JPH035822B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to an adsorbent for removing harmful components from blood. More specifically, the present invention relates to 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. [Problems to be solved by conventional technology/invention] Of the lipoproteins present in the blood, VLDL,
LDL contains a lot of cholesterol and is known to cause arteriosclerosis. Especially in cases of hyperlipidemia such as familial hyperlipidemia and hypercholesterolemia, JLDL and/or
It shows LDL level and causes hardening of coronary arteries.
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
Plasma exchange therapy, in which the plasma of a patient with high VLDL and LDL levels is separated and replaced with normal plasma or replacement fluid containing albumin as a component to lower VLDL and LDL levels, is currently almost the only effective treatment. It is a treatment method. However, as is well known, plasma exchange therapy (1)
It is necessary to use expensive fresh plasma or plasma preparations. (2) There is a risk of infection with hepatitis viruses, etc.
(3) It has the disadvantage that not only harmful components but also useful components are removed at the same time, that is, in the case of lipoproteins, high-density lipoproteins (HDL), which are useful, are also removed at the same time. Attempts have been made to selectively remove harmful components using membranes in order to overcome the above-mentioned drawbacks, but no membrane has been found to be satisfactory in terms of selectivity. For the same purpose, attempts have been made to use so-called immunoadsorbents to which antigens, antibodies, etc. are immobilized.
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, 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, have also been attempted. The ligand used in the method is an antigen,
There are many substances that are easier to obtain than antibodies, etc.
Since many of the substances are derived from living organisms, there are almost no substances that can be used in extracorporeal circulation therapy that are satisfactory in terms of stability against sterilization, cost, safety, etc. [Means for Solving the Problems] As a result of further intensive research in order to overcome the above-mentioned drawbacks, the present inventors found that dextran sulfate and/or its salts having a specific viscosity and sulfur content were made water-insoluble. The present invention was completed based on the discovery that an adsorbent for extracorporeal circulation therapy that can adsorb and remove lipoproteins with high efficiency, safety, and selectivity can be obtained by immobilizing it on a porous material through covalent bonds. I came to the conclusion. That is, the present invention provides water-insoluble porous dextran sulfate and/or its salts having an intrinsic viscosity (measured at 25°C in a 1M saline solution, the same shall apply hereinafter) of 0.12 dl/g or less and a sulfur content of 15 to 22% by weight. Formula in body: (where O ^A is dextran sulfate and/or
The oxygen atom derived from the hydroxyl group of the salt, O ^B is the oxygen atom derived from the surface hydroxyl group of the water-insoluble porous body) is fixed via a covalent bond of 0.2 to 100 mg per ml of column volume. The present invention relates to a therapeutic lipoprotein adsorbent. [Examples/Effects of the Invention] Dextran sulfate and/or its salt are the sulfate ester of dextran, which is a polysaccharide produced by Leuconostoc mesenteroides, and/or its salt. Dextran sulfate and/or its salts are known to form precipitates with lipoproteins in the presence of divalent cations such as calcium, and are usually used for this purpose with a molecular weight of 500,000 (intrinsic viscosity of about 0.20 dl/ g)
Dextran sulfate and/or its salts are used. However, as shown in the comparative example, even if dextran sulfate and/or its salts are immobilized on a water-insoluble porous material, LDL
and/or the adsorption capacity of VLDL is low and cannot be put to practical use. As a result of various studies, the present inventors found that the intrinsic viscosity is 0.12 dl/g or less, more preferably
It has been found that dextran sulfate and/or its salts with a sulfur content of 0.08 dl/g or less and a sulfur content of 15 to 22% by weight or more exhibit high LDL and/or VLDL adsorption ability and selectivity. Furthermore, surprisingly, whereas the precipitation method described above requires 10-40mM of divalent cations, the adsorbent of the present invention has high adsorption capacity and selectivity without necessarily adding divalent cations. We found that this shows that Furthermore, although the toxicity of dextran sulfate and/or its salts is low, it is known that the toxicity increases when the molecular weight increases beyond a certain level, and from this point of view, it is preferable that the intrinsic viscosity is 0.12 dl/g or less.
By using dextran sulfate and/or its salt having a relatively low molecular weight of 0.08 dl/g or less, it is possible to prevent the danger in the event that the fixed dextran sulfate and/or its salt should detach. Furthermore, since most of dextran sulfate and/or its salts are α-1,6-glycosidic bonds, there is little change even when operations such as high-pressure steam sterilization are performed. There are various methods for measuring the molecular weight of dextran sulfate and/or its salts, but viscosity measurement is generally used. However, since dextran sulfate and/or its salts are polyelectrolytes, they may vary depending on the ionic strength of the solution, pH, and the sulfur content (i.e., the amount of sulfonic acid groups) of dextran sulfate and/or its salts. Viscosity also differs depending on the molecular weight. In the present invention, the intrinsic viscosity refers to dextran sulfate and/or its salt as a sodium salt in a neutral 1M saline solution.
Measured at 25℃. Dextran sulfate and/or its salt used in the present invention may be linear or branched, and the salt is preferably a water-soluble salt such as sodium or potassium. The water-insoluble porous carrier used in the present invention preferably has the following properties. (1) It has relatively high mechanical strength, and when it is packed into a column and body fluids such as blood and plasma are passed through it, the pressure loss is small and it does not cause clogging. (2) There must be a large number of pores of sufficient size, that is, the substance to be adsorbed and removed must be able to enter the pores, and the exclusion limit molecular weight measured using globular proteins and viruses must be 1 million to 1
(However, the exclusion limit molecular weight refers to the molecular weight of the smallest molecular weight of molecules that cannot enter (exclude) the pores). (3) A functional group that can be used for immobilization reaction or a functional group that can be easily activated on the surface, such as an amino group,
There are carboxyl groups, hydroxyl groups, thiol groups, acid anhydride groups, succinylimide groups, chlorine groups, aldehyde groups, amide groups, epoxy groups, etc. (4) 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), if a carrier with an exclusion limit molecular weight of less than 1 million is used, the amount of VLDL and LDL removed will be Although it is too small to withstand practical use, the exclusion limit molecular weight is
Even carriers with a molecular weight of 1 million to several million, which is close to that of VLDL and LDL, 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. Typical examples of water-insoluble porous materials with the above-mentioned properties include styrene-divinylbenzene copolymer, cross-linked polyvinyl alcohol, cross-linked polyacrylate, cross-linked vinyl ether-maleic anhydride copolymer, and cross-linked styrene. -Porous bodies of synthetic polymers such as maleic anhydride copolymers and crosslinked polyamides, porous cellulose gels, porous silica gel glass, porous alumite, porous silica alumina, porous hydroxyapatite,
Examples include, but are not limited to, inorganic porous materials such as porous calcium silicate, porous zirconia, and zeolite. Further, the surface of the water-insoluble porous body may be coated with polysaccharides, synthetic polymers, or the like. Generally speaking, the smaller the particle size of the water-insoluble porous material, the better in terms of adsorption capacity, but if the particle size is too small, the pressure drop will increase when packed in a column, which is undesirable, and it is in the range of 1 to 5000μ. It is preferable. Further, the water-insoluble porous material may be used alone or in combination of two or more types. Among the representative examples mentioned above, porous cellulose gel not only has the properties (1) to (4) above, but also can efficiently fix dextran sulfate and/or its salts, so it is suitable for the present invention. It is one of the most suitable water-insoluble porous materials. There are various methods for fixing dextran sulfate and/or its salts to a water-insoluble porous material, but since it is important that the ligand used for extracorporeal circulation therapy does not detach, Preferably, it is fixed to a water-insoluble porous body. Examples of immobilization methods include the cyanogen halide method, epichlorohydrin method, bisepoxide method, and triazine halide method, but the epichlorohydrin method is most suitable for the present invention because of its strong binding and low risk of detachment of the ligand. There is. However, the epichlorohydrin method has low reactivity, and in particular when immobilizing dextran sulfate and/or its salts, the reactivity is even lower because the functional group of the ligand is a hydroxyl group. It is difficult to quantify. As a result of various studies, the present inventors found that the concentration of dextran sulfate and/or its salt (water-insoluble A sufficient amount of dextran sulfate and/or its salt can be obtained by keeping the concentration (based on the total weight of the reaction system excluding the porous body (dry weight)) at 3% by weight or more, more preferably 10% by weight or more. I found that it is fixed. The amount of dextran sulfate and/or its salt immobilized is preferably 0.2 mg or more per ml of column volume in order to obtain a significant adsorption amount of lipoproteins, and desirably 100 mg or less in consideration of economic efficiency. Furthermore, when using a porous cellulose gel, the amount of dextran sulfate and/or its salts is fixed even under the same conditions, which is advantageous compared to other water-insoluble porous materials. The adsorbent obtained by the reaction of a water-insoluble porous material activated by epichlorohydrin with dextran sulfate and/or its salt has the formula: (where O ^A is dextran sulfate and/or
The oxygen atom derived from the hydroxyl group of the salt (O ^B is the oxygen atom derived from the surface hydroxyl group of the water-insoluble porous body) is fixed to the water-insoluble porous body through a bond. Incidentally, after the immobilization reaction is completed, unreacted dextran sulfate and/or its salt can be recovered and reused through steps such as purification. After fixing dextran sulfate, it is desirable to seal unreacted active groups (epoxy groups when epichlorohydrin is used) with monoethanolamine or the like. There are various methods for using the adsorbent according to the present invention in extracorporeal circulation therapy, but one method is to use a column equipped with a filter, mesh, etc. at the inlet and outlet through which body fluid components (blood cells, proteins, etc.) can pass, but not the adsorbent. A typical method is to fill the column, incorporate the column into an extracorporeal circulation circuit, and pass body fluids such as blood and plasma through the column. Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited to these Examples. Comparative Example 1 Cellulofine A-3 (porous cellulose gel manufactured by Chitsuso Co., Ltd., exclusion limit molecular weight 50000000, particle size
45-105μm) 20%, 4g NaOH, heptane in 10ml
12 g and 1 drop of the nonionic surfactant Tween 20 were added, and the mixture was stirred at 40° C. for 2 hours, and then 5 g of epichlorohydrin was added and stirred for 2 hours.
After standing still, the supernatant was discarded, and the gel was washed with water and filtered to obtain an epoxidized cellulose gel. Next, dextran sulfate sodium, which is commercially available for LDL precipitation, has an intrinsic viscosity of 0.20 dl/g, an average degree of polymerization (average degree of polymerization of raw material dextran, hereinafter referred to as average degree of polymerization) of 3500, and a sulfur content of 17.7% by weight.
0.5 g was dissolved in 2 ml of water, 2 ml of the epoxidized cellulose gel obtained as described above was added, and the pH was adjusted to 12 (the concentration of sodium dextran sulfate was approximately 10% by weight). After shaking this at 40°C for 16 hours, the gel was separated and washed with 2M saline, 0.5M saline, and water to obtain a cellulose gel on which dextran sodium sulfate was immobilized. Unreacted epoxy groups were sealed using monoethanolamine. The amount of immobilized sodium dextran sulfate was 4.2 mg per ml column volume. Comparative Example 2 Sodium dextran sulfate with intrinsic viscosity 0.124
dl/g, an average degree of polymerization of 140, and a sulfur content of 5.7% by weight, in the same manner as in Comparative Example 1 to obtain a cellulose gel on which dextran sodium sulfate was fixed. The amount of immobilized sodium dextran sulfate was 2.5 mg per ml column volume. Example 1 As sodium dextran sulfate (1) Intrinsic viscosity 0.027 dl/g, average degree of polymerization 12, sulfur content 17.7% by weight (2) Intrinsic viscosity 0.055 dl/g, average degree of polymerization 40, sulfur content 19% by weight (3) (4) In the same manner as in Comparative Example 1, using four types with an intrinsic viscosity of 0.083 dl/g, an average degree of polymerization of 140, and a sulfur content of 19.2% by weight. A cellulose gel with immobilized sodium dextran sulfate was prepared. The amount of immobilized sodium dextran sulfate was 2.0 mg and 1.5 mg per ml column volume, respectively.
mg, 4.0 mg, and 4.3 mg. Example 2 Toyopearl, a cross-linked polyacrylate gel
HW65 (manufactured by Toyo Soda Co., Ltd., exclusion limit molecular weight 5,000,000,
Particle size: 50-100 μm) 10 ml of saturated NaOH aqueous solution 6
After adding 15 ml of epichlorohydrin and stirring at 50° C. for 2 hours, the gel was washed with alcohol and water to obtain an epoxidized gel. To 2 ml of the resulting gel were added 0.5 g of sodium dextran sulfate having an intrinsic viscosity of 0.55 dl/g, an average degree of polymerization of 40, and a sulfur content of 19% by weight, and 2 ml of water (the concentration of sodium dextran sulfate was approximately 13% by weight). The pH was then adjusted to 12, shaken at 40°C for 16 hours, and the gel was separated and washed with 2M saline, 0.5M saline, and water to obtain a gel on which dextran sodium sulfate was immobilized. Unreacted epoxy groups were sealed using monoethanolamine. The amount of immobilized sodium dextran sulfate is 0.4 mg per ml column volume.
It was hot. Example 3 Intrinsic viscosity 0.55 dl/g, average degree of polymerization 40, sulfur content
Using 19% by weight sodium dextran sulfate,
A gel on which dextran sodium sulfate was immobilized was obtained in the same manner as in Comparative Example 1, except that the concentration of dextran sodium sulfate in the immobilization reaction was changed to 2.5% by weight. The amount of immobilized sodium dextran sulfate was 0.15 mg per ml column volume. Test Example 1 ml of each of the dextran sodium sulfate-immobilized gels obtained in Comparative Examples 1 to 2 and Examples 1 to 3 was packed into a column, and 6 ml of plasma from a hyperlipidemic patient (total cholesterol concentration 300 dl/g) was charged. flowing,
The amount of adsorbed LDL was measured using total cholesterol as an index (because most of the cholesterol in the plasma used is derived from LDL). The results are shown in Table 1.

[Table] Example 4 Among the adsorbents obtained in Example 1, the intrinsic viscosity
Dextran sulfate sodium fixed at 0.027 dl/g, average degree of polymerization 12, and sulfur content 17.7% by weight was dispersed in physiological saline and subjected to high-pressure steam sterilization at 120°C for 20 minutes.
When the amount of adsorbed LDL was measured, it was found that there was only a slight decrease in the amount of adsorbed due to the sterilization procedure. Example 5 Of the adsorbents obtained in Example 1, the intrinsic viscosity
Fill the column with 1 ml of fixed dextran sodium sulfate with 0.027 dl/g, average polymerization degree of 12, and sulfur content of 17.7% by weight, and add 6 ml of normal human plasma (the ratio of LDL cholesterol to HDL cholesterol is approximately 1:1) to this column. As a result, LDL was significantly reduced, but HDL was hardly adsorbed. Example 6 A column was filled with 1 ml of the adsorbent used in Example 5, and 6 ml of normal rabbit plasma containing VLDL, LDL, and HDL was passed through the column. The results were investigated using disk electrophoresis. FIG. 1 is a chart showing the results. In Figure 1, curves A and B
are the results of electrophoresis before and after passing through the column, respectively, the vertical axis is the absorbance at 570 nm, and ↑ indicates the position where the VLDL, LDL, and HDL bands appeared, respectively. As shown in Figure 1, VLDL and LDL were adsorbed, but HDL was hardly adsorbed.

[Brief explanation of the drawing]

FIG. 1 is a chart showing the results of disk electrophoresis using polyacrylamide gel.

Claims (1)

[Claims] 1. The intrinsic viscosity is 0.12 dl/g or less and the sulfur content is
15-22% by weight of dextran sulfate and/or its salts are formulated into a water-insoluble porous body: (where O ^A is dextran sulfate and/or
The amount fixed via a covalent bond represented by the oxygen atom derived from the hydroxyl group of the salt (O ^B is the oxygen atom derived from the surface hydroxyl group of the water-insoluble porous material) is 0.2 to 100 mg per ml of column volume. Lipoprotein adsorbent for extracorporeal circulation treatment. 2 Exclusion limit molecular weight of water-insoluble porous material is 1 million or more
10. The adsorbent according to claim 1, wherein the adsorbent is in the range of 100 million. 3. The adsorbent according to claim 1 or 2, wherein the water-insoluble porous body is a porous cellulose gel.