JPS59168858A - Adsorptive cleaner of sugar-containing substance - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

Detailed Description of the Invention The present invention is for extracorporeal circulation therapy that adsorbs and purifies sugar-containing substances from body fluids, such as sugars, glycoproteins, and glycolipids, which are thought to be closely related to various pathological conditions. Regarding adsorption purifiers.

With recent advances in carbohydrate chemistry and complex carbohydrate chemistry, it is becoming clear that sugar-containing substances in the blood play important biological functions. In particular, acute phase reactants during inflammation, immunosuppressive substances for malignant swelling, mediators of immune responses (lymphin, monokine), etc. are closely related to the causes and progression of inflammation, malignant swelling, and immune diseases. It was expected that the drug would be absorbed and removed from body fluids, prevent the progression of the above-mentioned diseases, alleviate symptoms, and accelerate healing.

Conventionally, plasma exchange therapy has been used for this purpose,
Attempts have been made to non-specifically remove the increased sugar-containing substances. However, there are restrictions on widespread use due to the need to secure replacement fluids and the risk of hepatitis virus infection.

In addition, attempts have been made to adsorb and purify using glass beads with a controlled size, but the adsorption capacity is low, non-specific adsorption of substances other than the target object, and furthermore, activation of coagulation factors, etc. It has the following disadvantages.

The present invention has been made in view of the problems based on the prior art as described above.
The present invention aims to provide a safe adsorption purifier for extracorporeal circulation treatment of sugar-containing substances that has high adsorption capacity and adsorption selectivity.

The present inventors have completed the present invention as a result of intensive research in accordance with the above objectives.

That is, the present invention provides a sugar-containing substance for extracorporeal circulation therapy, characterized in that an adsorbent binding a lectin or/and its fragment to an insoluble carrier is housed in a container having a fluid inlet/outlet. This relates to an adsorption purifier.

The target substances targeted by the present invention are sugar-containing substances such as glycoproteins, mucopolysaccharides, ribopolysaccharides, peptidoglycans, their metabolic products, and sugar-containing complexes thereof, which exist or are mixed in body fluids. Intercellular transmitters such as immunoglobulin, immunoglobulin light chain, immune complexes, interferon, interleukin, lymphin, and monokine in immune diseases; clotting factors such as fibrinoken and prothrombin in blood diseases; hemoglobin, transferrin, etc. Glucose protein, α-fetoprotein in malignant swelling, carcinoembryonic glycoproteins such as carcinoembryonic antigen (CEA), α1-acetate F
Glycoprotein, (χ2-macroglobulin, α1-
Immunosuppressive glycoproteins such as antitrypsin, O-reactive glycoproteins in inflammation, sulfated sugars in stones, Tam-phosphorus (Ta+nm-Hors),
Examples include glycoproteins, hyaluronic acid, chondroitin sulfate, mucopolysaccharides such as heparin, lipopolysaccharides such as lipid A, and peptidoglycan of bacterial cell walls.

In the present invention, lectin refers to proteins or glycoproteins extracted from plant seeds, animal liver cells, microorganisms, etc., which aggregate cells and precipitate complex carbohydrates. Specific examples include urexone lectin (Ulex 1) extracted from gorse, and peanut agglutinin (PN) extracted from peanuts.
A), Bean lectin (SB) extracted from soybean
A), red kidney bean lectin (PHA) extracted from kidney beans, jack bean lectin (OonA) extracted from sea peas, hawk leido mitogen (PWM) extracted from American wildflower dow,
Wheat germ lectin (WG) extracted from wheat germ
A) etc. can be exemplified. The present invention is not limited to the above examples.

In the present invention, a fragment refers to a chemical or enzymatic degradation product of the lectin. Either a method of binding a mixture of decomposition products to an insoluble carrier or a method of separating a specific component using a separation method such as gel filtration and binding it to an insoluble carrier can be effectively used.

The insoluble carrier used in the present invention may be either a hydrophilic carrier or a hydrophobic carrier as long as it can bind lectin and/or its fragments. However, when a hydrophobic carrier is used, albumin may be added to the carrier. Hydrophilic carriers give more favorable results since non-specific adsorption of molecules may occur.

The shape of the insoluble carrier may be any known shape such as particulate, fibrous, hollow fiber, or membrane.
In view of the retained amount of fragments thereof and ease of handling as an adsorbent, particles or fibers are preferred.

As a particulate carrier, the average particle size is 25μ to 2500μ.
The range is preferably 50, more preferably 50.
The thickness is between 1500μ and 1500μ. The average particle size is J Is -Z
After classification in running water using a sieve specified in -8801, the particle size of all classes is determined to be the median value between the upper limit particle size and the lower limit particle size of each class, and the average particle size is calculated as the weight average. Also,
The particle shape is preferably spherical, but is not particularly limited. If the average particle size is 25007z or more, the adsorption amount and adsorption rate of the sugar-containing compound will decrease, and if it is 25μ or less, the pressure loss will be large and it will be difficult to pass liquid under normal conditions. Particulate carriers that can be used (7) include agarose-based, dextran-based, cellulose-based, polyacrylamide-based, glass-based, silica-based, and activated carbon-based carriers, but hydrophilic carriers with a glue structure have yielded good results. Furthermore, immobilized enzymes and known carriers commonly used in affinity chromatography can be used without any particular limitations.

As particulate carriers, it is also possible to use porous particles, in particular porous polymers. The porous polymer particles used in the present invention are capable of immobilizing lectins and/or fragments thereof on their surfaces.

Polymer composition is polyamide type, polyester type, rf
l) Known polymers with a porous structure such as urethane and vinyl compound polymers can be used, but vinyl compound porous polymer particles made hydrophilic with a hydrophilic monomer are particularly preferred. give.

As a carrier material, a copolymer having a hydroxyl group gives favorable results, and a copolymer having vinyl alcohol units as a main component gives extremely good results as a carrier. Due to its hydrophilic nature, the carrier made of a crosslinked copolymer mainly composed of vinyl alcohol units has a small interaction with solutes such as proteins in plasma, and reduces nonspecific adsorption to a minimum. It also has extremely excellent properties such as having little interaction with the complement system and coagulation system in plasma. In terms of physical properties, it has heat resistance, enables heat sterilization, and has excellent physical and mechanical strength, which is a characteristic of synthetic polymers.

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 adsorption amount and adsorption rate of the sugar-containing compound will be reduced, and if it is too small, the pressure loss will be too large and it will be difficult to pass liquid through the fiber under normal conditions. As the fibrous carrier that can be used, generally known fibers such as recycled reulose fibers, nylon, acrylic, polyester, and polyvinyl alcohol can be used.

Any known method can be used to immobilize lectin and/or its fragments on the surface of an insoluble carrier, such as covalent bonding, ionic bonding, physical adsorption, embedding, or insolubilization by precipitation on the polymer surface. Considering this, it is preferable to use it after being fixed and insolubilized by covalent bonding. For this purpose, it is possible to use an immobilized enzyme, a known method for activating an insoluble short form, and a method for binding commonly used in affinity chromatography. Furthermore, if necessary, a molecule of arbitrary length (subaser) can be introduced between the insoluble carrier and the lectin and/or its fragment. For example, the hydroxyl group of agarose and the incyanate group on one side of hexamethylene diinocyanate are reacted and bonded, and the remaining incyanate group is reacted with the amino group, hydroxyl group, sulfhydryl group, calzexyl group, etc. of the lectin and/or its fragment. , each combination can be carried out.

In the present invention, the amount of lectin and/or its fragment bound to the carrier is 0.05 to 5
A range of 0■ gives good results.

More preferably, it is in the range of 0.5 to 30 Tng.

Although the method for activating a carrier and binding lectin and/or its fragments has been described above in detail as method 7 for producing the adsorbent used in the present invention, the present invention is not limited thereto.

For example, lectin and/or
Alternatively, a method in which the fragments thereof are bonded and then polymerized (copolymerized), a method in which a crosslinking agent bonded with lectin and/or its fragments is used during post-crosslinking, etc. can also be used. Furthermore, there is also a method of coating an insoluble substance with a polymer capable of binding lectin and/or its fragment and then binding the lectin and/or its fragment, or a method of binding a lectin and/or its fragment to a polymer and then coating the insoluble substance. Can be used. At this time, the coat polymer may be post-crosslinked if necessary. It is also possible to adopt a method in which the lectin and/or its fragment is activated and then bound to a carrier.

That is, the present invention exhibits its effects as long as lectin and/or its fragments are bound to the adsorbent, and is not dependent on the manufacturing method.

The adsorption purifier of the present invention is made by filling and holding the adsorbent for sugar-containing compounds as described above in a container equipped with an inlet and outlet for body fluids.

In FIG. 1, reference numeral 1 indicates an example of the sugar-containing compound adsorption purifier of the present invention, in which a body fluid inlet 5 is connected to an opening at one end of a cylinder 2 through a backing 4 with a filter 3 stretched inside. A cap 6 having a cylindrical shape is fitted with a screw, and a filter 3' is placed inside the opening at the other end of the cylinder 2.
A cap 8 having a body fluid lead-out lower part is screw-fitted through the cap 8 to form a container, and an adsorbent layer 9 is formed by filling and holding an adsorbent in the gap between the filters 3 and 3'.

The adsorbent layer 9 may be filled with the adsorbent of the present invention entirely alone, or may be mixed or laminated with other adsorbents. As other adsorbents, for example, activated carbon having a wide range of adsorption capacities can be used. A wide range of clinical effects can be expected due to the synergistic effect of the adsorbent. When used for extracorporeal circulation, the appropriate volume of the adsorbent layer 9 is about 50 to 400.

When the adsorption purifier of the present invention is used in extracorporeal circulation, blood taken from the body is separated into plasma components and blood cell components using a centrifuge or a model plasma separator, and then the plasma components are removed by the adsorption. A method can be used in which the blood is passed through a purifier, purified, and then returned to the body together with blood cell components.

The method for passing body fluids may be either continuous or intermittent, depending on clinical needs or equipment conditions.

As described above, the adsorption purifier of the present invention selectively adsorbs and purifies sugar-containing compounds in body fluids at a high rate, has little non-specific adsorption, is extremely compact, and is safe.

The present invention is applicable to the general use of purifying and regenerating body fluids such as autologous blood and autologous plasma, and is applicable to the safe and reliable treatment of acute and chronic inflammation and diseases related to the body's immune function.
It is particularly effective in treating malignant swelling, collagen disease, autoimmune disease, blood disease, etc., and in removing foreign contaminants that are harmful to living organisms.

Example 1 Jack bean lectin (concanavalin A) extracted from sea cucumber was mixed with bromycean-activated Sepharose 4B (
(manufactured by Pharmacia) by a conventional method to prepare an adsorbent. The amount retained was 2.1 μ/me Sepharose. The adsorbent is placed in a 2-
was packed into a column.

An adsorption experiment apparatus was constructed as shown in Figure 2, and an adsorption experiment was conducted. Put 6 d of serum obtained from a gastric cancer patient into a container 11 and add 0.0 ml of serum.
It was circulated for 1 hour at a flow rate of 2 ml/min, and the serum before and after was analyzed. α2-macroglobulin (sugar tan, ξ
ri) is immunodiffusion method, albumin, total protein tlBO
It was measured by the G method and the Villette method. While α2-macroglobulin was 126 m97 dl before adsorption, it decreased to 60 η/dl after adsorption. albumin, total tannoξ
The former values were 2°4 g-/dt and 5.39-/dL, but the latter values were 2.3 f/dt and 5.0 f/dt.
There was almost no decline.

Example 2 The same as Example 1 except that Phaseolus zolgaris lectin was used as the lectin and the amount retained was 2,3 m9/vrl Cepharose. α Zoo Ashi'tsu F
Glycoproteins were measured by immunodiffusion method. αl-acid glycoprotein before adsorption is 68■
/dt, but after adsorption it decreased to 36 /dt. Changes in albumin and total tan were slight.

Example 3 Vinyl acetate 1OO51-, triallylisocyanurate 5L07, ethyl acetate 1007. 100g of heptane,
Polyvinyl acetate (degree of polymerization 500) 7.57-A uniform mixture of 3.8z and 2,27-azopisinbutyronitrile, 1 part by weight of polyvinyl alcohol, 0.0 parts by weight of sodium dihydrogen phosphate dihydrate. Dissolve 05% by weight of disodium hydrogen phosphate dihydrate and 1.5% by weight of disodium hydrogen phosphate dihydrate and add 400 g of water to a flask, stir thoroughly, and dissolve 65% by weight of disodium hydrogen phosphate dihydrate.
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. After filtration, washing with water, and extraction with acetone, the copolymer was transesterified in a solution consisting of 46.5% caustic soda and 2 tons of methanol at 40°C for 18 hours.

The average particle size of the obtained gel was 150 μm, and the vinyl alcohol unit per unit weight (q OH) was 10. Omcq
/1, specific surface area by BFiT method is 60 m2/fl,
The exclusion limit molecular weight with dextran was 6 x 105.

The resulting gel o,s P (dry weight 9) was then suspended in dimethyl sulfoxide 6 me, and epichlorohydrin 3.9 mA! , 30% sodium hydroxide 0.
5 ml was added and the activation reaction was carried out while shaking at 30°C for 5 hours. After the reaction, the mixture was washed with dimethyl sulfoxide, water, and dehydrated under suction. Next, 5 m/i of this activated gel was suspended in 10 d of 051M Fl [sodium powder (PH 9,8) containing 100 mg of concanavalin A. 5
The immobilization reaction was carried out with stirring at 0°C for 14 hours, and then 1.5m6f of a 6m9/mA' tris(hydroxyethyl)aminomethane solution was added, and the mixture was further heated at 50°C.
Blocking reaction (residual activity M
i-Block) was performed. Thereafter, it was thoroughly washed with water to obtain an adsorbent for extracorporeal circulation treatment. The amount of concanavalin immobilized on this adsorbent was 12 mm'ml gel.

The adsorbent was packed into a column in the same manner as in Example 1, and 7 adsorption experiments were conducted. Pour 6Mtt of rheumatism patient plasma into container 11 and make 0.2ml! The solution was circulated for 1 hour at a flow rate of /min.

Immune complexes (glycoprotein complexes) were analyzed using the solid phase 01q enzyme analysis method, fibrinogen was analyzed using the immunodiffusion method, and the rest was analyzed in the same manner as in Example 1. Immune complex before adsorption is 12μg/-
Whereas after adsorption it was 37. Z, decreased to /rd. Albumin, total tannin, and fibrinogen were the previous values of 2.2 f/dt, 5.6 f/-/dt,
It was 176 VaA, but after adsorption it was 2.1 f/dt.
, 5.37-/dt.

It was 153 mg/dL and hardly decreased.

[Brief explanation of the drawing]

FIG. 1 is a sectional view showing an example of the sugar-containing substance adsorption purifier of the present invention, and FIG. 2 is an explanatory diagram of the apparatus for an adsorption experiment in an example of the present invention. l...Sugar-containing substance adsorption purifier, 2...
・Cylinder, 3.3′...Filter, 4.4′・
... Nogutsuking, 5 ... Body fluid inlet,
6...Camping, 7...Body fluid outlet, 8
...Camping, 9...Adsorbent, 10...
...liquid supply bond, 1] ...container, 12...
...Drip channel-113...Sampling port, 14...Tube, 15...
Subject body fluid. Patent applicant: Asahi Kasei Industries, Ltd. Figure 1 Figure 2 Figure 4

Claims (1)

[Claims] An adsorption purifier for sugar-containing substances for extracorporeal circulation treatment, characterized in that an adsorbent binding a lectin or/and a fragment thereof to an insoluble carrier is housed in a container having a fluid outlet/outlet.