Soda). In addition, in order to evaluate storage stability, Protein A-Sepharose CL-
A similar experiment was conducted using 4B suspended in physiological saline and treated at 100°C for 60 minutes. Sepharose CL-4B was used as a control. Table 5 shows the results.
It was shown to. [Table] As is clear from the above table, Protein A-Sepharose has a lower removal rate than indole ring compounds, and when heat treated, isomerism decreases, making it undesirable. Example 3 The same procedure as in Example 2 was carried out except that systemic lupus erythematosus patient plasma was used instead of rheumatism patient plasma. The results are shown in Table-6. [Table] From the above table, it is clear that compounds containing an indole ring adsorb immune complexes and anti-DNA antibodies in the plasma of systemic lupus erythematosus patients at a higher rate and specifically. For the measurement of anti-DNA antibodies, formalin-fixed chicken blood cells were sensitized with DNA.
This method measures the ability of chicken blood cells to agglutinate when exposed to patient plasma containing antibodies. Other procedures were carried out similarly to the latex agglutination test.
Measure the amount of DNA antibody. The measurement was performed using a DNA test kit (Fuji Organ Pharmaceutical Co., Ltd.). Example 4 1:1 oligopeptide of d, l tryptophan, d, l phenylalanine and d, l lysine
It was synthesized by a carboxylic anhydride method using n-hexylamine as an initiator. The ε-amino group of lysine was protected in advance with a carbobenzoxy group, and removed by a conventional method after oligopeptide synthesis. The degree of polymerization (number average) of the produced oligopeptide was determined by fluorescence analysis of the terminal amino group in the same manner as in Reference Example 1. Each oligomer was bound to activated CH-Sepharose 4B by a conventional method, blocked with glycine buffer and washed, and the same experiment as in Example 2 was conducted. In addition, controls were blocked with ethanolamine.
CH-Sepharose 4B. The results are shown in Table-7. [Table] From the above table, it is clear that oligopeptides containing hydrophobic amino acids specifically adsorb and remove globulins, as well as rheumatoid factors and immune complexes. Reference Example 4 Experiments were conducted using terpolymer copolymers of hydroxylethyl methacrylate-ethylene glycol dimethacrylate-glycidyl methacrylate with various average particle sizes and average pore sizes as vinyl compound-based porous polymers. I went. The epoxy density in the polymer is 1 mol%. As a hydrophobic compound, l-tryptophan methyl ester was used and fixed by a conventional method. Unreacted epoxide was blocked using ethanolamine. The amount retained was 4.3 mg/ml resin. As a control, the entire amount of epoxide was blocked with ethanolamine. Similar to Reference Example 1, the sample was thoroughly washed and used for the experiment. Add 5 ml of the adsorbent to the first
A globulin removal device was created by storing it in a container as shown in the figure. Globulin adsorption experiments were conducted using the experimental system shown in FIG. That is, 25 healthy human blood 11 is placed in a container 10.
ml, pumped out at a flow rate of 1 ml per minute by pump 12, sent to immunoglobulin removal device 1, drip chamber 15 and sampling port 13.
tube 1 to be returned to container 10 through
4 was installed. In addition, there is 250U of heparin in the blood.
was added. After blood was circulated for 1 hour using the above device, the blood was sampled and the amount of globulin in the plasma was measured. The results are shown in Table-8. In both cases, there was little decrease in white blood cells and platelets before and after circulation. [Table] Reference Example 5 Using 0.5 denier Bemberg long fiber (manufactured by Asahi Kasei Industries), CNBr was activated by a conventional method, and l-
Tryptophan methyl ester was immobilized. Blocking and washing were carried out as in Reference Example 1. The amount retained is
It was 9.8 mg/g Benherg. An experiment similar to Example 2 was conducted using the adsorbent. The container shown in Figure 1 contains 1 g (Bemberg dry weight) of the adsorbent.
(packing density 0.2 g/ml). Separately, untreated Bemberg was used as a control for comparison. The results are shown in Table-9. Furthermore, there was a relatively small decrease in leukemia and platelet counts before and after circulation. [Table] Example 5 CNBr activated Cepharose 4B (Sweden,
Hydrophobic compounds such as 1-tryptophan, 1-phenylalanine, and adenine were immobilized on the adsorbent (manufactured by Pharmacia) by a conventional method to prepare an adsorbent. Blocking and washing were performed in the same manner as in Reference Example 1, and the amount retained was also measured in the same manner. The retained amounts were 2.3 mg/ml Cepharose (wet column volume) for l-tryptophan, 2.0 mg/ml Cepharose for l-phenylalanine, and 2.1 mg/ml Cepharose for adenine. In the adsorption experiment, 1 volume of myasthenia gravis patient plasma was mixed with 1 volume of drained adsorbent, and the mixture was incubated at 37°C for 3 hours.
It was incubated while shaking. Anti-acetylcholine receptor antibodies (autoantibodies) in the plasma after adsorption were measured by Lindstrom's two-antibody method. Also, unactivated Cefarrows
Adsorption experiments were conducted using 4B as a control. The results are shown in Table 10 as the removal rate when the control is set as 0. From Table 10, Seph Arrows
It is clear that the adsorbent in which l-tryptophan, l-phenylalanine, and adenine are immobilized on 4B selectively and highly adsorbs anti-acetylcholine receptor antibodies, which are autoantibodies for myasthenia gravis. [Table] Examples 6 to 10 and Comparative Examples 2 to 4 Tyrosine (Example 6), tryptophan (Example 7), phenylalanine (Example 8), histidine (Example 9), valine ( Example 10) was used, and DL was also used as a hydrophilic compound.
- An experiment similar to Example 3 was conducted using threonine (Comparative Example 2), hydroxyproline (Comparative Example 3), and proline (Comparative Example 4). The results are shown in Table 11. These results show that an adsorbent bound to a hydrophobic compound with a solubility in physiological saline of 100 mmol/dl or less at 25°C selectively targets globulins, rheumatoid factors, and immune complexes, and
It can be seen that it is adsorbed and removed at a high rate. 【table】

[Brief explanation of the drawing]

FIG. 1 is a cross-sectional view showing one example of the globulin compound adsorption apparatus of the present invention, and FIG. 2 is an explanatory diagram of a model experiment in an example. 1... Autoantibody and/or immune complex removal device, 2... Cylinder, 3,3'... Filter,
4, 4'... Packing, 5... Body fluid inlet, 6
... Cap, 7 ... Body fluid outlet, 8 ... Cap, 9 ... Adsorbent, 10 ... Container, 11 ... Blood, 12 ... Pump, 13 ... Sampling port,
14...Tube, 15...Drippu Chimber.

Claims (1)

[Scope of Claims] 1. A hydrophobic compound whose solubility in physiological saline at 25°C is 100 mmol/dl or less and/or an oligomer containing the hydrophobic compound with a molecular weight of 1000 or less is insoluble in an insoluble carrier. 1ml carrier
An adsorbent for body fluid purification treatment of autoantibodies and/or immune complexes, characterized in that 0.1 to 30 mg of autoantibodies and/or immune complexes are bound to each other. 2. The adsorbent according to claim 1, wherein the hydrophobic compound is a compound containing at least one aromatic ring. 3. The adsorbent according to claim 2, wherein the aromatic ring is an indole ring. 4. The adsorbent according to claim 1, wherein the hydrophobic compound is a hydrophobic amino acid or a derivative thereof. 5. The adsorbent according to claim 4, wherein the hydrophobic amino acid or its derivative is tryptophan or its derivative. 6. The adsorbent according to any one of claims 1 to 5, wherein the insoluble carrier is a hydrophilic carrier. 7 The insoluble carrier is particulate, and the average particle size is
The adsorbent according to any one of claims 1 to 6, having a particle size in the range of 150μ to 2500μ. 8. The adsorbent according to claim 7, wherein the insoluble carrier is a porous polymer particle, and the average pore diameter is in the range of 300 Å to 9000 Å. 9. The adsorbent according to any one of claims 1 to 6, wherein the insoluble carrier is fibrous and has a fiber diameter in the range of 0.02 denier to 10 denier. 10. A hydrophobic compound having a solubility in physiological saline at 25°C of 100 mmol/dl or less and/or an oligomer containing the hydrophobic compound is bound to the insoluble carrier by a covalent bond. adsorbent.