JP2665526B2 - β2-microglobulin adsorbent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial applications)   The present invention can be used in body fluids such as blood, plasma, serum, ascites, and pleural effusion.
Selectively adsorb and remove more malignant substances related to disease
The present invention relates to a body fluid purifying adsorbent. More specifically, renal failure
Increases in the body fluids of elderly and malignant tumors, causing carpal tunnel syndrome,
Miloidosis, fingering, shoulder and knee arthropathy, pruritus cutaneous,
Β causing bone disorders etc.₂-Microglobulin adsorbent
About. (Conventional technology)   Hemodialysis has been performed on patients with renal failure, and about 10 years have passed
Abnormalities such as carpal tunnel syndrome have become apparent. recent years,
This substance is relatively difficult to remove by dialysis.₂-Micro
It is a globulin that causes various symptoms due to its accumulation in the body.
It is clear that it will appear.   Conventionally, for the purpose of removing such medium molecular weight substances, blood
Filtration and diafiltration are used, but the removal rate is low.
If removed effectively, there is a problem that a large amount of replacement fluid is required.
To increase the removal rate, increase the pore size of the membrane.
However, if the pores are slightly larger, useful proteins
Of albumin, and the pore size is controlled
Can not achieve effective selective removal of medium molecular weight substances
It is the current situation.   On the other hand, recently, β₂-Microglobulin adsorbent is
Nos. 62-204761 and 62-240068.
ing. (Problems to be solved by the invention)   The object of the present invention is based on the therapeutic polymer membrane technology as described above.
In general, it is widely available and has a medium molecular weight
Substances, especially β₂-Select microglobulin with high efficiency
Adsorption, and non-specific adsorption, especially albumin
No, no rehydration required, safe and sterile
Operation is easy, and whole blood or plasma
Providing adsorbents suitable for body fluid purification or regeneration
Is what you do. (Means to solve the problem)   The present inventors have conducted intensive studies for the above purpose.
As a result, certain polyamino acids and / or polysaccharides and / or
Or an adsorbent having a synthetic polymer electrolyte on its surface is β₂
-Selective and surprisingly high microglobulin
Adsorption was found to be efficient. About this,
After a thorough study of polyamino acids, polysaccharides and
The molecular weight of the electrolyte body of the synthetic polymer and the electrolyte body has
Relationship with the number of carbon atoms, hydrophilic groups, and ionic groups in the skeletal structure
Only if satisfies certain conditions, β₂-Microglob
It is clear that phosphorus is adsorbed selectively and with high efficiency.
As a result, the present invention has been completed.   That is, the present invention provides a method for producing a polymer having a molecular weight on the surface of a water-insoluble carrier.
Is 1000 or more, and X represented by the formula (1) is 2.
Polyamino acids and / or polysaccharides exhibiting 0 <X and / or
Or characterized by having a synthetic polymer electrolyte body
β₂-A microglobulin adsorbent, A + B = X Equation (1)   The water-insoluble carrier has a contact angle of at least 20 degrees or more.
At least two of a water-insoluble material and a blood-compatible polymer
The above β having a layer structure₂-With microglobulin adsorbent
is there.   Β of the present invention₂-The adsorbent for microglobulin is
The particular polyamino acids and / or polysaccharides mentioned and / or
Or an adsorbent having a synthetic polymer electrolyte on its surface.
Is not limited to the following examples, but
In other words, the specific polyamino acids and / or
Or polysaccharide and / or synthetic polymer electrolytes (these
Is called a ligand), but some method (for example,
Water-insoluble carrier by lorhydrin method, cyanogen halide method)
What is fixed to the surface of is preferred. Furthermore,
Defined ligand makes the surface of the water-insoluble carrier
It is better to have a long, elongated form, rather than covering
New The reason is that β₂-Contact with microglobulin
The surface area of the adsorbent of the present invention that can be
This allows the adsorbent of the present invention to be more efficient
Β₂-Be able to adsorb microglobulin
And the like.   Β in the present invention₂-Microglobulin is usually
Β measured by enzyme immunoassay in floor examination₂-Micro
It is a globulin, but has the following physical properties
You. Sedimentation constant 1.6S Partial specific volume 0.72 ~ 0.73ml / g Molecular weight 11000-12000 Nitrogen content 16-17%   Β of the present invention₂-For microglobulin, β
₂-With microglobulin itself and other proteins
Comprising a complex of β₂-Amino acid sequence of microglobulin
Includes some mutations in the sequence.   In the present invention, X is a polyamide on the surface of a water-insoluble carrier.
Identifying electrolytes of no acids, polysaccharides and synthetic polymers
And electrolytes of polyamino acids, polysaccharides and synthetic polymers
Number of carbon atoms, number of hydrophilic groups, and cations in the skeleton structure contained in the body
The relational expression between the number of anionic groups and the number of anionic groups is defined by the following equation.
Say what was justified. A + B = X Equation (1)   In this formula, the number of carbon atoms in the skeletal structure refers to polyamino acids, polysaccharides,
Structural formulas of main and synthetic polymer electrolytes (backbone and side)
Chain), the carbon of the characteristic group having a negative charge,
That is, all carbons except the carboxyl carbon are referred to.
U. Here, the carbon in the carboxyl group was removed
Boxyl group is hydrophilic, mainly showing only the effect of negative charge
Because it is. Carbon number of characteristic group other than carboxyl group,
That is, an alkoxyl group, an aldehyde group, an alkoxy group
Carbon included in the carbonyl group, amide, etc. is counted.   In this formula, the number of hydrophilic groups refers to polyamino acids, polysaccharides,
The chemical structural formula (main chain and side chain) of the polymer electrolyte body is
Among the characteristic groups shown, polar properties that interact strongly with water
Say all of the group. For example, the carboxyl group COO^-, COO
H, COONa), sulfonic acid group (SO_Three ^-, SO_ThreeH, SO_ThreeNa), phosphoric acid
Group (PO_Three ^-, PO_ThreeH, PO_ThreeNa) primary, secondary, tertiary, quaternary
And a hydroxyl group (OH).   In this formula, the number of cationic groups refers to polyamino acids, polysaccharides and
Structural formulas of the polymer body and the synthetic polymer electrolyte body (backbone and side)
Chains, primary, secondary, tertiary and quaternary amines
Like neutral group, etc. in neutral electrolytes such as blood and body fluids.
Refers to all of the characteristic groups that exhibit charge.   In this formula, the number of anionic groups refers to polyamino acids, polysaccharides and
Structural formulas of the polymer body and the synthetic polymer electrolyte body (backbone and side)
Carboxyl group (COO)^-, COOH,
COONa), sulfonic acid group (SO_Three ^-, SO_ThreeH, SO_ThreeNa) phosphate group
(PO_Three ^-, PO_ThreeH, PO_ThreeIn blood, body fluid, etc.
Refers to all of the characteristic groups that exhibit a negative charge in a neutral electrolyte solution.   Shown by this formula Is the number of hydrophilic groups subtracted from the number of carbon atoms in the skeletal structure shown above.
Is the ratio of the number of hydrophilic groups Is the number of anionic groups from the number of cationic groups shown above.
It is the ratio of the absolute value of the subtracted value to the number of hydrophilic groups, and A + B = X
Is the sum of the two.   In the present invention, X needs to be larger than 2.
Preferably in the range of 3 to 50, more preferably 4 to
It is in the range of 25, more preferably in the range of 5 to 15. Book
When X of the invention is 2 or less, polyamino acid, polysaccharide, synthetic
Polymer electrolyte and β₂-Interaction with microglobulin
Weakened, surfaced with polyamino acids, polysaccharides, synthetic polymers
Β of the adsorbent₂-Microglobulin adsorption capacity
Lower.   These are the synergies of the A and B terms of X defined above.
This is considered to be a strategic effect. Item A is based on the number of carbon atoms in the skeletal structure.
It is the ratio of the number of aqueous groups minus the number of hydrophilic groups,
Β-amino acids, polysaccharides and synthetic polymer electrolytes₂−Miku
A term that expresses the hydrophobic interaction force acting with the globulin
it is conceivable that. Item B is anionic to cationic groups
The ratio of the absolute value of the value obtained by subtracting the number of bases to the number of hydrophilic groups,
Amino acids, polysaccharides and synthetic polymer electrolytes and β₂−Mi
Represents the ionic interaction force acting with globulin
Term. Synergistic effect of A and B,
That is, the hydrophobic interaction force in item A and the ionic interaction force in item B
By acting synergistically, β₂-Microglob
Phosphorus and polyamino acids, polysaccharides and electrolytes of synthetic polymers
Interaction with polyamino
Β of adsorbent having acid, polysaccharide and synthetic polymer on the surface₂−Mi
Chromoglobulin adsorption capacity is increased, and albumin
Adsorption selectivity for substances other than the target substance such as
it is conceivable that.   In the present invention, polyamino acids, polysaccharides and synthetic polymers
The electrolyte body of the child has a molecular weight of 1000 or more, blood,
Characteristics showing positive or negative charge in neutral electrolyte such as body fluid
One or more groups in one molecule, and X shown above is
2.0 <X for polyamino acids, polysaccharides and synthetic polymers
Say everything. The electrolyte body of the polyamino acid of the present invention is exemplified.
To make polyaspartic acid, polyglutamic acid,
Gin, polyhydroxylysine, etc.
As a characteristic group having, a carboxyl group, primary, secondary, etc.
Polymer of aliphatic amino acid containing amino group, polyg
Glutamic acid / alanine, polyglutamic acid / lysine,
Riaspartic acid / lysine, polylysine / phenylalan
Nin, polylysine / tryptophan, polyglutamic acid
・ Lysine / Tyrosine, Polyglutamic acid / Tyrosine / A
As a characteristic group having a negative charge or a positive charge such as Lanin,
Fatty acids containing carboxyl, primary, secondary, etc. amino groups
A copolymer of an aliphatic amino acid and an aliphatic amino acid, or
Represents aliphatic amino acids and other amino acids such as aromatic amino acids.
Having a positive charge such as polyhistidine.
Contains tertiary, quaternary, etc. amino groups
Polymer of heterocyclic amino acid, polyhistidine
Has a positive charge such as polyhistidine, tryptophan, etc.
Tertiary and quaternary amino groups
Copolymers of cyclic and heterocyclic amino acids, poly
Histidine / phenylalanine, polyhistidine / tyro
Heterocyclic amino acids such as syn and other groups such as aromatic amino acids
Copolymers with amino acids can be mentioned. Among these
Then, polyglutamic acid / alanine, polyglutamic acid
・ Tyrosine, polyglutamic acid, alanine, tyrosine, etc.
Contains a carboxyl group as a characteristic group having a negative charge
Copolymerization of Aliphatic Amino Acids with Aliphatic Amino Acids
Aroma with body and aliphatic amino acids and benzene ring
Copolymers with group amino acids give particularly favorable results.   Examples of the polysaccharide electrolyte of the present invention include xylan,
Milas, glycogen, dextran, pullulan, a
Homopolysaccharides such as galose, mannan, inulin and chitin
A positively charged characteristic group such as a dimethylaminoethyl group
Introduced, chondroitin, chondroitin sulfate,
Heteropolysaccharides such as hyaluronic acid, heparin and gum arabic
Dimethylaminoethyl and diethylaminoethyl groups
What introduces a characteristic group with a positive charge such as
Can be. Among these, dimethylaminoethyl-de
Kistran, like diethylaminoethyl-dextran
As described above, homopolysaccharides such as dextran
Features having a positive charge such as a tyl group and a diethylaminoethyl group
Those having a functional group give particularly favorable results.   Examples of the synthetic polymer electrolyte of the present invention include polymers.
Negative charge of tacrylic acid, polystyrene-sulfonic acid, etc.
Carboxyl groups and sulfonic groups as characteristic groups having
Polymer of vinyl compound contained, polypropylene
Crylic acid, polybutadiene / acrylonitrile / acrylic
Luic acid, polyethylene methacrylic acid, polymethyl meth
Acrylate / methacrylic acid, polyacrylonitrile
・ Maleic anhydride, polybutadiene ・ Maleic anhydride,
Polymethyl vinyl ether to maleic anhydride, polystyrene
Has a negative charge with vinyl compounds such as len and maleic anhydride.
Vinyl group containing carboxyl group
Copolymers with compounds can be mentioned.   Among these, polystyrene-sulfonic acid, police
Has a negative charge as shown in Tylene / maleic anhydride
Vinyl containing a characteristic group and having a benzene ring
Polymer of compound and vinyl system having benzene ring
Compound containing a compound and a characteristic group having a negative charge
Give particularly favorable results.   Further, polyvinyl pyridine, poly (N-alkyl-4)
-Vinylpyridinium chloride), polyvinylbenzyl
Features having a positive charge such as trimethylammonium chloride
Contains tertiary and quaternary amino groups
And vinyl polymer having a benzene ring
Can also be usefully used.   Furthermore, dimethylaminoethyl polymethacrylate
Methacrylate, polystyrene sulfonic acid, N-Al
Kill-4-vinylpyridinium chloride, poly-4-bi
Nyl-N-alkylsulfonates such as pyridinium
Characteristic groups with positive and negative charges
Polymers or copolymers of vinyl compounds contained in
Can be used for   The molecular weight specified in the present invention is on the surface of the water-insoluble carrier.
Electrolytes of polyamino acids, polysaccharides and synthetic polymers
The principle of light scattering [(for example,
Course 19, Polymer Chemistry II, P514-518, Chemical Society of Japan, Maruzen
(Published September 20, 1978)]
Weight average molecular weight. The molecular weight of the present invention is 1000 or less.
Must be above, preferably between 2000 and 1,000,
000, more preferably in the range 5000 to 500,000,
More preferably, it is in the range of 10,000 to 200,000.   If the molecular weight is less than 1000, it will be on the surface of the water-insoluble carrier.
Of polyamino acids, polysaccharides, and synthetic polymer electrolytes
The length becomes short, and enough β₂-Micro gro
Bling cannot be absorbed. Efficient and sufficient amount
Β₂-Polyamino acids for adsorbing microglobulins
The molecular weight of the electrolytes of acids, polysaccharides and synthetic polymers must be 1000 or more.
is necessary.   In the present invention, the water-insoluble carrier has a molecular weight of 10
00 or more, and X shown above represents 2.0 <X
Solid electrolytes of polyamino acids, polysaccharides and synthetic polymers.
If it can be determined, cellulosic, vinyl polymer,
Cryamide, polyhydroxyethyl methyl acryle
Organic polymer compounds such as glass, glass, silica, etc.
Or all inorganic compounds can be used, but β₂-Micro
Selectively adsorbs globulin with higher efficiency and
When used in whole blood with blood cells such as platelets
The above-mentioned water-insoluble carrier has a contact angle of at least 20 degrees or more.
At least the water-insoluble material and the blood-compatible polymer
It preferably has a two-layer structure.   The above contact angle refers to air bubbles on a solid surface in water
Is the contact angle of W.C.Hamilton, J. Colloid Interface
  Sci., 40, 219-222 (1972) [Double Sea Hamilt
, Journal of Colloid Interface
Science, 40, 219-222 (1972)] J.D.Andrade, J.Poly
m. Sci. Polym. Symp., 66, 313-336 (1979) [J.
-Andrade, Journal of Polymer Sai
Ens Polymer Symposium, 66, 313-336 (197
9) Contact measured according to the principle and method described in
Say the antennae. Solids in air, well known in the past
The method of measuring the contact angle of a droplet on a surface is
The value of the contact angle changes with the passage of time,
It is difficult to adopt. In addition, the sample
An illuminated molded product was manufactured, and the measurement temperature of the contact angle was 25 ° C.
10 times or more, and the average value is taken as the value of the contact angle of the material.
did.   In the present invention, a water-insoluble material having a contact angle of 20 degrees or more
As the material, the contact angle measured by the method shown above was 20 degrees
If it is above, all inorganic compounds and organic polymer compounds
But the safety of eluate etc. as a body fluid purification material
Organic polymer materials are preferred from the viewpoint of
It is.   As an organic polymer material, its β₂-Microglobuly
The contact angle is preferably 20 degrees or more due to the affinity for adsorption with
More preferably, a material having a temperature of 30 degrees or more, particularly preferably a temperature of 40 degrees or more.
Fees are used.   Preferred organic polymer materials include polyethylene and polyethylene.
Polio such as propylene and polytetrafluoroethylene
Lefin compound, polystyrene, polymethacrylate
Vinyl compounds such as esters and polyacrylate esters
Products, polyamide compounds such as nylon 6, 66,
Polyester compounds such as polyethylene terephthalate
And the like. Methacrylate
Stele, acrylate ester, styrene and polystyrene
Homopolymers, comonomer and cross-linking agent
Are preferably used. Especially methyl meta
Crosslinking weight mainly composed of acrylate or styrene
Coalesced particles are preferably used. Known crosslinking agents
Any cross-linking agent can be used, but is exemplified.
And divinylbenzene, ethylene glycol di (meth)
Acrylate, polyethylene glycol di (meth) ac
Related may be mentioned.   In the present invention, the blood-compatible polymer has a molecular weight
Is 1000 or more, and X shown above is 2.0 <X
Polyamino acids, polysaccharides and synthetic polymer electrolytes showing
Can be fixed, all commonly known blood-compatible materials
Includes, but prevents the generation of fine particles, that is, water-insoluble
From the ease of coating on the material, safety and sterility, (meta)
Acrylic ester polymer, acrylamide polymer
, Polyvinyl pyrrolidone polymer, polyvinyl alcohol
Polymer, ethylene-vinyl alcohol polymer,
Ethylene-vinyl acetate polymer, cellulose nitrate, and
And gelatin.   Prevent the generation of fine particles and further improve blood compatibility
For blood-compatible polymers, especially nitrogen-containing basic
A polymer having a functional group is preferably used.   The above "nitrogen-containing basic functional group" refers to an acidic aqueous solution
Functional group that has a positive charge on the nitrogen atom and can be a cation
It is. Such functional groups include primary amino groups,
Secondary amino group, tertiary amino group, quaternary ammonium group
And nitrogen-containing aromatic rings such as pyridyl and imidazolinyl groups
And the like. Therefore, the inclusions used in the present invention
Examples of the polymer having a nitrogen basic functional group include, for example,
Nylamine; 2-vinylpyridine, 4-vinylpyridine,
2-methyl-5-vinyl pyridine, 4-vinyl imidazo
, N-vinyl-2-ethylimidazole, N-vinyl
Of nitrogen-containing aromatic compounds such as 2-methylimidazole
Vinyl derivative; dimethylaminoethyl (meth) acryle
, Diethylaminoethyl (meth) acrylate, di
Methylaminopropyl (meth) acrylate, 3-dimethyl
Tylamino-2-hydroxypropyl (meth) acry
Acrylic acid and methacrylic acid derivatives such as
Tylaminoethyl (meth) acrylamide, N-die
Acrylics such as tylaminoethyl (meth) acrylamide
Amide and methacrylamide derivatives; p-dimethyl
Tylaminomethylstyrene, p-diethylaminoethyl
Styrene derivatives such as styrene; and the above vinyl compounds
With a quaternary ammonium salt by an alkyl halide or the like
And polymers containing the above-mentioned derivatives.   Of these, particularly preferred is diethylaminoethyl
(Meth) acrylate, diethylaminoethyl (meth)
Acrylate, p-dimethylaminomethylstyrene, p
-A polymer containing diethylaminoethylstyrene or the like
No.   Furthermore, it has a nitrogen-containing basic functional group used in the present invention.
Polymer has a vinyl compound and a nitrogen-containing basic functional group.
A copolymer with a monomer having a nitrogen content of 0.1 is preferred.
It is preferably from 0.05 to 3.5% by weight. In addition, nitrogen
Amounts between 0.1 and 2.5% by weight give better results
You. The nitrogen content here means the nitrogen atom in the above functional group
% In the prepolymer.   As the above vinyl compound, 2-hydroxyethyl
Methacrylate, methyl (meth) acrylate, ethyl
(Meth) acrylate, n-butyl (meth) acryle
(Meth) acrylates such as
Acrylamide, N-methyl (meth) acrylamide, etc.
Amides, N-vinylpyrrolidone, vinyl acetate, styrene
And the like.   Furthermore, it has a vinyl compound and a nitrogen-containing basic functional group.
Examples of copolymers with monomers include block copolymers and copolymers.
Graft copolymer, random copolymer, etc.
The copolymer and block copolymer have an average length of 100 長 to 100μ.
What has a microdomain structure is
Is more preferable.   Further, the vinyl compound and the nitrogen-containing basic functional group
The copolymer with the monomer having is a random copolymer
Is difficult to manufacture copolymer, stability of two-layer structure, blood compatibility
It is most preferable from the viewpoint of sex.   In the present invention, the two-layer structure refers to the water-insoluble material of the present invention.
Material is coated with a blood-compatible polymer by a generally known method.
The first layer is a water-insoluble material and the second layer is a blood-compatible polymer.
Say that structure. The second or higher layer is the same or different
Multi-layer coating by coating with blood-compatible polymer
It can be structured. To have such a structure
Can prevent the generation of fine particles of water-insoluble material,
And other safety issues. Select blood compatible polymer
To prevent the generation of fine particles and improve blood compatibility
It can be further improved. Furthermore, a multi-layer structure
Prevents the generation of fine particles and improves blood
Improves protein compatibility and absorbs useful proteins such as albumin.
Without wearing, β₂-Affinity with microglobulin
It can be further improved.   Hereinafter, the method for producing the adsorbent of the present invention,
(1) Polyamino acids, polysaccharides and synthetic polymers are converted to water-insoluble carriers
(2) Water-insoluble material and blood compatible polymerization
Of water-insoluble carrier consisting of at least two-layer structure with body
Although the method and conditions are exemplified, the present invention is limited to this example.
Of course, it is not specified. (1) In the present invention, polyamino acids, polysaccharides, synthetic polymers
To fix the spores to a water-insoluble carrier   The shape of the water-insoluble carrier is particulate, fibrous, hollow fiber,
Any known shape such as a film shape can be used.
Retention and adsorption of polyamino acids, polysaccharides and synthetic polymers of the invention
From the viewpoint of material handling, particles and fibrous
preferable.   The average particle size of the spherical or particulate carrier is 25 ~ 2500μm.
Can be used, but its specific surface area (adsorption as adsorbent)
Capacity) and the distribution of bodily fluids, especially those with 50 ~ 1500μm
preferable.   The specific surface area of the carrier is 5m^Two/ g or more is preferred, 55m^Two/ g or more
Is desirable.   As the particulate carrier, porous particles are preferred. The present invention
The porous particles used in the above process are provided on the surface of the porous particles of the present invention.
It can immobilize amino acids, polysaccharides, synthetic polymers,
Furthermore, β₂-Increase the efficiency of microglobulin adsorption
To the inside of the pores of the porous particles₂-Microglobuly
It is preferable that the pores of the porous particles be filled.
The uniform hole diameter is preferably in the range of 20 mm to 5000 mm.
Good.   If a fibrous carrier is used, the fiber diameter should be 0.02 denier.
To 10 denier, more preferably 0.1 denier
It is better to have one in the range of 5 denier. Large fiber diameter
If too long, β₂-Absorption of microglobulin compounds
If the loading and adsorption speed decrease or are too small
Easily causes coagulation activation, blood cell adhesion, and clogging
No. As the fibrous carrier to be used, regenerated cellulose fiber
Known fibers such as fibers, nylon, acrylic, polyester, etc.
It can be used generally.   In the present invention, polyamino acids, polysaccharides, synthetic polymers
The method of immobilizing on a water-insoluble carrier can be either covalent or ionic.
, Physical adsorption, embedding or precipitation on the surface of water-insoluble carrier
Any known method such as insolubilization can be used
Are immobilized by covalent bonds,
It is preferable to use it after being solubilized. Therefore, usually fixed
Enzyme, used in affinity chromatography
Uses known carrier activation methods and ligand binding methods
Can be.   Illustrative activation methods include the cyanogen halide method,
Chlorhydrin method, bisepoxide method, trihalide
Azine method, bromoacetyl bromide method, ethyl chloropho
Lumat method, 1,1'-carbonyldiimidazole method, etc.
Can be mentioned. The activation method of the present invention comprises the steps of:
Amino, hydroxyl, carboxyl, thiol, etc.
Substitution and / or addition with nucleophilic reactive group having active hydrogen
It is sufficient that the reaction can be performed, and the reaction is not limited to the above examples.
However, considering chemical stability and thermal stability, etc.
Preference is given to the method using oxides, especially epichlorohydrido.
Method is recommended.   Polyamino acids, polysaccharides and synthetics used in the present invention as carriers
It does not matter if two or more kinds of polymers are combined.   As described above, in the present invention, polyamino acids, polysaccharides, synthetic
As a method of fixing molecules to a water-insoluble carrier, a water-insoluble carrier is used.
After activating the body, polyamino acids, polysaccharides, synthetic polymers
Has been described in detail in the method of combining, the present invention,
It is not limited to this. For example, polyamino
Acid, polysaccharide, a polymerizable monomer having a synthetic polymer monomer or
Method of polymerizing (copolymerizing) using a cross-linking agent, cross-linked polymer particles
Polyamino acids, polysaccharides, and synthesis at the point of further post-crosslinking
A method using a crosslinking agent having a polymer monomer is also used.
Can be In addition, polyamino acids,
After coating the polysaccharide or synthetic polymer with a bondable polymer,
Methods for bonding amino acids, polysaccharides, and synthetic polymers
Polymers containing amino acid, polysaccharide and synthetic high polymer monomers
A method of coating with an insoluble substance can also be used. That
If necessary, post-crosslink the polymer used for coating.
Can also be. In addition, polyamino acids, polysaccharides, and synthetic polymers
It is also possible to adopt a method of binding to a carrier after activation.
Wear.   Also, if necessary, a water-insoluble carrier and a polyamino acid,
A molecule of any length between the sugar and the synthetic polymer (spacer
-), For example, aminoethyl group, aminopentyl group,
It is also possible to introduce a minooctyl group, aminododecyl group, etc.
it can. That is, the present invention relates to polyamino acids, polysaccharides,
The effect is achieved by the presence of the synthetic polymer on the surface of the adsorbent.
And does not depend on the manufacturing method.
No. (2) At least a water-insoluble material and a blood-compatible polymer
Manufacturing method and conditions of adsorbent with two-layer structure   All known coating methods such as spraying and dipping are used.
It is. Here, the details of the immersion method will be described.   Dissolve the blood compatible polymer in the polymer solvent beforehand.
Make a solution. The polymer solvent dissolves the polymer uniformly.
Dissolve and impregnate or apply polymer to water-insoluble material
Is a solvent that facilitates
Can be used as long as the solvent can dissolve the polymer.
Noh. Suitable solvents are easy to remove and remain in traces
Should be selected in consideration of safety in the event of
In the present invention, such solvents include methanol and ethanol.
Lower alcohols such as knol, acetone and dimethyl
Formamide and mixtures of these with water are preferred
No.   Water-insoluble materials of various sphere diameters are added to this solution under a nitrogen atmosphere.
After soaking at room temperature for about 5 minutes (with occasional stirring),
Aspirate excess solution on the filter and then transfer
While balancing the amount of nitrogen gas and the amount of sucked nitrogen gas,
Dry with nitrogen on a glass filter for 20 minutes. Then true
Dry for 24 hours at room temperature and above 755mmHg in an air dryer.
Dry.   When producing two or more layers of adsorbent,
Bilayer structure of manufactured water-insoluble material and blood compatible polymer
Adsorbent consisting of water-insoluble material,
Achieved by repeating the operation. Used here
Blood compatible polymer used in the second layer
The same type or different types of the hydrophilic polymer can be used.   Hereinafter, β of the present invention₂-Use of microglobulin adsorbent
The present invention is limited to this example.
Of course it is not something that is done.   The adsorbent of the present invention may be used alone,
You may mix and use with a body fluid purifying material or a storage tank. other
As a body fluid purifying material, the activity used in adsorption-type artificial kidneys
Used for charcoal, dialysis type artificial kidney, filtration type artificial kidney
A hollow fiber membrane and a flat membrane can be exemplified. This allows adsorption
Broader clinical effects can be expected due to the synergistic effect of the materials.
When used for extracorporeal circulation, the adsorbent volume is about 50-600 ml
Is appropriate.   When the adsorbent of the present invention is used in extracorporeal circulation,
There are three ways. For one thing, take it out of the body
Blood directly passed through the device, a method of purifying,
Second, the blood removed from the body is centrifuged or
Uses a membrane-type plasma separator to separate plasma components and blood cell components.
After separation, the plasma components were passed through the device and purified
After that, it is a method of returning to the body together with blood cell components,
The blood taken from the body is adsorbed artificial kidney,
Through a body fluid purifier such as artificial kidneys and filtration artificial kidneys
After passing through the device to purify. vice versa
After passing the blood through the device, the blood is passed through a body fluid purifier.
You may.   In addition, regarding the passage speed of blood or plasma,
Since the adsorption efficiency of the adsorbent is very high, the viscosity of the adsorbent
And the filling degree can be lowered,
Gives a high passage speed regardless of the shape of the material layer
Can be Therefore, it is necessary to process a large amount of body fluid
it can.   As a method for passing bodily fluids such as blood and plasma,
Continuous according to the needs of
The liquid may be passed continuously or may be used continuously.
No.   The adsorbent of the present invention, as described above,
β₂-High efficiency and specific adsorption and removal of microglobulin
Leave and convenient and secure.   The present invention purifies and regenerates body fluids such as autologous blood and plasma
It can be used for general use, and patients with renal failure or malignant tumors
Increased in body fluids of patients, carpal tunnel syndrome, amyloidosis
Sources, fingering / shoulder / knee arthropathy, pruritus, bone disorders
Causative medium molecular weight substance, amyloid protein, especially β
₂− Effective and safe use for microglobulin adsorption and removal
It can be used. (Example)   Next, the present invention will be described in more detail by way of examples. Example 1   Β of the present invention₂-On microglobulin adsorbent surface
Polyamino acid and / or polysaccharide and / or synthetic high
Molecule and β₂-To check the binding ability to microglobulin
Utilizes enzyme immunoassays commonly used in clinical tests
And plastic plate (elizer plate) table
Β fixed to the surface₂-Microglobulin and polyamino
The ability to bind to acids, polysaccharides and synthetic polymers was evaluated. Less than
The evaluation method and conditions are described below. (1) β on the surface of the ELISA plate₂-Microglob
Fixing phosphorus   Β obtained by purification from human urine₂-Microglobuly
(Sigma, USA) with PBS (phosphate buffered physiological diet)
Salt water) and 10μg β₂-Microglobulin / ml PBS
A solution was made. 100 μl of this solution was used for enzyme immunoassay.
Plate (product name IMMU)
Ron, 600, West Germany, Greiner) at 4 ° C, 24
Left for hours. (2) β₂-Free microglobulin is not fixed
Of bovine serum albumin on the surface of a fresh ELISA plate
Fixed (blocking operation)   Free ELISA plate surface and polyamino acids
In order to suppress the binding to sugar and synthetic polymer, the following operation
do.   10 μg β added₂-Microglobulin solution 100μ
After aspiration and removal, 0.5% bovine serum albumin PBS solution 200
Add μ to the ELISA plate and leave at 25 ℃ for 2 hours
I do. (3) Free and not fixed to the ELISA plate
Removal of bovine serum albumin   Bovine serum albumin solution from the above ELISA plate
After removing by suction, 100 μl of PBS is added. Then PBS
Is removed by suction, and 100 μl of PBS is added again. Add PBS
Then, the operation of removing by suction is called a washing operation.
Repeat the washing operation three times, and fix the plate to the ELISA plate.
Remove any unreacted bovine serum albumin. (4) Polyamino acids, polysaccharides, synthetic polymers and ELISA
Β fixed on the rate surface₂-Connection with microglobulin
Combination   Dissolve polyamino acids, polysaccharides and synthetic polymers in PBS and add 0.1
Make up each mg / ml PBS solution. After the operation of (3),
Aspirate the PBS remaining on the ELISA plate and remove
0.1mg / ml PBS solution of lamino acid, polysaccharide, synthetic polymer 100μ
Was added to each ELISA plate, and the mixture was added at 37 ° C, 2
Leave for a time. (5) β fixed on the surface of the ELISA plate₂−Miku
Free polyamino acids not bound to logoglobulin,
Removal of polysaccharides and synthetic polymers Suction removal of PBS solution of polyamino acids, polysaccharides and synthetic polymers
After that, the washing operation with PBS shown in (3) was performed three times.
Yes, β₂-Free and not bound to microglobulin
Removes polyamino acids, polysaccharides and synthetic polymers. (6) It is fixed on the surface of the ELISA plate and
Β not bound to amino acids, polysaccharides and synthetic polymers₂−
Microglobulin measurement   This operation is similar to the usual enzyme immunoassay.
Now. (A) After the operation of (5), the anti-human β₂-Microglobulin
Add 100μ of rabbit antibody to the ELISA plate and add
After leaving at ℃ for 1 hour, the same PBS washing as in (3) is performed.
By this operation, polyamino acids, polysaccharides and synthetic polymers are linked.
Β on the surface of the unmixed ELISA plate₂-Micro
Globulin and anti-human β₂-Antigen and antibody against microglobulin
The reaction takes place. (B) Next, enzyme-labeled (peroxidase-labeled)
Add 100μ of anti-rabbit IgG (Vectorstin ABC kit)
In addition, anti-human β₂-Microglobulin rabbit antibody and anti-rabbit
Perform antigen-antibody with protein IgG. (C) Next, 2,2'-azino-bis (3-ethyl bench
Azoline) -6-sulfonic acid (molecular weight 514, manufactured by Wako Pure Chemical Industries)
And an aqueous solution of hydrogen peroxide
Let it develop color. After color development, measure absorbance at 405nm wavelength
I do.   The value of this absorbance is β₂-Binds microglobulin
Anti-human β₂-Correlate with the amount of microglobulin antibody. Ma
This review of polyamino acids, polysaccharides, and synthetic polymers themselves
Considering the effect on multivalent system, β₂−Mi
Perform steps (2) to (6) without adding globulin.
Made separately.   In addition, polyamino acids, polysaccharides, and synthetic polymers are
No addition to the plate, ie (1), (2),
Only the operations (3) and (6) were separately performed.   By performing the above operations, the ELISA plate
Immobilized on the surface and polyamino acids, polysaccharides, synthetic
Β not combined with child₂-Percentage of microglobulin
(Y) [C]; when all the operations of (1) to (5) are performed
Anti-human β₂-Microglobulin antibody binding amount [D]; β in the operation of (1)₂-Eliminate microglobulin
Perform steps (2) to (5) without adding to the plate.
Anti-human β when all is done₂-Microglobulin antibody binding
Combined quantity [E]; do not add polyamino acids, polysaccharides, or synthetic polymers.
That is, without performing the operations of (4) and (5),
(1), (2), (3), when performing the operation of (6)
Anti-human β₂-Microglobulin antibody binding amount Then Can be represented by   That is, the smaller the Y, the more the ELISA plate
Β fixed on the surface of₂-Microglobulin and polyamide
High binding capacity with no acids, polysaccharides and synthetic polymers
Become.   Tables 1, 2, and 3 show the results obtained using this evaluation method.
Show. Comparative Example 1   The following samples were evaluated using the same evaluation method as in Example 1.
did. The results are shown in Tables 4 and 5.   The results of Example 1 and Comparative Example 1 are collectively graphed.
FIG. 1 shows the result. The vertical axis is Y and the horizontal axis is X. this
The results show that X is 2.0, polyamino acids showing <X, polysaccharides and
And synthetic polymer is β₂-Strong binding ability with microglobulin
It turns out that it shows power. Example 2   The following samples were evaluated using the same evaluation method as in Example 1.
did. The results are shown in Table 6.Comparative Example 2   The following samples were evaluated using the same evaluation method as in Example 1.
did. The results are shown in Table 7.   The results of Example 2 and Comparative Example 2 are collectively graphed.
FIG. 2 shows the result. The vertical axis is Y and the horizontal axis is polystyrene ~
The molecular weight of sodium sulfonate was used. From this result, the molecular weight
Good β over 1000₂-Shows microglobulin binding ability
You can see that Example 3   Toyopearl HW-60 (Toyo Soda Industry Co., Ltd., particle size 30 ~
60 μm), washed with water, dehydrated, and treated with dimethyl sulfoxide.
Suspend in. Next, the dimethyl sulfoxide is removed,
The operation of suspending HW-60 in dimethyl sulfoxide again
Repeat. 30 ml of HW-65 obtained after this operation was
Suspend in 36 ml of tyl sulfoxide and add
Add 2 ml of drin and 3.0 ml of 50% sodium hydroxide, and at 30 ° C
The activation reaction was performed with stirring for 5 hours. After the reaction,
Washed with ethanol, washed with water and suction dehydrated. The obtained activity
30 ml of sexualized HW-60 is divided into 10 ml, and each HW-60 is
-L-aspartic acid [molecular weight 6,000] solution (3 mg / ml, pH
9.0), PL-glutamic acid / alanine [molecular weight 145,0
00, glutamic acid: alanine = 1: 1] solution (3 mg / ml, pH 9.
0), polystyrene / maleic anhydride [molecular weight 50,000,
Tylene: maleic anhydride = 1: 1] solution (3mg / ml, pH4.5) 5
Suspended in 0 ml and immobilization reaction with stirring at 50 ° C for 14 hours
Was performed. Then, 0.1M sodium carbonate buffer,
Washed alternately with 0.1M sodium citrate buffer
Thereafter, the plate was sufficiently washed with PBS and physiological saline to obtain an adsorbent. P
-L-aspartic acid, PL-glutamic acid / alani
Each primary amino group was converted to 4-phenyl
Spiro [furan-2 (3H), 1'-phthalane] -3,3'-
Zeon (“Fulrum "Roche)
Issued. In addition, the retention amount of polystyrene and maleic anhydride
Measures the absorption of the benzene ring at a wavelength of 259.5 nm and calculates
did.   The adsorption experiment was performed using the adsorbent and pre-fixation
After mixing HW-60 at a volume ratio of 6: 1, shake at 37 ° C. for 1 hour,
Β in plasma before and after₂-Microglobulin and albumin
Quantified. β₂-Microglobulin is ria method, albumin
The BCG method was used. Table 8 shows the results. Comparative Example 3   Activated in the same manner as in Example 3 to obtain P-L-aspartic acid
[Molecular weight 600] solution (3mg / ml, pH 9.0), polyacrylic acid
[Molecular weight 5,000] solution (3mg / ml, pH3.0)
The reaction was performed, and the adsorption experiment was performed in the same manner as in the example. Conclusion
The results are shown in Table 9.   From the results of Example 3 and Comparative Example, the molecular weight was 1000 or more.
Wherein X is 2.0 <X on the surface
The material is β₂-High adsorption capacity for microglobulin
And it has adsorption selectivity for albumin
Recognize. Example 4   Methyl methacrylate to divinyl as water-insoluble material
Sheet of rubenzene copolymer (80: 20% by weight) and 420
~ 800μ particles are produced and the contact angle of air bubbles in water
Was measured. Next, 2-hydroxyethyl methacryle
-Diethylaminoethyl methacrylate copolymer
Make a 2% wt / V methanol solution of
On the other hand, after immersing 30 ml of the above particles for 5 minutes (with occasional stirring)
A), aspirate off excess solution on a glass filter
The balance between the amount of nitrogen gas to be sent in and the amount of nitrogen gas to be suctioned.
While drying on a glass filter for 20 minutes.
Then, in a vacuum dryer, room temperature, 755mmHg or more conditions
For 24 hours. By this operation, methyl methacrylate
-Divinylbenzene copolymer and 2-hydroxye
Cyl methacrylate to diethylaminoethyl methac
Thus, a water-insoluble carrier having a two-layer structure composed of a relate is obtained.   This water-insoluble carrier is heat-treated at 125 ° C for 45 minutes and ethanol
And then wash with water. Then dehydrate and dimethyl
Suspend in sulfoxide. Next, dimethyl sulfoxy
And remove it again in water-insoluble dimethyl sulfoxide.
Repeat the operation of suspending the body. Water obtained after this operation
Suspend 30 ml of insoluble carrier in 36 ml of dimethyl sulfoxide
Then add 24ml of epichlorohydrin, 50% sodium hydroxide
Add 3.0 ml of lithium and activate while stirring at 30 ° C for 5 hours
The reaction was performed. After the reaction, wash with methanol and water
And dehydrated by suction.   30 ml of the obtained activated water-insoluble carrier is mixed with polystyrene
Water maleic acid [molecular weight 50,000, styrene: maleic anhydride
Acid = 1: 1] solution (3 mg / ml, pH 4.5)
Water-soluble carbodiimide [1-ethyl-3- (3-dimethyl)
Ruaminopropyl) -carbodiimide hydrochlora
750 mg) and stirring at 25 ° C. for 14 hours.
An immobilization reaction of polystyrene / maleic anhydride was performed.
Then, 0.1 M sodium carbonate buffer, 0.1 M citric acid
After washing alternately with sodium buffer, PBS, physiological
After being sufficiently washed with a saline solution, an adsorbent was obtained.   The retention amount of polystyrene and maleic anhydride is benzene
Ring absorption was measured at a wavelength of 259.5 nm and calculated.   The adsorption experiments consisted of renal failure patient plasma and water-insoluble
After mixing the mixture at a volume ratio of 24: 1, shake for 1 hour at 37 ° C,
Β in plasma after₂-Define microglobulin and albumin
Weighed. β₂-Microglobulin is RIA method, albumin
Used the BCG method. Table 10 shows the results.   Next, as an evaluation of blood compatibility, evaluation of platelet permeability
Was performed. The evaluation method is described below, and the results are shown in Table 11.
Show. Evaluation of platelet permeability (A) Creating an evaluation column   First, the prepared adsorbent was placed in a cylinder with a diameter of 5 mmφ and a length of 10 mm.
Saline in the shape column under vacuum, suction of 200-300Hg
While flowing. (Population of the column, 80 me at the exit
A mesh polyester mesh is attached. Next
And let stand for 16 hours. (B) Calculation of human blood flow and platelet permeability Evaluation above
At a rate of 0.1 ml / min into a column for use (column capacity 0.2 ml)
For 20 minutes, add heparinized diet (1 unit / ml; heparin concentration)
Flow with a syringe-type micropump. Next, the end of blood
(15 units / ml; heparin concentration) at room temperature (20 ° C)
Flow at min speed. Raw food in the column is pushed from the column outlet
The time at which the column was output is defined as 0 hours of sampling, and
Sun with ethylenediaminetetraacetic acid = sodium from mouth
Sampling of blood with pull bin. sampling
After finishing, within 2 hours by BRECHER-CRONKITE method,
Column population and platelet V at column exit and morphological changes were measured.
Specified.   From the above results, the contact angle is at least 20 degrees or more
Water-insoluble carrier with two-layer structure of water-insoluble material and blood compatibility
Has a molecular weight of 1000 or more and X is 2.0 <X
The adsorbent having the indicated is β₂-For microglobulin
High adsorption capacity and high adsorption capacity for albumin
Be selective and even better for whole blood use
Recognize.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the results of Example 1 and Comparative Example 1.
FIG. 2 is a graph showing the results of Example 2 and Comparative Example 2.

Claims (1)

(57) [Claims] The surface of the water-insoluble carrier has a polyamino acid and / or polysaccharide and / or synthetic polymer electrolyte having a molecular weight of 1000 or more and wherein X represented by the formula (1) represents 2.0 <X. A characteristic adsorbent for β ₂ -microglobulin. A + B = X Equation (1) 2. The β according to claim 1, wherein the water-insoluble carrier has at least a two-layer structure of a water-insoluble material having a contact angle of at least 20 degrees and a blood-compatible polymer.
_2- Adsorbent for microglobulin.