JPH04227268A - Manufacture of adsorbing body - Google Patents

Abstract

PURPOSE:To manufacture a lipoprotein-adsorbing body for ex vi vo circulation therapy in which the harmful components (particularly extreme low density lipoprotein and/or low density lipoprotein) in blood are selectively eliminable by adsorption thereto. CONSTITUTION:A lipoprotein-adsorbing body for ex vi vo circulation therapy is manufactured by fixing a dextran sulfuric acid and/or its salt with a water- insoluble porous body through covalent bond.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing an adsorbent for removing harmful components from blood. For more details,
The present invention relates to a method for producing an adsorbent for selectively adsorbing and removing lipoproteins, particularly very low density lipoproteins (VLDL) and/or low density lipoproteins (LDL) from blood, plasma, or serum.

0002

BACKGROUND OF THE INVENTION It is known that among lipoproteins present in blood, VLDL and LDL contain a large amount of cholesterol and cause arteriosclerosis. In particular, in cases of hyperlipidemia such as familial hyperlipidemia and hypercholesterolemia, VLDL and/or LDL values are several times higher than normal values, leading to hardening of coronary arteries.

[0003] Dietary therapy and drug therapy are used to treat these diseases, but their effectiveness is limited and there are concerns about side effects. Especially for familial hyperlipidemia, VLD
At present, so-called plasma exchange therapy is almost the only effective treatment, in which plasma from a patient containing a large amount of L and LDL is separated and then replaced with normal plasma or replacement fluid containing albumin, etc., to lower VLDL and LDL levels. It is a treatment method.

However, as is well known, plasma exchange therapy (1) requires the use of expensive fresh plasma or plasma preparations, (2) there is a risk of infection with hepatitis viruses, and (3) it contains only harmful ingredients. However, it has the disadvantage that useful components are also removed at the same time, that is, in the case of lipoproteins, useful high-density lipoproteins (HDL) are also removed at the same time.

[0005] In order to overcome the above-mentioned drawbacks, attempts have been made to selectively remove harmful components using membranes, but no membrane has been found to be satisfactory in terms of selectivity.

[0006] Furthermore, attempts have been made to use so-called immunoadsorbents to which antigens, antibodies, etc. are immobilized for the same purpose.
Although this method is almost satisfactory in terms of selectivity, it has the disadvantage that the antigens and antibodies used are difficult and expensive to obtain.

Furthermore, attempts have been made to develop adsorbents based on the principle of so-called affinity chromatography, in which a substance (hereinafter referred to as a ligand) having a specific affinity for the substance to be removed is immobilized on a carrier. Many of the ligands used in this method are easier to obtain than antigens, antibodies, etc., but since many of them are derived from living organisms, their stability against sterilization, price, safety, etc. must be considered before they can be used in extracorporeal circulation therapy. There is not much to be satisfied with in this respect.

Among these, dextran sulfate and/or its salts are excellent as ligands, and by fixing them to a water-insoluble porous material through covalent bonds, lipolysis can be carried out with high efficiency, safety, and high selectivity. An adsorbent for extracorporeal circulation therapy that can adsorb and remove proteins is obtained.

However, dextran sulfate and (
or) The salt has only a hydroxyl group with low reactivity as a functional group available for the immobilization reaction, making it difficult to immobilize the required amount of dextran sulfate and/or its salt.

As a result of extensive research, the present inventors have discovered that dextran sulfate and/or its salts can also be efficiently immobilized depending on the carrier, and have completed the present invention.

[0011]

[Means for Solving the Problems] That is, the present invention relates to a method for producing a lipoprotein adsorbent for extracorporeal circulation therapy, in which dextran sulfate and/or its salts are fixed to a water-insoluble porous material through covalent bonds.

0012

[Example] Dextran sulfate and/or its salts are Leuconostoc mesenteroides (Leuco
It is a sulfate ester of dextran, which is a polysaccharide produced by S. nostoc mesenteroides, and/or its salt, and may be linear or branched, and the salt is preferably a water-soluble salt such as sodium or potassium.

[0013] The dextran sulfate and/or its salt used in the present invention is desirably one with a relatively low molecular weight and a high sulfur content from the viewpoint of lipoprotein adsorption efficiency and safety. As a guide, the intrinsic viscosity is 0.12 dl/
g or less, and the sulfur content is 15% by weight or more.

[0014] The intrinsic viscosity as used in the present invention is measured at 25°C using dextran sulfate and/or its salt as a sodium salt in a neutral 1M saline solution.

[0015] Examples of the water-insoluble porous material used as a carrier include porous cellulose gel. Compared to other carriers (especially synthetic polymer carriers), porous cellulose gel is advantageous in that it can immobilize a large amount of dextran sulfate and/or its salts per unit active group on the carrier. It has the following advantages.

(1) It has high mechanical strength, and when it is packed in a column or the like and body fluids such as blood and plasma are passed through it, the pressure loss is small and clogging is less likely to occur.

(2) A large number of pores of sufficient size are present, allowing the substance to be adsorbed and removed to penetrate into the pores. The exclusion limit molecular weight measured using globular proteins and viruses is appropriately in the range of 1 million to 100 million.
(referring to the molecular weight of the one with the smallest molecular weight among the excluded molecules).

(3) Compared to synthetic polymer hard gels having similar pore diameters and porosity, it is stronger and less likely to break.

(4) A hydroxyl group is present on the surface as a functional group that can be used in an immobilization reaction or a functional group that can be easily activated.

(5) There is little change due to sterilization operations such as high-pressure steam sterilization.

Regarding the exclusion limit molecular weight (hereinafter referred to as exclusion limit molecular weight) measured using the globular protein and virus in (2), when a carrier with an exclusion limit molecular weight of less than 1 million is used, VLDL, The amount of LDL removed is too small to be practical, but the exclusion limit molecular weight is 10.
Even carriers with molecular weights close to VLDL and LDL, ranging from 1,000,000 to several million, can be used to some extent for practical use. on the other hand,
When the exclusion limit molecular weight exceeds 100 million, the amount of immobilized ligand decreases, resulting in a decrease in the amount of adsorption, and the strength of the gel also decreases, which is not preferable. For this reason, it is appropriate that the water-insoluble porous material used in the present invention has an exclusion limit molecular weight in the range of 1 million to 100 million.

[0022] Furthermore, the surface of the water-insoluble porous body may be coated with a synthetic polymer or the like.

[0023] The smaller the particle size of the water-insoluble porous material is, the better from the point of view of adsorption capacity, but if the particle size is too small, the pressure loss will increase when packed in a column, which is undesirable. Preferably, the range is within the range.

There are various methods for immobilizing dextran sulfate and/or its salts on a water-insoluble porous material, but it is important that the ligand does not detach when used for extracorporeal circulation therapy. It is necessary that the material be fixed to the water-insoluble porous material through a covalent bond.

Typical examples of immobilization methods include the cyanogen halide method, the epichlorohydrin method, the method using polyoxirane compounds such as bisepoxide, and the halogenated triazine method. The less hazardous epichlorohydrin method or the method using polyoxirane compounds such as bisepoxides are suitable for the present invention. Dextran sulfate and (
or) The amount of the salt immobilized is preferably 0.2 mg or more per ml of column volume in order to obtain a significant amount of lipoprotein adsorption, and desirably 100 mg or less in consideration of economic efficiency.

[0026] After completion of the immobilization reaction, unreacted dextran sulfate and/or its salt can be recovered and reused through purification and other steps.

[0027] Also, dextran sulfate and/or
In the water-insoluble porous material having the salt fixed thereon, it is desirable to inactivate unreacted active groups with monoethanolamine or the like.

There are various methods for using the adsorbent according to the present invention for extracorporeal circulation therapy, but filters, meshes, etc., which allow body fluid components (blood cells, proteins, etc.) to pass through but not the adsorbent, are attached to the inlet and outlet. A typical method is to fill a column with a sample of 1,000 ml, incorporate the column into an extracorporeal circulation circuit, and pass body fluids such as blood and plasma through the column.

Next, the method of the present invention will be explained in more detail with reference to Examples, but the present invention is not limited to these Examples.

Example 1 Crosslinked polyacrylate Toyopearl HW65 (manufactured by Toyo Soda Co., Ltd., exclusion limit molecular weight 5,000,000,
Particle size: 50-100 μm) Add saturated NaOH to 10 ml.
After adding 6 ml of an aqueous solution and 15 ml of epichlorohydrin and reacting at 50° C. for 2 hours with stirring, the gel was washed with alcohol and water in that order to obtain an epoxidized gel. The amount of epoxy group introduced is 1 ml column volume.
It was 250 μmol per portion.

Intrinsic viscosity (measured with sodium dextran sulfate and/or its salt in a neutral 1M saline solution at 25°C; the same shall apply hereinafter) of 0.027 dl/g was added to 2 ml of the resulting gel. 0.5 g of sodium dextran sulfate with a sulfur content of 17.7% by weight and 2 ml of water
ml (dextran sodium sulfate concentration approximately 13% by weight). Then, the pH was adjusted to 11, and the pH was adjusted to 16 at 45°C.
After shaking for an hour, the gel was filtered and washed in the order of 2M saline, 0.5M saline, and water to obtain a gel on which dextran sodium sulfate was immobilized. The amount of immobilized sodium dextran sulfate was 0.4 mg per ml column volume.
The amount of immobilization per 1 μmol of epoxy group is 1
．． It was 6 μg.

Example 2 Porous cellulose gel CSKA-3 (manufactured by Chisso Corporation, exclusion limit molecular weight 50,000,000, particle size 4)
5-105 μm) 4g of 20% NaOH to 10ml
, heptane 12g and nonionic surfactant TW
One drop of EEN20 was added. After stirring at 40°C for 2 hours, 5 g of epichlorohydrin was added and stirred for 2 hours, and the gel was washed with water and filtered to obtain an epoxidized cellulose gel. The amount of epoxy groups introduced was 30 μm per ml of column volume.
It was ol.

2 ml of the obtained gel has an intrinsic viscosity of 0.027.
dl/g, 0.12 g of dextran sodium sulfate with a sulfur content of 17.7% by weight and 2 ml of water (the concentration of dextran sodium sulfate is approximately 2.5% by weight).
, the pH was adjusted to 11, and the mixture was shaken at 45° C. for 16 hours. The gel was filtered and washed with 2M saline, 0.5M saline, and water in this order to obtain a cellulose gel on which dextran sodium sulfate was immobilized.

The amount of dextran sodium sulfate immobilized was 0.15 mg per ml column volume, and the amount immobilized per 1 μmol of epoxy group was 5 μg.

Example 3 The concentration of dextran sodium sulfate during the immobilization reaction was
The immobilization reaction was carried out in the same manner as in Example 2, except that the amount was changed to 3% by weight. The amount of immobilized sodium dextran sulfate was 2.3 mg per ml of column volume, and the amount immobilized per 1 μmol of epoxy group was 76 μg.

Test Example After inactivating the unreacted epoxy groups of the gels obtained in Examples 1 to 3 using monoethanolamine, 1 ml of each gel was packed into a column, and the column was filled with hyperlipidemia. Patient's plasma (total cholesterol concentration 308 mg/dl) 6 m
The LDL concentration in the effluent plasma was measured using total cholesterol as an index (because most of the cholesterol in the plasma used was derived from LDL).

The results are shown in Table 1.

[0038]

[Table 1]

[0039]

Effects of the Invention When the adsorbent produced by the method of the present invention is used for extracorporeal circulation treatment, lipoproteins, especially very low density lipoproteins (VLDL) and/or low density lipoproteins, can be extracted from blood, plasma, and semen. Protein (LDL) can be selectively adsorbed and removed.

Claims (1)

[Claims] 1. A method for producing a lipoprotein adsorbent for extracorporeal circulation therapy, in which dextran sulfate and/or its salt is immobilized on a water-insoluble porous material via a covalent bond.