JPH06237995A - Blood purifying/adsorbing material - Google Patents

Abstract

PURPOSE:To provide a blood purifying/adsorbing material which has the blood adaptability to eliminate troubles, such as clotting of blood and destruction and decrease of blood cell components and has the adsorptivity and adsorption/ selection performance to selectively, easily and effectively adsorb the specific components in blood by directly perfusing the blood. CONSTITUTION:This blood purifying/adsorbing material is formed by introducing polycarboxylic acid having 400 to 40000mol.wt. into a water-insoluble non-fabric type carrier consisting of fibers having 0.1 to 100mum fiber diameter in such a manner that the carboxyl group content attains 10 to 10<4>mueq/g and by immobilizing an org. compd. having affinity for the materials to be adsorbed thereto via a hydrophilic spacer having epoxy rings at both terminals and consisting of polyalkylene oxide skeleton.

Description

Detailed Description of the Invention

[0001]

The present invention relates to autoimmune diseases (myasthenia gravis, chronic rheumatism, idiopathic thrombocytopenic purpura, etc.), immune-related diseases (bronchial asthma, glomerulonephritis, etc.),
Treatment of cancer (leukemia, etc.), hyperlipidemia, and stem cells,
The present invention relates to a blood purification adsorbent for the purpose of separating blood cell components such as B cells and T cells, and separating specific components in blood by directly contacting blood without separating plasma from blood.

[0002]

2. Description of the Related Art Conventionally, the plasmapheresis method has been effective and widely used for the treatment of immunodeficiency, tumor, hyperlipidemia and the like. However, this therapy replaces all plasma, so
(1) Not only unnecessary components but also necessary components are removed. (2) A large amount and long-lasting plasma is lacking due to lack of plasma or plasma preparations to replace the removed plasma. Difficult to obtain, (3) Serum hepatitis, allergies, and AIDS (acquired immunodeficiency syndrome)
There are many problems involved in plasmapheresis, such as the risk of infection.

Against this background, in recent years, therapy by selective removal of pathogen-related substances has become popular. Such a method is described, for example, in Cascade (Siebelth, H .;
G. , Plasma Exchange, P .; 29,
F. K. Schattauer Verlog, Stu
ttgart-New York, 1980) or double filtration method (Aketsu Tetsuzo et al., Kidney and dialysis, 10 (3), 4).
75, 1981), a freeze filtration method (L'Abatte).
A. , Et al. , Proc. Eur. Dial. T
ranplant. Assoc. , 14 , 486, 1
977), salting-out plasma treatment method (Hiroaki Oe et al., Artificial organ, 1
40 (1), 472-475 (1985)).

As other treatment methods, various drugs have been used, but generally there is a risk of side effects, and it is necessary to pay close attention to the amount used and the period of use.

As a therapeutic method capable of solving such a problem, a method of purifying own blood and then reinjecting it, that is, an extracorporeal blood purifying method is desirable. In adopting this method, a purification material and a blood purification therapy capable of sufficiently and selectively removing a pathogenic substance from own blood without side effects have been desired.

Heretofore, as blood purification materials which can be used for this purpose, (1) affinity adsorption materials (for example, Japanese Patent Publication No. 2-50)
96, JP-B-2-26988, JP-A-1-158970.
(2) Porous resin (for example, JP-A-56-147710,
Japanese Examined Patent Publication No. 62-2543, "Amberlite XAD-7" manufactured by Rohm & Haas, etc.) (3) Ion exchanger (for example, carboxymethyl cellulose, diethylaminoethyl agarose, etc.) (4) Inorganic porous substance (for example, Japanese Examined Patent Publication No. Sho 62-2543, porous) Glass, ceramics, etc.).

However, porous resins and ion exchangers have low adsorption ability and low adsorption specificity, and when they are used as blood purification materials, not only are the effects insufficient, but they also adsorb essential components in blood. There is a possibility that it will end up, and there is a problem in terms of safety. In addition, although the inorganic porous material is relatively good in adsorption ability and adsorption characteristics, it is still insufficient for practical use and poor in blood compatibility, so it is necessary to add a large amount of heparin, and bleeding tendency, etc. Side effects of.
From these points, it is desired to develop a useful blood purification material using an affinity adsorbent, which has the greatest possibility in the future.

The affinity adsorbent is a bioaffinity adsorbent (which binds to a pathogenic substance in blood by a biological interaction such as antigen-antibody bond, complement bond, Fc bond, etc. and adsorbs it) and physical chemistry. Affinity adsorbents (electrostatic bond, hydrogen bond, van der Waals force such as physical or chemical interaction with the pathogen in the blood,
This can be roughly classified into the following (6) and the conventionally known physicochemical affinity adsorbents can be classified into the following 6 types. (1) Porous material having a carboxyl group or a sulfonic acid group on its surface (for example, JP-A-56-147710, JP-A-57-56038, JP-A-57-75141, and JP-A-5-75141).
7-170263, JP-A-57-197294, (2) Hydrophilic carrier to which a hydrophobic amino acid is bound (for example, JP-A-57-122875, JP-A-58-159).
24, JP-A-58-165859, JP-A-58-165.
861, Asahi Medical Imsorber) (3) A carrier to which other hydrophobic low molecular weight compounds are fixed (for example, JP-A-1-158970) (4) A hydrophilic carrier to which modified immunoglobulin (IgG) is bound (for example, Kai 57-77624, JP-A-5
7-77625, JP-A-57-156035) (5) Porous substance to which methylated albumin is bound (for example, JP-A-55-10875 and JP-A-55-1258).
72) (6) Carrier to which sugar or modified sugar is bound (for example, JP-A-57-134164 and JP-A-58-13325)
7. Kanegafuchi Chemical Liposorber) (7) Porous body to which purine base or pyrimidine base or sugar phosphate is bound (for example, JP-A-57-1925).
60, JP-A-58-61752, JP-A-58-9814
2)

However, these conventional physicochemical affinity adsorbents cannot be said to be sufficient for the treatment of autoimmune diseases and the like by extracorporeal blood purification, and are also poor in blood compatibility, and therefore have higher efficiency and specificity. There is a demand for a purification material that can remove the etiological agent and that has less adverse effect on body fluid.

As a blood purification material developed based on such a concept, for example, a patent of Japanese Patent Laid-Open No. 1-158970 is disclosed. The blood purification material disclosed by this patent is a low molecular weight organic compound as a substance (hereinafter abbreviated as a ligand) having an affinity for an adsorbed substance through a hydrophilic spacer having an ethylene oxide skeleton having a polymerization degree of 1 to 90. Is immobilized on the surface of a water-insoluble porous solid, and blood is directly perfused to the surface to adsorb immunoglobulin.

One of the meanings of the hydrophilic spacer is to prevent adhesion of blood cell components in blood and proteins that are not adsorbed substances. The hydrophilic spacer adsorbs water around the hydrophilic spacer. Therefore, the surface of the adsorbent is covered with a layer of water, and this layer of water prevents adhesion of blood cell components (excluded volume effect). Further, the movement of the spacer forming the water layer makes it more difficult to attach the blood cell component, and the attached blood cell component is likely to be detached. It also has the advantage that the activation of coagulation factors and complement is suppressed. That is, it is possible to improve blood compatibility by introducing a hydrophilic spacer.

However, when relying solely on the excluded volume effect of the hydrophilic spacer as a method for improving blood compatibility, it cannot be said that the degree of polymerization of the spacer is 90 or less, and in JP-A-1-158970, improvement of blood compatibility is not achieved. Therefore, acrylic acid derivative is coated. Such an operation is not only complicated, but may cause the elution of a toxic substance.

The second significance of the hydrophilic spacer is that it is expected that the adsorption ability can be improved by increasing the mobility of the ligand. When the substance to be adsorbed is a macromolecule, it is difficult to approach the substance to be adsorbed and the ligand by directly immobilizing the ligand, and even if some of them are bound, the number of binding sites is not sufficient and it is easy to do so. There is a high possibility that they will be removed. By interposing a hydrophilic spacer, proximity to the substance to be adsorbed is promoted, and since the movement of the ligand is flexible, it is easy for the surrounding ligand to participate in binding with the substance to be adsorbed. Can be created.

With respect to such an effect of the hydrophilic spacer, it is undeniable that the effect will be reduced to half by coating as described in JP-A-1-158970.

Japanese Patent Publication No. 4-29396 discloses a blood purification adsorbent characterized in that an insoluble carrier having a negative charge is bound to an organic compound having a functional site capable of binding to a substance to be adsorbed. The fact that the negatively charged surface has a function of suppressing blood coagulation and complement activation was discovered by Sawyer et al. In 1951, and has been investigated by many researchers thereafter (for example, Sawyer et al. , Amer. J. Sur
g. , 114 , 42, 1967, Kasai et al. , Artificial organ 1
2 , 327, 1983). Japanese Examined Patent Publication (Kokoku) No. 4-29396 also intends to improve blood compatibility by negative charges, as in these studies.

However, even when a carrier having a negative charge is used as disclosed in Japanese Patent Publication No. 4-29396, it is sufficient when a whole non-woven fabric having a small fiber diameter is selected as a carrier for treating whole blood. It is difficult to achieve effective suppression of blood cell adhesion.

[0017]

DISCLOSURE OF THE INVENTION The present invention solves the above-mentioned drawbacks of the prior art and brings out the effect of the ligand sufficiently,
By providing a blood purification adsorbent that has good adsorption ability and adsorption characteristics and that does not require complicated operations such as coating and yet has sufficient blood compatibility, there are no side effects and the pathogenic substance from own blood The present invention is intended to realize an effective extracorporeal blood purification treatment capable of sufficiently and selectively removing or selectively adsorbing and collecting a specific component in blood.

[0018]

The blood purification adsorbent of the present invention is a water-insoluble non-woven fabric composed of fibers having a fiber diameter of 0.1 to 100 μm, into which a polycarboxylic acid having a molecular weight of 400 to 40,000 is introduced, and epoxy rings at both ends. It is characterized in that an organic compound having an affinity with the substance to be adsorbed is immobilized via a hydrophilic spacer having The content of the carboxyl group derived from the polycarboxylic acid on the surface of the carrier used in the present invention is characterized by being 10 to 10 ⁴ μeq / g. The hydrophilic spacer used in the present invention has 2 carbon atoms.
To 6 having a polyalkylene oxide skeleton, a polymerization degree of 2 to 400, and an epoxy ring at both ends, which is the structure shown in Chemical formula 1.

The water-insoluble nonwoven fabric used in the nonwoven fabric-type blood purification adsorbent of the present invention includes polyester, polyethylene,
Synthetic organic polymer compounds such as polypropylene, polystyrene, polymethacrylic acid and its derivatives or copolymers thereof, natural organic polymer compounds such as cellulose, sepharose, dextran, chitin and chitosan, or modified acylcellulose, acyl sepharose and the like. It is preferably a natural organic polymer compound. Among these polymer compounds, considering mechanical strength, easiness of modification, etc., polyester, polyethylene, polystyrene and derivatives thereof or copolymers thereof, ethylene-vinyl alcohol copolymer, or cellulose, chitosan Desirably, more preferably polyester, polyethylene, or cellulose.

One of the features of the present invention is the use of a water-insoluble nonwoven fabric as a carrier. In the present invention, since it is intended to directly perfuse whole blood without separating plasma from blood to separate a specific component in blood,
A major problem is how to reduce the flow resistance when the adsorbent comes into contact with blood. Although many of the prior patents exemplify various shapes such as particles, fibers, hollow fibers, and membranes as the shape of the carrier, it is premised that the particulate carrier is substantially used. There are many. However, although it is relatively easy to obtain an effective surface area with a particulate carrier, it is difficult to say that it is necessarily suitable for keeping the flow resistance low. In this way, when considering both the pressure loss and the effective surface area, it was concluded that it is preferable to use a fibrous carrier, and it is preferable that the fibrous carrier is a non-woven fabric.

The diameter of the fibers forming the non-woven fabric type carrier is 0.1
It is necessary to be in the range of -100 μm, preferably 1-50 μm, more preferably 3-20 μm. When the fiber diameter is smaller than this, the circulation resistance of blood cells increases, and when the fiber diameter increases, the effective surface area decreases. Regarding the shape of the fiber, it is not preferable that spiny protrusions are present in view of damage to cells, but it is preferable that micropores are present for the purpose of increasing the effective surface area. The average pore size is 20Å ~ 3
It is 000Å, preferably 100Å to 2000Å. If the average pore diameter is smaller than this, the measured value of the surface area will be large, but since the substance to be adsorbed cannot reach the inside of the micropores, the effective surface area will not be expanded, and if it is larger than this, the strength of the carrier may be reduced. The presence of crimps in the fibers is also preferable because it leads to a reduction in flow resistance and an improvement in contact efficiency with blood. The nonwoven fabric of the present invention is 0.6 g /
cm ³ or less, preferably 0.4 g / cm ³ or less, and the lower limit is preferably 0.01 g / cm ³ .

Molecular weight used in the present invention 400-400
The polycarboxylic acid of 00 is a polymer of a carboxyl group-containing vinyl compound such as acrylic acid, vinyl benzoic acid, methacrylic acid, maleic acid or fumaric acid, or a copolymer containing a carboxyl group-containing vinyl compound as a copolymerization component. Examples of the compound include acidic polyamino acids such as polyglutamic acid and polyaspartic acid, and acidic copolymers containing these as copolymerization components; acidic polysaccharides such as alginic acid and pectic acid. Also, polymethylmethacrylate,
Polyacrylamide, polyacrylonitrile, hydrolysates of polymaleic anhydride, etc. may also be used. Of these polycarboxylic acids, carboxyl group-containing vinyl compound polymers such as acrylic acid, vinyl benzoic acid, methacrylic acid, maleic acid and fumaric acid are preferred from the viewpoint of ease of introduction and stability after introduction, hydrophilicity and the like. , And more preferably polyacrylic acid and polymethacrylic acid.

In the present invention, the significance of immobilizing a ligand by introducing a polycarboxylic acid into a carrier can be broadly divided into two. The first is to suppress blood coagulation and complement activation by introducing a negative charge on the surface of the carrier to improve blood compatibility. Since the blood cells are negatively charged, electrostatic repulsion from the surface having a negative charge inhibits adsorption, and coagulation is less likely to occur. Moreover, since the cascade of complement activation is inhibited by the trap of the negatively charged material, an effect of suppressing complement activation can be expected.

To impart a negative charge, a sulfonic acid group, a phosphoric acid group, etc. can be considered in addition to the carboxyl group. As a result of examining the blood compatibility of these various negatively charged substituents, especially regarding blood cell adhesion. The most preferable result was to introduce a carboxyl group.

Further, compared with the case where a negative charge is introduced on the carrier surface with a low molecular weight compound containing a carboxyl group, when a polycarboxylic acid having a molecular weight in the range of 400 to 40,000 is introduced into a water-insoluble carrier, Good blood compatibility,
Adsorption performance can be obtained. The reasons for this are as follows. That is, in a liquid such as blood, it can be expected that a part of the molecular chain of polycarboxylic acid exists so as to rise from the surface of the carrier and swing near the surface. Since this raised polycarboxylic acid molecular chain has a negative charge and is hydrophilic, an excluded volume effect can be expected,
At the same time as contributing to the improvement of blood compatibility, it also improves the adsorption ability by increasing the mobility of the ligand. That is, the second effect of the polycarboxylic acid can be expected to act as a hydrophilic spacer.

The above-mentioned excluded volume effect cannot be expected simply by immobilizing a carboxyl group-containing low molecular weight compound or using a material having a negative charge as a carrier, and it is difficult to obtain sufficient blood compatibility. Yes, the effect of the ligand is not fully elicited. That is, in order to obtain good blood compatibility and adsorption performance, it is essential to use a polycarboxylic acid having a molecular weight of 400 to 40,000 to impart a negative charge to the carrier surface. From this point of view, the more preferable molecular weight of the polycarboxylic acid is 500 to 20,000,
It is more preferably 1000 to 10,000. If the molecular weight is smaller than this, sufficient excluded volume effect and ligand mobility cannot be obtained, and if the molecular weight is higher than this, the introduction efficiency decreases, which is not preferable.

However, it cannot be said that the effect as a hydrophilic spacer is still sufficient with polycarboxylic acid alone, and a nonionic hydrophilic spacer is interposed in order to obtain better adsorption performance and blood compatibility. There is a need.

The hydrophilic spacer used in the present invention is characterized by having the structure shown in Chemical formula 1. Examples of the basic skeleton include polyethylene glycol, polypropylene glycol, polytetramethylene glycol and the like, but the basic skeleton is not limited to these as long as the structure conforms to the general formula shown in Chemical formula 1.

The purpose of the present invention is to immobilize a ligand on a non-woven fabric type carrier into which polycarboxylic acid has been introduced via hydrophilic spacers having epoxy rings at both ends. A method of introducing a hydrophilic spacer and further immobilizing a ligand; a method of introducing a polycarboxylic acid to a carrier and then introducing a hydrophilic spacer to which a ligand is immobilized in advance; a polycarboxylic acid having a hydrophilic spacer immobilized to a carrier After that, a method of immobilizing a ligand; a method of introducing a polycarboxylic acid having a ligand immobilized thereto through a hydrophilic spacer into a carrier may be employed, and any of the polycarboxylic acids present in the finally obtained blood purification adsorbent may be used. Acid-derived carboxyl group content is 10
The production method is not limited as long as it is 10 ⁴ μeq / g, and the ligand is immobilized through hydrophilic spacers having epoxy rings at both ends.

Furthermore, a method of independently introducing a hydrophilic spacer to the surface of the carrier separately from the polycarboxylic acid introduced to the surface of the carrier and immobilizing a ligand on one end of this hydrophilic spacer is also adopted. obtain. In this case, the molecular chain of polycarboxylic acid contributes to the improvement of blood compatibility of the carrier by imparting a negative charge and the excluded volume effect, and the hydrophilic spacer introduced on the surface of the carrier contributes to the improvement of blood compatibility by the excluded volume effect. At the same time, it contributes to improving the mobility of the ligand. In other words, the polycarboxylic acid molecular chain and the hydrophilic spacer are responsible for improving blood compatibility and ligand mobility to some extent.

The polycarboxylic acid for the water-insoluble non-woven fabric type carrier used in the present invention is not particularly limited as long as it is not liberated during use, but covalent bond by a chemical reaction, ionic bond, electron beam. Grafting by irradiation with or ultraviolet rays is preferable. Particularly, when the material of the non-woven fabric type carrier is cellulose, chitosan or the like, immobilization by a covalent bond by a chemical reaction is preferable, and when the material is polyethylene, polyester or the like, grafting by electron beam irradiation is preferable.

The cellulose carrier includes an aldehyde group introduced by a reaction with periodate, a carboxyl group introduced by a reaction with monochloroacetic acid, a carboxyl group introduced by a reaction with succinic anhydride, and paratoluenesulfonyl chloride. By using an active substituent such as an active ester introduced by the reaction with and as a foothold, the polycarboxylic acid can be immobilized by a chemical bond through a reaction of several steps.

When polyacrylic acid is introduced into the non-woven fabric carrier by electron beam irradiation, polyacrylic acid can be dissolved, and the polyacrylic acid is dissolved in a volatile solvent, and this solution is applied to the non-woven fabric carrier. The method of irradiating with an electron beam after drying is preferable. The volatile solvent is not particularly limited, but water, methanol, ethanol,
Methylene chloride, chloroform, acetone, dioxane,
Tetrahydrofuran or a mixed solvent thereof is preferable.

However, even if this method is used, if the boiling point of the polycarboxylic acid is low, a part or all of the polycarboxylic acid will evaporate during the solvent drying step, making it difficult to control the graft amount. there is a possibility. On the other hand, if the melting point of the polycarboxylic acid is high, the polycarboxylic acid will precipitate as a solid after the solvent is dried, so that the contact efficiency with the non-woven fabric type carrier will decrease, and the graft amount may decrease. is there.

In order to avoid such a situation and improve the grafting efficiency of the polycarboxylic acid by electron beam irradiation, it is preferable to add a high boiling point compound capable of dissolving the polycarboxylic acid in the coating solution. Such high boiling point compounds are not particularly limited, but polyhydric alcohols such as ethylene glycol, propylene glycol, glycerin, and dehydration condensates thereof having a degree of polymerization of 1
Those having a fluidity of 0 or less are preferable. The addition amount of these high boiling point additives is preferably about 1/10 to 10/1 with respect to the weight of the polycarboxylic acid.

A crosslinkable vinyl compound is an additive that promotes the introduction of the polycarboxylic acid more effectively than the high boiling point compound. Examples of the crosslinkable vinyl compound include methylenebisacrylamide, trimethylolpropane diacrylate, triallyl isocyanurate, trimethylolpropane triacrylate, tetramethylolmethane tetraacrylate, and the like, more preferably as shown in Chemical formula 2 below. Compounds are preferred. The amount of the additive having the structure shown in Chemical formula 1 is 1/50 to 50/1, preferably 1/50 to the weight of the polycarboxylic acid.
It is 30 to 30/1.

In the chemical formula 2, the integers R ³ , R ⁴ and R ⁵ of p = 1 to 30 are hydrogen atoms or methyl groups and may be the same or different.

[Chemical 2]

In the method of introducing the hydrophilic spacer having epoxy rings at both ends into the water-insoluble non-woven fabric carrier used in the present invention, no release is observed during use such as covalent bonding and grafting by electron beam irradiation. It is not particularly limited as long as it is fixed by the covalent bond by utilizing the reactivity of the epoxy ring.

It is known that an epoxy ring causes a coupling reaction by reacting with a nucleophilic substituent. Immobilization is preferably performed by utilizing this reaction. That is, when immobilizing a hydrophilic spacer having epoxy rings at both ends on a carrier into which a polycarboxylic acid has been introduced, a reaction of reacting -OH of a carboxyl group with an epoxy ring and immobilizing it as shown in the following chemical formula 3 is performed. Recommended. However, since it is hard to say that the nucleophilicity of the carboxyl group is large, it is preferable to add a tertiary amine, a quaternary ammonium salt, an imidazole compound or the like as a catalyst. It is also recommended that an appropriate buffer solution be used for the reaction, or that the reaction be performed by applying heat, if necessary.

[0040]

[Chemical 3]

As shown in Chemical formula 3, when the immobilization reaction is carried out by utilizing the reactivity of the epoxy ring, the epoxy ring is opened. Therefore, in the present invention, the immobilization via hydrophilic spacers having an epoxy ring at both ends means that the immobilization reaction is performed by utilizing the reactivity of the epoxy ring as a result of the structure of the hydrophilic spacer. It also includes the case where a part is modified.

The substances to be adsorbed targeted by the adsorbent of the present invention are various specific substances in blood. More specifically, ordinary immunoglobulins (A, D, E, G, M) and anti-DNA antibodies are used. , Anti-acetylcholine receptor antibody, anti-blood group antibody,
Autoantibodies such as antiplatelet antibodies; antigen-antibody complexes; endotoxins; rheumatoid factors; lipoproteins such as LDL and VLDL; stem cells, B cells, T cells, monocytes, macrophages, TILs (cancer tissue infiltrating T cells), etc. Examples include cell components in blood.

The ligand to be immobilized needs to be appropriately changed depending on the substance to be adsorbed, but it may be antigen-antibody binding, complement binding, Fc
Various biological ligands that utilize biological interactions such as binding; or hydrophobic amino acids, methylated albumin, modified immunoglobulins (IgG), sugars or modified sugars, purine bases, pyrimidine bases, sugar phosphates Various physicochemical ligands utilizing physical or chemical interactions such as electrostatic bond, hydrogen bond, van der Waals force, etc. are considered.

The method of immobilizing the ligand is not particularly limited, such as covalent bond, physical adsorption, ionic bond, biochemical specific bond, etc. However, considering the bond stability in blood, the covalent bond utilizing the reactivity of the epoxy ring is used. Binding is preferred. That is, a method of immobilizing by utilizing the reaction between the nucleophilic substituent contained in the ligand and the epoxy ring is recommended. In this case, it is preferable to use a substituent having a relatively high nucleophilicity such as an amino group, an imino group and a thiol group rather than a carboxyl group from the viewpoint of the immobilization rate.

The blood purification adsorbent of the present invention can be used by filling it in a suitable container having a body fluid outlet port. The material of the container is not particularly limited, such as glass, stainless steel, polyethylene, polypropylene, polycarbonate, polystyrene, polymethylmethacrylate, etc., but polypropylene and polycarbonate are preferable in consideration of handling such as sterilization. The form of the container is also not particularly limited, but a cylindrical column type with both ends having a blood inflow part and a blood outflow part, or a sheet-like shape and then sandwiching the container may be suitable.

The blood purification adsorbent of the present invention may be used alone, or may be mixed and laminated with another adsorbent such as activated carbon, porous glass or silica gel. Also, 2 as a ligand
There is no limitation on immobilizing one or more kinds of organic compounds and mixing and laminating adsorbents having different ligands immobilized thereon.

[0047]

EXAMPLES The present invention will be described below with reference to examples. Example 1 (1) Synthesis of Carrier A nonwoven fabric made of polyethylene terephthalate (hereinafter abbreviated as PET) having a fiber diameter of 3.5 μm and a basis weight of 45 g / m ² was cut into a size of 15 cm × 12 cm (weight: about 810).
mg). This PET non-woven fabric was thoroughly washed with acetone before modification. 15 g of polyacrylic acid having a molecular weight of 4000 (hereinafter abbreviated as PAA), 5 g of methylenebisacrylamide (hereinafter abbreviated as MBAA), and 2 g of glycerin are dissolved in 2 l of ethanol, and a PET non-woven fabric is dipped in this solution to apply PAA. I went. After drying it sufficiently, it was irradiated with an electron beam (hereinafter abbreviated as EB) at a dose of 5 Mrad on one side to PA on the PET surface.
Grafting of A was performed. It was thoroughly washed with water and methanol to obtain a PAA-introduced PET nonwoven fabric (PET-PAA). The obtained PET-PAA was 120 sheets. The acid content of this PET-PAA is COMMITTE1 made by Hiranuma Sangyo.
As determined by acid-base titration with 01, 0.32 m
It was eq / g.

(2) Introduction of Spacer Containing Terminal Epoxy Group PET-PAA obtained by the above operation in a separable flask.
40 sheets (about 32.5 g) were taken, and a thermometer and a cooling tube were attached. 39 g of polyethylene glycol having an epoxy ring at both ends (the molecular weight of polyethylene glycol is 400, this compound is abbreviated as PEOG hereinafter) and 13 g of pyridine were taken and dissolved in 650 ml of ion-exchanged water. This solution was poured into a separable flask containing PET-PAA so that PET-PAA was immersed therein. The separable flask was immersed in a water bath, and the cooling tube was allowed to stand at 70 ° C. for about 14 hours while being perfused with tap water. PET-PAA
Was taken out, thoroughly washed with ion-exchanged water, and dried under reduced pressure. PET-PAA with PEOG immobilized in this way
(PET-PAA-PEOG) was obtained. When the residual carboxyl group was quantified by acid-base titration,
It was 0.18 meq / g. Also fixed PEO
The epoxy group content derived from one end of G is 0.11 meq
/ G.

The terminal epoxy group was quantified by the following method. That is, PET-PAA-PEOG
About 200 mg was accurately weighed, 10 ml of a 1/10 N hydrochloric acid dioxane solution and about 50 ml of ion-exchanged water were added, and the mixture was heated at 60 ° C. for 1 hour. By this operation, the epoxy group reacts with hydrochloric acid to open the epoxy ring, and at the same time,
Hydrochloric acid is consumed. Since the reaction between the epoxy ring and hydrochloric acid proceeds at a molar ratio of 1: 1, the amount of hydrochloric acid consumed is equal to the content of epoxy groups. The amount of consumed hydrochloric acid is calculated by titrating the heated sample with a 1/10 N sodium hydroxide aqueous solution and quantifying excess hydrochloric acid. Since this consumed hydrochloric acid amount is equal to the epoxy group content, the epoxy group content in the sample is calculated.

(3) Immobilization of ligand An oligopeptide having an affinity for an antiplatelet antibody, which is a causative agent of idiopathic thrombocytopenic purpura (hereinafter abbreviated as ITP), is added to the carrier synthesized by the above-mentioned operation. Was immobilized as a ligand. This oligopeptide is Glu-Thr
With a heptapeptide consisting of the sequence -Arg-Asn-Val-Gly-Ser (hereinafter abbreviated as Pep), the immunogenicity against anti-platelet antibody was confirmed by an enzyme immunoassay (hereinafter abbreviated as ELISA method).

420 mg of the above Pep was taken in a glass bottle and 150 ml of pH 10.0 carbonate buffer was added and dissolved. PET-PAA-P obtained by the above operation was added to this solution.
Soak 10 sheets of EOG (about 8.4g) at 40 ℃ for about 18
The mixture was shaken for a reaction. The product is taken out, washed thoroughly, and the target product, Pep-immobilized PET non-woven fabric PE
T-PAA-PEOG-Pep was obtained. When the nitrogen content in the reaction residual liquid was calculated from the value quantified by the micro Kjeldahl method, the Pep immobilization rate was 69%.

(4) Evaluation of adsorption performance of ligand-immobilized adsorbent An evaluation module was prepared using the PET-PAA-PEOG-Pep described above, and its anti-platelet antibody adsorption performance was evaluated as follows.
It was evaluated using the serum of ITP patients. Although the form of the evaluation module is shown in FIG. 1, between the funnel-shaped molded body 3 having the body fluid introduction port 5 and the funnel-shaped molded body 3 ′ having the body fluid discharge port 6, the nonwoven fabric is provided at the portion 4 It has a structure in which a type blood purifying material is sandwiched and tightened by a cap 1 and a cylinder 2 which can be fitted to each other with screws. Using this module, I
50 ml of TP patient serum was perfused for 30 minutes at 37 ° C.

The amount of antiplatelet antibody in serum before and after perfusion was determined by Ac
ta. Haemat. , 66, 251, 1981, and the adsorption rate was calculated. The results are as shown in Table 1. Also, for non-specific adsorption of albumin, the amount of albumin before and after perfusion was adjusted to albumin B.
-Quantification was performed using Test Wako (manufactured by Wako Pure Chemical Industries), and the adsorption rate was calculated from the value. The results are as shown in Table 2. Furthermore, regarding the adhesion of blood cell components to the sample due to perfusion, Toa Medical Electronics automated blood cell counter Sysmex
It measured using F-800. Regarding blood cell adhesion, it depends on the blood cell content (white blood cells, platelets) in the blood before perfusion,
Percentage of blood cell content in blood after perfusion (blood cell residual rate)
Was calculated as shown in Table 3.

[0054]

[Table 1]

[0055]

[Table 2]

[0056]

[Table 3]

Example 2 (1) Synthesis of Carrier A rayon nonwoven fabric having a fiber diameter of 8.3 μm and a basis weight of 120 g / m ² was cut into a size of 15 cm × 12 cm (weight: about 2.16 g). This rayon nonwoven fabric was thoroughly washed with acetone before modification. 40 g of sodium periodate was placed in a glass bottle and 3.5 L of 1N-sulfuric acid was added to dissolve it. Rayon non-woven fabric 120 in this glass bottle
The plate was immersed and shaken at room temperature for about 18 hours for reaction. The product was thoroughly washed with ion-exchanged water and dried under reduced pressure to obtain an aldehyde-introduced rayon nonwoven fabric (CA). The aldehyde content was determined by the oxime method to be 0.41 meq / g
Met.

60 CA sheets obtained above were placed in a glass bottle,
2.0 l of carbonate buffer pH 9.5 was added. 20 g of diethylenetriamine was added to this, and it was made to react by shaking at room temperature for about 18 hours. After taking out the product and thoroughly washing it with ion-exchanged water, the product was placed in a beaker containing 2.0 l of carbonate buffer having pH 9.0, and sodium borohydride 1
The Schiff base was hydrogenated by adding 00 g. Thus, an amino group-introduced rayon nonwoven fabric Cen was obtained. When the content of amino groups in Cen was quantified by acid-base titration, it was 0.38.
It was meq / g.

1.6 g of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (hereinafter abbreviated as EDC) was placed in a glass bottle and 300 ml of a phosphate buffer having a pH of 4.5 was added and dissolved. . P in this glass bottle
Add 34 g of AA and p with 30% sodium hydroxide aqueous solution.
H was adjusted, and 10 sheets of Cen obtained above were immersed in this and shaken for about 18 hours to react. Take out the product,
Fully washed and PAA-immobilized rayon non-woven fabric C-PA
I got A. The acid content of C-PAA was determined by acid-base titration to be 0.40 meq / g.

(2) Introduction of Spacer Containing Terminal Epoxy Group C-PAA20 obtained by the above operation in a separable flask.
A sheet (about 44.0 g) was taken, and a thermometer and a cooling tube were attached. 58 g of polyethylene glycol having epoxy rings at both ends (molecular weight of polyethylene glycol is 400, hereinafter this compound is abbreviated as PEOG) 58 g, pyridine 19
g was taken and dissolved in 900 ml of ion-exchanged water. This solution was poured into a separable flask containing C-PAA, and C
-PAA was allowed to soak. Soak the separable flask in a water bath and irrigate it with tap water.
It was left to stand at ℃ for about 14 hours. The nonwoven fabric was taken out, thoroughly washed with ion-exchanged water, and dried under reduced pressure. Thus PEO
C-PAA (C-PAA-PEOG) having G immobilized thereon was obtained. The residual carboxyl group was determined by acid-base titration to be 0.16 meq / g. The epoxy group content derived from one end of the fixed PEOG was 0.12 meq / g.

(3) Immobilization of Ligand PET of C-PAA-PEOG obtained in the above
Pep was immobilized by the same method as that for introducing Pep into PAA-PEOG to obtain C-PAA-PEOG-Pep. When the nitrogen content in the reaction residual liquid was calculated from the value quantified by the micro Kjeldahl method, the immobilization rate of Pep was 70.
%Met.

(4) Evaluation of adsorption performance of ligand-immobilized adsorbent C-PAA-PEOG-Pep was prepared in the same manner as in Example 1.
Was evaluated for anti-platelet antibody adsorption performance, non-specific albumin adsorption, and blood cell survival rate after perfusion. The results are shown in Table 1, Table 2 and Table 3, respectively.

Comparative Example 1 (1) Synthesis of Carrier The CA having an aldehyde content of 0.41 meq / g obtained in Example 2 was used as it was as a carrier.

(2) Introduction of ligand Ten CA sheets were taken in a glass bottle and 400 ml of a carbonate buffer solution having a pH of 9.5 was added. The same heptapeptide Pep used in Example 1 was previously dissolved in a small amount of a pH 9.5 carbonate buffer solution, this solution was added to a container containing CA, and the mixture was reacted by shaking at room temperature for about 18 hours. . After taking out the product and thoroughly washing it with ion-exchanged water, pH 9.0
The solution was placed in a beaker containing 400 ml of the carbonate buffer solution, and 20 g of sodium borohydride was added to the beaker to hydrogenate the Schiff base. The product is taken out and thoroughly washed to obtain the desired product, Pep-immobilized rayon nonwoven fabric C-Pe.
got p. When the nitrogen content in the reaction residual liquid was calculated from the value quantified by the micro Kjeldahl method, the Pep immobilization rate was 78%.

(3) Evaluation of adsorption performance of ligand-immobilized adsorbent In the same manner as in Example 1, the anti-platelet antibody adsorption performance of C-Pep, albumin non-specific adsorption, and blood cell survival rate after perfusion were evaluated. The results are shown in Table 1, Table 2 and Table 3, respectively.

Comparative Example 2 (1) Synthesis of Carrier 60 sheets of CA having an aldehyde content of 0.41 meq / g obtained in Example 2 were placed in a glass bottle and 2.5 l of a carbonate buffer solution having a pH of 9.5 was added. 20 g of L-glutamic acid was added to this, and it was made to react by shaking at room temperature for about 18 hours. After taking out the product and thoroughly washing it with ion-exchanged water, pH 9.0
Into a beaker containing 1.2 liters of the carbonate buffer solution (1) was added 100 g of sodium borohydride, and the Schiff base was hydrogenated. Thus, a carboxyl group-introduced rayon nonwoven fabric CG was obtained. The carboxyl group content of CG was determined by acid-base titration to be 0.36 meq / g.

(2) Introduction of ligand 220 mg of EDC was placed in a glass bottle and 400 ml of a phosphate buffer having a pH of 4.5 was added and dissolved. After adding Pep to the glass bottle to dissolve it, 10 CGs (about 22.1
g) was soaked and shaken for about 18 hours to react. The product was taken out and thoroughly washed to obtain a Pep-immobilized rayon non-woven fabric CG-Pep which was a target product. When the nitrogen content in the reaction residual liquid was calculated from the value quantified by the micro Kjeldahl method, the Pep immobilization rate was 72%.

(3) Evaluation of adsorption performance of ligand-immobilized adsorbent In the same manner as in Example 1, CG-Pep was evaluated for anti-platelet antibody adsorption performance, albumin non-specific adsorption, and blood cell survival rate after perfusion. The results are shown in Table 1, Table 2 and Table 3, respectively.

Comparative Example 3 (1) Synthesis of Carrier Carboxyl group content obtained in Example 1 0.32 meq / g
PET-PAA of was used as a carrier as it was.

(2) Immobilization of ligand 80 mg of EDC was placed in a glass bottle, and 150 ml of phosphate buffer having a pH of 4.5 was added and dissolved. P in this glass bottle
After adding and dissolving ep, 10 pieces of PET-PAA (about 8.1 g) were dipped and reacted by shaking for about 18 hours.
Take out the product, wash it thoroughly, and remove the target Pe.
A non-woven fabric PET-PAA-Pep made of p-immobilized PET was obtained. When the nitrogen content in the reaction residual liquid was calculated from the value quantified by the micro Kjeldahl method, the immobilization rate of Pep was 77.
%Met.

(3) Evaluation of adsorption performance of ligand-immobilized adsorbent In the same manner as in Example 1, PET-PAA-Pep was evaluated for anti-platelet antibody adsorption performance, albumin non-specific adsorption, and blood cell residual rate after perfusion. The results are shown in Table 1, Table 2 and Table 3, respectively.

As is clear from the above example, the non-woven blood purification adsorbent of the present invention was able to selectively and easily remove a specific component (antiplatelet antibody) in blood. In the case where no polycarboxylic acid is present on the carrier (Comparative Example 1), the anti-platelet antibody adsorption ability is greatly inferior. In addition, when compared with Example 2 in which the materials are the same, the nonspecific adsorption of albumin and the blood cell residual rate after treatment are also inferior to the adsorbent of the present invention. Further, when a low molecular weight carboxyl group-containing compound (L-glutamic acid) is introduced onto the carrier (Comparative Example 2)
The non-specific adsorption of albumin and the blood cell residual rate after treatment are not significantly inferior to those of the non-woven blood purification adsorbent of the present invention, but the anti-platelet antibody adsorption capacity is still not at a satisfactory level. When a polycarboxylic acid is present on a carrier and a ligand is immobilized without a hydrophilic spacer (Comparative Example 3), both blood compatibility and adsorption performance are considerably improved as compared with Comparative Examples 1 and 2. It's still hard to say.

As compared with Comparative Examples 1 to 3, the molecular weight is 400 to 40.
The data of the example in which 000 polycarboxylic acids were introduced onto a carrier, and a ligand was immobilized via hydrophilic spacers having epoxy rings at both ends, both excellent blood compatibility and adsorption performance were obtained.

[0074]

INDUSTRIAL APPLICABILITY The non-woven blood purification adsorbent of the present invention has both good adsorption performance and blood compatibility due to the negative charge of the polycarboxylic acid having a molecular weight of 400 to 40,000 and the function of the nonionic hydrophilic spacer. can do. Also,
Since the shape of the carrier is a non-woven type, it is possible to suppress the pressure loss when perfusing whole blood to a low level, and by directly perfusing blood without the complicated method of separating plasma components from blood and perfusing in advance. A sufficient effect can be expected. For this reason, various diseases can be treated by removing pathogenic substances in blood, or B cells, T
It can be widely used for selective separation of specific components such as cells.

[Brief description of drawings]

FIG. 1 illustrates a form of an evaluation module used in an example.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Masakazu Tanaka 2-1-1 Katata, Otsu City, Shiga Prefecture Toyobo Co., Ltd.

Claims (3)

[Claims] 1. A molecular weight of 400 to 40,000 as a carrier.
Diameter of the fiber introduced with the polycarboxylic acid of 0.1 to 100 μm
A blood purification adsorbent comprising an organic compound having an affinity for a substance to be adsorbed, which is fixed to a water-insoluble non-woven fabric made of the above fibers via hydrophilic spacers having epoxy rings at both ends. 2. The blood purification adsorbent according to claim 1, wherein the content of the carboxyl group derived from the polycarboxylic acid on the surface of the carrier is 10 to 10 ⁴ μeq / g. 3. The blood purification adsorbent according to claim 1, wherein the hydrophilic spacer having epoxy rings at both ends is a compound having a structure of the following chemical formula 1. In Chemical formula 1, n = 0 to 10 integer m = 2 to 400 integer l = 1 or 2 R ¹ and R ² are hydrogen atoms or methyl groups, and may be the same or different. [Chemical 1]