JPS638789B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for manufacturing an immunoadsorption device that removes high molecular weight substances dissolved in blood and purifies blood. More specifically, the method for manufacturing an immunoadsorption device that can safely and efficiently adsorb and remove immunoglobulins and/or their complexes such as harmful autoantibodies found in the blood of patients with diseases related to abnormalities in the immune function of the living body. Regarding. As is well known, immunoglobulins such as autoantibodies and/or their complexes expressed in blood,
It is believed that there is a close relationship with the causes and progression of conditions of autoimmune diseases such as cancer, rheumatoid arthritis, and systemic lupus erythematosus, as well as diseases that are deeply related to the immune system of the body, such as allergies and rejection reactions during organ transplants. It is considered. Recently, plasmapheresis therapy, which removes malignant substances such as antigens, autoantibodies, and immune complexes from patient plasma, and replaces the patient's plasma with other fresh frozen plasma or albumin preparations, has been used to significantly alleviate symptoms. Progress prevention or curative effects have been confirmed. However, this plasmapheresis therapy has two drawbacks: (1) difficulty in obtaining large quantities of fresh frozen plasma or plasma components in a sustained manner to replenish the removed plasma;
(2) Because it uses someone else's blood plasma, it has the disadvantage of being at high risk of infection with hepatitis viruses, etc., and cannot be widely used. Additionally, to remove malignant substances from patient plasma, there is a method using an ultrafiltration membrane, which has the advantage of not requiring supplementation of plasma components; however, (1) it cannot be separated cleanly due to molecular weight; (2) There is no selectivity for removal other than molecular weight, so useful substances in plasma are also removed. (3) Problems such as decreased overspeed due to membrane clogging and fluctuations in cut-off molecular weight. have. Furthermore, a method has been proposed in which biopolymers such as protein A, DNA, the first component of complement, denatured biopolymers, synthetic nucleic acid polymers, etc. are immobilized on a carrier to remove autoantibodies and their complexes. There is. However, in these methods, the immobilized substance is expensive and has antigenicity, so there is a risk of side effects when it elutes into the blood, and at the same time, its activity is unstable, so It had the disadvantage that it was easily deactivated due to handling, storage after fixation, etc. A particularly critical problem is that, due to its instability, sterilization operations cannot be performed to achieve sterility, which is an essential requirement for a therapeutic device. One type of sterilization operation is radiation sterilization, and its purpose is achieved by irradiation with gamma rays or electron beams; however, during the irradiation, the immobilized substance is deactivated, causing binding to substances such as autoantibodies. Conventionally, radiation sterilization could not be used because decomposition and dissociation of the immobilized substance occur at the same time as the loss of its ability to produce antigenic decomposition products. In order to overcome these problems, the present inventors
As a result of extensive research into an adsorbent that is simple and safe, can efficiently remove malignant substances such as autoantibodies from patient blood or plasma, and can be sterilized by radiation, we have discovered that it is a crosslinked polymer compound with hydroxyl groups that can be used for storage. By covalently bonding an organic low-molecular compound that can bind to an adsorbed substance to a carrier with a water content of 0.5 to 6 g/g, it is possible to remove the target adsorbed substance, and at the same time, radiation irradiation is possible. The inventors have discovered that sterilization can be easily performed with little denaturation of the immobilized substance and decrease in strength of the carrier, and have completed the present invention. This made it possible to perform extracorporeal circulation safely and overcome the difficulties in actual clinical application. That is, the present invention is a crosslinked polymer compound having a hydroxyl group and having a water retention capacity of 0.5 to 6 g/g, by covalently bonding an organic low molecular compound containing a functional site capable of bonding with an adsorbed substance. After combining, fill it into a container that allows the flow of the liquid to be treated,
This is a method for manufacturing an immunoadsorption device, which further comprises performing radiation sterilization treatment. In the present invention, a functional site capable of binding to an adsorbed substance is a chemical structure that interacts with the adsorbed substance through Van der Waals force or/and Coulomb force and has the function of adsorbing the adsorbed substance to an adsorbent. means. The cross-linked insoluble carrier having a hydroxyl group used in the present invention can be in any known shape such as particulate, fibrous, hollow fiber, or membrane, but the amount of organic low-molecular compound retained and the ease of production can be used. From the viewpoint of ease of handling as an adsorbent, particles or fibers are preferred. When using a fibrous carrier, the fiber diameter is
0.02 denier to 10 denier, more preferably
It is preferable to use one in the range of 0.1 denier to 5 denier. If the fiber diameter is too large, the amount and rate of adsorption will decrease, and if the fiber diameter is too small, clogging is likely to occur. The carrier used in the present invention is a crosslinked polymer compound that is insoluble in water and has a hydroxyl group, and has a water retention capacity of 0.5 to 6 g/g, more preferably 1.0 to 5.0 g/g. can be used suitably. The water retention capacity is defined as the amount of saline that can be contained in the carrier per unit dry carrier when the carrier is equilibrated with physiological saline. Water retention capacity is 6g/g
The larger the size, the lower the mechanical strength of the carrier;
Particles may be destroyed during manufacturing, sterilization, transportation, etc., which is undesirable. If the water retention amount is less than 0.5 g/g, the pore volume and surface area of the carrier particles will decrease, resulting in a decrease in adsorption capacity, which is not preferable.
The carrier must have an average particle diameter of 25 to 2500μ, in order to be able to stably flow liquids with high viscosity and high solute concentration, such as body fluids such as blood and plasma, at high flow rates for long periods of time, and at the same time maintain high adsorption performance. More preferably 40~
A diameter of 1000μ is preferably used, but a diameter of 400μ or more is desirable for blood circulation. The carrier used in the present invention suppresses interaction with plasma proteins, blood cell components, etc., and at the same time retains a large amount of an organic low-molecular compound having a site capable of binding to immunoglobulins and/or immunoglobulin complexes. Therefore, a carrier having a hydroxyl group density of 5 meq/g or more is preferably used. The carrier used in the present invention is composed of a compound having a hydroxyl group and a crosslinkable compound, and by containing the crosslinkable compound in an amount of 5% by weight or more, more preferably 5 to 50% by weight, based on the compound having a hydroxyl group,
A suitable carrier is obtained. If the amount of the crosslinkable compound is small, the physical strength of the carrier will decrease and it will not be able to withstand radiation sterilization treatment, which is not preferable. Too much crosslinking compound reduces the hydroxyl group density and increases unfavorable interactions;
The amount of water retained decreases and is not suitable. The carrier can be synthesized by polymerizing a monomer having a hydroxyl group or by introducing a hydroxyl group through a chemical reaction of a polymer. It is also possible to synthesize both in combination. As the polymerization method, known polymerization methods such as condensation polymerization, radical polymerization, ionic polymerization, and ring-opening polymerization can be used. The crosslinking agent is preferably introduced by copolymerization during polymerization. Alternatively, it may be introduced through a chemical reaction of the polymer (polymer and crosslinking agent). For example, it can be made by copolymerizing a vinyl monomer or vinylene monomer with a vinyl or allyl crosslinking agent. In this case, the carriers include crosslinked polyvinyl alcohol, crosslinked 2-hydroxyethyl acrylate, crosslinked 2-hydroxyethyl acrylate, and crosslinked 2-hydroxyethyl acrylate.
- Crosslinked vinyl polymers such as hydroxyethyl methacrylate can be exemplified. Examples of crosslinking agents include allyl compounds such as triallyl isocyanurate and triallyl cyanurate, di(meth)acrylates such as ethylene glycol dimethacrylate and diethylene glycol dimethacrylate, butanediol divinyl ether, diethylene glycol divinyl ether, and tetravinyl. Polyvinyl ethers such as glyoxal, polyallyl ethers such as diarylidene pentaerythrite and tetraallyloxyethane, and glycidyl acrylates such as glycidyl methacrylate can be used. In particular, triallylisocyanurate units are preferred from the viewpoints of mechanical strength, hardness, micropore structure, and chemical properties. If necessary, copolymerized comonomers such as vinyl esters and vinyl ethers may also be used. In the case of vinyl-based or vinylene-based copolymers, polyvinyl alcohol is obtained by copolymerizing a vinyl ester of carboxylic acid with a vinyl compound having an isocyanurate ring (allyl compound), and then cross-hydrolyzing the copolymer. Allyl isocyanurate crosslinkers provide particularly good supports in terms of mechanical strength, hardness, pore stability, and chemical properties. All known methods such as covalent bonding, ionic bonding, physical adsorption, embedding, and insolubilization by precipitation on the polymer surface can be used to bond the organic low-molecular compound to the carrier. It is preferable to use it by holding and insolubilizing it by covalent bonding. For this purpose, known methods for activating and binding carriers commonly used in immobilized enzymes, affinity chromatography, and affinity chromatography can be used. Examples of activation methods include cyanogen halide method, epichlorohydrin method, bisepoxide method,
Examples include the halogenated triazine method, the bromoacetyl bromide method, the ethyl chloroformate method, and the 1,1'-carbonyldiimidazole method. The activation method of the present invention only needs to be able to perform a substitution and/or addition reaction with a nucleophilic reactive group having active hydrogen such as a cyano group, a hydroxyl group, a carboxyl group, or a thiol group of the organic low-molecular compound, and is not limited to the above-mentioned examples. It's not something you can do. The organic low-molecular compound used in the present invention is preferably a compound that does not have antigenicity in the body when released from a carrier during clinical use, and has a molecular weight of 10,000 or less, particularly a polypeptide compound with a molecular weight of 1000 or less. It is preferable that there be. By using organic low-molecular compounds, compared to the case of using conventional high-molecular-weight physiologically active substances, the immunoadsorption device can be used without impairing its adsorption properties during handling such as preparation, drying, and sterilization of the adsorbent. It can be manufactured. Examples of organic low molecular compounds include aliphatic, aromatic,
Organic low molecular compounds such as alicyclic compounds, amino acids, peptides, glycopeptides, sugars, oligosaccharides, polysaccharides, nucleobases such as purine bases and pyrimidine bases, and nucleic acid-related compounds such as nucleosides and nucleotides are used. be able to. To give examples by disease and malignant substance, for the adsorption of rheumatoid factors and immune complexes that are detected at a high rate in the plasma of patients with rheumatoid arthritis, a solubility in physiological saline of 100 mmol/d (25°C) or less is more preferable. A hydrophobic organic compound having a concentration of 30 mmol/d or less and a polymer containing the compound can be used. 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, polyvalent nitrogen-containing 6-membered rings such as pyridazine, pyrimidine, sym-triazine, sym-tetrazine, quinazoline, 1,5-naphthyridine, buteridine, phenazine, pyrazole, iminazole, Polyvalent nitrogen-containing five-membered rings such as 1,2,4-triazole, 1,2,3-triazole, tetrazole, benziminazole, imidazole, purine, norharman ring, perimidine ring, benzofuran, isobenzofuran, dibenzofuran, etc. Oxygen aromatic ring, sulfur-containing aromatic ring such as benzothiophene, thienothiophene, thievine, oxazole, isoxazole, 1,
Aromatic rings such as nitrogen-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-weight organic compound having at least one of these gives preferable results. Among them, compounds containing an indole ring such as tryptamine can be particularly preferably used. Hydrophobic amino acids and their derivatives can also be preferably used. What are hydrophobic amino acids and their derivatives?
Tanford, Nozaki [J.Am.Chem.Soc., 184 , 4240
(1962), J.Biol.Chem., 246 , 2211 (1971)]
Amino acids and their derivatives with a hydrophobicity scale of 1500 cal/mol or more as defined by Journal of Biological Chemistry, 246 , 2211 (1971), and compounds with a solubility in physiological saline of 100 mmol/d or less. do. Examples include lysine, valine, leucine, tyrosine, phenylalanine, isoleucine, tryptophan and derivatives thereof. Among these hydrophobic amino acids and their derivatives, tryptophan and its derivatives give particularly good results. The polymer containing a hydrophobic compound is a polymer having a molecular weight of 10,000 or less, more preferably a polymer having a molecular weight of 1000 or less. This allows protein A (molecular weight
Compared to natural polymers such as 42000), it is easier to handle during immobilization and to store after immobilization. Furthermore, even when the substance is eluted from the insoluble carrier, polymers with a molecular weight of 10,000 or less are safe and have negligible antigenicity to living organisms.
Sterilization operations can also be easily performed. The polymer 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 tributamine, or a hydrophobic amino acid such as tryptophan can be used. In addition, adenine, cytosine, guanine, uracil, thymine, Bases such as hypoxanthine and xanthine, nucleosides such as adenosine, cytidine, guanosine, uridine, isosine, xanthosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxyuridine, and thyminedine, adenosine 5'-phosphate, cytidine
5′-phosphate, guanosine 5′-phosphate, isosine
5′-phosphoric acid, uridine 5′-phosphate, and deoxyribose derived from these riboses, as well as diphosphoric acid, triphosphoric acid, and those with phosphoric acid at the 2′ and 3′ positions. Nucleotides such as, nucleotides bound to sugars such as glucose or mannose, oligonucleotides with 10 or less nucleotides, nicotinamide adenine dinucleotide (NAD), frapin adenine dinucleotide (FAD), coenzyme A, coenzyme B ₁₂ , etc. nucleotide coenzymes, and derivatives of all these can be used. In addition, anti-lymphocyte antibodies, anti-platelet antibodies, anti-erythrocyte antibodies, anti-acetylcholine receptor antibodies, anti-smooth muscle antibodies, anti-epidermal intercellular antibodies, anti-basement membrane antibodies, anti-proteoglycan antibodies, anti-collagen antibodies, anti-gastric factor antibodies, For adsorption of autoantibodies such as anti-thyroid microsomal antibodies, anti-microsomal antibodies, and anti-colon antibodies, glycolipids in body tissues and the surface of somatic cells,
Monosaccharides and their derivatives that make up glycoproteins, proteoglycans, etc. As a monosaccharide,
N-acetyl-D-glucosamine, N-acetyl-D- with pyranose or furanose structure
Hexosamines such as galactosamine, hexoses such as D-galactose, D-mannose, and D-glucose, and 6- such as L-fucose and L-rhamnose.
Pentoses such as deoxyhexose, D-xylose, and D-arabinose, and sialic acids such as N-acetylneuraminic acid and N-glycolylneuraminic acid are used. Both α-type and β-type isomers can be used without particular limitation. As the oligosaccharide, a single oligomer or two or more types of oligomers of the above monosaccharides can be used, regardless of whether they are linear or branched. In particular, dimers to dodecamers give good results. The present invention is not limited to the above examples, but exhibits its effects in a wide range of organic low-molecular compounds. The immunoadsorption device of the present invention is one in which the above-mentioned adsorbent is filled and held in a container through which a liquid to be treated can flow, and sterilized by radiation. FIG. 1 shows an embodiment of the immunoadsorption device of the present invention, in which a cap 5 having a fluid inlet 4 is screwed into an opening at one end of a cylinder 1 through a packing 3 with a filter 2 stretched inside. A cap 5' having a fluid outlet 4' is screwed into the opening at the other end of the cylinder 1 through a packing 3' with a filter 2' stretched inside. The adsorbent layer 7 is formed by filling and holding the adsorbent in the gap.
It is formed by forming. The adsorbent layer 7 may be filled with the adsorbent of the present invention alone, or may be mixed or laminated with other adsorbents. The appropriate volume of the adsorbent layer 7 is about 50 to 400 ml when used for extracorporeal circulation. As a method for radiation sterilization, electron beams or gamma ray irradiation, which are highly penetrating radiation, are used, but gamma ray irradiation is more preferable from the viewpoint of operability of sterilization treatment. The larger the irradiation dose, the greater the sterilization effect, but it also has a greater effect on the carrier and the container of the immunoadsorption device, which is undesirable as it causes a decrease in strength. 2.5MR (Mega Roentgen) ~ 4.5MR
It is appropriate to select an irradiation dose within the range of (mega Roentgen). When the device of the present invention is used 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 of the present invention for purification. One method is to return the blood 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 device of the present invention for purification. The method for passing body fluids may be continuous or intermittent depending on clinical needs or the installation status of the equipment. As described above, the adsorption device of the present invention can remove immunoglobulins such as autoantigens and autoantibodies in body fluids or their complexes very efficiently and can be used clinically with safety. Hereinafter, embodiments of the present invention will be explained in more detail with reference to Examples. Example 1 100g of 2-hydroxyethyl methacrylate,
25g of ethylene glycol dimethacrylate, 12g of glycidyl methacrylate, 124g of ethyl acetate,
124g heptane, polyvinyl acetate (degree of polymerization 500)
A homogeneous mixture of 3.1 g and 3.1 g of 2,2'-azobisisobutyronitrile, 1% by weight of polyvinyl alcohol, 0.05% by weight of sodium dihydrogen phosphate dihydrate, and disodium hydrogen phosphate dodecahydrate. Pour 400 ml of water containing 1.5% by weight of the compound into a flask, stir thoroughly, and then heat at 60°C for 18 hours.
Suspension polymerization was carried out by heating and stirring at 75° C. for 5 hours to obtain a particulate copolymer. After washing with water, classification was performed to obtain a carrier with an average particle size of 180μ. The water holding capacity of the obtained particulate carrier was 4.5 g/g, and the specific surface area was 10 m ² /g. The exclusion limit molecular weight of the standard globular protein measured using phosphate buffered saline was approximately 1.5 million. The organic low molecular weight compounds shown in Table 1 were dissolved in 0.1 M sodium hydrogen carbonate adjusted to pH 9.5 using an aqueous sodium hydroxide solution, added to 10 ml of carrier, and heated at 25°C.
After shaking and reacting for 16 hours, unreacted activated functional groups were blocked with glycine, and washing with physiological saline was repeated to obtain an immunoadsorbent. The adsorbent was filled into an 8 ml container as shown in Fig. 1 to prepare an immunoadsorbent, and then sterilized with gamma rays at an irradiation dose of 3.5 megaroentgen. From the observation results of the adsorbent and internal solution in the adsorber using an optical microscope, no destruction such as rupture or cracking of the adsorbent due to the sterilization treatment was observed. Adsorption experiments were conducted using the model experimental system shown in Figure 2, using immunoadsorbers before and after sterilization. That is, 15 ml of systemic lupus erythematosus patient's plasma 9 is put into the container 8, and the pump 10 pumps the blood at 0.5 ml per minute.
ml at a flow rate and sent to the immunoadsorber 11, drip chamber 12 and sampling port 13.
tube 14 to be returned to container 8 through
was installed. After circulating plasma for 3 hours using the above experimental system, the plasma was sampled, and anti-DNA antibodies, which are autoantibodies in the plasma, were measured by a hemagglutination method, and anti-nuclear antibodies were measured by an enzyme-linked antibody method. Immune complexes were determined by measuring complement consumption in polyethylene glycol precipitates. The results are shown in Table 1.

[Table] Example 2 Various organic low-molecular compounds shown in Table 2 were retained on a carrier prepared in the same manner as in Example 1 to prepare an immunoadsorbent. An adsorption experiment similar to that in Example 1 was conducted using chronic rheumatism patient plasma. Rheumatoid factor, an autoantibody, was measured by latex agglutination method and Waller-Rose method.
Immune complexes were measured in the same manner as in Example 1. The results are shown in Table 2.

【table】

[Table] Example 3 Average particle size 450 μ produced in the same manner as Example 1
L-tryptophan methyl ester is added to the carrier.
In the experimental system shown in Figure 2, 25 ml of blood from a chronic rheumatoid patient was recirculated for 3 hours using an immunoadsorbent device filled with adsorbent bound to 28 μmol/ml and sterilized by gamma rays. The reduction rate of red blood cells and white blood cells after circulation is less than 6%, and the reduction rate of platelets is
It was below 38%. Rheumatoid factor (latex agglutination) was reduced by one-eighth.

[Brief explanation of the drawing]

FIG. 1 is a cross-sectional view showing one example of the immunoadsorption device of the present invention, and FIG. 2 is an explanatory diagram of a model experiment using the immunoadsorption device of the present invention. 1...Cylinder, 2, 2'...Filter, 3,
3'...Packing, 4,4'...Body fluid inlet/outlet,
5, 5'... Cap, 6, 6'... Screw, 7...
Adsorbent material, 8... Container, 9... Patient plasma or blood, 10... Pump, 11... Immunoadsorber, 12
...Drip yambar, 13...Sampling port, 14...Tube.

Claims (1)

[Claims] 1 A crosslinked polymer compound having a hydroxyl group,
After covalently bonding an organic low-molecular compound containing a functional site capable of binding with an adsorbed substance to a carrier with a water retention capacity of 0.5 to 6 g/g, the carrier is filled into a container through which the liquid to be treated can flow. A method for manufacturing an immunoadsorption device, which further comprises performing radiation sterilization treatment.