JPH01280469A - Absorbent - Google Patents

Abstract

PURPOSE:To safely absorb-remove a lipoprotein with a high efficiency and moreover, with a good selectivity by fixing a dextran sulfuric acid having specific viscosity and sulfur containing quantity and the salt through a covalent bond to a water insoluble porous body. CONSTITUTION:By using a liprotein absorbent for an external circulation remedy made after the dextran sulfuric acid, in which an intrinsic viscosity is 0.12dl/g or below and simultaneously, the sulfur containing quantity is 15weight% or above, and the salt are fixed through the covalent bond to the water insoluble porous body, harmful components in a blood are removed. The dextran sulfuric acid and the salt may be a straight chain shape or branched chain shape, and for the salt, a soluble salt such as a natrium and a potassium is preferable. For a method for fixing the dextran sulfuric acid and the salt to the water insoluble porous body, since the fact that a is not remove-separated the ligand for using it for the external circulation remedy is important, the fact that the ligand is fixed through the covalent bond with a firm bond to the water insoluble porous body is hoped, and an epichlorohydrine method is most suitable in which the bond is firm and the danger of the removal separation of the ligand is small.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to an adsorbent for removing harmful components from blood. More specifically, riboproteins, especially very low density protein (VLDL) and (
or) relates to an adsorbent for selectively adsorbing and removing low density riboprotein (LDL).

[Prior Art/Problems to be Solved by the Invention] Among the riboproteins present in the blood, VLDL and LDL are known to 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.

Diet, therapy, and drug therapy are used to treat these diseases, but their effectiveness is limited, and there are concerns about side effects. In particular, for familial hyperlipidemia, the patient's plasma containing a large amount of VLDL% LDL is separated and then replaced with normal plasma or a replacement fluid containing albumin, etc., to lower the VLDL and LDL levels. Plasma exchange therapy is currently almost the only effective treatment.

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, etc. (3) Not only harmful components but also useful components are removed at the same time.
DL) is also removed at the same time.

In order to overcome the above-mentioned drawbacks, attempts have been made to selectively remove harmful components using membranes, but none have been found that are satisfactory in terms of selectivity.

For the same purpose, attempts have been made to use so-called immunoadsorbents on which antigens, antibodies, etc. are immobilized.Although this method is generally satisfactory in terms of selectivity, it is difficult and expensive to obtain the antigens and antibodies used. It has its drawbacks.

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. 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, etc. are required for use in extracorporeal circulation therapy.
There are very few that are satisfactory in terms of safety.

[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. We have discovered that it is possible to replace an adsorbent for extracorporeal circulation therapy that can adsorb and remove riboproteins with high efficiency, safety, and high selectivity by immobilizing them on a porous material through covalent bonds, and have completed the present invention. Ta.

That is, the present invention has a limiting viscosity (in 1M saline solution, 25
Measured at °C, the same applies hereafter) is 0.12 dl! 7g or less and the sulfur content is 15fffm5 or more, preferably 15~
The present invention relates to a riboprotein adsorbent for extracorporeal circulation therapy, in which 22% by weight of dextran sulfate and/or its salt is fixed to a water-insoluble porous material through covalent bonds.

[Examples/Effects of the Invention] Dextran sulfate and/or its salts are Leuconostoc mesenteroides (Leuconostoc mesenteroides).
It is a sulfate ester of dextran, a polysaccharide produced by A. mesenteroldes, etc., and/or a salt thereof.

It is known that dextran sulfate and/or its salts form a precipitate with riboproteins 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 dR1g). ) of dextran sulfate and/or its salts are used. However, as shown in the comparative example, even when dextran sulfate and/or its salts are immobilized on a water-insoluble porous material, the tracheid capacity of LDL and/or VLDL is low;
Not practical. As a result of various studies, the present inventors found that
L with high dextran sulfate and/or its salts having an intrinsic viscosity of 0.12dN 7g or less, more preferably 0.08dN 7g or less and a sulfur content of 15% by weight or more
It was found that it exhibits DL and/or VLDL adsorption capacity and selectivity.

Even more surprisingly, in the precipitation method described above,
While 40 mM of divalent cations are required, the adsorbent of the present invention has been found to exhibit high adsorption capacity and selectivity without necessarily adding divalent cations. Furthermore, although the toxicity of dextran sulfate and/or its salts is low, it is known that the toxicity increases when the molecular mouth becomes larger than a certain degree, and from this point of view, the intrinsic viscosity is 0.1.
2dN/g or less, more preferably o, osdfI/g
By using the following relatively low molecular weight dextran sulfate and/or its salt, it is possible to prevent danger in the event that the immobilized dextran sulfate and/or its salt are separated.

Furthermore, since most of dextran sulfate and/or its salts are α-1,6-glycosidic bonds, there is little change even after operations such as high-pressure steam sterilization.

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, the ionic strength of the solution, pl
Furthermore, the viscosity differs even if the molecular weight is the same, depending on the sulfur content (ie, the amount of sulfonic acid groups) of dextran sulfate and/or its salt. The intrinsic viscosity as used in the present invention is determined by determining the sodium salt of dextran sulfate and/or its salt in a neutral 1M saline solution at 25°C.

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 or the like and blood, plasma, and other body fluids flow 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 (excluded) into the pores.)
.

(3) functional groups that can be used for immobilization reactions on the surface or functional groups that can be easily activated, such as amino groups, carboxyl groups, hydroxyl groups, thiol groups, acid anhydride groups, succinylimide groups, chlorine groups, aldehyde groups, There are amide groups, epoxy groups, etc.

(4) Little change due to sterilization operations such as high-pressure steam sterilization.

In addition, the exclusion limit molecular weight (hereinafter referred to as exclusion limit molecular weight) measured using ('2J globular protein and virus)
As for the exclusion limit molecule ffi to.

If less than 10,000 carriers are used, the amount of VLDL and LDL removed will be too small to be practical, but the exclusion limit molecular weight will be 10,000.
Even a carrier having a molecular weight close to VLDLSLDL, 00,000 to several million, can be used practically to some extent. 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 100,000 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 alumina, porous silica alumina, porous hydroxyapatite, porous calcium silicate Examples include, but are not limited to, inorganic porous materials such as porous zirconia and zeolite. Further, the surface of the water-insoluble porous body may be coated with polysaccharide, synthetic polymer, or the like.

Generally speaking, the smaller the particle size of the water-insoluble porous material, the better from the viewpoint of adsorption capacity, but if the particle size is too small, the pressure drop will increase when packed in a column, which is undesirable. It is preferable that

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 is the above-mentioned (1)
It is one of the most suitable water-insoluble porous materials for the present invention because it not only has the properties described in ) to (4), but also can efficiently fix dextran sulfate and (or) its salts.

There are various methods for fixing dextran sulfate and/or its salts to water-insoluble porous materials, but for use in extracorporeal circulation therapy it is important that the ligand does not detach.
It is desirable that the ligand is fixed to the water-insoluble porous material through strong covalent bonds.

Typical examples of immobilization methods include the cyanogen halide method, epichlorohydrin method, bisepoxide method, and halogenated triazine method, but the epichlorohydrin method is the most preferred in the present invention because it provides strong binding and has little risk of detachment of the ligand. suitable for 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 in the step of reacting an epoxidized water-insoluble porous material activated with epichlorohydrin with dextran sulfate and/or its salt,
By keeping the concentration of dextran sulfate and/or its salt (concentration based on the weight of the total reaction system excluding the water-insoluble porous material (dry weight), the same applies hereinafter) at 3% by weight or more, more preferably 10% by weight or more, sufficient It has been found that amounts of dextran sulfate and/or its salts are 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 amount of riboprotein adsorption, 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 is
The salt has a bond represented by the formula: % formula % (where OA is an oxygen atom derived from the hydroxyl group of dextran sulfate and/or its salt, and 08 is an oxygen atom derived from the surface hydroxyl group of the water-insoluble porous material). It is fixed to the water-insoluble porous body through.

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 only to these examples.

Comparative Example 1 Cellulofine A-3 (Porous cellulose gel manufactured by Chisso ■, exclusion limit molecular weight 50,000.OOO, particle size 4
5 to 105 cylinders) 4g of 20% NaOH, 12g of hebutane and the nonionic surfactant Tween (T
veen) 20 was added thereto, 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, the intrinsic viscosity 0.
20 dI/g, average degree of polymerization (average degree of polymerization of raw material dextran, hereinafter referred to as average degree of polymerization) 3,500, sulfur content 1
0.5 g of 7.7% by weight sodium dextran sulfate 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 from the furnace.
It was 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 had an intrinsic viscosity of 0.124 d
l/g, average degree of polymerization 140. A cellulose gel on which dextran sodium sulfate was fixed was obtained in the same manner as in Comparative Example 1 except that the sulfur content was changed to 5.7% by weight. The amount of immobilized sodium dextran sulfate was 2.5+ag per ml column volume.

Example 1 As dextran sodium sulfate (1) Intrinsic viscosity 0.027 dR/g, average degree of polymerization 12
, sulfur content 17.7 weight 96 ('2J intrinsic viscosity 0.055 dN/g, average degree of polymerization 40°, sulfur content 19 weight % (3) intrinsic viscosity 0.083 djl!/g, average degree of polymerization 140, sulfur content 19.2% by weight (4) Intrinsic viscosity 0.118 dfl/g, average degree of polymerization 2
A cellulose gel on which dextran sodium sulfate was fixed was obtained in the same manner as in Comparative Example 1 using four types of 7o1 with a sulfur content of 17.7%. The amount of fixed dextran sodium sulfate is 1 column volume
2.0 mg each per ml5 1.51℃1gs
4. Oa+g was 4.3 mg.

Example 2 Toyovar 11W65, a cross-linked polyacrylate gel
(manufactured by Toyo Soda ■, exclusion limit molecular weight 5,000,000,
Particle size 50~LOO) Saturated Na OI in 10ml
Add 6 ml of I aqueous solution and 15 ml of epichlorohydrin,
After reacting at 50° C. for 2 hours with stirring, the gel was washed with alcohol and water to obtain an epoxidized gel.

2 ml of the resulting gel has an intrinsic viscosity of 0.055 djJ
/g.

0.5 g of dextran sodium sulfate having an average degree of polymerization of 40 and a sulfur content of 19% by weight and 2 ml of water were added (the concentration of dextran sodium sulfate was approximately 13% by weight). The gel was then adjusted to p1112, immersed at 40°C for 16 hours, and the gel was filtered and washed with 2M saline, 0.5M saline, and water to obtain a gel on which dextran sodium sulfate was fixed.

Unreacted epoxy groups were sealed using monoethanolamine. The amount of immobilized sodium dextran sulfate was 0.4 mg per ml column volume.

Example 3 Intrinsic viscosity 0.055 dfI/g, average degree of polymerization 40. A gel on which sodium dextran sulfate was fixed was obtained in the same manner as in Comparative Example 1, except that sodium dextran sulfate containing 19% by weight of sulfur was used and the concentration of sodium dextran sulfate in the fixation reaction was changed to 2.5% by weight.

The amount of immobilized sodium dextran sulfate was 0.15 mg per ml of column body N.

Test Examples Comparative Examples 1 to 2 and Examples 1 to 3 were obtained by filling 1 ml of each of the gels fixed with dextran sulfate sodium into a column, and applying the plasma of a hyperlipidemic patient (total cholesterol concentration: 300 I1 g/dI). 6 ml was poured into the tube, and the amount of adsorbed LDL 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.

Example 4 Of the adsorbents obtained in Example 1, the intrinsic viscosity was 0.027.
Fixed dextran sodium sulfate with dfI/g, average degree of polymerization of 12, and sulfur content of 17.7% by weight was dispersed in physiological saline and subjected to high-pressure steam sterilization at 120°C for 20 minutes, in the same manner as in Example 3. When the amount of LDL adsorption was measured, it was found that the amount of adsorption decreased only slightly due to the sterilization operation.

Example 5 Of the adsorbents obtained in Example 1, the intrinsic viscosity was 0.027.
d#/g, average degree of polymerization 12, sulfur content 17.7% by weight fixed dextran sodium sulfate 1 m
1 of normal human plasma (the ratio of LDL cholesterol to IIDL cholesterol is approximately 1:1).
When I passed 6ml, LDL decreased significantly, but I
Almost no IDL was adsorbed.

Example 6 A column was filled with 1 ml of the adsorbent used in Example 5, and 1.
: 1: 6 ml of normal rabbit plasma containing VLDL, LDL, SHD[, . 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 absorbance at 570 nm, ↑ is VLDL,
The positions where the LDL and IIDL bands appear are shown.

As shown in FIG. 1, VLDL and LDL were adsorbed, but IIDL was hardly adsorbed.

[Brief explanation of the drawing]

FIG. 1 is a chart showing the results of disk electrophoresis using polyacrylamide gel. 21 territories

Claims (1)

[Scope of Claims] 1. Dextran sulfate and/or its salts having an intrinsic viscosity of 0.12 dl/g or less and a sulfur content of 15% by weight or more are fixed to a water-insoluble porous material through covalent bonds. Lipoprotein adsorbent for extracorporeal circulation treatment. 2. The adsorbent according to claim 1, wherein the water-insoluble porous material has an exclusion limit molecular weight in the range of 1 million to 100 million. 3. The adsorbent according to claim 1 or 2, wherein the water-insoluble porous body is a porous cellulose gel. 4 Dextran sulfate and/or its salts have the formula: ▲Mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, 0^A is an oxygen atom derived from the hydroxyl group of dextran sulfate and/or its salts, and 0^B is The adsorbent according to claim 1, 2, or 3, which is fixed to a water-insoluble porous material through a bond represented by (which is an oxygen atom derived from a surface hydroxyl group of the water-insoluble porous material) . 5. The adsorbent according to claim 1, 2, 3, or 4, wherein the fixed amount of dextran sulfate and/or its salt is 0.2 mg or more per ml of column volume.