JPS61107942A - Adsorbent for lipoprotein - Google Patents

Abstract

PURPOSE:To selectively remove LDL and VLDL, by converting at least a part of a porous cellulose gel having a specific range of an exclusion limit MW contacted with a liquid to sulfuric ester to form an adsorbent for lipoprotein. CONSTITUTION:A porous cellulose gel, o which the exclusion limit MW measured by using spherical protein is one million - one billion, is coupled by sulfuric acid and carbondiimide in a solvent such as dimethylformamide to convert at least a part of the porous cellulose gel contacted with a liquid to sulfuric ester. As the porous structure of the porous cellulose gel, a full porous one is more suitable than a surface porous one and it is pref. that a void volume is 20% or more. The shape if a carrier can be selected from an arbitrary shape and, when a granular carrier is used, the particle size thereof is desirably 1-5,000mum.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to an adsorbent for lipoproteins for removing lipoproteins, which are harmful components in blood. More specifically, the present invention relates to a lipoprotein adsorbent for selectively adsorbing and removing lipoproteins, particularly low density lipoproteins (LDL) and very low density lipoproteins (VLDL) from blood, plasma, or serum.

[Prior Art/Problems to be Solved by the Invention] Among the lipoproteins present in blood, LDL and VLDL contain a large amount of cholesterol and are known to cause arteriosclerosis. In particular, in cases of hypercholesterolemia such as familial hyperlipidemia, the LDL level is several times higher than the normal value, leading to hardening of the coronary arteries.

For the treatment of hypercholesterolemia such as familial hyperlipidemia, diet therapy and drug treatments such as probucol and cholestyramine are used to lower LDL and VLDL levels in the blood, but their effectiveness is limited. There are also concerns about side effects. In particular, for familial hyperlipidemia, so-called plasma exchange therapy, in which the patient's plasma is separated and replaced with normal plasma or a replacement fluid containing albumin, is currently almost the only effective treatment. be.

However, as is generally known, plasma exchange therapy (1) requires the use of expensive fresh plasma or plasma preparations, (a) it removes not only harmful components but also useful components, and (3) hepatitis It has drawbacks such as the risk of infection with viruses, etc.

Attempts have been made to remove harmful components using membranes in order to overcome these drawbacks, but none of them are satisfactory in terms of selectivity, and some plasma proteins are also removed at the same time. It still has drawbacks such as the need for

For the same purpose, attempts have been made to use so-called immunoadsorbents on which antibodies, etc. are immobilized, and although these are mostly satisfactory in terms of selectivity, the antibodies used are difficult and expensive to obtain, and the sterilization of the adsorbents is difficult. There are many problems such as difficulty and poor storage stability.

Furthermore, adsorbents using the principle of so-called affinity chromatography, in which compounds having an affinity for harmful components (so-called ligands) are immobilized, have also been attempted. Although this adsorbent has good selectivity and the ligand used therein is not very expensive, it is necessary to further reduce the price in order to use it in large quantities for extracorporeal circulation therapy. Furthermore, since these adsorbents that apply the principles of affinity chromatography use soft gels such as agarose as carriers, they have problems such as poor flow of body fluids and frequent clogging.

The object of the present invention is to eliminate the above-mentioned drawbacks, to provide an adsorbent for extracorporeal circulation therapy that selectively removes LDL and VLDL, is safe, inexpensive, and has good flow.

[Means for Solving the Problems] The present invention provides excellent selectivity and stable lipolysis without using an expensive Gantt by sulfuric acid esterification of the surface of a porous cellulose gel having a specific pore size. The purpose is to scale adsorbents for proteins, and the exclusion limit molecule m measured using globular proteins is 1 million to 1.
The present invention relates to an adsorbent for lipoproteins, which is a porous cellulose gel of 100 million yen, characterized in that at least a portion of the porous cellulose gel that is in contact with a liquid is sulfuric esterified.

[Example] Unlike soft gels such as agarose, porous cellulose glue does not change due to pressure and is less prone to compaction even when flowing highly viscous fluids such as body fluids, and is also suitable for handling substances other than the target substance. It is the most suitable carrier for adsorbing and removing harmful components from body fluids through extracorporeal circulation because it has a low amount of non-specific adsorption (so-called non-specific adsorption).

The first property required of the porous cellulose gel used in the present invention is that it has continuous pores with a large diameter. That is, VLDL, L[lL is a large molecule with a molecular size of at least 1 million or more, and in order to adsorb and remove it, it is necessary that LD'L 5VLDL can easily penetrate into the gel.

There are various methods for measuring the pore diameter, and the mercury intrusion method is the most commonly used, but it is difficult to apply to porous cellulose gels. Exclusion limit molecular weight is often used as a guideline for the pore diameter of hydrophilic gels such as porous cellulose gels.

Exclusion limit molecular weight is a molecular weight that cannot enter the pores (excluded ) Refers to the molecular weight of the substance with the smallest molecular weight among molecules.

It is known that the exclusion limit molecular weight varies depending on the target compound, and in general, it has been well investigated for globular proteins, dextran, polyethylene glycol, etc., but it has hardly been investigated for lipoproteins. Therefore, it is appropriate to use the values obtained using the most similar globular proteins (including viruses).

As a result of studies conducted by the present inventor using various carriers with different exclusion limits, the exclusion limits molecular weights were unexpectedly lower than LDL and VL.
Even molecules with a molecular weight of about 1 million, which is smaller than that of DL, exhibit a certain degree of adsorption ability, and those with larger pore diameters do not necessarily have greater adsorption ability, but rather have a lower ability.
It has become clear that proteins other than 1VLDL can be adsorbed, that is, that there is an optimal pore size range. In other words, when a carrier with an exclusion limit molecular weight of less than 1 million is used, the amount of LDL and VLDL adsorbed is small and cannot be put into practical use;
It has become clear that even if a carrier with a molecular m of LDL or VLDL close to 10,000 to several million molecule is used, an adsorbent which can be used practically to some extent can be obtained. On the other hand, as the exclusion limit molecular weight increases, the adsorption amount of VLDL and LDL increases, but eventually reaches a plateau.When the exclusion limit molecular weight exceeds 100 million, the cellulose gold color is too small and sufficient sulfate groups are not introduced. , the amount of adsorption decreases noticeably. Therefore, the preferable exclusion limit molecule m of the carrier used in the present invention is 1 million to 100 million,
More preferably, it is 3 million to 70 million.

As for the porous structure of the porous cellulose gel that is the carrier used in the present invention, total porosity is preferable to surface porosity.
It is preferable that the pore volume is 20% or more. Further, the shape of the carrier can be selected from any shape such as granules, fibers, membranes, and hollow fibers. When using a particulate carrier, the particle size is 1 to 5000 μm.
It is desirable that

The porous cellulose gel may be crosslinked with epichlorohydrin or the like, or may be non-crosslinked.

In the present invention, at least a portion of the cellulose molecules in contact with the liquid of the porous cellulose gel are sulfuric acid esterified.

Methods for sulfuric acid esterification of cellulose constituting porous cellulose gel include a method in which chlorosulfonic acid, sulfuric anhydride, etc. are reacted with porous cellulose gel in the presence of pyridine, dimethylformamide, etc., and a method in which chlorosulfonic acid, sulfuric anhydride, etc. Although there are various methods such as coupling cellulose and ms with carbodiimide and direct reaction with concentrated sulfuric acid, it is preferable to conduct the reaction under anhydrous conditions or near-anhydrous conditions because decomposition of cellulose can be suppressed.

The amount of sulfuric acid residue introduced is 1 m of porous cellulose gel.
Desirably 0.1 μmal to 10 mmol per 1
More preferably 0 μmol to 1 lallio+. If the content is less than 1μmol1, the adsorption capacity is insufficient and 10m
If n+ol is exceeded, there will be too much non-specific adsorption, especially of fibrinogen, etc., making it difficult to put it to practical use. Furthermore, if the amount of sulfuric acid residues is too large, there is a risk of changing the pH of body fluids.

There are various ways in which the adsorbent of the present invention can be used therapeutically.

The simplest method is to draw the patient's blood out of the body and store it in a blood bag, mix it with the adsorbent of the present invention to remove LOL and VLOL, and then pass it through a filter to remove the adsorbent and collect the blood. There is a way to get it back to the patient. Although this method does not require complicated equipment, it has the drawbacks that the amount of treatment per treatment is small, the treatment takes time, and the operation is complicated.

Another method is to fill a column with an adsorbent and incorporate it into an extracorporeal circulation circuit to perform online adsorption and removal. Treatment methods include a method in which whole blood is directly perfused, and a method in which plasma is separated from blood and then passed through a column. Although the adsorbent of the present invention can be used in either method, it is most suitable for on-line processing as described above.

When removing LDL and VLDL using the adsorbent of the present invention, the removal efficiency and selectivity can be improved by adding polyvalent metal ions to the blood or plasma to be treated. Polyvalent metal ions used for this purpose include alkaline earth metal ions such as calcium, magnesium, barium, and strontium, group III element ions such as aluminum, group III element ions such as manganese,
Examples include group II element ions such as cobalt.

Hereinafter, the adsorbent for lipoproteins of the present invention will be explained in more detail with reference to Examples.

Example 1 Gel A-3 (Chisso ■) was added to C, which is a porous cellulose gel.
Exclusion limit for globular proteins: 5oooooooo, particle size: 45
-105μ setting) 10t+ti! was removed and dried by critical point drying in ethanol. The dried gel was suspended in 10 d of well-dried pyridine and cooled on ice.

2 d of chlorosulfonic acid was added dropwise to this while stirring, and stirring was continued for 10 minutes after the dropwise addition was completed. After the reaction is complete, the gel is filtered and washed with pyridine and then water to remove sulfuric acid residues on the surface.
A cellulose gel was obtained in which the amount shown in the table was introduced.

Example 2 Cellulofine GCL-2, a porous cellulose gel
000 (manufactured by Chisso ■, exclusion limit for globular proteins 30,000
00G, particle size 45-105μ milk, cross-linked gel) 10- was taken and dried by critical point drying in ethanol. The dried gel was suspended in 10 m of well-dried lysine and cooled on ice. Chlorosulfonic acid 2Id was added dropwise to this solution while stirring, and stirring was continued for 10 minutes until the end of the dropwise addition. After the reaction was completed, the gel was filtered and washed with pyridine and then water to obtain a cellulose gel with sulfuric acid residues introduced onto the surface in the amount shown in Table 1.

Example 3 1Od of CK gel A-3 was dried by critical point drying in ethanol. Each dried gel was suspended in 10 ae of well-dehydrated dimethylformamide and added with 4 molar H,
N-Dicyclohexylcarbodiimide was cooled. Six ml of a 2 molar sulfuric acid/dimethylformamide solution was added dropwise to this while stirring, and stirring was continued at 0°C for 2 hours. After the reaction was completed, the gel was filtered and washed with dimethylformamide and then water to obtain a cellulose gel with sulfuric acid residues introduced onto the surface in the amount shown in Table 1.

Example 4 1Od of Cellulophane GCL-2000 was dried in ethanol by critical point drying. Each dried gel was suspended in 10 ml of well-dehydrated dimethylformamide, and 12 ml of a 4 molar N,N-dicyclohexylcarbodiimide/dimethylformamide solution was added thereto and cooled on ice. Add to this a 2 molar sulfuric acid/dimethylformamide solution wi6I1.
1 was added dropwise with stirring, and stirring was continued at 0° C. for 2 hours. After the reaction was completed, the gel was filtered and washed with dimethylformamide and then water to obtain a cellulose gel with sulfuric acid residues introduced onto the surface in the amount shown in Table 1.

Examples 5-6 The amounts of chromium sulfonic acid were 6d and 8IIr1, respectively.
(corresponding to Examples 5 and 6, respectively) in the same manner as in Example 1 to obtain cellulose gels into which sulfuric acid residues were introduced in the amounts shown in Table 1.

Comparative Example 1 Cellulose gel was used as Cellulofine GC 70G (manufactured by Chisso ■, exclusion limit of globular protein 400,000, particle size 45
A cellulose gel having sulfuric acid residues introduced on the surface as shown in Table 1 was obtained in the same manner as in Example 1, except that the thickness was changed to -105 μm, ).

Examples 7 to 12 and Comparative Examples 2 to 3 Each gel 11n1 synthesized in Examples 1 to 6 and Comparative Example 1
was placed in a test tube, 6 volumes of plasma from a familial hyperlipidemia patient were added thereto, and the mixture was incubated at 37°C for 2 hours with stirring (corresponding to Examples 7 to 12 and Comparative Example 2, respectively).

LDL, VLDLlHDLDL/Suf0-At,
The amount of fibrinogen was measured. The results are shown in Table 1.

LDL also applies to those without gel.

The amounts of VLDL, HOL cholesterol, and fibrinogen were measured (Comparative Example 3). The results are shown in Table 1.

[Margins below] [Effects of the invention] By using the adsorbent of the present invention, it is possible to selectively and efficiently remove harmful LDL and VLDL from patient's body fluids, and it is possible to remove harmful LDL and VLDL from the patient's body fluids without using relatively expensive ligands. Treatment can be performed at a lower cost than using an adsorbent based on the principle of affinity chromatography.

Claims (1)

[Claims] 1 Exclusion limit molecular weight measured using globular protein is 10
1. An adsorbent for lipoproteins, which is a porous cellulose gel of 00,000 to 100 million, characterized in that at least a portion of the porous cellulose gel in contact with a liquid is sulfuric esterified.