JPH01158970A - Immunoglobulin adsorbing material for direct blood perfusion and adsorbing apparatus - Google Patents

Abstract

PURPOSE:To enhance blood compatibility, by bonding a low HW org. compound having high compatibility with immunoglobulin to the surface of a water- insoluble porous solid substance through a hydrophilic spacer. CONSTITUTION:As a water-insoluble porous solid substance, an acrylic polymer and chitosan are especially pref. from the aspect of the absorbing capacity to immunoglobulin and, as the shape thereof, a granular carrier is especially pref. The average particle size thereof is pref. 0.5-2mm and the average pore size thereof is pref. 150-3000Angstrom . A low MW org. compound having high compatibility with immunoglobulin is selected from a group consisting of heterocyclic compounds, peptides and amino acids. As a spacer 15, a substance, which forms a molecular chain and is interposed between the surface 13 of the water- insoluble porous solid substance and a compound 14 to make it possible to provide an arbitrary length and can suppress the bonding of a blood corpuscle, is used and, for example, a hydrophilic substance having an ethylene oxide skeletal having a polymerization degree of 1-90 is especially pref.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention is applicable to autoimmune diseases (myasthenia gravis, rheumatoid arthritis, systemic lupus erythematosus, etc.), immune-related diseases,
(glomerulonephritis, bronchial asthma, polyneuritis, etc.), organ transplantation (transplant kidney rejection, blood type incompatible bone marrow transplantation, etc.),
The present invention relates to an immunoglobulin adsorbent that allows direct blood perfusion without separating plasma from blood, and to an immunoglobulin adsorption device using the adsorbent, for the purpose of treating cancer (leukemia, etc.).

[Conventional technology]

Conventionally, plasma exchange therapy has been used to treat conditions such as immunodeficiency, tumors, hypercholesterolemia, and liver failure. However, since this therapy indiscriminately removes all plasma components, not only are necessary plasma components lost, but there are also many problems such as a lack of plasma as a replacement fluid or plasma preparations, and complications such as serum hepatitis and allergies. It contains the problem of. Therefore, the trend is toward methods for selectively removing pathogen-related substances, and double filtration methods and cold filtration methods are being used. High molecular weight substances such as IgM, immune complexes (1), and β-lipoprotein can be separated relatively well, and their clinical usefulness has been recognized.

Other treatment methods include the use of various drugs, but
Generally, there are side effects, so care must be taken when using them, such as keeping the amount and period of use to the minimum necessary.

Therefore, there has been a desire for an adsorbent and an adsorption therapy that have no side effects and can selectively remove specific pathogen-related substances in plasma by membrane separation.

Adsorbents that can be used for such purposes include (1) affinity adsorbents (for example, JP-A-55-1 2087;
5, 57-1 228 7 5, 5
9-1 7 3 5 4, 59-19673
8, 6 1-(2) Porous resins (e.g., JP-A-56-1477101, JP-A-62-2543) (3) Inorganic porous bodies (e.g., JP-A-62-254).

In addition, affinity adsorbents include cellulose particles bound with dextran sulfate (Ribosorber manufactured by Kanebuchi Chemical Co., Ltd.) for the treatment of hypercholesterolemia, polyvinyl alcohol particles bound with phenylalanine (Imusorber manufactured by Asahi Medical Co., Ltd.) for the treatment of autoimmune diseases, and Quartz crystal particles bound to blood type A or B antigens (Biosynsorb, manufactured by Chembiomed, manufactured by Biosynsol, Inc., for removal of A anti-B antibodies. However, in both cases, blood was separated into blood cells and plasma using a plasma separator. This is for plasma perfusion, in which only the plasma is treated with an adsorbent afterwards.

On the other hand, various auxiliary artificial livers are commercially available as adsorbents for direct blood perfusion that can easily purify blood without using a plasma separator.

Examples of these include cellulose and poly-1(H
MA-coated activated carbon (Asahi Medical Hem)
osorba, Kuraray D) IP-1, Tijin H
emocels, etc.), heparin-treated activated carbon (Terumo Hemocolumn), or styrene resin (Extra
XR-004) manufactured by Corporate Corporation. However, activated carbon and styrene resin have the disadvantage of low adsorption specificity, and furthermore, activated carbon has small pore diameters, so it is not suitable for removing all medium- and high-molecular substances.

As described above, the current adsorbents used for the purpose of removing medium- and high-molecular-weight pathogen-related substances and protein-bound pathogen-related substances from blood are for plasma perfusion. Therefore, the development of adsorbents for direct blood perfusion has become active (for example, for artificial organs,
Kamiyu (2), 993 (1984)). but,
Surface improvement of adsorbents to increase their blood compatibility is a very difficult task, and on the other hand, the use of antithrombotic agents such as PGI2 (prostaglandin I2) is undesirable because it leads to high treatment costs.

[Problem that the invention seeks to solve]

An object of the present invention is to solve the above-mentioned problems of the prior art.After bonding a ligand to the surface of a porous solid material, a mesh-like or porous structure is formed that prevents blood cells from entering and allows immunoglobulin to enter. By covering with a structure,
Another object of the present invention is to provide an immunoglobulin adsorbent for direct blood perfusion and an adsorption device that has improved blood compatibility by binding a ligand via a hydrophilic spacer.

Means for Solving Problem C] The present invention involves bonding a low-molecular-weight organic compound having high affinity with immunoglobulins to the surface of a water-insoluble porous solid material via a hydrophilic spacer, or After bonding a low-molecular-weight organic compound that has a high affinity with immunoglobulins to the surface of a solid substance, the outer surface is coated with a mesh or porous structure that prevents blood cells from entering and allows immunoglobulins to enter. This invention relates to an immunoglobulin adsorbent for direct blood perfusion with improved blood compatibility and an adsorption device using the same.

The substances to be adsorbed and removed by the adsorbent of the present invention are pathogen-related substances in blood, but to explain in more detail, ordinary immunoglobulins (A, D, E, G%M), anti-DNA antibodies autoantibodies such as anti-acetylcholine receptor antibodies,
These include antigen/antibody complexes.

The water-insoluble porous solid materials used in the present invention include synthetic organic polymer materials such as polyvinyl alcohol, methacrylic acid ester polymers, and styrene-divinylbenzene copolymers; natural organic polymer materials such as cellulose, agarose, and chitosan; Examples include inorganic porous materials such as porous glass, alumina, and ceramics, but acrylic polymers and chitosan are particularly preferred from the viewpoint of adsorption ability for immunoglobulin, mechanical strength, operability, and the like.

The shape of the water-insoluble porous solid substance may be any known shape such as particulate, fibrous, hollow fiber, or membrane. A shaped carrier is particularly preferably used.

The average particle diameter of the particle carrier is preferably in the range of 0.05 mm to 2 cm; however, as the particle diameter decreases, the flow resistance of blood cells increases, and as the particle diameter increases, the carrier filling amount decreases. Average particle size is from 0.5mm to 2
Particularly preferred is a range of mm.

Further, the particulate carrier is preferably spherical because it is less likely to damage cells and is less likely to crack or chip due to physical force.

The particulate carrier used in the present invention is a porous material capable of binding a large number of ligands (substances that specifically bind to pathogenic substances) on its surface, and has an average pore diameter of 50 to 5,000 molecules, more preferably immunoglobulin or immunoglobulin. It is in the range of 150 to 3000 people that many complexes can invade.

The low-molecular-weight organic compound having high affinity with immunoglobulin used in the present invention is selected from the group consisting of heterocyclic compounds, peptides, and amino acids.

A heterocyclic compound is a cyclic compound that contains a petro atom along with a carbon atom in the ring, and in a three-membered heterocyclic compound,
Furan and its derivatives, thiophene and its derivatives and dithiolane derivatives, virol and its 8-conductor, azoles,
Six-membered heterocyclic compounds include pyridine and its derivatives, quinoline and related compounds, pyrazine and related compounds, biran and toron and related compounds, phenoxazine, phenothiazine, pterin and alloxazine compounds, purine bases, nucleic acids, hemin, chlorophyll, Refers to vitamin B12 and phthalocyanines or alkaloids, and fused ring heterocyclic compounds.

Among the heterocyclic compounds, sulfa drugs (sulfonamides) or their derivatives give preferable results. Furthermore, among sulfa drugs, sulfathiazole gives particularly favorable results.

The peptides used in the present invention include at least two
It is a synthetic compound in which more than 100 amino acids are obtained through peptide bonds, and oligopeptides with about 2 to 10 amino acids give preferable results. Furthermore, among these oligopeptides, those imitating the affinity sites of known natural substances such as protein A, which have specific binding properties with immunoglobulin molecules, are preferred, and in particular, oligopeptides based on lysine and tyrosine, or aspatylphenylalanine methyl ester. Compounds such as aspatyl phenylalanine alkyl esters give very good results.

In addition, the amino acids used in the present invention are car-type amino acids or two or more amino acids used at the same time, such as hydrophobic valine, leucine, tyrosine, phenylalanine, isoleucine, tryptophan, basic lysine, arginine, or Particularly good results are obtained when acidic proline and aspartic acid and their derivatives are used alone or in combination. Note that it does not matter whether these substances are in the L form or not.

The low-molecular-weight organic compounds that have high affinity with immunoglobulins and are preferably used in the present invention are characterized by the hydrophobicity of the main part of the compound, the electrostatic properties of heteroatoms, the hydrogen near the main part, and the like. The binding properties exert interaction forces with the amino acid residues at adsorption sites in the immunoglobulin molecular chain, and in addition, the three-dimensional structure of the compound appropriately fits into the intramolecular pocket of the immunoglobulin, so that non-antigen/antigen This is thought to be due to the high affinity of the system.

Methods for immobilizing the low-molecular-weight organic compound having high affinity with immunoglobulin used in the present invention on a water-insoluble porous solid material include covalent bonding, physical adsorption, ionic bonding,
Although carrier binding methods such as biochemical specific binding, crosslinking methods, entrapping methods, and composite methods can be carried out, covalent bonding is preferred from the viewpoint of binding stability in blood.

Furthermore, in order to covalently bond the compound to the water-insoluble porous solid material, various methods are used depending on the functional groups of both materials. For example, cyanogen bromide activation method, condensation reagent method using carbodiimide reagent, acid azide derivative method, diazo method, alkylation method, carrier crosslinking method using dialdehyde, bisepoxide, diisocyanate, etc., γ-glycidoxypropyltritri A silane coupling agent activation method using methoxysilane, β-(3,4-epoxycyclohexyl)ethyltrimethoxysilane, etc. is used.

The immunoglobulin adsorbent for direct blood perfusion of the present invention is characterized in that a low-molecular organic compound having a high affinity for immunoglobulin is bonded to the surface of a water-insoluble porous solid material via a hydrophilic spacer. It is.

The spacer of the present invention is in the form of a molecule and is interposed between the surface of a water-insoluble porous solid substance and the compound, and can have any length, and can suppress the adhesion of blood cells. Wood-loving spacers are preferred.

Among hydrophilic spacers, those having an ethylene oxide skeleton with a degree of polymerization of 1 to 90 are particularly preferred, and examples include those having the following structures.

[In the formula, R1 and R2 are each hydrogen or an alkyl group having 1 to 4 carbon atoms, and n is a number of 0 to 90, more preferably 3 to 29. ] When such a spacer is interposed between the surface of a water-insoluble porous solid substance and the compound, the fluidity of the spacer and the excluded volume effect suppress the adhesion of blood cells to the adsorbent surface. it is conceivable that.

In addition, the surface density of the spacer that suppresses the adhesion of blood cells is 1
-100 μmol/g, more preferably 5-40
It is μmol/g. Therefore, the compound via the spacer and the compound not via the spacer may coexist and be bonded to the surface of the water-insoluble porous solid substance.

The mechanism by which the use of the wood-philic spacer makes it difficult for blood cells to adhere to the surface of the adsorbent is thought to be as described below.

(1) Water is solvated by hydrogen bonding with the oxygen atom of the ether bond of a hydrophilic spacer, for example, ethylene oxide. In this way, a layer of water is formed on the surface of the adsorbent, and the distance (thickness of the water layer) depends on the length of the spacer. This water layer prevents blood cells from adhering to the carrier (adsorbent) surface. This is called the excluded volume effect.

Furthermore, the movement of the spacer forming the water layer makes it difficult for blood cells to adhere to the carrier and makes it easier for them to detach from the carrier, and this movement of the spacer is referred to as fluidity.

(2) At the portion of the carrier surface where the spacer polymers overlap, it can be considered that two polymer solutions (the hydrophilic spacer of the adsorbent and the sugar chains/proteins of the blood cells) are mixed. As a result, the following effects are thought to occur.

■ Osmotic pressure increases as the concentration of the polymer solution increases.

■Each segment within the polymer moves irregularly,
The volume through which this movement takes place is narrowed for mixing.

Therefore, the configuration entropy of the polymer segment decreases. ■ is called the osmotic pressure effect, and ■ is called the entropy effect. Both effects create a repulsive effect, making it difficult for blood cells to adhere to the carrier.

Another immunoglobulin adsorbent for direct blood perfusion of the present invention is
After bonding a low-molecular-weight organic compound that has a high affinity with immunoglobulins to the surface of a water-insoluble porous solid material, a network or porous structure (hereinafter referred to as immunoglobulin) that is difficult for blood cells to enter and that allows immunoglobulins to enter is formed. It is characterized by having its outer surface coated with a globulin-invading structure.

The immunoglobulin entry structure used in the present invention is preferably a methacrylic acid copolymer, which is a sustained release film coating that swells in an aqueous medium and allows entry of immunoglobulins but not blood cells. -Exhibits a net-like structure. In addition, it is preferably used because it has excellent blood compatibility and low toxicity.

Among the methacrylic acid copolymers, copolymers of methacrylic ester and acrylic ester are particularly preferred, and those having the following structure (R6hm Pharm
Eudragit RL/RS) manufactured by company a can be cited as an example.

[In the formula, R1=H, CH3 R2=CH3, C2Hs] Coating methods for methacrylic acid copolymer include pan coating method, fluidized bed method, dipping method, etc., and any known method that can form a uniform layer can be used. It can be used without any problems.

Substances that can be used other than methacrylic acid copolymers include:
Polydroxyethyl methacrylate, collodion, N
- Vinylpyrrolidone, polymethacrylic acid, polypropylene glycol, polyvinyl alcohol, polyethylene oxide, etc. may be mentioned. These coating methods are also the same as above.

The immunoglobulin adsorption device for direct blood rinsing of the present invention is made by filling and holding the above-mentioned adsorbent in a container provided with a blood inlet/outlet.

As the material of the container, glass, stainless steel, polyethylene, polypropylene, polycarbonate, polystyrene, polymethyl methacrylate, etc. can be used, but polypropylene and polycarbonate are particularly preferable because they can be sterilized in an autoclave and are easy to handle. Further, it is preferable that a filter is provided between the adsorbent in the container and the inlet/outlet portion, which has a mesh or pores large enough to allow blood to pass through but not allow the adsorbent to pass through. This material may be physiologically inert and strong, but especially polyester,
Polyamide mesh is preferably used.

Next, the immunoglobulin adsorption device for direct blood perfusion of the present invention will be explained in more detail with reference to the drawings.

Figure 1 shows one of the immunoglobulin adsorption devices for direct blood perfusion.
FIG. 3 is a cross-sectional view of an example. Blood is introduced through the blood inlet 4, treated with the immunoglobulin adsorbent 2, and then
The blood is drawn out from the blood outlet 5. The immunoglobulin adsorbent 2 is held within the container by filters 3a and 3b.

FIG. 2 is a blood circuit diagram showing one example of a blood purification treatment method. Blood is introduced from the blood introduction side blood vessel connection part 6,
It is supplied to the immunoglobulin adsorption device 1 from a drip chamber 8 having a pressure gauge 9 through a pump 7 .
The adsorbed blood passes through the filter 10,
After being heated by the heat exchanger 11, the blood introduction blood vessel connection part 12
It is derived from

〔Example〕

Hereinafter, embodiments of the present invention will be explained in more detail with reference to Examples.

Examples and Comparative Examples (Preparation of adsorbent) Chitosan beads with three types of average particle sizes (Chitovar manufactured by Fujibo Co., Ltd., average particle size 1.0, 1, 5, 2.0 mmφ) were mixed into 1
00 g each was placed in a polyethylene bottle, and 200 ml of pH 7.4-0.1M phosphate buffer containing 5% glutaraldehyde was added to each. After degassing under reduced pressure, the mixture was stirred overnight at room temperature using a blood mixer (Model BM-161, manufactured by Kayagaki Medical Science Co., Ltd.) to react the amino groups of chitosan with glutaraldehyde. Next, distilled water volume 1f
After washing with dimethylformamide and pH 10-0
,0 dissolved in a mixture (2/3) of 2M carbonate buffer
, 200 mu of 1M sulfathiazole solution were added. After degassing under reduced pressure, the mixture was stirred overnight at room temperature using a blood mixer to react the aldehyde group of glutaraldehyde bonded to chitosan with the amino group of sulfathiazole. After washing with 11 volumes of distilled water, p.
200 volumes each of H8-IM 1,000 sonoethanolamine aqueous solution was added and degassed under reduced pressure. This was set in a blood mixer and stirred overnight at room temperature to block remaining aldehyde groups. Next, after washing with distilled water at 1°C, pH 7,4- containing 1% sodium borohydride was added.
0.1M phosphate buffer was added in an amount of 2 o mjl each, and the mixture was allowed to stand at room temperature for 3 hours with occasional stirring to reduce and stabilize the azomethine bond. Next, distilled water and pH 10
It was washed repeatedly with 1) H4-0,002M acetate buffer containing -0.2M carbonate buffer and 0.5M sodium chloride, and finally washed with distilled water and filtered with suction. The adsorbent thus produced having three types of average particle diameters is referred to as an adsorbent (A).

Next, 30 g of the adsorbent (A) was placed in a polyethylene bottle, and bis-epoxide having an ethylene oxide skeleton with a degree of polymerization of 22 (Dena manufactured by Nagase Chemical Industries: 7-LEX 86) was added.
100mM of p)110-0.2M carbonate buffer containing 10% of 1) was added to each. After degassing under reduced pressure, the mixture was stirred overnight at room temperature using a blood mixer, and further stirred in an 80°C water bath (Yamato Scientific Water Bath BM-4).
Type 1) for 1 hour to cause the hydroxyl groups of chitosan to react with the glycidyl groups of bisepoxide. Subsequently, after washing with 11 volumes of distilled water, dimethylformamide and pH 10
A solution of 0.1M sulfathiazole dissolved in a mixture (2/3) of -0.2M carbonate buffer was added in 10ml portions. After degassing under reduced pressure, it was heated in an 80°C water bath for 3 hours,
Further, stir overnight at room temperature using a blood mixer,
The glycidyl group of bisepoxide bonded to chitosan was reacted with the amino group of sulfathiazole.

After washing with 1 ft of distilled water, 1 ooIII5 I of a monoethanolamine aqueous solution having a pH of 8 to 1 M was added thereto, and the mixture was degassed under reduced pressure. This was set in a blood mixer and stirred overnight at room temperature to block remaining glycidyl groups. Next, add distilled water, pH 10-0, 2M carbonate buffer, pH 4-0 containing 0.5M sodium chloride.
, 02M acetate buffer, and finally with distilled water, followed by suction filtration. The adsorbent thus produced having three types of average particle diameters is referred to as an adsorbent (B).

Separately, 30 g of each adsorbent (A) was taken and washed with acetone. and 6.25% methacrylic acid copoly7-
(Rt;hm Eudragit R5 manufactured by Pharma
) in a dichloromethane solution, and then the solvent was evaporated using a dryer. Finally, it was washed with distilled water tIt and filtered with suction. The adsorbent thus produced having three types of average particle diameters is referred to as an adsorbent (C).

(Direct blood perfusion experiment using domestic rabbits) After soaking the adsorbents (A), (B), and (C) in physiological saline and degassing them, they were packed into a 20 m capacity column with polyester mesh attached to both ends. . And 1 o o
Physiological saline containing 11/ndl of heparin, followed by 10/ndl of heparin, followed by saline too containing 10/ndl of heparin.
The inside of the column and the inside of the blood circuit were washed with mU. Next, blood was introduced into the blood circuit from the carotid artery of a domestic rabbit, and a perfusion experiment was conducted for 3 hours in which the blood was returned to the body from the jugular vein through the column. The blood flow rate was set at 5 mU/min, and changes in column pressure, adsorption amounts of albumin, total protein, IgG, and IgM, and changes in blood cell components were observed. Albumin was measured using the bromcresol green method, total protein was measured using the biuret method, and IgG
and IgM were measured by one-way radial immunodiffusion method, and the blood cell count was measured using an automatic blood cell counting device (Ortho Instruments, ELT-
8). In addition, during the experiment, heparin, an anticoagulant, was administered into the blood at a rate of 300 u/Kg at the start of blood perfusion and then at a rate of 50 u/Kg every 30 minutes.

The results are shown in Table 1. From this, the adsorbent of the present invention
It is clear that direct blood perfusion can efficiently remove immunoglobulins.

〔Effect of the invention〕

The immunoglobulin adsorbent material and adsorption device for direct blood perfusion of the present invention can selectively and easily remove proteins such as immunoglobulins and immune complexes that are pathogen-related substances in blood.

Furthermore, since the ligand is a low-molecular organic compound, sterilization can be performed easily and reliably.

Since the adsorbent of the present invention has good blood compatibility,
The present invention enables a direct blood perfusion method that is easier to operate and causes less pain to the patient than the plasma perfusion method.

Furthermore, the adsorbent and adsorption device of the present invention can be effectively used for separation and purification of proteins such as immunoglobulins and immune complexes, and as materials for their testing.

[Brief explanation of the drawing]

FIG. 1 is a schematic cross-sectional view showing an example of an immunoglobulin adsorption device for direct blood perfusion. FIG. 2 is a blood circuit diagram showing one example of a blood purification treatment method. FIG. 3 is a schematic diagram of the present invention, and FIG. 4 is a schematic diagram of the present invention. Explanation of symbols 1: Immunoglobulin adsorption device for direct blood rinsing, 2: Immunoglobulin adsorbent for direct blood rinsing, 3a,
3b...Filter, 4...Blood inlet, 5...Blood outlet, 6...Blood inlet side blood vessel connection part, 7...Pump, 8...Drip chamber, 9... Pressure gauge, 10... Filter, 11... Heat exchanger, 12... Blood outlet side blood vessel connection part, 13... Porous carrier, 14... Ligand, 15... Hydrophilic spacer, 16...Methacrylic acid copolymer

Claims (6)

[Claims] (1) An immunoglobulin adsorbent for direct blood perfusion, characterized in that a low-molecular-weight organic compound having a high affinity for immunoglobulin is bonded to the surface of a water-insoluble porous solid substance via a hydrophilic spacer. (2) After bonding a low-molecular-weight organic compound that has a high affinity with immunoglobulins to the surface of a water-insoluble porous solid material, a mesh-like or porous structure is created that is difficult for blood cells to penetrate and that allows immunoglobulins to penetrate. An immunoglobulin adsorbent for direct blood rinsing, characterized in that its outer surface is coated. (3) The immunoglobulin adsorbent according to claim 1, wherein the hydrophilic spacer has an ethylene oxide skeleton with a degree of polymerization of 1 to 90. (4) Claim 1 or 2, wherein the low-molecular-weight organic compound having high affinity with immunoglobulins is selected from the group consisting of heterocyclic compounds, peptides, and amino acids. Immunoglobulin adsorbent for direct blood perfusion. (5) The immunoglobulin for direct blood perfusion according to claim 1 or 2, wherein the water-insoluble porous solid substance is a particle consisting of a synthetic organic polymer substance, a natural organic polymer substance, or an inorganic substance. adsorbent. (6) An immunoglobulin for direct blood perfusion, characterized in that the immunoglobulin adsorbent according to any one of claims 1 to 5 is housed in a container having a fluid inlet and outlet. Adsorption device.