JPS5917356A - Adsorbing material and apparatus for purifying body liquid having anti-thrombotic property - 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 an adsorbent for body fluid purification having antithrombotic properties and an adsorption device using the adsorbent. For more details,
Malignant substances such as autoantibodies and immune complexes that are expressed in the blood and other body fluids of patients in diseases related to abnormalities in the body's immune function and are thought to be closely related to the cause or progression of the disease. The present invention relates to an adsorbent that adsorbs and removes blood and has excellent antithrombotic properties, and an adsorption device using the adsorbent.

Conventionally, as an adsorbent for body fluid purification treatment, one coated with activated carbon lubrication or activated carbon k k% aqueous polymer has been used as an artificial liver mainly for liver diseases. However, in many diseases, various malignant substances that are closely related to the cause or progression of the disease have come to be known, and there has been an increasing demand for selectively removing these malignant substances from body fluids. At present, adsorbents based on activated carbon have low adsorption selectivity and cannot meet this standard.

Furthermore, in body fluid purification treatments using adsorbents, blood coagulation induced by contact between adsorbents such as activated carbon, which are foreign substances to living organisms, and body fluids such as blood has been a major problem. In order to prevent this blood coagulation, attempts have been made to coat activated carbon with hydrophilic polymers, but sufficient antithrombotic properties have not been achieved, and the anticoagulant heparin is supplied into the blood. The current situation is that blood clotting is prevented2.

In this case, since a large amount of heparin is used, there are problems such as side effects such as bleeding in some cases.

As a result of intensive research in response to the demand for selective adsorption and removal of malignant substances, the present inventors have found that the carrier retains a special substance that selectively interacts chemically with the adsorbed substance through chemical bonds. He discovered various adsorbents and filed patent applications for them (Japanese Patent Applications 1971-7152, 1982-76776, 1982-159444, and 1982-18923).

The present invention was arrived at as a result of intensive research into the above-mentioned blood coagulation problems in connection with the previous invention.

That is, in view of the problem of blood coagulation due to contact between the adsorbent and blood based on the prior art as described above, the present inventors
As a result of extensive research into adsorbents that selectively adsorb malignant substances in body fluids and do not cause blood to coagulate when they come into contact with them, we have discovered that the insoluble carrier contains functional sites that can bind to adsorbed substances. If it is a compound that combines both an organic low-molecular compound (hereinafter referred to as an organic low-molecular ligand) and an antithrombotic agent, and the ligand can be bound through some kind of bond, the shape , can be used regardless of composition. Preferred shapes include particles and fibers from the viewpoint of ease of handling. In addition, the composition includes natural polymers such as cellulose, agarose, and dextran, polyvinyl alcohol, acrylamide, and polyacrylic acid ester.
Examples include synthetic polymers such as polystyrene, and inorganic materials such as glass and silica, but the material is not particularly limited to those exemplified here.

The organic low-molecular ligand used in the present invention is based on the previous patent application (%Application No. 56-71.52, Japanese Patent Application No. 56-71).
6776, patent application 1984-15944, patent application 1982-18
923) Ic includes the disclosed organic low molecular compounds.

That is, examples thereof include hydrophobic low-molecular compounds, low-molecular compounds containing sugar phosphate as a constituent, monosaccharides, and oligosaccharides.

These ligands bind to autoantibodies through hydrophobic interactions, hydrogen bonds, etc., and adsorb and remove the autoantibodies.

The organic low-molecular compound referred to in the present invention refers to a molecular weight of 10,000 or less;
More preferably, it is an organic compound with a molecular weight of 1000 or less. The hydrophobic low-molecular compound as used in the present invention refers to a low-molecular compound having a solubility in physiological saline of 100 mmo/, /d/ or less (at 25° C.), more preferably 30 mmot/dl or less. Among these hydrophobic compounds, compounds containing hydrophobic amino acids and purine bases or pyrimidine bases as constituent elements are practically preferred because they safely and efficiently adsorb malignant substances.

Examples of hydrophobic amino acids include lysine, valine, leucine, tyrosine, phenylalanine, isoleucine,
Examples include tryptophan, especially tryptophan,
Aromatic amino acids such as phenylalanine and tyrosine give good results. Substances containing purine bases and pyrimidine bases as constituents include adenine, cytosine,
Examples include bases such as guanine, uracil, and thymine, and derivatives thereof.

In the present invention, the low molecular weight compounds containing sugar phosphate as a component include tetroses such as erythrose and threose;
Aldopentoses such as arabinose, xylose, lyxose, and ribose; ketohendoses such as xylulose, pentulose, and refulose; aldohexoses such as galactose, glucose, talose, and mannose;
Ketohendos such as sorbose, tagatose, psicose, fructose, sosamines such as N-acetyl-glucosamine, aldoheptose, ketoheptose, ketooctose, ketononose, etc. 11L'1A14.2
-deoxypentose, 6-deoxyhexose, 2-
Deoxyhexose, 2,6-sibuoxyhexose,
Deoxy sugars such as 3,6-sibuoxyhexose, sugar alcohol anhydrides, acidic sugars such as uronic acid, ketoaldonic acid, and ascorbic acid, sugar methyl ethers, branched sugars, amino sugars, and mono- or di-orthophosphorus of sialic acids. These include acid esters, mono- or divirophosphate esters, triphosphates, etc. In the present invention, monosaccharides and oligosaccharides refer to body tissues and cells, surface glycolipids, glycoproteins,
Monosaccharides and their derivatives that make up proteoglycans, etc. Monosaccharides include 7'CN-acetyl-D-glucosamine, N
-7 Hexosamines such as ceteru-D galactosamine, D-
Galactose, D-mannose, D-glucose, hexose, L-fucose, L-rhamnose, 6-deoxyhexose, D-xylose, D-arabinose, pentose, N-acetylneuraminic acid, N-glycolyl Sialic acids such as neuraminic 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.

Examples of antithrombotic agents used in the present invention include streptokinase, urokinase, plasmin, purinase, fibrinolytic activators such as synthetic fibrinolytic activators, heparin, anticoagulants such as coumarin derivatives, and boulostaglandin derivatives. Known antithrombotic agents such as platelet inhibitors can be used.

In the present invention, the organic small molecule ligand and the antithrombotic agent can be immobilized on the surface of the insoluble carrier by any known method such as covalent bonding, ionic bonding, physical adsorption, embedding, or insolubilization by precipitation on the carrier surface. However, considering the dissolution properties of these compounds, it is preferable to use them after being covalently bonded, immobilized, or insolubilized. Therefore, it is possible to use commonly known methods for activating immobilized enzymes, insoluble carriers used in affinity chromatography, and methods for binding to ligands. Also, if necessary, a molecule (spacer) of arbitrary length can be added between the insoluble carrier and the ligand.
It is also possible to introduce and use it.

Examples of functional groups that can form a covalent bond include hydroxyl group, amine group, carboxyl group, epoxy group, chloroformyl group, azide group, formyl group, bromoacetyl group, incyanato group, cyano group, acid chloride group, and acid anhydride group. However, if these functional groups have low reactivity and it is difficult to directly form a covalent bond with a ligand, highly reactive functional groups can be used using known activation methods. The converted edge may form a covalent bond with the ligand. For example, the hydroxyl group of the carrier can be prepared using the rhodanide cyanide method.
Activation can be performed by the epichlorohydrin method, bisepoxide method, halogenated triazine method, bromoacetyl bromide method, ethyl chloroformate method, carbonyldiimidazole method, etc. These functional groups are organic small molecule ligands as well as amine groups, hydroxyl groups, carboxyl groups, and antithrombotic agents.
It can react with nucleophilic reactive groups such as thiol groups to form covalent bonds.

Binding of an organic low-molecular-weight ligand and an antithrombotic agent to an insoluble carrier can be achieved by introducing both into the reaction system at the same time, or by reacting the antithrombotic agent after reacting with the organic low-molecular-weight ligand. let

Alternatively, the reaction may be performed in the opposite order. Further, when the insoluble carrier has an ion exchange group, the organic low molecular weight ligand and the antithrombotic agent can be immobilized by ionic bonding.

Examples of ion exchange groups include anionic sulfonic acid groups, carboxyl groups, cationic amine groups, and quaternary ammonium groups. As a method of binding an organic small molecule ligand and an antithrombotic agent to an insoluble carrier, one method is to covalently bond one to the other,
It is also possible to use two different methods together, such as using ionic bonding as the other.

The adsorption device for body fluid purification of the present invention is made by filling and holding the above-described adsorbent for body fluid purification in a container provided with a body fluid inlet and outlet, as shown in the drawings. Body fluids such as blood are passed through this device continuously or intermittently to selectively adsorb and remove malignant substances. In the drawing, (1) is a cylinder with a cap (5) and a screw (6) having a body fluid inlet/outlet (4) via a parakink (3) with a filter (2) placed inside the opening at both ends of the cylinder.
The gaps between the filters at both ends are filled with adsorbent (7).

The adsorbent and adsorption device of the present invention have excellent ability to remove malignant substances and excellent antithrombotic properties, and can be applied to general usage for purifying and regenerating body fluids such as autologous blood, and are useful for improving biological immune function. It is effective in treating related diseases.

Embodiments of the present invention will be explained in more detail with reference to Examples below.

Example 1 CNBr activated Sepharose 4B (manufactured by Pharmacia)
Take 10di and add hepari/sodium 1. Ov and t-
A pH 9.5 carbonate buffer containing 0.129 tryptophan 20- was added, and the mixture was reacted for 2 hours at room temperature and then overnight at 4°C. Excess active groups were blocked by shaking at room temperature for 2 hours with a 0.1 mot/4 solution of tris(hydroxymethyl)aminomethane. After thorough washing with carbonate buffer, acetic acid buffer, and physiological saline, the sample was subjected to an evaluation test.

The adsorption performance of the adsorbent thus obtained was evaluated using plasma from a human patient with chronic arthritic arthritis.

That is, 5 volumes of patient plasma and 1 volume of adsorbent were mixed and heated at 37°C.
After incubation for 2 hours, the adsorbent and plasma were separated, and the adsorbed plasma was analyzed to calculate the adsorbed amount of rheumatoid factor, which is a malignant factor in rheumatoid arthritis. The method for evaluating rheumatoid factor is passive sensitization hemagglutination test (RAHA).
The RAHA titer of the patient's plasma, which was positive at 620-fold dilution, became negative at 40-fold after adsorption. Furthermore, in order to evaluate the anti-blood coagulability of this adsorbent, 3 volumes of fresh human blood and 1 volume of the adsorbent were mixed and left for 2 hours, but the blood did not coagulate. For comparison, we conducted the same blood contact experiment as above using an adsorbent prepared using a heparin-free solution under the same other conditions, and the blood coagulated.

As described above, the adsorbent of the present invention in which t-)ributophane is bound to an organic low-molecular-weight ligand and heparin as an antithrombotic agent was found to have excellent antithrombotic properties and to be effective in removing lymphomatosis factor. .

Example 2 0.5 denier Penpelg long fiber (manufactured by Asahi Kasei Industries) 1
2 was used to activate CNBr by a conventional method, and β-(p
-aminophenyl)-ethylated Ichida-galactose 0
．． 05 mol/l and 20 ml of physiological saline containing 1200 units/- of urokinase were added, and the mixture was reacted and fixed at 7°C for 24 hours to obtain an adsorbent. Blocking cleaning is Example 1
I followed the instructions.

The fibrinolytic activity of the obtained adsorbent was determined by adding 10 dK of human fibrinogen aqueous solution (5 m9/me) and a physiological saline solution of human plasma thrombin (25 units/mj) to a fibrin plate with a thickness of about 2 all. Measured at That is, the adsorbent was placed on a fibrin plate, and 37
After standing at ℃ for 24 hours, fibrinolytic activity was measured by the degree of dissolution of the fibrin film around the adsorbent piece. As a result, β-(p-aminophenyl)-ethylated D-(t)-
An adsorbent containing galactose and urokinase dissolved fibrin membranes.

In the adsorption experiment, 6 volumes of systemic lupus erythematosus patient plasma and 1 volume of adsorbent were mixed and incubated for 2 hours. The amount of anti-T cell antibodies in the plasma after adsorption was measured by a fluorescent antibody method using normal T cells as a detector.

The measurement operations and conditions are as follows.

(1) Lymphocytes in peripheral blood of healthy people by specific gravity centrifugation 1) 1
1t [t] and sort T cells using the nylon wool column method.

(2) Add 0.1 - of the plasma after the adsorption experiment to 106 isolated healthy human T cells, and treat at 4°C for 1 hour.

(3) After washing, apply FITC-labeled anti-human immunoglobulin rabbit antiserum to count the number of fluorescence-positive cells.

As a result, the patient's plasma before adsorption was 25 positive, but after treatment with this adsorbent, it was less than 5 positive.

From the above, the adsorbent of the present invention had excellent antithrombotic properties and was effective in removing anti-T cell antibodies.

Example 3 Vinyl acetate 100f, ) diallyl isocyanurate 41
f1 Ethyl acetate 100 f, polyvinyl acetate (polymerization degree 5
00) A homogeneous liquid mixture consisting of 3.6 tons of 7F and 2.2'-azobisisobutyronitrile, 1% by weight of polyvinyl alcohol, and 0.05% of sodium dihydrogen phosphate dihydrate.
Weight % and disodium hydrogen phosphate dodecahydrate 1.5
Put 0.4 t of water dissolved at 65% by weight into a flask.
Suspension polymerization was carried out by stirring at 75° C. for 18 hours and then at 75° C. for 5 hours to obtain a granular copolymer. This polymer was hydrolyzed with caustic soda to obtain a polyvinyl alcohol crosslinked polymer. The average particle size of this polymer was 150 μm. Using this polymer as a carrier, an adsorbent was prepared in the following manner. That is, the dried carrier 152 is mixed with dimethyl sulfoxide 180 and epichlorohydrin 120
-, a 30% aqueous sodium hydroxide solution 15- was added thereto, and the mixture was stirred at 30°C for 75 hours to obtain an epoxy activated carrier. The activated carrier 107! Take it,
Adenine total 0.02 s mat/L, urokinase 1
Add 20 g of physiological saline containing 200 units/- and heat at 7°C.
The reaction was carried out for 24 hours to obtain an adsorbent on which adenine and urokinase were immobilized. Blocking and washing were performed according to Example 1.

The fibrinolytic activity of the obtained adsorbent was measured using the same method as in Example 2. As a result, this adsorbent completely dissolved fibrils/membranes. The adsorption experiment was carried out by mixing 3 volumes of systemic lupus erythematosus patient plasma and 1 volume of adsorbent and incubating at 67°C for 2 hours*.

The anti-DNA antibody titer is determined to be positive or negative based on the presence or absence of an agglutination reaction (at room temperature) that occurs when formalin-fixed chicken blood cells sensitized with DNAt are mixed with serial dilutions of treated or untreated patient plasma. I asked for it. For the measurement, Kiratra from ``rDNA Test'' (manufactured by Fuji Organ Pharmaceutical Co., Ltd.) was used.

The antinuclear antibody titer is determined by dropping a serially diluted sample (-antibody) onto a slide glass smeared with cells, performing an antigen-antibody reaction, and measuring peroxidase-labeled anti-human immunoglobulin antibody (
A secondary antibody) was added dropwise, and the color reaction of the enzyme was observed using an optical microscope. For the measurement, "Enzyme ANA Test" (manufactured by Medical Biology Research Co., Ltd.) Kiratra was used. The results showed that both anti-DNA antibody and anti-nuclear antibody were positive in the patient's plasma, but negative in the plasma treated with the adsorbent.

As described above, the adsorbent of the present invention has excellent antithrombotic properties and anti-D
Example 4 A carrier prepared in the same manner as that used in Example 3, which is effective in removing NA antibodies and antinuclear antibodies, was activated with CNBr by a conventional method and used. Activated carrier 10-
pH 7,6 containing heparin sodium, 0.59 and deoxyribose 3,5-syric acid 0.29 i!
20 grams of phosphoric acid buffer was added, and the mixture was reacted day and night at room temperature to obtain an adsorbent retaining the above-mentioned ligand. Blocking and washing were performed according to Example 1.

In an adsorption experiment using the obtained adsorbent, adsorption of antinuclear antibodies was measured in the same manner as in Example 3 using systemic lupus erythematosus patient plasma, and the results showed that the adsorption of antinuclear antibodies was positive in the patient's plasma, but negative after treatment with the adsorbent. Met. Further, a contact experiment with blood was conducted in the same manner as in Example 1 for anticoagulant properties, but the blood did not coagulate.

As described above, it was confirmed that this adsorbent exhibited excellent antithrombotic properties and was effective in adsorbing antinuclear antibodies.

Example 5 A polyvinyl alcohol carrier prepared in the same manner as that used in Example 3 was used and activated with epichlorohydrin in the same manner as in Example 3. Take the activated carrier 1076 and add heparin sodium 0.59. Add 20 g of pH 9 carbonate buffer containing t-phenylalanine o, 2rt,
The reaction was carried out at 0° C. for 16 hours to obtain an adsorbent retaining the above-mentioned ligand.

Blocking was performed according to Example 1. The adsorption experiment of the obtained adsorbent was evaluated in the same manner as in Example 1 using human rheumatoid arthritis patient plasma. As a result, the RAHA titer was positive in patient plasma diluted 320 times, but after adsorption it was 4 times positive.
It was negative at 0x dilution.

Furthermore, anticoagulant properties were tested in the same manner as in Example 1, and as a result, blood did not coagulate.

As described above, this adsorbent was found to have excellent antithrombotic properties and to be effective in adsorbing lymphomatosis factors.

[Brief explanation of the drawing]

The drawing is a sectional view showing an example of the adsorption device for body fluid purification having antithrombotic properties of the present invention.

Claims (2)

[Claims] (1) An adsorbent for body fluid purification characterized in that an organic low-molecular compound containing a functional site capable of binding to an adsorbed substance and an antithrombotic agent are bound to an insoluble carrier. (2) An adsorbent in which an antithrombotic agent and an organic low-molecular-weight compound containing a functional site capable of binding to an adsorbed substance are bound to an insoluble carrier are housed in a container having a fluid inlet and outlet. Adsorption device for body fluid purification 1゜ characterized by