JPS5854960A - Production of immune adsorbing apparatus - 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 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, we manufacture an immunoadsorption device that can safely and efficiently adsorb and remove harmful immunoglobulin and/or immunoglobulin complexes such as autoantibodies found in the blood of patients with diseases related to abnormalities in the immune function of the living body. Regarding the law.

As is well known, immunoglobulins such as autoantibodies and/or their complexes expressed in the blood can cause autoimmune diseases such as cancer, rheumatoid arthritis, and systemic lupus erythematosus, as well as allergies and organ transplant rejection reactions. It is thought that there is a close relationship with the causes and progression of pathological conditions of diseases that are deeply related to immune function.

Recently, plasmapheresis therapy, which removes malignant substances such as antigens, autoantibodies, and immune complexes from patient plasma, and replaces patient plasma with other fresh frozen plasma or albumin preparations, has been used to significantly alleviate symptoms and prevent progression. , and its therapeutic effects have been confirmed.

However, this plasma exchange therapy has problems such as (1) difficulty in obtaining large quantities of fresh frozen plasma or plasma components in a sustained manner to replenish the removed plasma; It has disadvantages such as a high risk of infection, and is not widely used.

In addition, 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 (11). (2) There is no selectivity for removal other than molecular weight, so useful substances in plasma are also removed, (3) Filtration rate decreases due to membrane clogging, and cut-off molecular weight changes, etc. It has the following problems.

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 identified substance is expensive and has antigenicity, so there is a risk of side effects when it is eluted into the blood, and at the same time, its activity is unstable, so It has the disadvantage that it tends to be inactivated due to handling, storage after fixation, etc. A particularly fatal problem is
Due to its instability, it cannot be sterilized to achieve sterility, which is an essential requirement for a therapeutic device. One type of sterilization operation is chemical sterilization, which achieves its purpose by reacting with ethylene oxide, propylene oxide, formalin, etc.
During this reaction, a reaction occurs not only with the bacteria but also with the immobilized substance, and the ability to bind to substances to be bound such as autoantibodies is lost, making it impossible to use chemical sterilization in the past.

In addition, when chemical sterilization with formalin is performed in an aqueous solution, it is necessary to wash out the formalin after sterilization, which incurs the inconvenience of having to wash with water for a long time. Oxide, propylene oxide, etc. have come to be used favorably, and washing with water after sterilization is no longer necessary. However, sterilization under a gas atmosphere requires that the material to be sterilized be dry. The carriers used in conventional adsorbents have the disadvantage that their structure changes during drying, resulting in a decrease in effective surface area and a decrease in adsorption performance.

In order to overcome these problems, the present inventors have conducted extensive research into adsorbents that are simple and safe, can efficiently remove malignant substances such as autoantibodies from patient blood or plasma, and can be chemically sterilized. As a result, by bonding an organic low-molecular compound capable of binding to an adsorbed substance to a crosslinked insoluble carrier having a hydroxyl group, it is possible to remove the target adsorbed substance, and at the same time, it is possible to remove the immobilized substance. It was discovered that the denaturation of the carrier and the change in the surface area of the carrier were small, and chemical sterilization treatment could be easily performed, and the present invention 8A was completed.

This made it possible to perform extracorporeal circulation safely and overcome the difficulties in actual clinical application.

That is, in the present invention, a crosslinked insoluble carrier having a hydroxyl group is bonded with an organic low-molecular compound containing a functional moiety that can bind to an adsorbed substance, and after drying, a solution to be treated can be distributed. This is a method for producing an immunoadsorption device, which is characterized in that the immunoadsorption device is filled into a container, or after being filled into a container, a drying process is performed, and further a chemical sterilization process is performed.

In the present invention, the functional site capable of binding to an adsorbed substance is:
It refers to a chemical structure that interacts with an adsorbed substance by van der Waals force or/and Coulomb force and has the function of adsorbing the adsorbed substance to an adsorbent.

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, particulate or fibrous materials are preferred.

When a fibrous carrier is used, the fiber diameter 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 amount and rate of adsorption will decrease, and if the fiber diameter is too small, clogging is likely to occur.

The particulate 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 69/9°, preferably 1
．． A material having a specific surface area in the range of 0 to s, o y7y and 5 g/f or more can be suitably used. Water holding capacity is defined as the amount of saline that can be contained within the carrier per unit dry carrier when the carrier is equilibrated with saline. When the water retention amount is greater than 6 f/l, the mechanical strength of the carrier decreases, and destruction of particles occurs during manufacturing, sterilization, transportation, etc., which is preferable. If the water retention amount is less than 0.5 f/f, the pore volume and surface area of the carrier particles will decrease, resulting in a decrease in adsorption capacity, which is undesirable. The surface area is measured by the BET adsorption method, and if it is less than 5/2, the adsorption performance is low (unfavorable). The carrier must have an average particle diameter of 25 to 2500 pm, or more, 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 to maintain high adsorption performance. Preferably, those having a diameter of 40 to 1000Q are suitably used, but in order to allow blood to circulate, it is desirable that the diameter is 400μ or more.

The carrier used in the present invention suppresses interaction with plasma proteins, blood cell components, etc., and at the same time contains a large amount of an organic low-molecular compound having a site capable of binding to immunoglobulin and/or A, □ immunoglobulin complex. For retention, a carrier having a hydroxyl group density of 5 meq/f 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 the amount of the crosslinkable compound to the compound having a hydroxyl group is 10% by weight or more, more preferably 10% by weight.
A suitable carrier is obtained by containing .about.50% by weight. If the amount of the crosslinking compound is small, the physical strength of the carrier will decrease and it will not be able to withstand chemical sterilization treatment, which is preferable. If there is too much crosslinking compound, the hydroxyl group density will decrease,
Unfavorable interactions increase and, at the same time, water retention decreases, making it unsuitable.

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, a vinylene thread binder, and a vinyl or allyl crosslinking agent. Examples of the carrier in this case include crosslinked vinyl polymers such as crosslinked polyvinyl alcohol, crosslinked 2-(l')oxyethyl acrylate, and crosslinked i1z-hydroxyethyl methacrylate.

Examples of crosslinking agents include allyl compounds such as triallyl cyanurate and triallyl cyanurate, di(meth)acrylates such as ethylene glycol dimethacrylate and diethylene glycol dimethacrylate, butanediol divinyl ether, and diethylene glycol divinyl ether. , polyvinyl ethers such as tetravinylglyoxal, polyallyl ethers such as diarylidene pentaerythritol, and tetraallyloxyethane.

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. Also, vinyl ester, if necessary.
It can be made by copolymerizing comonomers such as vinyl ether.

In the case of vinyl or vinylene copolymers, polyvinyl alcohol is obtained by copolymerizing a vinyl ester of carboxylic acid with a vinyl compound (allyl compound) having an incyanurate ring, and then cross-decomposing the copolymer. Triallyl in cyanurate crosslinkers provide particularly good supports in terms of mechanical strength, hardness, pore stability, and chemical properties.

Methods for binding organic low-molecular compounds to carriers include covalent bonding,
Any known method such as ionic bonding, physical adsorption, embedding, or precipitation and insolubilization on the polymer surface can be used, but in view of the elution properties of the bound substance, it is preferable to hold and insolubilize 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 the rhodanated cyanide method, the epichlorohydrin method, the biseboxyte method, the norodanated triazine method, the bromoacetylbromide method, the ethyl chloroformate method, and the 1,1'-carbonyldiimidazole method. I can do it. The activation method of the present invention includes a 7-mino group, a hydroxyl group, a carboxyl group,
It is not limited to the above examples as long as it can undergo a substitution and/or addition reaction with a nucleophilic reactive group having active hydrogen such as a thiol group.

The organic low molecular weight compound used in the present invention is desirably a compound that does not have antigenicity in the body when released from a carrier during clinical practice. It is preferable that the molecular weight is 1000 or less. By using organic low-molecular-weight compounds, handling such as preparation, drying, and sterilization of adsorbents is easier than when using conventional high-molecular-weight physiologically active substances.
An immunoadsorption device can be manufactured without impairing its adsorption properties.

Examples of organic low molecular compounds include organic low molecular compounds such as aliphatic, aromatic, and alicyclic compounds, amino acids, peptides, glycopeptides, sugars, oligosaccharides, polysaccharides, and nucleic acid bases such as purine bases and pyrimidine bases. Examples of diseases and malignant substances such as nucleic acid-related compounds such as nucleosides and nucleotides include rhifmatoid factor, which is detected at a high rate in the plasma of patients with rheumatoid arthritis, and immune complexes that have a solubility in saline of 100. mmol/dl (at 25° C.), preferably less than S G mmol/dl, and polymers containing such compounds can be used.

Among the hydrophobic compounds, compounds having at least one aromatic ring give particularly favorable results. Aromatic ring means a compound having an aromatic property, and any of them can be usefully used, including benzene-based aromatic rings such as benzene, naphthalene, and phenanthrene, pyridine, quinoline, acridine, and isoquinoline. , 6-membered nitrogen-containing ring of phenanthrene, indole, carbazole, isoindole, indolizine, porphyrin, 2, 5, 2',
! 5-membered nitrogen-containing rings such as '-pyrrolovirol, pyridazine, pyrimidine, sym-triazine, sym-tetrazine, quinazoline, 1,5-s7tyridine, pteridine,
Polyvalent nitrogen-containing 6-membered rings such as phenazine, pyrazole, imidazole, 1,2.4-)lyazole, 1,2. → polyvalent nitrogen-containing 5-membered rings such as lyazole, tetrazole, penziminazole, imidazole, purine, norhal! Reward,
Oxygen-containing aromatic rings such as perimidine, benzofuran, inobenzofuran, and dibenzofuran, sulfur-containing aromatic rings such as benzothiophene, chenothiophene, and chepine, oxazole, isoindole, 1,2.5-oxadiazole, penz Oxygen-containing heteroaromatics such as oquinazole, thiazole, isothiazole, 1,3,4-thiadiazole, benzothiazole and other hydrophobic compounds containing at least one aromatic ring and its derivatives, such as sulfur-containing aromatic rings such as benzothiazole, etc. Molecular organic compounds give favorable results. Among them, compounds containing an indole ring such as tryptamine can be particularly preferably used. Furthermore, hydrophobic amino acids and their derivatives 4 can be preferably used.

Hydrophobic amino acids and their derivatives are defined by Tanford
.

Nozaki (J, Am, Chem, 8oc,, 1
84, 4240 (1942), J. Biol, C.
hαξ, 246.2211 (1?A1)] [Tan 7 Odonozaki (Journal of American Chemical
Society, 1 (et al. 4240 (1?42), Journal of Biological Chemistry.

4, 2211 (1971)), amino acids and their derivatives with a hydrophobicity of 1500 m/mot or more, and compounds with a solubility in physiological saline of 100 mm/17 mol/dj or less. Examples include lysine, valine, leucine, tyrosine, phenylalanine, inleucine, tryptophan and derivatives thereof. Among these hydrophobic amino acids and their derivatives, tryptophan and its derivatives give 1IIIK 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 makes it easier to handle during immobilization and to store after immobilization compared to natural polymers such as protein A (molecular weight 4200.0). Furthermore, even when the substance is eluted from the insoluble carrier, polymers with a molecular weight of 10,000 or less have negligible antigenicity to living organisms and are safe, and can be sterilized into containers. The polymer can be obtained by copolymerizing the hydrophobic compound monomer alone or with other compounds. Hydrophobic compounds! As -, for example, vinyl derivatives of indole-containing compounds such as Tributa 57, and hydrophobic amino acids such as tryptophan can be used.

In addition, anti-nuclear antibodies and anti-DN, which are frequently found in the blood of patients with autoimmune diseases such as systemic lupus erythematosus,
For the removal of A antibodies and their immune complexes, adenine, cytosine, guanine, uracil, thymine, hypoxanthine, xanthine, adenosine, cytidine, guanosy/! Nucleosides such as lysine, inosine, xanthosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxyuridine, thyminedine, adenosine 5'-phosphate, cytidine 5'-phosphate, guanosine 5'-phosphate, inosine 5'-phosphate ,
Nucleotides such as uridine 5'-phosphate, deoxyliposes of these riboses, diphosphoric acids, triphosphoric acids, and nucleotides with phosphates attached at the 9, 2', and 3' positions Nucleotides bound to sugars such as glucose or mannose, oligonucleotides with 10 or less nucleotides, nicotinamide adenine dinucleotide (NAD), fucibin adenine dinucleotide (FAD)
), coenzyme A, coenzyme 1ltt, 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-basement membrane antibodies, anti-thyroid microsomal antibodies, anti-microseam antibodies, anti-colon antibodies, etc. For adsorption of antibodies, monosaccharides and their derivatives that constitute glycolipids, glycoproteins, proteoglycans, etc. on the surface of body tissues and cells are used. Monosaccharides include di-acetyl-D-glucosamine with a pyranose or furanose structure, hexosamine in the N-acetyl-D-glucosamine group, D-galactose, D-mannose, D-
Hexoses such as glucose, L-fucose, L-rhamnose and other deoxyhexoses, D-xylose, D
Pentoses such as -79binose and sialic acids such as N-acetyl quinic dinitric acid and N-glycolylneuraminic acid are used. Any of the α type and /II 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. 4IK dimer to dodecamer give favorable results.

The present invention is not limited to the above-mentioned examples, but exhibits its effects in a wide range of organic low-molecular compounds.

In the immunoadsorption device of the present invention, the adsorbent as described above is subjected to a drying process and then filled into a container through which the liquid to be treated can flow, or alternatively, after being filled into a container, the adsorbent is subjected to a drying process, and then It has been chemically sterilized.

For the drying process, known drying methods such as freeze drying, hot air drying, vacuum drying, etc. can be used. In order to shorten the time required for drying, a method of drying after replacing with a low boiling point organic solvent such as acetone or ethanol can also be preferably used.

FIG. 1 shows an embodiment of the immunoadsorber of the present invention, in which a filter (2
Fluid inlet (4) through backing (3) covered with
Screw-fit the cap (5) with re)L, cylinder (1)
At the other end opening of the and (the adsorbent layer is filled and held in the gap between the two) (
7).

The adsorbent layer (7) may be filled with the adsorbent of the present invention alone, or mixed or stacked with other adsorbents. The volume of the adsorbent layer (7) is 5 when used for extracorporeal circulation.
Approximately 0 to 400- is an additional tooth.

As the chemical sterilization method, the methods and conditions specified in the "Japanese Pharmacopoeia Law" can be used.

To illustrate the case of ethylene oxide gas sterilization, ethylene oxide gas concentration 11o o w9/As temperature 4
It can be carried out under conditions such as 0° C., humidity of 53 to 601 i, and time of 4 hours.

When using the adsorbent of the present invention for extracorporeal evaporation, there are two methods as shown below by Ohji. 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. One method is to purify the blood and then return it to the body together with blood cell components.The other method is to directly pass the blood that has been eliminated from the body through the absorber of the present invention and purify it.

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 very efficiently remove immunoglobulins such as autoantigens and autoantibodies in body fluids, or their complexes, and can be used clinically safely.

Hereinafter, embodiments of the present invention will be described with reference to Examples. Example 1 2-hydroxymethacrylate 100f1 ethylene/glycol dimethacrylate 25f, glycidyl methacrylate 12t1 ethyl acetate 124t.

Heptane 124 f, polyvinyl acetate (degree of polymerization 5OO)
A homogeneous mixture of 3.1 t and 5.12 t of 2,2'-azobisisobutyronitrile, 1 weight of polyvinyl alcohol, 0.05 weight of sodium dihydrogen phosphate dihydrate, and 5.12 weight of 2,2'-azobisisobutyronitrile, 4,005 g of water in which 1.5 weight of sodium dodecahydrate was dissolved was placed in a flask, thoroughly stirred, and heated and stirred at 60°C for 18 hours and then at 75°C for 5 hours to perform suspension polymerization. A copolymer was obtained. After filtration and washing with water, classification was performed to obtain carriers with an average particle size of 180 μm.

Well, the water retention capacity of the obtained particulate carrier was 4.5 f/f, and its specific surface area was 10 g/l. The exclusion limit molecular weight of the standard globular protein measured using phosphate buffered saline was approximately 1.5 million.

The organic low-molecular compounds shown in Table 1 were adjusted to pH 9.5 using a sodium hydroxide + 79 μM aqueous solution tube, dissolved in IM sodium bicarbonate, added to 10 m of carrier, and incubated at 25°C for 16 hours. After shaking and reaction, unreacted activated functional groups were blocked with glycine, and then the F separation and washing with physiological saline were repeated to obtain an immunoadsorbent.

The skeleton immune adsorbent was washed and replaced with acetone, and then dried in a hot air dryer at 60°C for 6 hours. The obtained dried immunoadsorbent was filled into an 8-yd container as shown in Fig. 1 to form an immunoadsorption device, and then sterilized using an ethylene oxide gas sterilizer.
Ethylene oxide gas concentration 11o any/l, temperature [
40℃, humidity Sos.

Chemical sterilization treatment was performed for 4 hours.

Adsorption experiments were conducted using the model experimental system shown in FIG. 2, using immunoadsorbers before and after sterilization.

That is, 15 times of systemic lupus erythematosus patient plasma (9) is placed in a container (8), pumped out at a flow rate of 0.5 times per minute by the pump shaft, and sent to the immunoadsorber I), drip chamber a, and sampling. The tube a4 is arranged so that it is returned to the container (8) through the mouth.

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 antibody method.

Immune complexes were determined by measuring complement consumption in polyethylene glycol precipitates.

The results are shown in Table 1.

Example 2 Various organic low molecular weight 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.

Example 3 28 μm of L-trypto-7ammethyl ester was added to a carrier with an average particle size of 450 μm prepared in the same manner as in Example 1.
Using an immunoadsorbent device filled with adsorbent bound with /- and sterilized with ethylene oxide, blood from a patient with chronic rheumatoid arthritis (25-) was recirculated for 3 hours in the experimental system shown in Figure 2. The reduction rate of red blood cells and white blood cells after accumulation was less than 4%, and the reduction rate of platelets was less than 71'55%. Rheumatoid factor (latex agglutination) was reduced by one-eighth.

[Brief explanation of drawings]

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'...Backing, 4.4'...
...Body fluid inlet/outlet, 5.5'... Cap, 6
．． 6'...Screw, 7...Adsorbent Fig. 1 4 Fig. 2 4

Claims (1)

[Claims] An organic low-molecular compound containing a functional site capable of binding to an adsorbed substance is bonded to a cross-linked insoluble carrier having a hydroxyl group, and after drying, the carrier is filled into a container through which the liquid to be treated can flow. , or a method for manufacturing an immunoadsorption device, which is characterized in that after being filled into a container, a drying process is performed, and further a chemical sterilization process is performed.