JPH05285381A - Low-specific gravity lipoprotein adsorptive material - Google Patents

Abstract

PURPOSE:To provide the low-sp. gr. lipoprotein adsorptive material having the blood adaptability to avert troubles, such as coagulation of blood and destruction and decrease of blood cell components, by direct perfusion of the blood and the adsorption power adsorption characteristic capable of selectively, easily and effectively adsorbing the low-sp. gr. lipoprotein and (or) ultra-low-sp. gr. lipoprotein. CONSTITUTION:This low-sp. gr. lipoprotein adsorptive material is constituted by fixing N-sulfo PEI onto the surface of a water insoluble solid in such a manner that the acid content derived from a sulfamino group attains 50mueq/ml to 2.0meq/ml (wet state) by which the low-sp. gr. lipoprotein and (or) ultra-low- sp. gr. lipoprotein in the blood is removed.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a low-density lipoprotein (hereinafter abbreviated as LDL) and / or a very low-density lipoprotein (hereinafter abbreviated as VLDL), which are causative agents of arteriosclerosis and hyperlipidemia. The present invention relates to a low-density lipoprotein adsorbent that adsorbs LDL and / or VLDL in blood by contacting blood or blood components for the purpose of adsorbing and removing and treating diseases.
In the present invention, N-sulfopolyethyleneimine (hereinafter abbreviated as N-sulfoPEI) refers to a compound having a structure of the following chemical formula 1.

[0002]

[Chemical 1] X in Chemical formula 1 is a salt-forming group such as H or Na.

Further, in the present invention, -N (R) SO ₃
H, —NHSO ₃ H, and all of these salts are called sulfamino groups. (However, R represents a polyethyleneimine residue.)

[0004]

2. Description of the Related Art Various drugs are used for the treatment of hyperlipidemia that causes arteriosclerosis, but in general, there is a risk of side effects, so pay close attention to the amount and duration of use. There is a need. In addition, in many cases familial hyperlipidemia (FH), which shows LDL and / or VLDL levels that are several times higher than the normal values and is at risk of inducing the most serious symptoms, the effect of the drug cannot be expected sufficiently. ..

As another treatment method, the plasma exchange method is effective and is widely used. However, since all plasma is exchanged in this therapy, (1) not only unnecessary components but also necessary components are removed, (2) plasma that is replaced by the removed plasma, or plasma that is supplemented in the body, or plasma. Inadequate preparations make it difficult to obtain large quantities and continuously, (3) serum hepatitis, allergies, and the risk of AIDS (acquired immunodeficiency syndrome) infection, etc. There are many problems involved.

[0006] 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, there has been a demand for a purification material and a blood purification therapy capable of sufficiently and selectively removing a pathogenic substance from own blood without causing side effects.

Against this background, LD
Therapy by adsorptive removal of L and / or VLDL has become popular. Such a method is, for example, an adsorbent (S. Moorja) in which heparin is immobilized on agarose gel.
ni et al. , Clin. Chim. Acta. ，
77 (1977) 21-30) and the like,
This material does not have sufficient adsorption capacity, and it cannot be said that it has sufficient mechanical strength. In addition, attempts have been made to adsorb and remove LDL and / or VLDL using an immunoadsorbent on which an antibody or the like is immobilized. It has the disadvantage of being expensive.

The adsorbent provided for the purpose of improving such problems has a porous material (silica or the like) having affinity for harmful components (Japanese Patent Laid-Open No. 59-139935).
JP-A-63-160669, etc.), and compounds having an affinity for harmful components (ligand: sulfonic acid group, carboxyl group, etc.) are immobilized. The latter is a so-called affinity adsorbent and can be roughly classified into the following two. (1) A polyanion immobilized as a ligand on an insoluble carrier (JP-B-62-59975, JP-A-1-145)
071, Limiting the ligand to dextran sulfate: Tokuhei 3
-5822, Limiting ligand to heparin: JP-A-59-59
139937) (2) Insoluble carrier to which a monomer having a sulfonic acid group and / or a carboxyl group is fixed as a ligand (Japanese Patent Publication No. 2-51346, JP-A-58-27559, etc.)

Ligands used for affinity adsorbents are relatively inexpensive and have relatively satisfactory levels of selectivity and adsorption capacity. However, since these adsorbents have poor blood compatibility, it is necessary to add a large amount of heparin. Therefore, there is a possibility that side effects such as bleeding tendency may occur. From such a point, there is a demand for a purifying material that can remove a pathogenic substance with higher efficiency and specificity and that has less adverse effects on body fluids.

Turning to an adsorbent that adsorbs substances other than LDL and / or VLDL, for example, Japanese Patent Laid-Open No. 1-158970 describes a blood purification material developed based on such a concept. It is disclosed.
The blood purification material disclosed by this patent fixes a low molecular weight organic compound as a ligand to a water-insoluble porous solid surface through a hydrophilic spacer having an ethylene oxide skeleton having a polymerization degree of 1 to 90, and It is characterized in that blood is directly perfused to adsorb immunoglobulin.

The significance of the hydrophilic spacer can be broadly divided into two. First, the blood cell components in blood,
This is to prevent the attachment of proteins that are not adsorbed substances. The hydrophilic spacer adsorbs water around the hydrophilic spacer.
For this reason, 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.

Second, it is expected that the adsorptivity can be improved by increasing the mobility of the ligand. Particularly when the substance to be adsorbed is a macromolecule, it is difficult to approach the substance to be adsorbed and the ligand by directly fixing the ligand to the surface of the carrier, and even if some of them are bound, the number of binding sites is not sufficient. There is a high possibility that they will be easily removed. By interposing a hydrophilic spacer, the approach between the substance to be adsorbed and the ligand is promoted, and the flexibility of the movement of the ligand makes it easy for surrounding ligands to participate in the binding with the substance to be adsorbed. Many tight bonds can be generated.

In JP-A-1-158970, the degree of polymerization is 1
Although 90 hydrophilic spacers having an ethylene oxide skeleton are interposed, the effect of this spacer is not essentially limited to a substance having an ethylene oxide skeleton. If the spacer is a long-chain hydrophilic compound, improvement in blood compatibility due to the excluded volume effect can be expected.

Further, it is difficult to realize sufficient blood compatibility simply by introducing a hydrophilic spacer having an ethylene oxide skeleton having a degree of polymerization of up to 90. In JP-A-1-158970, improvement of blood compatibility is also achieved. Therefore, acrylic acid derivative is coated. Such an operation is not only complicated, but may cause the elution of a toxic substance.

[0015]

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 having good adsorbability and adsorption properties and omitting complicated operations such as coating, and having sufficient blood compatibility, there are no side effects and LDL and (Or) It is intended to provide an effective adsorbent capable of sufficiently and selectively removing VLDL.

[0016]

The low-density lipoprotein adsorbent of the present invention contains N-sulfoPEI in a water-insoluble solid and an acid content derived from a sulfamino group of 50 μeq / ml (wet state) to 2.0 meq / ml ( It is characterized in that the low-density lipoprotein and / or the extremely low-density lipoprotein in the blood are removed by fixing it so that it is in a wet state. The blood purification adsorbent of the present invention is a water-insoluble solid polystyrene, cross-linked polyvinyl alcohol,
Synthetic organic polymer compounds such as polymethacrylic acid and its derivatives or copolymers thereof, natural organic polymer compounds such as cellulose, chitin and chitosan, or modified natural polymer compounds such as acylcellulose and acyl chitin Is preferred. Considering the mechanical strength and ease of ligand introduction among these polymer compounds,
Properly cross-linked polystyrene, polymethacrylic acid and derivatives thereof or copolymers thereof, and cellulose are desirable, and cellulose is more desirable.

The following N-sulfoPEI used in the present invention is
Has a molecular weight of 100 to 300,000, preferably 300 to 100,000. If the molecular weight is smaller than this, blood compatibility and adsorption performance cannot be sufficiently obtained, and if it is larger than this, the introduction rate tends to decrease, and improvement in performance cannot be expected.

The introduction of N-sulfoPEI in the present invention is carried out by introducing polyethyleneimine (hereinafter abbreviated as PEI) into a water-insoluble solid and then sulfonation of amino groups and imino groups. Turned P
Any method of introducing EI into a water-insoluble solid can be used. When the former is adopted, the sulfonation rate of the amino group and imino group contained in N-sulfoPEI is 30% or more, preferably 50% or more, more preferably 70% or more. When the latter is adopted, the sulfonation rate of PEI is preferably 30% or more, preferably 50% or more, more preferably 70% or more, but if all the amino groups and imino groups of PEI are sulfonated (sulfonated Rate 100
%), And it becomes impossible to introduce a residual amino group or imino group into the water-insoluble solid by a chemical bond, which is not preferable. However, the presence of a large amount of residual amino groups and imino groups may have an unfavorable effect on LDL adsorption. Therefore, when the immobilization on the water-insoluble solid is completed, the residual amino groups and imino groups are 10% or less of the total. Is desirable. When an amount of residual amino group or imino group exceeding this is present, acetic anhydride, succinic anhydride, 1,
Blocking with 3-propane sultone or the like is recommended. Chlorosulfonic acid, concentrated sulfuric acid, fuming sulfuric acid, sulfur trioxide / dimethylformamide complex, sulfur trioxide / pyridine complex, etc. may be used for N-sulfonation of PEI in the present invention. / Pyridine complex is preferred. The sulfonation rate can be measured by a method of titrating amino groups and imino groups contained in PEI before and after sulfonation, or by quantifying nitrogen and sulfur contents by elemental analysis.

The N-sulfoPEI of the present invention is not only used as a ligand for adsorbing and removing L-and / or VLDL by its affinity with LDL and / or VLDL.
It also has a function as a hydrophilic spacer disclosed in 58970. The N-sulfoPEI chain in the vicinity of the binding point with the water-insoluble solid surface mainly contributes to the improvement of blood compatibility and the mobility of the ligand as a hydrophilic spacer,
N-Sulfo P at a relatively long distance from the surface of a water-insoluble solid
EI is LDL and / or VLDL as a ligand
Has the role of adsorption removal. However, it improves ligand motility and results in better LDL and / or
The immobilization of N-sulfoPEI on a water-insoluble solid via a spacer in order to realize the adsorptivity of VLDL and blood compatibility is not limited in the present invention.

N-sulfoPEI also functions as an analog (heparinoid) showing the function of heparin, which is well known as an anticoagulant. The basic skeleton of heparin is D-glucosamine and D-glucuronic acid, which has a structure in which this amino group and some hydroxyl groups are sulfonated. Among these structures, the sulfamino group particularly contributes greatly to the anticoagulant property. Therefore, N-sulfoPEI having a large number of sulfamino groups in its molecule can be expected to have a great effect as a heparinoid. Therefore, JP-A-1-
158970 makes it possible to achieve blood compatibility that was still insufficient.

It is toxic to be noted when discussing the anticoagulant properties of such heparinoids, but in the present invention, since N-sulfoPEI is immobilized on the surface of a water-insoluble solid, the anticoagulant property is The risk is extremely reduced compared to when it is directly administered as a drug. In addition, toxicity is greatly affected by the chain length and molecular weight of N-sulfoPEI. When N-sulfoPEI having a molecular weight in the range as described above is immobilized on the surface of a water-insoluble solid, even if N-sulfoPEI is released and released into the blood, it may cause serious side effects. Is small.

The shape of the water-insoluble solid used in the present invention may be any known shape such as particle shape, fibrous shape, and film shape. From the viewpoint of easiness and the like, a particulate or fibrous form is desirable. The average particle size of the particulate carrier is preferably in the range of 10 μm to 5 mm, but the smaller the particle size, the greater the flow resistance of blood cells, and the larger the particle size, the smaller the carrier loading amount and, consequently, the effective surface area. Therefore, it is desirable that the average particle size is in the range of 30 μm to 1 mm. More preferably, the average particle size is 50 μm to 500 μm. Regarding the particle shape, a spherical shape is desirable because it is less likely to damage cells, has strength against physical external force, and is easy to prepare. For the same reason, the fiber diameter of the fibrous carrier is preferably 0.1 μm to 50 μm, preferably 0.3 μm to 25 μm, and more preferably 0.5 μm to 15 μm.

Micropores are preferably present on the surface of the carrier in order to expand the effective surface area. In the case of a particulate carrier, the average pore size is 20Å to 20000Å, preferably 10Å
0 Å to 15000 Å, more preferably 1000 Å to 12000 Å, in case of fibrous carrier 20 Å to 3000
Å, preferably 100Å to 2000Å. If the average pore diameter is smaller than this, the substance to be adsorbed cannot reach the inside of the fine pores, and if it is larger than this, the effect of expanding the effective surface area is insufficient and the strength of the carrier may be reduced.

N- to water-insoluble solid used in the present invention
The method of introducing sulfoPEI is not particularly limited, such as covalent bonding and grafting by electron beam irradiation, but when [cellulose]-[N-sulfoPEI] is obtained, the following method is preferable. After introducing PEI into cellulose, a method of sulfonation of amino groups and imino groups of this PEI (1) Modification of cellulose Sodium periodate 0.1 to 5.0 normal, preferably 0.5 normal to 1 In a solution of 0.5 N sulfuric acid, the average particle size is 20 μm to 200 μm, preferably 30
μm to 70 μm, average pore size 500Å to 10000
Å, preferably 1000 Å to 8000 Å granular porous cellulose is added, and 10 ℃ to 50 ℃, preferably 2
0 to 30 ° C., 5 to 30 hours, preferably 1
React for 0 to 24 hours. The sodium periodate concentration of the sodium periodate-sulfuric acid solution is 1% by weight to 15% by weight, preferably 3% by weight to 10% by weight. The bath ratio of the granular porous cellulose to the sodium periodate solution is 10% by volume to 30% by volume, preferably 15% by volume to 25% by volume. The reaction mixture was filtered to recover the product, washed thoroughly with water, and the aldehyde content in the swollen state was 0.050 meq / g to 2.00 m.
eq / ml, preferably 0.10 meq / g to 1.5
0 meq / ml of aldehyde cellulose is obtained. This aldehyde cellulose has a structure in which some glucose units are ring-opened, as shown in the chemical formula 2 below.

[0025]

[Chemical 2]

(2) Introduction of PEI Molecular weight 100 to 200,000, preferably 300 to 10
0000 PEI was dissolved in a pH 9.5 buffer solution, to which the aldehyde cellulose obtained in (1) above was added and stirred at 10 ° C to 50 ° C, preferably 20 ° C.
The reaction is carried out at 30 to 30 ° C. for 5 to 30 hours, preferably 10 to 24 hours. The concentration of the above PEI-buffer is 0.2% to 5.0% by weight, preferably 0.4% to 3.0% by weight, and the bath ratio of aldehyde cellulose to this solution is 3% to 20% by volume. %, Preferably 5% to 15% by volume. The reaction mixture is filtered to recover the product, washed with water, dispersed in a buffer having a pH of 9.0, sodium borohydride is added, and the mixture is heated at 10 ° C. to 50 ° C., preferably 20 ° C. to 30 ° C. 30 from time
The Schiff base is hydrogenated by reacting for a time, preferably 10 to 24 hours. Schiff-containing base [cellulose]
-The bath ratio of [PEI] to the buffer solution is 3% to 20% by volume, preferably 5% to 15% by volume, and the charge ratio of the Schiff-containing base [cellulose]-[PEI] to sodium borohydride is 20%. / 1 to 3/2, preferably 15/1 to 3/1 (both in volume / weight ratio). Thus, [cellulose]-[PEI] having an amino group content of 0.010 to 2.50 meq / ml, preferably 0.050 to 1.50 meq / ml is obtained.

(3) N-Sulfonation of PEI [1] Water is added to the [cellulose]-[PEI] obtained in (2) above.
/ 3 to 1/12, preferably 1/5 to 1/10, so that the volume ratio is added and dispersed, and the concentration is 10% by weight.
To 50% by weight, preferably 20% to 40% by weight
The pH of the reaction solution is adjusted to 9 by adding sodium hydroxide aqueous solution.
To 10 and then adding sulfur trioxide / pyridine complex little by little, and adding the above sodium hydroxide aqueous solution little by little to keep the pH at 9 to 10 and from 10 ° C to 50 ° C.
℃ preferably from 20 ℃ to 30 ℃, 0.5 hours to 12
The reaction is carried out for a time, preferably 1 to 8 hours. The product was recovered from the reaction mixture and washed sufficiently until the washing water had a pH of 7.0, and then [cellulose]-[N-sulfoPEI].
To get The volume ratio of [cellulose]-[PEI] / water suspension to the sodium hydroxide aqueous solution added thereto is 3/1.
To 20/1, preferably 7/1 to 15/1, the charge ratio of [cellulose]-[PEI] / water suspension and sulfur trioxide / pyridine complex is 5/1 to 30/1, preferably 10
/ 25 to 25/1 (both volume / weight ratio).

Method of introducing PEI in which amino group and imino group are sulfonated in advance onto the surface of a water-insoluble solid (1) N-sulfonation of PEI Molecular weight 100 to 200,000, preferably 300 to 10
Water is added to 0000 PEI at a weight ratio of 1/3 to 1/12, preferably 1/5 to 1/10, and dissolved, and the concentration thereof is 10% by weight to 50% by weight, preferably 20% by weight. 40 wt% sodium hydroxide aqueous solution is added to adjust the pH of the reaction solution to 9 to 10, and sulfur trioxide / pyridine complex is added little by little, and the sodium hydroxide aqueous solution is added little by little. pH 9 ~
Keep at 10 to 10 ° C to 50 ° C, preferably 20 ° C to 3
The reaction is carried out at 0 ° C. for 0.5 to 12 hours, preferably 1 to 8 hours. N-sulfo PE thus obtained
The sulfonation ratio of I is 30% to 99%, preferably 50
% To 95%, more preferably 70% to 95%. This reaction mixture can be used as it is for the introduction reaction into cellulose, but if sufficient purification is required, concentrate the reaction mixture to a volume of about 1/3, then put it in a cellulose semipermeable membrane and dialyze with distilled water. The low molecular weight impurities can be removed by, and this can be concentrated to dryness. The weight ratio of the PEI aqueous solution to the sodium hydroxide aqueous solution added thereto is 3/1 to 20/1, preferably 7/1 to 15/1,
Charge ratio of chitosan and sulfur trioxide / pyridine complex is 1/3
To 10/1, preferably 2/3 to 5/1 (all by weight).

(2) Introduction of N-SulfoPEI into Cellulose The N-sulfoPEI obtained in the above (1) was dissolved in a buffer solution having a pH of 9.5 and obtained by the method of the above (1). Add aldehyde cellulose and stir at 10 ℃
To 50 ° C., preferably 20 ° C. to 30 ° C., for 5 hours to 30 hours, preferably 10 hours to 24 hours. The concentration of the N-sulfoPEI-buffer is 0.2 wt% to 5.0 wt%, preferably 0.4 wt% to 3.0 wt%.
% By weight, the bath ratio of aldehyde cellulose to this solution is 3
It is from 20% by volume to 20% by volume, preferably from 5% by weight to 15% by weight. The reaction mixture is filtered to recover the product,
Wash with water, disperse this in a pH 9.0 buffer, add sodium borohydride, and heat at 10 ° C to 50 ° C, preferably 20 ° C to 30 ° C for 5 hours to 30 hours, preferably 10 hours to 24 hours. The reaction is carried out to hydrogenate the Schiff base. The bath ratio of the Schiff-containing base [cellulose]-[N-sulfoPEI] to the buffer is 3 to 20% by volume, preferably 5 to 15% by volume. The Schiff-containing base [cellulose]-
The charging ratio of [N-sulfoPEI] and sodium borohydride is 10/1 to 3/2, preferably 7/1 to 3/1.
(Both are capacity / weight ratio). The product was recovered from this reaction mixture and washed thoroughly with water to remove [cellulose]-
[N-sulfoPEI] is obtained.

For example, after the particulate or fibrous [water-insoluble solid]-[N-sulfoPEI] is obtained by the above-mentioned method, the blood purification adsorption of the present invention is carried out by filling this into a suitable container. The material is obtained. The material of the container is glass, stainless steel, polyethylene, polypropylene, polycarbonate, polystyrene, polymethylmethacrylate, etc.
Although not particularly limited, polypropylene and polycarbonate are preferable in consideration of handling such as sterilization. The form of the container is not particularly limited, but in the case of a particulate carrier, a cylindrical column type with both ends of the blood inflow part and the blood outflow part,
In the case of a fibrous carrier, it is suitable to use the same column type as in the case of a particulate carrier, or a sheet-shaped product and then sandwiching it.

[0031]

EXAMPLES The present invention will be described below with reference to examples. Parts in the examples mean parts by weight unless otherwise noted. Example 1 Granular porous cellulose (average particle size 50 μm, average pore size 5
000 Å) 1000 parts (volume) was added to a solution of 125 parts of sodium periodate dissolved in 4000 parts of 1N-sulfuric acid, reacted at 25 ° C for 18 hours, filtered, washed with water,
Aldehyde cellulose (CA-1) having an aldehyde content of 0.98 meq / ml was obtained. The aldehyde was quantitatively determined by the oxime method. That is, CA-1 about 1.0 ml
Was accurately weighed (this amount is V ₁ ml), and ₁₀ ml of 0.5N hydroxylamine hydrochloride solution (35 g of hydroxylamine hydrochloride dissolved in 160 ml of water and diluted with 95% ethanol to 1 l) , Pyridine bromphenol blue solution (95% of a mixture of 0.25 ml of 4% bromphenol blue and 20 ml of pyridine)
35 ml, which had been diluted to 1 l with ethanol) was added. This suspension (hereinafter referred to as “sample”) was treated with 0.5 ml of 0.5N hydroxylamine hydrochloride solution without adding CA-1.
1 and 35 ml of a pyridine bromphenol blue solution (hereinafter referred to as blank) were put together and left in the ultrasonic cleaner for 15 minutes. The sample and the blank were taken out, and a 0.1N sodium hydroxide-methanol solution was added dropwise until the color exhibited by the sample became the same as the blank. When the amount of sodium hydroxide dropped at this time is W ₁ ml and the titer is F ₁ , the aldehyde content X ₁ meq / g is obtained by the following formula. X ₁ = (0.1 × F ₁ × W ₁ ) / V ₁

100 parts (by volume) of CA-1 obtained above was taken and dispersed in 2000 parts (by volume) of a carbonate buffer having a pH of 9.5, and 1200 parts of PEI having a molecular weight of 10,000 was added to the solution and stirred at 25 ° C. And reacted for 18 hours. The reaction mixture is filtered to recover the product, washed with water and then pH adjusted.
A monoethanolamine-containing carbonate buffer solution of 8.0 was added and the mixture was stirred at 25 ° C. for 3 hours to block residual aldehyde groups. The product is collected by filtration, washed thoroughly and pH adjusted.
Disperse in 2000 parts (volume) of 9.0 carbonate buffer, add 40 parts of sodium borohydride, and add 18 parts at 25 ° C.
The Schiff base was hydrogenated by reacting for a period of time. The product was recovered and thoroughly washed with ion-exchanged water to obtain a PEI-derived amino group content of 1.88 meq / ml [cellulose]-[P
EI] (CE-1) was obtained. The amino group was quantified by the following method. Accurately weigh about 0.1 ml of CE-1 (this amount is V ₂ g), and 0.1 N hydrochloric acid aqueous solution 1
0 ml (titer F ₂ ) is added and the suspension is diluted with dioxane to a total volume of 40 ml (this suspension is referred to below as sample). After stirring the sample for about 30 minutes, an automatic titrator (COMITITE 101 manufactured by Hiranuma Sangyo)
0.1 N sodium hydroxide aqueous solution (with a titer of F
₂ ') and perform titration. The amount of 0.1N sodium hydroxide aqueous solution required to neutralize the sample was changed to W ₂ ml.
Then, the amino group content X ₂ meq / g of CE-1 is obtained by the following equation. V ₂ × X ₂ + 0.1 × F ₂ '× W ₂ = 0.1 × F ₂ × 10

100 parts (volume) of the CE-1 obtained above was taken and dispersed in 850 parts (volume) of a carbonate buffer having a pH of 9.5, and a 30 wt% sodium hydroxide aqueous solution was added little by little to adjust the pH to 9 While adjusting to -10, 55 parts of sulfur trioxide / pyridine complex was added little by little over 4 hours at 25 ° C, and the mixture was further stirred at 25 ° C for 1 hour. The sodium hydroxide aqueous solution required here was 90 parts (volume). The reaction mixture was recovered and sufficiently washed with ion-exchanged water until the washing water had a pH of 7.0, and the acid content derived from the sulfamino group contained in N-sulfoPEI was 1.76 meq / ml [cellulose]-[N- SulfoPEI] (CSE-1) was obtained. The acid content was quantified by the following method. After washing the sample with an N / 10 perchloric acid aqueous solution, it was thoroughly washed with ion-exchanged water until the washing water reached pH 7.0, and the same method as in the case where the amino group of CE-1 was quantified as described above. Then, 0.1N aqueous hydrochloric acid solution was added to 0.1N sodium hydroxide aqueous solution, and 0.1N sodium hydroxide aqueous solution was added to 0.1N sodium hydroxide aqueous solution.
It was quantified by replacing with 1N perchloric acid-dioxane solution and measuring.

The CSE-1 obtained above was packed in a 100 ml column, 100 ml of a physiological saline solution containing 100 U / ml heparin, and subsequently 100 ml of a physiological saline solution containing 1 U / ml heparin were placed in the column and in the blood circuit. The inside was washed. On the other hand, a blood circuit was set up so that 1 l of citrated pig blood was placed in a beaker and returned to this beaker through a column. Using this device, perfusion experiment was carried out continuously at a blood flow rate of 50 ml / min for 3 hours, and blood LDL before and after the column was analyzed.
Was measured. In addition, albumin amount, total protein amount, and platelet amount in whole blood were measured before and after the start of perfusion. LDL was quantified by heparin precipitation / colorimetric method using β-lipoprotein C-Test Wako manufactured by Wako Pure Chemical Industries. The amount of albumin was determined by the bromcresol green method, the amount of total protein was determined by the Biuret method, and the amount of platelets was determined by an automatic hemocytometer. The results are shown in Tables 1 and 2. The amount of LDL was shown as the actual measurement value, and albumin, total protein, and platelets were expressed as the residual rate. Unit mg / dl in Table 1, LDL (before)
Indicates the amount of LDL in blood immediately before flowing into the column, and LDL (after) indicates the amount of LDL in blood immediately after flowing out of the column. The residual rate in Table 2 was determined by the concentration of albumin total protein and the number of platelets.

[0035]

[Table 1]

[0036]

[Table 2]

Example 2 PEI2 having a molecular weight of 10,000
While being dissolved in 6000 parts (volume) of carbonate buffer solution having a pH of 9.5, a 30% by weight aqueous solution of sodium hydroxide was added little by little to maintain the pH at 9 to 10,
650 parts of sulfur trioxide / pyridine complex was added little by little over 5 hours at 25 ° C., and the mixture was further stirred at 25 ° C. for 1 hour. The aqueous solution of sodium hydroxide required here was 200 parts (volume). The reaction mixture was concentrated to a volume of about 1/3, put into a cellulose dialysis membrane having an exclusion limit molecular weight of 1000, and ion-exchanged water was flowed to the reaction mixture for 24 hours.
Dialysis was performed for an hour. This solution was concentrated and diluted again with a carbonate buffer solution having a pH of 9.5 to obtain an N-sulfoPEI (SE-2) solution having a concentration of about 250 g / l.

100 parts (volume) of CA11 obtained in Example 1 was taken and 800 parts (volume) of carbonate buffer having a pH of 9.5.
SE-2 solution 12 obtained above
00 parts (volume) was added, stirred, and reacted at 25 ° C. for 18 hours. The reaction mixture was filtered to collect the product, which was washed with water, added with a monoethanolamine-containing carbonate buffer having a pH of 8.0, and stirred at 25 ° C. for 3 hours to block residual aldehyde groups.

The product obtained by the above operation was collected by filtration, washed thoroughly, dispersed in a carbonate buffer having a pH of 9.0, and 30 parts of sodium borohydride was added. Two
The Schiff base was hydrogenated by stirring at 5 ° C. for 18 hours. The product was recovered and thoroughly washed with ion-exchanged water to obtain [cellulose]-[N-sulfoPEI] (CSE-2) having a sulfamino group-derived acid content of 1.65 meq / ml. The acid content was quantified by the same method as the sulfonic acid content of CSE-1 in Example 1.

[Cellulose]-[N-sulfoPE]
I] (CSE-2) was used to perform a blood perfusion experiment in the same manner as in Example 1 to measure the blood LDL amount before and after the column, the albumin amount in the whole blood before and after the perfusion start, the total protein amount, and the platelet amount. .. The results are shown in Tables 1 and 2.

Example 3 27 parts of diethylenetriamine was dissolved in 2000 parts (volume) of carbonate buffer having a pH of 9.5, and 100 parts (volume) of CA-1 obtained in Example 1 was added thereto. The mixture was stirred and reacted at 25 ° C. for 18 hours. The reaction mixture is filtered to collect the product, washed with water,
The Schiff base was hydrogenated by dispersing in 2000 parts of a carbonate buffer of pH 9.0, adding 30 parts of sodium borohydride, and reacting at 25 ° C. for 18 hours. The product is recovered and thoroughly washed with ion-exchanged water to obtain an amino group content of 1.0
8 meq / ml amino group-introduced cellulose (Cen-
3) was obtained. The amino group content was determined by the same method as in Example 1.

100 parts of the above Cen-3 was taken to obtain a pH of 9.
Disperse in 2000 parts (volume) of carbonate buffer solution of 5,
To this, add 25 parts of a 60% glutaraldehyde aqueous solution,
The reaction was allowed to stir at 25 ° C. for 18 hours. The reaction mixture was collected by filtration, washed with water, and washed with a carbonate buffer solution of pH 9.5.
After being dispersed in 0 part (volume), 1200 parts of the SE-2 solution obtained in Example 2 was added. After stirring and reacting at 25 ° C. for 18 hours, the product was collected and washed with ion-exchanged water.
A carbonate buffer containing monoethanolamine having a pH of 8.0 was added to this product, and the mixture was stirred at 25 ° C. for 3 hours to block residual aldehyde groups.

The product obtained by the above operation was dispersed in 2000 parts of carbonate buffer of pH 9.0, 30 parts of sodium borohydride was added, and the mixture was reacted at 25 ° C. for 18 hours to hydrogenate the Schiff base. .. The product was recovered and thoroughly washed with ion-exchanged water to obtain a sulfamino group-derived acid content of 1.42.
[Cellulose]-[Glutaraldehyde]-[N-sulfoPEI] (CGSE-3) of meq / ml was obtained.
The acid content was quantified by the same method as in Example 1.

Using the above [cellulose]-[glutaraldehyde]-[N-sulfoPEI] (CGSE-3), a blood perfusion experiment was carried out in the same manner as in Example 1, and the blood LDL amount before and after the column and before and after the perfusion start. The amount of albumin in the whole blood of
The total protein amount and the platelet amount were measured. The results are shown in Tables 1 and 2.

Comparative Example 1 600 parts of a polyacrylic acid having a molecular weight of 4000 was dissolved in 2500 parts of a citrate buffer solution having a pH of 4.5 to prepare N-cyclohexyl-N '-(2-morpholinoethyl) carbodiimide-meth-p-. Add 63 parts of toluene sulfonate (hereinafter abbreviated as CMEC), and add 0
The mixture was stirred at 0 ° C for 20 minutes. Cen-3 obtained in Example 3 was added to this.
Was added to 100 parts (volume), the reaction temperature was gradually raised from 0 ° C. to room temperature, and the reaction was stirred for 18 hours for reaction. The product was recovered by filtration, washed thoroughly with a 3% aqueous sodium hydroxide solution and then with ion-exchanged water, and the acid content derived from the carboxyl group of polyacrylic acid was 1.36 meq / ml.
[Cellulose]-[Polyacrylic acid] (CPA-4)
Got The acid content was quantified by the same method as in the example.

A blood perfusion experiment was carried out in the same manner as in Example 1 using the above CPA-3, and the blood LDL amount before and after the column, the albumin amount in the whole blood before and after the start of perfusion, the total protein amount, and the platelet amount were measured. .. The results are shown in Tables 1 and 2.

<Comparative Example 2> 1 of CA-1 obtained in Example 1
2000 parts carbonate buffer solution of pH 9.5 by taking 00 parts (volume)
26 parts of taurine was added thereto, stirred, and reacted at 25 ° C. for 18 hours. The product was collected by filtration, washed with ion-exchanged water, dispersed in a monoethanolamine-containing carbonate buffer having a pH of 8.0, and stirred at 25 ° C. for 3 hours to block residual aldehyde groups. The product was recovered by filtration, washed with ion-exchanged water, and then washed with carbonate buffer 2000 having a pH of 9.0.
Parts (volume), 30 parts of sodium borohydride was added, and the mixture was stirred and reacted at 25 ° C. for 18 hours to hydrogenate the Schiff base. The product is recovered, washed thoroughly with ion-exchanged water, and then [cellulose]-[taurine] (CT-
5) was obtained. When measured by the same method as in the example, C
The content of sulfonic acid derived from taurine of T-5 is 0.94 m
It was eq / ml.

[Cellulose]-[Taurine] (CT
-5) was used to conduct a blood perfusion experiment in the same manner as in Example 1,
The amount of LDL in blood before and after the column, the amount of albumin in the whole blood before and after the start of perfusion, the amount of total protein, and the amount of platelets were measured. The results are shown in Tables 1 and 2.

Comparative Example 3 100 parts by volume of granular porous cellulose (average particle size 50 μm, average pore size 5000 Å), 40 parts by volume 20% by weight sodium hydroxide aqueous solution, 120 parts by volume heptane, gelatin 8 After stirring 2 parts of the mixed suspension at 40 ° C. for 2 hours, 50 parts of epichlorohydrin was added and stirred at 40 ° C. for another 2 hours. The product was recovered from this reaction mixture and thoroughly washed to obtain epoxidized cellulose (CE-6). Intrinsic viscosity 0.08
5 parts of sodium dextran sulfate having 3 dl / g, an average degree of polymerization of 140, and a sulfur content of 19.2% by weight was dissolved in 20 parts of water (volume), and 20 parts (volume) of the above CE-5 was added thereto. The pH was adjusted to 12 and reacted at 40 ° C. for 18 hours. After blocking unreacted glycidyl groups with monoethanolamine, the product was recovered and washed thoroughly to give a sulfonic acid content of 0.30 meq / ml [cellulose]-
[Dextran sodium sulfate (CDS-6) was obtained.

A blood perfusion experiment was carried out in the same manner as in Example 1 using the above-mentioned [cellulose]-[dextran sodium sulfate] (CDS-6), and the amount of LDL in blood before and after the column and albumin in whole blood before and after the start of perfusion. The amount, total protein amount, and platelet amount were measured. The results are shown in Tables 1 and 2.

As is clear from the above examples, the constant density lipoprotein adsorbent of the present invention was able to selectively and simply remove LDL in blood. When focusing only on the adsorption capacity, the adsorbent having dextran sulfate immobilized was at the same level as the adsorbent of the present invention, but when considering the effect on other blood components such as albumin and platelets, the adsorption of the present invention was considered. The wood was found to be extremely good. The reason for this is as follows. The N-sulfoPEI chain immobilized on the water-insoluble solid acts as a hydrophilic spacer and a good blood compatibility is realized by the excluded volume effect. In addition, N-sulfoPEI also has an effect as a heparinoid, and it is considered that this also contributes to the improvement of blood compatibility. The sulfamino group that acts as a ligand is derived from N-sulfoPEI, which is relatively distant from the surface of the solid, and the N-sulfoPEI chain near the surface of the solid acts as a hydrophilic spacer, so its mobility increases the mobility of the ligand. , The adsorption capacity is improved.

[0052]

INDUSTRIAL APPLICABILITY The low specific gravity lipoprotein adsorbent of the present invention exhibits the effect as a heparinoid in addition to the excluded volume effect for the N-sulfoPEI chain near the solid surface to act as a hydrophilic spacer. It is possible to achieve both good adsorption ability and blood compatibility, and it is possible to expect a sufficient effect by directly perfusing blood without the complicated method of separating plasma components from blood and perfusing in advance. Therefore, it can be used for amelioration and treatment of symptoms by removing low-density lipoprotein with a simple operation.

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

Claims (1)

[Claims] 1. A water-insoluble solid having N-sulfopolyethyleneimine and a sulfamino group-derived acid content of 50 μeq /
A low specific gravity lipoprotein adsorbent, which is fixed so as to have a volume of from 2.0 ml (wet state) to 2.0 meq / ml (wet state).