JP2002102692A - Manufacturing method for lipoperoxide-adsorbing material - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a manufacturing method for lipoperoxide-adsorbing material capable of adsorbing and removing a peroxide lipid. SOLUTION: In the manufacturing method for lipoperoxide-adsorbing material, a cationic polymer is immobilized on a support by irradiating a radiation to the support in the state that the cationic polymer is deposited thereon.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for producing a lipid peroxide adsorbent.

[0002]

2. Description of the Related Art Conventionally, patients with heart disease and dialysis patients often exhibit arteriosclerosis, and the concentration of lipid peroxide in blood has increased.

[0003] Among lipid peroxides, oxidized low-density lipoprotein (hereinafter, referred to as LDL) has various biological actions. In addition to the action of inhibiting the production of nitric oxide (NO) from endothelial cells, it has a single effect. The sphere is transported under the subendothelium and accumulated,
It plays an important role in the onset and progression of arteriosclerosis, such as promoting macrophages, taking oxidized LDL itself into foam cells, promoting plaque formation on the arterial wall, and promoting endothelial cell and smooth muscle cell damage. Play. Therefore, it is desired to remove lipid peroxides, particularly oxidized LDL, from blood. Until now, there has been no effective means for lowering the concentration of lipid peroxide in blood.

Since high-density lipoprotein (hereinafter referred to as HDL) has a function as an arteriosclerosis-protecting factor, the blood HDL concentration of a dialysis patient must not be lowered.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for producing an adsorbent capable of adsorbing and removing lipid peroxide by improving the disadvantages of the prior art.

[0006]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for adsorbing a lipid peroxide, comprising immobilizing a cationic polymer on a support by irradiating the support with the cationic polymer attached thereto. This is achieved by the method of manufacturing the material.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The lipid peroxide adsorbent of the present invention has a cationic polymer immobilized on a support. Examples of the cationic polymer include primary, secondary, and tertiary amino groups,
Examples include an amino group-containing polymer having a quaternary ammonium salt, a polymer having an aziridine (ethyleneimine) compound, chitin, chitosan, acrylamide-based polymers, and copolymers of these, or copolymers with nonions and anionic compounds. The polymer may be linear, branched or cyclic. In addition, the polymer here refers to a compound having a molecular weight of 600 or more.

Examples of the amino group-containing polymer include polyalkyleneimine, polyallylamine, polyvinylamine, diethylaminoethyldextran and those having a substituent introduced therein, and copolymers comprising monomer units constituting these. Can be

[0009] Examples of the polyethyleneimine derivative include those obtained by derivatizing polyethyleneimine at various ratios such as alkylation, carboxylation, phenylation, phosphorylation, and sulfonation.

A linear or branched polyethyleneimine having a molecular weight of 600 or more is used.

Low toxicity among cationic polymers,
Branched polyethyleneimine is preferably used in terms of availability, handling, and the like.

The method for producing a lipid peroxide adsorbent of the present invention comprises:
Irradiation is performed with the cationic polymer attached to the support. Specifically, for example, irradiation is performed with the support immersed in a solution of a cationic polymer. Alternatively, the support may be immersed in a cationic polymer solution, extracted from the solution, and irradiated with radiation while the support is sealed in nitrogen without drying. That is, irradiation is performed in a wet state under a nitrogen atmosphere. Alternatively, after immersion in a cationic polymer solution, the support may be washed with water and irradiated with water while immersed in water.
That is, the cationic polymer is adsorbed on a support and irradiated with radiation while immersed in water. Irradiation while immersed in a cationic polymer solution has the advantage of increasing the amount of cationic polymer immobilized. The method of irradiating a cationic polymer solution after immersing it in a solution of the cationic polymer, extracting the solution from the solution, and enclosing the support in nitrogen without drying it has an advantage that washing after irradiation is easy.
The method in which the support is washed with water after being immersed in the solution of the cationic polymer and irradiated with water while immersed in water has an advantage that the cationic polymer is hardly eluted from the support. These methods can be appropriately selected and used from the viewpoints of performance, safety and the like of the cationic polymer.

By irradiating the support with the cationic polymer adhered thereto, the support has a surface area of 1 m ^2.
When the adsorption operation was performed under the condition that the plasma volume per blood was 2.8 × 10 ² ml / m ² , the adsorption removal rate of oxidized LDL having an initial concentration of 2 μg / ml contained in the plasma was 5%. We have found that we can achieve the above.

The concentration of the cationic polymer solution into which the support is immersed depends on the amount of the cationic polymer immobilized and the oxidation L
It is desirably 0.01 wt% or more from the rate of DL adsorption removal.

The shape of the support may be any shape such as a flat membrane, a hollow fiber membrane, a fibrous shape, and a granular shape. However, a hollow fiber membrane is preferable because it allows adsorption and removal of oxidized LDL during dialysis. . The film thickness of the hollow fiber membrane is 10 to 80
μm is preferable, and 20 to 60 μm is more preferable. As for the average pore diameter of the membrane, the albumin transmittance in a 1 wt% albumin solution is preferably 0.5% or more, more preferably 1% or more. Inside diameter is 100-8
It is preferably 00 μm, more preferably 150 to 300 μm. Further, the hollow fiber membrane preferably has a function as an artificial kidney. Here, having the function as an artificial kidney means that it belongs to the type I or type II in the functional classification of the artificial kidney (Dialysis Society of Japan, 32 (12): 1465-1469, 199).
9).

The material of the support in the present invention is preferably a material used for medical purposes, and examples thereof include polyvinyl chloride, cellulosic polymers, polystyrene, polymethyl methacrylate, polycarbonate, polysulfone, and polyurethane. Among them, polysulfone is particularly preferably used because it is easy to mold.

The polysulfone used in the present invention has an aromatic ring, a sulfonyl group and an ether group in the main chain. For example, polysulfone represented by the following formulas (1) and (2) is preferably used. . N in the formula is 50-8
It is an integer of 0.

[0018]

Embedded image

As specific examples of polysulfone, "Udel" P-1700, P-3500 (manufactured by Teijin Amoko), "Ultrason" S3010, S6010 (BA
SF), Victrex (Sumitomo Chemical), Radel A (manufactured by Teijin Amoko), Ultrason E
(Manufactured by BASF) and the like. The polysulfone used in the present invention is preferably a polymer consisting of only the repeating unit represented by the above formula (1) and / or (2), but is preferably used together with other monomers as long as the effects of the present invention are not impaired. It may be polymerized. It is preferable that the other comonomer is not more than 10% by weight.

In the present invention, it is preferable to blend a hydrophilic polymer in order to improve the blood compatibility of the support. As the hydrophilic polymer, polyethylene glycol, polyvinylpyrrolidone and the like are preferable in view of blood compatibility. Further, the hydrophilic polymer is more preferably one that can be radiation-crosslinked with a cationic polymer.
Polyvinylpyrrolidone is particularly preferred because of its compatibility with polysulfone.

The polyvinylpyrrolidone may be laminated on a support as a support, but is preferably mixed or compatible.

As polyvinylpyrrolidone, commercially available ones having a weight average molecular weight of 360,000, 160,000, 40,000 and 10,000 are preferably used, but those having other molecular weights may be used. The weight average molecular weight of polyvinylpyrrolidone is preferably from 2,000 to 2,000,000, more preferably from 10,000 to 1500,000. The molecular weight is a molecular weight in a raw material stage, and in a final product, the molecular weight may be much larger than the above value due to radiation crosslinking or the like. Further, when the polyvinylpyrrolidone used in the present invention is used by blending with a support, a homopolymer is preferable,
It may be copolymerized with another monomer as long as the effects of the present invention are not impaired. It is preferable that the other comonomer is not more than 10% by weight.

It is to be noted that a polymer other than the above may be mixed as long as the effects of the present invention are not impaired. However, such other materials should be 10% by weight or less based on the total weight of the support. Is preferred.

The following method is an example of the method for producing a support in the present invention.

Polysulfone and polyvinylpyrrolidone (weight ratio 20: 1 to 1: 5, preferably 5: 1 to 1: 1) are mixed with a good solvent (N, N-dimethylacetamide, dimethylsulfoxide, dimethylformamide, N
-Methylpyrrolidone, dioxane, etc. are preferable) or a stock solution dissolved in a mixed solution containing a good solvent (concentration: 1
(0 to 30% by weight is preferable, and 15 to 25% by weight is more preferable.) When the injection liquid is discharged from the double annular die, the infusion liquid is flowed inside, the dry part is run, and then the coagulation bath is introduced. To obtain a support. At this time, since the humidity of the dry part has an effect, the water separation from the outer surface of the membrane during running in the dry part accelerates the phase separation behavior near the outer surface, enlarges the pore size,
As a result, permeation and diffusion resistance during dialysis can be reduced. However, if the relative humidity is too high, the coagulation of the undiluted solution on the outer surface becomes dominant, and the pore size is rather reduced, and as a result, the permeation and diffusion resistance during dialysis tends to increase. Therefore, the relative humidity is preferably from 60 to 90%. Further, it is preferable to use an injection liquid composition having a composition based on the solvent used for the stock solution from the viewpoint of process suitability. As the injection solution concentration, for example, when dimethylacetamide is used, an aqueous solution of 45 to 80% by weight, more preferably 60 to 75% by weight is suitably used.

The conditions for measuring the performance of the lipid peroxide adsorbent of the present invention are described below. (1) Preparation of antioxidant LDL antibody The one prepared by Itabe et al. Was used (H. Itabe et al.)
al. , J. et al. Biol. Chem. 269: 1527
4, 1994). That is, a human atherosclerotic lesion homogenate was injected into a mouse and immunized, a hybridoma was prepared from the spleen of the mouse, and a mouse that reacted with copper sulfate-treated LDL was selected. Antibody class is mouse IgM, untreated LDL, acetyl LDL, malondialdehyde LDL
Does not react with Reacts with some phosphatidylcholine peroxidation products, including phosphatidylcholine aldehyde derivatives and hydroperoxides. 150
10 mM borate buffer (pH 8.
5) was used (protein concentration 0.60 mg /
ml). (2) Preparation of oxidized LDL After desalting commercially available LDL (manufactured by Funakoshi), 0.2 mg was prepared.
/ Ml phosphate buffer (hereinafter referred to as PBS)
After dilution with 1 wt% of a 0.5 mM copper sulfate aqueous solution,
The reaction was performed at 37 ° C. for 16 hours. 1 wt% of 10 mM 25 mM ethylenediaminetetraacetic acid (hereinafter referred to as EDTA)
What added t% sodium azide so that it might become 0.02 wt% was used as oxidized LDL standard. (3) Adsorption operation The above oxidized LDL was added to healthy human plasma (Japanese, 30 years old) at 2 μl
g / ml.

From a hollow fiber membrane having an inner diameter of 200 μm and a thickness of 40 μm, mini modules (inner surface area: 53 cm ² ) having a length of 12 cm and 70 pieces were prepared, and an inner diameter of 7 mm (outer diameter: 10 m)
m), 2 cm long silicone tube (product name ARA)
M) and a silicone tube with an inner diameter of 0.8 mm (outer diameter of 1 mm) (product name ARAM, 37
cm at two ends) and the above plasma 1.5m
was perfused into the hollow fiber at a flow rate of 0.5 ml / min at 25 ° C. for 4 hours (the plasma volume per 1 m ^{2 of the} surface area of the hollow fiber membrane was 2.8 × 10 ² ml / m ² ).

Further, a perfusion operation was performed using only a silicone tube without attaching a mini-module.

Oxidized LDL in plasma before and after perfusion, LDL,
By quantifying the HDL concentration, the respective adsorption removal rates were calculated by the following equations.

Adsorption removal rate (%) = Adsorption removal rate (%) using mini-module-Adsorption removal rate only using silicone tube (%) Each adsorption removal rate (%) = 100 x (concentration before perfusion-Perfusion) (4) Measurement of oxidized LDL, LDL, and HDL concentrations The antioxidant LDL antibody was diluted to 5 μg / ml with PBS, and
100 μl / well was dispensed into a 6-well plate, shaken at room temperature for 2 hours, and adsorbed to the wall at 4 ° C. overnight or more.

Discard the antibody solution in the well, and add 1 wt% Bo
Tris-HCl buffer (pH 8.0) containing vine serum albumin (BSA, "Fraction V", Seikagaku Corporation) was dispensed at 200 μl / well, and shaken at room temperature for 2 hours to block the wall. The BSA solution is discarded, and plasma containing oxidized LDL and a standard for preparing a calibration curve (0 to 2 μg / ml of oxidized LD
L containing PBS buffer) was dispensed at 100 μl / well. Then, after shaking at room temperature for 30 minutes, it was left at 4 ° C. overnight.

After returning to room temperature, the solution in the well was discarded.
The wells were washed three times with a Tris-HCl buffer (pH 8.0) containing 05 wt% "Tween-20" (Katayama Chemical). Sheep anti-apoB antibody (THEBINDING SITE) diluted 2000-fold with PBS in the washed wells
Was dispensed at 100 μl / well and shaken at room temperature for 2 hours. Then, the anti-apoB antibody in the well was discarded, and 0.05 wt.
% "Tween-20" in Tris-HCl buffer (p
H8.0) were washed three times. 100 μl / well of alkaline phosphatase-labeled donkey anti-sheep IgG antibody (CHEMICON) diluted 2000-fold with Tris-HCl buffer (pH 8.0) containing 2 wt% “Block Ace” (Dainippon Pharmaceutical) in the washed wells Pour and shake at room temperature for 2 hours. Thereafter, the labeled antibody in the well is discarded, and the well is washed three times with a Tris-HCl buffer solution (pH 8.0) containing 0.05 wt% "Tween-20", and further washed with a Tris-HCl buffer solution (pH 8.0). Was washed twice. Subsequently, p-nitrophenyl phosphate (Boeh
1 mg of Ringer Mannheim GmbH)
/ Ml solution (0.0005 M MgCl ₂ , 1 M diethanolamine buffer, pH 9.8) was dispensed at 100 μl / well and allowed to react at room temperature for an appropriate time.
The absorbance in nm was measured with a plate reader. A calibration curve was drawn from the results of the standard to determine the oxidized LDL concentration.

LDL was quantified using a β-lipo assay kit (Wako Pure Chemical Industries, Ltd.).

Quantification of HDL was performed using an HDL-C measurement kit (Wako Pure Chemical Industries, Ltd.).

[0035]

EXAMPLES Example 1 Polysulfone ("Udel" P-3 manufactured by Teijin Amoko)
500) 18 parts by weight, polyvinylpyrrolidone (BASF
9 parts by weight of K30) were added to 72 parts by weight of N, N-dimethylacetamide and 1 part by weight of water and dissolved by heating at 90 ° C. for 14 hours. This membrane-forming stock solution has an outer diameter of 0.3 mm and an inner diameter of 0.2 m
m orifice-type double cylindrical die, discharge a solution consisting of 58 parts by weight of dimethylacetamide and 42 parts by weight of water as a core liquid, and after passing a dry length of 350 mm,
The mixture was led to a coagulation bath containing 100% water to obtain a hollow fiber.

The obtained polysulfone hollow fiber separation membrane was 70
This bundle, both ends are fixed to the glass tube module case with an epoxy potting agent so as not to block the hollow portion of the hollow fiber, a mini-module is prepared, and a 1 wt% aqueous solution of polyethyleneimine (molecular weight 10,000, manufactured by Wako Pure Chemical Industries) And irradiated with γ rays at 25 kGy. The mini-module had a diameter of about 7 mm and a length of 12 cm. The irradiated membrane was washed again with distilled water at 37 ° C. for 30 minutes.
It was subjected to an adsorption experiment.

The experimental results are shown in Table 1.

Comparative Example 1 A mini-module was prepared in the same manner as in Example 1, and 1 wt.
% Polyethyleneimine aqueous solution instead of water and filled with γ-rays at 25 kGy to prepare a sample.

Example 2 A mini-module was prepared in the same manner as in Example 1, and 1 wt.
% Polyethyleneimine aqueous solution, the aqueous solution was extracted, nitrogen gas was charged into the container, and the material was irradiated with γ-rays at 25 kGy in a wet state.

Example 3 A mini-module was prepared in the same manner as in Example 1, and 1 wt.
% Polyethyleneimine aqueous solution, the aqueous solution is withdrawn, the material is washed with water, water is filled, and polyethyleneimine is adsorbed on the material, and γ is applied at 25 kGy.
Irradiation was performed.

Oxidation L in Examples 1 to 3 and Comparative Example
Table 1 shows the results of the adsorption and removal rates of DL, LDL, and HDL.

[0042]

[Table 1]

[0043]

Industrial Applicability According to the present invention, the present invention is used for blood purifiers and the like, and is particularly preferably used for preventing the development of arteriosclerosis and for preventing its onset in patients with heart disease and dialysis patients. A lipid peroxide adsorbent can be provided.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) B01J 20/30 B01J 20/30 // B01J 19/12 19/12 DF term (Reference) 4C077 AA13 BB03 MM01 MM09 NN07 PP05 PP08 PP09 PP10 PP13 PP15 4D006 GA13 HA01 MA01 MA03 MA33 MB06 MB14 MC11 MC21 MC24 MC27 MC36 MC40 MC40X MC49 MC53 MC62 MC62X NA04 NA42 PB09 PB52 PC41 PC47 4G066 AC14C AC17B AC17C AC21C AC24C AC31CA13 CA03 FA20 AA32 BD16 CA38 EB21 FA11 FC02

Claims (6)

[Claims] 1. A method for producing a lipid peroxide adsorbent, wherein a cationic polymer is immobilized on a support by irradiating the support with the cationic polymer attached thereto. 2. The method for producing a lipid peroxide adsorbent according to claim 1, wherein the state in which the cationic polymer is attached to the support is a state in which the support is immersed in a solution of the cationic polymer. 3. A state in which the cationic polymer is adhered to the support according to claim 1. After the support is immersed in a solution of the cationic polymer, the support is extracted from the solution, and the support is sealed in nitrogen without drying. A method for producing a lipid peroxide adsorbent, characterized in that the adsorbent is in a state of being adsorbed. 4. The state in which the cationic polymer is attached to the support according to claim 1, wherein the support is immersed in a solution of the cationic polymer, and then the support is washed with water and immersed in water. A method for producing a lipid peroxide adsorbent, comprising: 5. A method for producing a lipid peroxide adsorbent, wherein the concentration of the cationic polymer solution according to claim 1 is 0.01 wt% or more. 6. The method for producing a lipid peroxide adsorbent according to claim 1, wherein the support is a hollow fiber membrane.