JPS5917355A - Immune adsorbent - 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 that specifically adsorbs and removes autoantibodies and/or immune complexes that are thought to be closely related to various diseases caused by biological immune function. As is well known, autoantibodies and/or immune complexes expressed in the blood are associated with cancer, immunoproliferative syndromes, chronic arthritis, autoimmune diseases such as systemic lupus erythematosus, allergies, and rejection during organ transplants. It is thought that there is a close relationship with the cause or progression of diseases and phenomena related to the body's immune function. Therefore, by specifically adsorbing and removing the above-mentioned autoantibodies and/or immune complexes from body fluid components such as blood and plasma, it is possible to prevent the progression of the above-mentioned diseases, alleviate symptoms, and even cure them.
It was hoped that this would be accelerated.

The present inventors have discovered that autoantibodies and/or immune complexes can be specifically adsorbed with high efficiency, non-specific adsorption is low, safety is achieved, and sterilization can be easily performed.
As a result of intensive research aimed at providing an adsorbent suitable for purification or regeneration of body fluids, we have developed a carrier that retains a substance with a special chemical structure that selectively interacts with the adsorbed substance. He discovered various adsorbents and filed patent applications for them (Japanese Patent Applications 1971-7152, 1987-18923,
(Patent application 1982-76776, Patent application 1984-159444)
The present invention was made as a result of a more detailed study of the carrier in relation to the previous invention, and relates to improvements to the above-mentioned adsorbent.

Desired properties of the adsorbent for body fluid purification used for the purpose of the present invention are: (2) difficulty in adsorbing substances other than the target substance, and (3) sufficient mechanical strength. Yes, and it is difficult to cause chips or cracks due to activation, immobilization, transportation, etc. In addition, by filling the column with adsorbent material, it is possible to flow high-viscosity solutions of body fluids such as blood and plasma at a high flow rate without causing clogging; (4) it is sterilizable; For example, it should not be activated.

The present inventors have an object to provide an adsorbent that can adsorb and remove autoantibodies and immune complexes with higher efficiency as an adsorbent for body fluid purification, that is, can be made into a compact adsorbent with a small priming volume. Further intensive research was conducted with the goal of providing an adsorbent that is mechanically strong, sterilizable, and has no adverse effect on blood or plasma.

As a result, it is mechanically strong, hard, and structurally superior, but it has a lot of nonspecific adsorption and activates the coagulation and fibrinolytic system and the complement system, so it is not used for body fluid purification. Surprisingly, by bonding an organic low-molecular-weight compound containing a hydrophobic compound to a porous material with silanol groups on the surface, which had not previously been used, the structural excellence was retained and the non-specificity was achieved. adsorption is suppressed, autoantibodies,
The present inventors have found that specific adsorption to immune complexes is imparted, and furthermore, activation of the coagulation fibrinolytic system and the complement system is suppressed, leading to the present invention.

That is, the present invention is an adsorbent for autoantibodies and/or immune complexes, characterized in that an organic low-molecular compound containing a hydrophobic compound is bonded to a porous substance having a silanol group on the surface.

The porous material with silanol groups on its surface is
Porous materials, such as silica gel and porous glass, have silanol groups on the surface and have a fine pore structure that extends into the inside like a network. The size of the pores is preferably such that proteins can freely enter the pores, and the average pore size is 250X to 250X.
(A range of ?'^ is desirable.Also, the larger the surface area of the pores is, the more surface areas can adsorb malignant substances, so it is preferable, but it is desirable that it be at least 10 rrt/?.Also, the silica content of the porous material The larger the silica, the more preferable it is because the number of silanol groups can be increased, but it is desirable to contain at least 50% of silica by weight.

Here, the average pore diameter is determined by the mercury intrusion method (for example, Catalyst Engineering Course-4, Catalyst Measurement Method, edited by the Catalysis Society, Chidaishokan, 69
The pore surface area is measured by the BET method using nitrogen gas (Pages 5 to 58) using nitrogen gas. Say the value.

The shape of the porous substance having silanol groups on the surface used in the present invention is not particularly limited, but considering the ease of handling during adsorbent production and handling during use, the particle size is 2.
Preferably, it is in the form of particles of 5 μm to 2500 μm.

The adsorbed substances targeted by the present invention are autoantibodies and immune complexes, but in more detail, rheumatoid factors, antinuclear antibodies, anti-DNA antibodies, anti-lymphocyte antibodies,
Autoantibodies such as anti-erythrocyte antibodies, anti-platelet antibodies, anti-acetylcholine receptor antibodies, serum demyelinating antibodies, anti-thyroglobulin antibodies, anti-microseam antibodies, anti-colon antibodies, between immunoglobulins or between immunoglobulins and other substances, especially antigens and antigens complexes with similar substances, etc. Among these, autoantibodies and immune complexes, which are closely related to the cause and progression of autoimmune diseases, are particularly preferred as adsorbent substances targeted by the present invention.

The hydrophobic compound used in the present invention has a solubility in physiological saline of 100 mmol/dl or less (at 25°C), more preferably 1
j Refers to a compound with 30 milJ mol/di or less. Solubility in physiological saline is 100 mmol/d7! A larger compound becomes too hydrophilic and has a reduced affinity for globulin compounds, resulting in an extremely reduced adsorption capacity. !
Furthermore, an affinity for albumin, which is more hydrophilic, is generated, and albumin is also non-specifically adsorbed, which is undesirable. Among hydrophobic compounds, compounds having at least one aromatic ring give particularly favorable results.

Aromatic rings refer to fcm-shaped gold compounds with aromatic properties, and any of them can be usefully used, but examples include benzene, naphthalene,
Benzene aromatic rings such as phenanthrene, 6-membered nitrogen-containing rings such as hyricine, quinoline, acridine, isoquinoline, phenanthrene, indole, carbazole, isoindole, indolizine, porphyrin, 2,3.2',
Nitrogen-containing 5-membered elements such as 3'-pyrrolopyrrole, pyridazine,
Pyrimidine, s5rm-) riazine, sym-tetrazine, quinazoline, 1,5-naphthyridine, pteridine, polyvalent six-membered crab-containing ring such as phenazine, pyrazole, imidazole, '*2+'-) lyazole, 1,2.3
−) Riazole, tetrazole, penziminazole,
Polyvalent nitrogen-containing five-membered rings such as imidazole and purines, norharman rings, perimidine rings, oxygen-containing aromatic rings such as benzofuran, inbenzofuran, and dibendofuran, sulfur-containing aromatic rings such as pendothiophene, chenothiophene, and chepine, and oxazole. , oxygen-containing heteroaromatic rings such as inoxazole, 1,2,5-oxadiazole, and benzoxazole; sulfur-containing heteroaromatic rings such as thiazole, inthiazole, 1,3,4-thiadiazole, and benzothiazole; Hydrophobic compounds having at least one aromatic ring and its derivatives give favorable results. Among these, compounds containing an indole ring such as tryptamine give particularly favorable results. This can be interpreted as a result of the hydrophobicity and molecular rigidity of the globulin compound effectively acting on the bond between the compound and the compound.

In addition, as a result of intensive research in search of inexpensive hydrophobic compounds that can be put to practical use in a safe manner, the present inventors have found that hydrophobic amino acids and their derivatives are highly effective and specific in producing globulin compounds. It was discovered that it can be adsorbed and removed.

Hydrophobic amino acids and their derivatives are defined by Tanford
.

Nozaki (J, Am, Chem, Soc,,
184 424 G (1962), J. Biol.
Chem, 246 2211 (1971) Tanford, Nozaki (Journal of American Chemical Science, 184.4240 (1962), Journal of Biological Chemistry 6
, 2211 (1971)), amino acids and derivatives thereof having a hydrophobicity of 1,500 cat/mot or more, and a compound having a solubility in physiological saline of 100 mmol/a or less. Examples include lysine, valine, leucine, tyrosine, phenylalanine, inleucine, tritophane and derivatives thereof. Among these hydrophobic amino acids and their derivatives, tryptamine/phenylalanine and its derivatives give particularly good results. Furthermore, the amino acids can be used without particular limitations on the t and d conformations. Furthermore, the term "organic low molecular compound" as used herein refers to an organic compound having a molecular weight of 10,000 or less, preferably 1,000 or less.

Protein A (
Compared to naturally occurring molecules such as molecular weight 42,000), it is easier to handle during immobilization and to store after immobilization. Furthermore, even if the substance stagnates, organic low-molecular compounds with a molecular weight of 10,000 or less are safe and have negligible antigenicity to living organisms, and can be easily sterilized.

The organic low-molecular compound can be obtained by copolymerizing the hydrophobic compound monomer alone or with other compounds.

As the hydrophobic compound, for example, a vinyl derivative of an indole mt-containing compound such as tryptamine, or a hydrophobic amino acid such as tryptophan can be used.

There are various known methods for bonding an organic low-molecular compound containing a hydrophobic compound to a porous material having a silanol group on its surface, such as covalent bonding, ionic bonding, physical adsorption, embedding, and insolubilization by precipitation on the surface of a porous material. However, considering the elution properties of organic low-molecular-weight compounds, it is preferable to use them after covalent bonding, immobilization, or insolubilization. Therefore, it is possible to use a known method for activating an immobilized enzyme, an insoluble carrier used in affinity chromatography, and a method for binding to a low-molecular-weight organic compound.

Moreover, if necessary, an insoluble carrier and an organic compound can be completely introduced for use. As for the spacer length, a range from no spacer to 20 atoms in the spacer gives particularly preferable results.

For example, when r-glycidoxypropyl trimethoxysilane is reacted with the silanol group of a porous material, and this epoxy group is reacted and bonded with the amine group of an organic low molecular compound containing a hydrophobic compound. <Can be implemented. Alternatively, after reacting a spacer with an organic low-molecular compound containing a hydrophobic compound, the spacer may be bonded to a porous substance having a silanol group.

As described above, the adsorbent of the present invention can highly efficiently and specifically adsorb and remove autoantibodies and/or immune complexes in body fluids, and is mechanically strong and sterilizable.
It is an adsorbent that does not have a negative effect on blood or plasma. That is, the adsorbent of the present invention can be applied to general uses for purifying and regenerating body fluids such as autologous plasma and autologous blood, and for collagen diseases such as cancer, immunoproliferative syndrome, rheumatoid arthritis, and systemic lupus erythematosus. , autoimmune diseases such as myasthenia gravis, allergies, diseases and phenomena related to the body's immune system such as organ transplant rejection, or kidney diseases such as nephritis,
It can be effectively used for extracorporeal circulation treatment of liver diseases such as hepatitis. Furthermore, the adsorbent of the present invention can be used not only for purification and regeneration of body fluids, but also for the separation and purification of autoantibodies and immune complexes.

The embodiments of the present invention will be explained in detail with reference to the following examples.

Example 1 Silica gel tot-1 with an average pore size of 400 X and a surface area of 15071 f/f is immersed in a 20% solution of γ-glycidoxyprobyltrimethoxysilane (acetone solvent) and
The reaction was allowed to proceed at 0°C for 60 hours with shaking. After the reaction, the reaction mixture was washed with 500 d of acetone, washed with water, replaced with 0.1M sodium carbonate buffer, and dehydrated by suction. Next, this gel was suspended in 20 tne of 0.1M sodium carbonate buffer containing 10frLl'JJ liptophan 500m9f. The immobilization reaction was carried out at 5'oC for 20 hours, then a 60~/d tris(human tetraxyethyl) aminomethane solution 5- was added, and further [50oC]
A blocking reaction (blocking remaining active groups) was carried out for 5 hours with stirring. After that, the adsorbent was thoroughly washed with water to obtain an adsorbent.

The amount of tryptophan fixed on this adsorbent is 4゜μm
I met with ol/gogel.

When the adsorbent was compared with the original silica gel particles and observed under an optical microscope, no damage such as chips or cracks was observed.

This adsorbent has an inner diameter of 1011111. Two columns with a length of 5Ω were filled, and one was filled with 0 ACD plasma.
．． 12-1 and ACD in the other bottle at a flow rate of 4ml/mix
The patient's blood fO was flowed at a flow rate of 7 me/min for 20 hours. No decrease in packing volume, clogging, or decrease in flow rate was observed in any of the columns, and the pressure difference before and after the column was less than 1011+aHg for plasma, and 10 to 2 degrees for blood.
It was in the Hg range.

Changes in plasma proteins and blood cell components before and after passing through the column'
In terms of kN, adsorption of albumin in plasma was slight, and the decrease in complement component C3 was also small, but the titer of rheumatoid factor (based on sensitized hemagglutination, RAHA test, Fuji organ layer) was 640 times higher than before adsorption. While it was positive after dilution, the titer after adsorption became negative at 40 times.

In addition, the immune complex (by C+q solid phase method EIAK) was 17 μm/- before adsorption, but it normalized after adsorption (
3μf/m/or less). Regarding blood, no significant differences were observed in the concentrations of red blood cells, white blood cells, and platelets before and after passing through the column.

Next, in order to investigate the activation of the coagulation system, activated partial thromboplastin time (A-PTT)'i57 was measured using the adsorbent of this example instead of kaolin. As a result, the A-PTT using the adsorbent was 80 seconds, the A-PTT using kaolin was 38 seconds, and the partial thromboplastin time (PTT) was 78 seconds. That is, A-PTT using an adsorbent and PTT took approximately the same time, and no activation of the coagulation system by the adsorbent was observed.

Silica gel 102 with an average pore diameter of 800X and a surface area of 7074/f was immersed in a 20% solution of γ-crisitoxyprobyltrimethoxysilane (acetone solvent) and heated at 50 °C for 3
The reaction was carried out at 0:00.

After the reaction, it was washed with acetone 500, washed with water, replaced with 0.1M sodium carbonate buffer, and then dehydrated.

Next, this gel 10- was added to phenylalanine 5QOIv'
20rn of 0.1M sodium carbonate buffer containing t-!
.

suspended in it. The immobilization reaction was carried out with shaking at 50°C for 20 hours, then 5d of a 601Q/ld tris(hydroxyethyl)aminomethane solution was added, and the
A blocking reaction (to block remaining active groups) was carried out at 0° C. for 5 hours with stirring. After that, the adsorbent was thoroughly washed with water to obtain an adsorbent. The amount of phenylalanine immobilized on this adsorbent was 50 μmol/gel.

When the adsorbent was compared with the original silica gel particles and observed under an optical microscope, no breakage such as chipping or crumbling was observed.

This adsorption phase was packed into two columns arranged in order of inner diameter tomu and length 50 (7.
, 12 bottles with a flow rate of 4 ml/min, and one bottle with A
CDCD medium gusset patient blood at a flow rate of 0.7-7 m20
ml was poured. No decrease in packing volume, clogging, or decrease in flow rate was observed in any of the columns, and the pressure difference before and after the column was less than 1g for plasma, and 10 to 20wrm for blood.
At 7 in the Hg range? :.

When we investigated changes in plasma proteins and blood cell components before and after passing through the column, we found that albumin was only slightly adsorbed in plasma and there was a small decrease in complement component C5, but rheumatoid factors (sensitized hemagglutination, RAHA test, Fuji organ layer), the titer before adsorption was positive at 1280 times dilution, while the titer after adsorption became negative at 40 times dilution.

In addition, the immune complex (by C+q solid-phase EIA) rarely reached 14 μy/- before adsorption, but normalized after adsorption (
3 μm/- or less). Regarding blood, no significant difference was observed in the number of red blood cells, white blood cells, and platelets before and after passing through the column.

Next, in order to investigate the activation of the coagulation system, the activated partial thromboplastin time (A-PTT) was measured using the adsorbent of this example instead of kaolin. As a result, A-PTT using an adsorbent was 82 seconds, A-PTT using kaolin was 38 seconds, and the partial thromboplastin time (PTT) was 78 seconds. That is, A-PTT using an adsorbent and PTT took approximately the same time, and no activation of the coagulation system by the adsorbent was observed.

Procedural amendment September 21, 1980 Director-General of the Patent Office Kazuo Wakasugi 1 Indication of the case Special No. 126822/1982 2 Name of the invention Immunoadsorbent 6 Relationship with the ministry case to be amended/Patent applicant (003) Asahi Kasei Kogyo Co., Ltd. 4 Agent 5th Floor, Toranomon Sangyo Building, 2-29 Toranomon-chome, Minato-ku, Tokyo Column 6 for Detailed Description of the Invention in the Specification Contents of Amendment The following amendments have been made to the description of the specification.

(11, page 10, lines 14-16, “Also, here...
It refers to an organic compound of... ” Please correct the following.

“The organic low-molecular compound containing a hydrophobic compound used in the present invention refers to a substance in which part or all of the organic low-molecular compound is composed of a hydrophobic compound, and the molecular weight is 1.
10,000 or less, preferably 1,000 or less. For example, an organic low-molecular compound containing a hydrophobic compound that can be used in the present invention can be obtained by copolymerization of a hydrophobic compound or copolymerization of a hydrophobic compound and another compound. In other words,
Examples include condensation products of hydrophobic amino acids such as tryptophan and phenylalanine and hydrophilic amino acids or peptides, and condensation products of hydrophobic amino acids such as tryptophan and phenylalanine. Furthermore, hydrophobic amino acids alone are also included in the organic low-molecular compounds. ”(21,
On page 10, line 17, "organic low-molecular compound" is corrected to "organic low-molecular compound containing a hydrophobic compound." (41, page 11, lines 4-9, "The organic low-molecular compound is...").
......can be used. ” Delete all.

(51, page 11, line 19, “organic low molecular compound” is corrected to “organic low molecular compound containing a hydrophobic compound”).

(6), “Organic low molecular compound” from negative line 20 of page 11 to line 1 of page 12 “organic low molecular compound containing a hydrophobic compound”

Claims (1)

[Claims] (]) An adsorbent for autoantibodies and/or immune complexes, characterized in that an organic low-molecular compound containing a hydrophobic compound is bound to a porous substance having a silanol group on the surface. . (2) The average pore diameter of the porous material is from 250X to 2
in the range of 500X, and the surface area of the pores is 10×72
The adsorbent according to claim 1, which is the above. (3) The adsorbent according to claim 1, wherein the porous substance contains 50 weight or more of silica. (4) The adsorbent according to claim 1, wherein the hydrophobic compound is a compound containing at least one aromatic ring. (5) The adsorbent according to claim 4, wherein the aromatic ring is an indole ring. (6) Claim 4 in which the aromatic ring is a benzene ring
Adsorbent material as described in section. (7) The adsorbent according to claim 1, wherein the hydrophobic compound is a hydrophobic amino acid or a derivative thereof. (8) The adsorbent according to claim 7, wherein the hydrophobic amino acid or its derivative is tryptophan or its derivative. (9) The adsorbent according to claim 7, wherein the hydrophobic amino acid or its derivative is phenylalanine.