JPH05285382A - Low-specific gravity lipoprotein adsorptive material - Google Patents

Abstract

PURPOSE:To obtain the adsorptive material which can remove the low-sp. gr. lipoprotein and ultra-low-sp. gr. lipoprotein in blood by fixing N- sulfopolyethyleneimine via a hydrophilic spacer consisting of polyalkylene oxide skeleton onto the surface of a water insoluble solid in such a manner that the acid content derived from a sulfamino group attains a specific ratio. CONSTITUTION:The N-sulfopolyethyleneimine is fixed via the hydrophilic spacer is fixed to the water insoluble solid (e.g. polystyrene) in such a manner that the acid content derived from the sulfamino group attains 50mulq/ml (wet state) to 2.0mlq/ml. The hydrophilic spacer is so formed as to have the polyalkylene oxide skeleton (e.g. polyethylene glycol) consisting of 1-4C repeating unit and 5 to 400 degree of polymn. Consequently, the low-sp. gr. lipoprotein adsorptive material which can selectively, easily and effectively adsorb the low-sp. gr. lipoprotein and/or ultra-low-sp. gr. lipoprotein in the blood is obtd.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a low-density lipoprotein (hereinafter abbreviated as LDL) and / or an extremely low-density lipoprotein (hereinafter VLD), which is a causative agent of arteriosclerosis and hyperlipidemia.
L) and (or) VLDL in blood by contacting blood or blood components for the purpose of adsorbing and removing LDL) and adsorbing low-density lipoprotein adsorbent. .. 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] In Chemical formula 1, X represents a salt-forming group such as H or Na.

Further, in the present invention, -N (R) SO ₃
H, both the -NHSO ₃ H or a salt thereof is referred to as a sulfamino group. (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. 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, 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.

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 is hard to say 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, but it is satisfactory in terms of adsorption selectivity and adsorption capacity, but it is difficult to obtain the antibody. 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. 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.

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 a hydrophilic spacer having an ethylene oxide skeleton of 90 is interposed, it is difficult to achieve sufficient blood compatibility simply by introducing a hydrophilic spacer having an ethylene oxide skeleton having a degree of polymerization of up to 90. Also in JP-A-1-158970, coating with an acrylic acid derivative is performed to improve blood compatibility. Such an operation is not only complicated, but may cause the elution of a toxic substance.

[0014]

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.

[0015]

The low-density lipoprotein adsorbent of the present invention has N-sulfoPEI added to a water-insoluble solid through a hydrophilic spacer and an acid content derived from a sulfamino group of 50.
It is characterized in that it is fixed so as to be μeq / ml (wet state) to 2.0 meq / ml (wet state), thereby removing low-density lipoprotein and / or extremely low-density lipoprotein in blood. Is. The hydrophilic spacer used in the low-density lipoprotein adsorbent of the present invention has a carbon number of 1
And a polyalkylene oxide skeleton having a degree of polymerization of 5 to 400. Examples of such a hydrophilic spacer include compounds having a skeleton such as polyethylene glycol, polypropylene glycol, and polytetramethylene glycol. The hydrophilic spacer used in the present invention is
It is preferably nonionic like the compounds exemplified here.

In the blood purification adsorbent of the present invention, the water-insoluble solid is a synthetic organic polymer compound such as polystyrene, crosslinked polyvinyl alcohol, polymethacrylic acid and its derivatives or copolymers thereof, and natural substances such as cellulose, chitin and chitosan. It is preferably an organic polymer compound or a modified natural organic polymer compound such as acyl cellulose or acyl chitin. Of these polymer compounds, considering mechanical strength and ease of ligand introduction, moderately cross-linked polystyrene, polymethacrylic acid and its derivatives or their copolymers, and cellulose are preferable, and cellulose is more preferable. ..

The N-sulfoPEI used in the present invention preferably has a molecular weight of 100 to 200,000, preferably 30.
0 to 10000 is desirable. 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 is impossible to introduce a chemical bond into the [water-insoluble solid]-[hydrophilic spacer] by utilizing the remaining amino group and imino group, 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, the residual amino groups and imino groups account for 10% of the total amount when fixing to the water-insoluble solid is completed. The following is desirable. When the amount of residual amino group or imino group exceeds this amount, it is recommended to block with acetic anhydride, succinic anhydride, 1,3-propane sultone or the like. PE in the present invention
For N-sulfonation of I, chlorosulfonic acid, concentrated sulfuric acid, fuming sulfuric acid, sulfur trioxide / dimethylformamide complex, sulfur trioxide / pyridine complex and the like can be used. Among them, sulfur trioxide / pyridine complex is preferable. preferable. 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, which is relatively close to the surface of the water-insoluble solid, mainly contributes to the improvement of blood compatibility and the mobility of the ligand as a hydrophilic spacer.
I has a role of adsorption removal of LDL and / or VLDL as a ligand. However, it cannot be said that the effect of the N-sulfoPEI chain as the hydrophilic spacer is sufficient. Furthermore, when the polyanion is directly introduced into the water-insoluble solid, regardless of the introduction method,
There is a great possibility that the polyanion is fixed to the water-insoluble solid at a large number of bonding points, and there is a drawback in that the effect of the polyanion is not sufficiently exerted during LDL and / or VLDL adsorption. Therefore, in order to achieve better adsorption performance and blood compatibility, it is necessary to interpose a nonionic hydrophilic spacer.

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.

The method of introducing the hydrophilic spacer and N-sulfoPEI into the water-insoluble solid used in the present invention is not particularly limited, such as covalent bonding and grafting by electron beam irradiation, but it is [cellulose]-[hydrophilic]. Spacer]-
When obtaining [N-sulfoPEI], the following method is preferable. A method in which PEI is introduced into cellulose through a hydrophilic spacer, and then amino groups and imino groups of this PEI are sulfonated. (1) Modification of cellulose Sodium periodate 0.1 to 5.0 normal, preferably An average particle size of 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 ° C to 30 ° C, 5 hours to 30 hours, preferably 1
React for 0 to 24 hours. The sodium periodate concentration of the sodium periodate-sulfuric acid solution is 0.5% by weight.
To 15% by weight, preferably 1.0% to 10% by weight. The bath ratio of the granular porous cellulose to the sodium periodate solution is 10% by volume to 50% by volume,
It is preferably 15% by volume to 35% by volume. The reaction mixture was filtered to collect the product, washed thoroughly with water, and swollen to give an aldehyde content of 0.010 meq / g to 2.
00 meq / ml, preferably 0.050 meq / g to 1.50 meq / ml aldehyde cellulose are obtained. This aldehyde cellulose has a structure in which some glucose units are ring-opened as shown in the following chemical formula 2.

[0025]

[Chemical 2]

(2) Introduction of hydrophilic spacer Molecular weight 300 to 8000, preferably 400 to 700
0, more preferably 1000 to 6500 polyethylene glycol having amino groups at both ends (hereinafter abbreviated as PEO amine) was dissolved in a buffer solution having a pH of 9.5, and the aldehyde cellulose obtained in the above (1) was added to the solution. Is added and stirred at 10 ° C to 50 ° C, preferably 20 ° C.
5 to 30 hours, preferably 10 to 30 ° C
Allow to react for 24 hours. The concentration of the PEO amine buffer is 5% to 60% by weight, preferably 10% to 40% by weight, and the bath ratio of aldehyde cellulose to this solution is 1% to 20% by volume, preferably 3% by volume.
To 15% by volume. The reaction mixture was filtered to collect the product, which was washed with water, dispersed in a buffer solution having a pH of 9.0, and sodium borohydride was added to the reaction mixture at 10 ° C to 50 ° C.
Hydrogenation of the Schiff base is carried out by reacting at a temperature of preferably 20 ° C. to 30 ° C. for 5 hours to 30 hours, preferably 10 hours to 24 hours. Schiff-containing base [cellulose]-
The bath ratio of [PEO amine] to the buffer is from 1% by volume to 20%.
% By volume, preferably 3% to 15% by volume, and the charge ratio of the Schiff base [cellulose]-[PEO amine] to sodium borohydride is 20/1 to 3/2, preferably 15/1 to 3/1. (Both are capacity / weight ratio).
Thus the amino group content is 0.010 to 2.50 meq /
ml, preferably 0.050 to 1.50 meq / ml
[Cellulose]-[PEO amine] of is obtained. The PEO amine used here has a structure of the following chemical formula 3.

[0027]

[Chemical 3]

(3) Introduction of PEI [cellulose]-[PEO amine] obtained in the above (2).
Is dispersed in a buffer solution of pH 9.5, glutaraldehyde is added to this, and the mixture is stirred at 10 ° C to 50 ° C.
Preferably at 20 ° C to 30 ° C for 5 hours to 30 hours,
The reaction is preferably carried out for 10 to 24 hours. The bath ratio of [cellulose]-[PEO amine] to the above buffer solution is 3% by volume to 20% by volume, preferably 5% by volume to 15% by volume, and the amount of glutaraldehyde added is 0.05% by weight to 5% by weight. 0.0% by weight, preferably 0.1% by weight to 3.
It is desirable to be 0% by weight, and [cellulose]-[P
It is necessary that an excess amount be added to the amino group derived from EO amine. Thus [cellulose]-[PE
O amine]-[glutaraldehyde] is obtained. The reaction mixture is filtered to collect the product, washed with water,
It is dispersed in a buffer solution of 9.5 to have a molecular weight of 100 to 20,000.
After adjusting the pH by adding PEI of 0, preferably 300 to 100,000, 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 bath ratio of [cellulose]-[PEO amine]-[glutaraldehyde] to the above buffer solution is 3% to 20% by volume, preferably 5% to 15% by volume, and the addition amount of PEI is 1.5%. % To 60% by weight, preferably 10 to 40% by weight, preferably [cellulose]-[PEO amine].
-It is necessary that an excess amount is added to the aldehyde groups derived from [glutaraldehyde]. 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. Hydrogenation of the Schiff base is carried out by reacting for 30 hours, preferably for 10 hours to 24 hours. The bath ratio of the Schiff-containing base [cellulose]-[PEO amine]-[PEI] to the buffer is 3% by volume to 20% by volume, preferably 5% by volume.
To 15% by volume, Schiff-containing base [cellulose]-[PE
The charge ratio of O amine]-[PEI] to sodium borohydride is 20/1 to 3/2, preferably 15/1 to 3
/ 1 (both volume / weight ratio). Thus, [cellulose]-[PEI] having an amino group content of 0.010 to 2.50 meq / ml, preferably 0.050 to 2.00 meq / ml is obtained.

(4) N-sulfonation of PEI [cellulose]-[PEO amine] obtained in the above (3).
-[PEI] buffer solution pH 9.5 from 1/3 to 1/1
2 Add to be dispersed preferably at a volume ratio of 1/5 to 1/10, and add sulfur trioxide / pyridine complex little by little to obtain a concentration of 10% by weight to 50% by weight, preferably 20% by weight. While adjusting the pH of the reaction solution to 9 to 10 by adding a 40 wt% sodium hydroxide aqueous solution, 10 ° C to 50 ° C, preferably 20 ° C to 30 ° C.
For 0.5 to 12 hours, preferably 1 to 8 hours
React for hours. The product is recovered from the reaction mixture and washed thoroughly until the washing water reaches pH 7.0 [cellulose].
-[PEO amine]-[N-sulfoPEI] is obtained.
The charging ratio of [cellulose]-[PEO amine]-[PEI] / buffer suspension and sulfur trioxide / pyridine complex was 5 /.
It is 1 to 30/1, preferably 10/1 to 25/1 (both in volume / weight ratio).

P having an amino group previously sulfonated
Method of introducing EI into water-insoluble solid (1) N-sulfonation of PEI Molecular weight 100 to 200,000, preferably 300 to 10
1/3 to 1 buffer of pH 9.5 in 0000 PEI
/ 12, preferably added so as to have a volume ratio of 1/5 to 1/10 and dissolved, and a sulfur trioxide / pyridine complex is added little by little to obtain a concentration of 10% by weight to 50% by weight, preferably 20% by weight. To 40 wt% sodium hydroxide aqueous solution is added to adjust the pH of the reaction solution to 9 to 10 ° C to 50 ° C, preferably 20 ° C to 30 ° C.
The reaction is carried out at 0 ° C. for 0.5 to 12 hours, preferably 1 to 8 hours. N-sulfoPEI thus obtained
Has a sulfonation rate of 30% to 99%, preferably 50%
To 95%, more preferably 70 to 95%. This reaction mixture is as it is [cellulose]-[PE
O amine], but when sufficient purification is required, the reaction mixture is concentrated to a volume of about 1/3, then put into a cellulose semipermeable membrane and dialyzed against distilled water to obtain a low molecular weight compound. The impurities of the above are removed, and this may be concentrated to dryness. The charge ratio of PEI and sulfur trioxide / pyridine complex is 1/3 to 10/1, preferably 2/3 to 5/1 (all are weight ratios).

(2) Introduction of N-sulfoPEI The N-sulfoPEI obtained in (1) above is converted to (2) above.
[Cellulose]-[PEO amine]-[N-sulfoPEI] is obtained by immobilizing [Cellulose]-[PEO amine] obtained by the above method by the same method as in the above (3).

For example, by the above-mentioned method, particulate or fibrous [water-insoluble solid]-[hydrophilic spacer]-
After obtaining [N-sulfoPEI], it is filled in an appropriate container to obtain the low specific gravity lipoprotein adsorbent of the present invention. 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 in the case of a particulate carrier, a cylindrical column type with both ends of blood inflow part and blood outflow part, in the case of a fibrous carrier, a column type similar to the case of the particulate carrier, or It is suitable that the sheet is formed into a sheet and then sandwiched.

[0033]

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
m, average pore diameter 5000 Å) 1000 parts (volume) was added to a solution of 125 parts of sodium periodate dissolved in 4000 parts of 1N-sulfuric acid and reacted at 25 ° C for 18 hours.
The crystals were separated by filtration and washed with water to obtain aldehyde cellulose (CA-1) having an aldehyde content of 0.98 meq / ml. 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 0.5 N hydroxylamine hydrochloride solution (35 g of hydroxylamine hydrochloride was dissolved in 160 ml of water and diluted with 95% ethanol to yield 1 liter). And what) 10
ml, pyridine bromophenol blue solution (mixture of 0.25 ml of 4% bromophenol blue and 20 ml of pyridine diluted to 95% ethanol to make 1 liter)
35 ml was added. This suspension (hereinafter referred to as sample)
Was mixed with 10 ml of 0.5 N hydroxylamine hydrochloride solution and 35 ml of pyridine bromphenol blue solution (hereinafter referred to as blank) without CA-1 and left in an 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. At this time, the amount of sodium hydroxide dropped is W
Aldehyde content X ₁ meq, assuming ₁ ml and titer F _1.
/ G is obtained by the following equation. X ₁ = (0.1 × F ₁ × W ₁ ) / V ₁

57 parts of diethylenetriamine was added to pH 9.5.
Dissolved in 5000 parts (volume) of carbonate buffer solution, and 250 parts (volume) of the above CA-1 was added and stirred,
The reaction was carried out at 25 ° C for 18 hours. The reaction mixture was filtered to collect the product, which was washed with water and dispersed in 5000 parts of a carbonate buffer solution having a pH of 9.0 to give 70 parts of sodium borohydride.
A portion of the Schiff base was added and hydrogenated by reacting at 25 ° C. for 18 hours. Thus the amino group content is 1.02m
Amino group-introduced cellulose (Cen-1) of eq / ml was obtained. The amino group was quantified by the following method. C
About 0.1 ml of en-1 was accurately weighed (this amount was V ₂ g
10 ml of 0.1 N hydrochloric acid aqueous solution (with a titer of 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, it is titrated with an aqueous 0.1 N sodium hydroxide solution (titer is F ₂ ') using an automatic titrator (COMITITE 101 manufactured by Hiranuma Sangyo). Assuming that the amount of 0.1 N sodium hydroxide aqueous solution required to neutralize the sample is W ₂ ml, Cen
The amino group content X ₂ meq / g of -1 is obtained by the following formula. V ₂ × X ₂ + 0.1 × F ₂ '× W ₂ = 0.1 × F ₂ × 10

Polyethylene glycol having glycidyl groups at both ends (hereinafter abbreviated as PEO epoxy) 300
Parts were dissolved in 5000 parts (volume) of carbonate buffer having a pH of 9.5, and 250 parts (volume) of the above Cen-1 was dissolved therein.
The mixture was added, stirred, and reacted at 25 ° C. for 18 hours. The reaction mixture was filtered to collect the product and washed with water to obtain a hydrophilic spacer-introduced cellulose (CPEO-1). CPE
O-1 was dispersed in 6000 parts (volume) of carbonate buffer having a pH of 9.5, 3500 parts of PEI having a molecular weight of 10,000 was added, and the mixture was reacted at 25 ° C. for 18 hours while stirring. The reaction mixture was filtered to collect the product, washed with water, and then adjusted to pH 8.
25 of the carbonate buffer containing monoethanolamine of 0 was added.
The mixture was stirred at 0 ° C for 3 hours to block residual glycidyl groups.
The product was collected by filtration, washed thoroughly and washed with [cellulose]-[PEO epoxy]-[PEI] (CPEOE
-1) was obtained. When the amino group content of CPEOE-1 was measured by the same method as above, it was 1.72 meq / ml. The structure of PEO epoxy used in this reaction is as shown in Chemical Formula 4 below.

[0036]

[Chemical 4]

100 parts (volume) of the CPEOE-1 obtained above was dispersed in 850 parts (volume) of 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 Adjusting to -10, sulfur trioxide /
50 parts of 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 aqueous solution of sodium hydroxide required here was 84 parts (volume). The reaction mixture was recovered, washed sufficiently with ion-exchanged water until the washing water reached pH 7.0, and the acid content derived from the sulfamino group contained in N-sulfoPEI was 1.52 meq / m 2.
l of [cellulose]-[PEO epoxy]-[N-sulfoPEI] (CPEOSE-1) was obtained. The acid content was quantified by the following method. After washing the sample with an N / 10 perchloric acid aqueous solution, the washing water is adjusted to pH 7.0 with ion-exchanged water.
Was thoroughly washed until it became, and a 0.1N aqueous solution of hydrochloric acid was added to a 0.1N aqueous solution of sodium hydroxide in the same manner as in the case of quantifying the amino group of Cen-1. The sodium oxide aqueous solution was replaced with a 0.1N perchloric acid-dioxane solution, and the measurement was performed.

100 ml of CPEOSE-1 obtained above
The column and the blood circuit were washed with 100 ml of a physiological saline solution containing 100 U / ml heparin, and subsequently with 100 ml of a physiological saline solution containing 1 U / ml heparin. 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. Blood flow of 50 ml / mi using this device
The perfusion experiment was carried out continuously for 3 hours by measuring the amount of LDL in blood before and after the column. In addition, albumin amount, total protein amount, and platelet amount in whole blood were measured before and after the start of perfusion. LD
Quantification of L was performed 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. The ratio was determined by the concentration for MATA and albumin total protein and the number for platelets.

[0039]

[Table 1] In the table, the unit is mg / dl, LDL (before) indicates the blood LDL amount immediately before the column inflow, and LDL (after) indicates the blood LDL amount immediately after the column outflow.

[0040]

[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 CPEO-1 obtained in Example 1 was taken and dispersed in 800 parts (volume) of carbonate buffer having a pH of 9.5, and the SE-2 solution 1200 obtained above was added thereto. Part (volume) was added, and the mixture was 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 the residual glycidyl group. The product is collected by filtration,
Thoroughly washed, acid content derived from sulfamino group 1.63m
eq / ml [cellulose]-[PEO epoxy]-
[N-sulfoPEI] (CPEOSE-2) was obtained. The acid content was quantified by the same method as the acid content of CPEOSE-1 in Example 1.

[Cellulose]-[PEO epoxy]
A blood perfusion experiment was carried out in the same manner as in Example 1 using [N-sulfoPEI] (CPEOSE-2), the blood LDL amount before and after the column, the albumin amount in whole blood before and after the perfusion start,
The total protein amount and the platelet amount were measured. The results are shown in Tables 1 and 2.

Example 3 400 parts of PEO amine having a molecular weight of 4500 was taken and dissolved in 1200 parts (volume) of a carbonate buffer solution having a pH of 9.5, and 1 part of CA-1 obtained in Example 1 was added thereto.
00 parts (volume) was added, and the mixture was stirred at 25 ° C. for 18 hours for reaction. The reaction mixture was filtered to collect the product, which was washed with water and suspended in 1200 parts (volume) of a carbonate buffer solution having a pH of 9.5.
00 parts was added and reacted by stirring at 25 ° C. for 18 hours. The product recovered by filtration and washed with water was suspended in 800 parts (volume) of a pH 9.5 carbonate buffer solution, and the suspension was added to Example 2
1200 parts (volume) of the SE-2 solution obtained in Step 2 was added, and 2
The reaction was allowed to stir at 5 ° C. for 18 hours. The reaction mixture obtained by the above operation is filtered to collect the product, which is washed with water and then adjusted to pH.
The mixture was dispersed in 400 parts (volume) of a carbonate buffer solution containing 8.0 monoethanolamine and stirred at 25 ° C. for 3 hours to block residual aldehyde groups. The product was collected by filtration, washed with ion-exchanged water, and then dispersed in 2000 parts of a carbonate buffer solution having a pH of 9.0 to give 30 parts of sodium borohydride.
A portion of the Schiff base was hydrogenated by reacting at 25 ° C. for 18 hours. The product was recovered and thoroughly washed with ion-exchanged water to obtain an acid content of [cellulose]-[PEO amine]-[N-sulfoPEI] having an acid content of 1.34 meq / ml.
(CPEASE-3) was obtained. The acid content was determined in Example 1
The same method was used.

[Cellulose]-[PEO amine]-
A blood perfusion experiment was conducted in the same manner as in Example 1 using [N-sulfoPEI] (CPEASE-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 platelets were measured. The quantity was measured. The results are shown in Tables 1 and 2.

Comparative Example 1 CPEO-1 obtained in Example 1
100 parts (volume) was taken and dispersed in 2500 parts of a citrate buffer solution having a pH of 4.5, 600 parts of polyacrylic acid having a molecular weight of 4000 was added thereto, and the mixture was reacted at 25 ° C. for 18 hours with stirring. The product was treated from this reaction mixture in the same manner as in Example to block residual glycidyl groups, then collected by filtration, washed thoroughly with 3% aqueous sodium hydroxide solution, and then ion-exchanged water. Cellulose]-[PEO epoxy]-[Polyacrylic acid] (CP
EOA-4) was obtained. When the carboxyl group derived from polyacrylic acid was quantified by the same method as in Example 1, it was 1.0
It was 8 meq / ml.

[Cellulose]-[PEO epoxy]
A blood perfusion experiment was carried out in the same manner as in Example 1 using [polyacrylic acid] (CPEOA-4), and blood L before and after the column was analyzed.
DL amount, albumin amount in whole blood before and after the start of perfusion, total protein amount, and platelet amount were measured. The results are shown in Tables 1 and 2.

<Comparative Example 2> 26 parts of taurine was added to pH 9.5.
Was dissolved in 2000 parts (volume) of the carbonate buffer solution, and 100 parts (volume) of CPEO-1 obtained in Example 1 was added and stirred, and the mixture was reacted at 25 ° C. for 18 hours. The product was treated from this reaction mixture in the same manner as in Example to block residual glycidyl groups, and then recovered by filtration.
Thoroughly wash with ion-exchanged water, then [cellulose]-[P
EO epoxy]-[taurine] (CPEOT-5) was obtained. When measured in the same manner as in the example, the CPEOT
-4 content of sulfonic acid derived from taurine is 0.91 me
It was q / ml.

[Cellulose]-[PEO epoxy]
A blood perfusion experiment was conducted in the same manner as in Example 1 using [taurine] (CPEOT-5), and blood LDL before and after the column was analyzed.
The amount of albumin in the whole blood, the amount of total protein, and the amount of platelets before and after the start of perfusion were measured. The results are shown in Tables 1 and 2.

<Comparative Example 3> 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, and then washed with carbonate buffer solution 4 containing monoethanolamine at pH 8.0.
It was dispersed in 00 parts (volume) and stirred at 25 ° C. for 3 hours to block residual aldehyde groups. The product was collected by filtration, washed with ion-exchanged water, dispersed in 2000 parts (volume) of carbonate buffer having a pH of 9.0, 30 parts of sodium borohydride was added, and the mixture was stirred and reacted at 25 ° C. for 18 hours. Then, the Schiff base was hydrogenated. Collect the product and wash it thoroughly with deionized water to remove [cellulose]-
[Taurine] (CT-6) was obtained. When measured by the same method as in Example, the content of sulfonic acid derived from taurine in CT-5 was 0.94 meq / ml.

[Cellulose]-[Taurine] (CT
-6) was used to perform 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 4 Granular porous cellulose (average particle size 50 μm, average pore size 5000 Å) 100 parts (volume), 20 wt% sodium hydroxide aqueous solution 40 parts (volume), heptane 120 parts (volume), 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-7). 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-7 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-7) was obtained.

A blood perfusion experiment was conducted in the same manner as in Example 1 using the above-mentioned [cellulose]-[dextran sodium sulfate] (CDS-7), 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 easily 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 hydrophilic spacer fixed on the water-insoluble solid surface works effectively, and 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 acting as a ligand is fixed away from the solid surface by a hydrophilic spacer, and the mobility of the hydrophilic spacer in the vicinity of the solid surface increases its mobility to improve its adsorptivity. It is clear from the fact that the sample of Example 3 has a very good performance that the effect of this hydrophilic spacer is extremely large.

[0055]

INDUSTRIAL APPLICABILITY The low-density lipoprotein adsorbent of the present invention exhibits good adsorption because N-sulfoPEI introduced as a ligand exerts an effect as a heparinoid in addition to the excluded volume effect by introducing a hydrophilic spacer. It is possible to achieve both high performance and blood compatibility, and it is possible to expect a sufficient effect by directly perfusing blood without taking the complicated method of separating the plasma component 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 (2)

[Claims] 1. An N-sulfopolyethyleneimine is added to a water-insoluble solid through a hydrophilic spacer, and an acid content derived from a sulfamino group is 50 μeq / ml (wet state) to 2.0 m.
A low-density lipoprotein adsorbent, which is fixed so as to have an eq / ml (wet state). 2. The hydrophilic spacer is composed of a repeating unit having 1 to 4 carbon atoms, and has a polyalkylene oxide skeleton having a polymerization degree of 5 to 400.
The low-density lipoprotein adsorbent described.