JPS6256782B2 - - Google Patents

Description

[Detailed description of the invention]

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 low-density lipoproteins (LDL), from blood, plasma, or serum. Among the lipoproteins present in the blood, LDL contains a large amount of cholesterol and is known to cause arteriosclerosis. In particular, in cases of hypercholesterolemia such as familial hyperlipidemia, the LDL value is several times higher than the normal value, leading to hardening of the coronary arteries. For this treatment, dietary therapy and drug treatments such as probucol and cholestyramine are being used to lower blood LDL, but their effectiveness is limited and there are concerns about side effects. Particularly 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, etc., is currently almost the only effective treatment. be. However, as is well known, plasmapheresis therapy requires the following: 1) It is necessary to use expensive fresh plasma or plasma preparations, 2) There is a risk of infection with hepatitis viruses, etc., and 3) It removes not only harmful components but also useful components. It has drawbacks such as being stowed away. In order to overcome these drawbacks, attempts have been made to remove harmful components using membranes, but no membranes 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 these are mostly satisfactory in terms of selectivity, they have the fatal problem of being difficult and expensive to obtain the antigens and antibodies used. It has some disadvantages. Furthermore, adsorbents based on 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. The ligands used for this are relatively inexpensive and have relatively good selectivity, but because the carrier is a soft gel such as agarose, it is difficult to obtain a sufficient flow rate when packed in a column. It was hot. In other words, if these adsorbents are to be used in blood or plasma perfusion therapy (so-called plasmapheresis, etc.) using the recently developed extracorporeal circulation circuit, special ingenuity is required in the column shape in order to obtain a high flow rate. However, there are many problems, such as the need to prepare spare columns because they often clog, and the situation has not been reached in which stable treatment can be performed. Although it is obvious that a carrier with high mechanical strength can be used to improve the flow characteristics of an adsorbent, it is known that the use of these carriers lowers the adsorption capacity compared to soft gels such as agarose. ing. On the other hand, it is known that polyanionic compounds such as sulfated polysaccharides have an affinity for lipoproteins and form precipitates in the presence of metal ions [for example, M.
Burnstein and HR Scholnick, Adv. in Lipid.
Res., 11 , 67 (1973)] and is used for clinical analysis, etc. However, with this method, the
To remove LDL, at least 0.05% of a polyanionic compound and 0.02M or more of metal ions must be added to the plasma to be processed, and the resulting precipitate must be separated by a method such as centrifugation. However, the operation was complicated and highly dangerous, and it was virtually impossible to apply. As a result of extensive research, the present inventors have found that by using a specific porous polymer hard gel and fixing a polyanion compound that has affinity for lipoproteins to it, it is possible to achieve a low cost, good flow characteristics, and a soft gel that can be used as a carrier. The present invention has been achieved by obtaining a lipoprotein adsorbent that has excellent removal ability without decreasing its adsorption ability compared to when the lipoprotein adsorbent was used. That is, the present invention is a lipoprotein adsorbent comprising a porous polymer hard gel having an exclusion limit molecular weight of 1 million or more and 1 or less for globular proteins, and a polyanionic compound having affinity for lipoproteins fixed thereon. The present invention will be explained in detail below. A carrier suitable for use in the present invention must 1) be pressure resistant and 2) have pores with a relatively large diameter, and polymer hard gel is the most suitable carrier for the present invention. The term "hard gel" as used herein refers to a gel that swells less with solvents than soft gels such as dextran, agarose, acrylamide, etc., and is less likely to deform under pressure. Hard gels and soft gels can be distinguished by the following method. In other words, as shown in the reference example below, when gel is uniformly packed into a cylindrical column and an aqueous liquid is flowed, the relationship between pressure loss and flow rate is almost a straight line for hard gel, but for soft gel there is a pressure drop. Beyond this point, the gel deforms and becomes compacted, and the flow rate no longer increases. In the present invention, when the column shown in the reference example below is used, a gel having the above linear relationship up to at least 0.3 Kg/cm ² is referred to as a hard gel. The next required property is to have pores with a relatively large diameter. That is, LDL is a large molecule with a molecular weight of at least 1 million or more, and in order to adsorb and remove it, it is necessary for LDL to be able to enter the pores. Next, even if LDL can enter the pores, its performance as an adsorbent will be low unless the probability of LDL entering the pores is high to some extent.In other words, the distribution ratio between the mobile phase and the stationary phase (inside the pores) is It is considered that a higher ratio (phase concentration/mobile phase concentration) is more preferable. Therefore, it seems that the larger the pore diameter is, the more advantageous it is. There are various methods for measuring pore diameter, and mercury intrusion method is the most commonly used, but it may not be applicable to polymer hard gels. Therefore, it is appropriate to use the exclusion limit molecular weight as a measure of the pore diameter. What is the exclusion limit molecular weight?As stated in books (for example, Hiroyuki Hatano and Toshihiko Hanai, Experimental High Performance Liquid Chromatography, Kagaku Doujin), the molecular weight cannot enter the pores (is excluded) in gel permeation chromatography. It refers to the molecular weight of the one with the smallest molecular weight among molecules. Phenomenologically, molecules with a molecular weight above the exclusion limit are eluted near the mobile phase volume Vo, so the exclusion limit molecular weight can be determined by examining the relationship with the elution volume using compounds of various molecular weights. It is known that the exclusion limit molecular weight varies depending on the type of 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 using various carriers with different exclusion limits, we found that, contrary to expectations, even carriers with an exclusion limit molecular weight of about 1 million, which is smaller than the molecular weight of LDL, have a certain degree of
It has become clear that there is an optimal pore size range because it exhibits LDL adsorption ability, and the larger the pore size, the greater the ability, rather proteins other than LDL are removed. In other words, when using a carrier with an exclusion limit molecular weight of less than 1 million,
Although the amount of LDL removed was too small to be practical, an adsorbent with an exclusion limit molecular weight of 1 million to several million, which is close to the molecular weight of LDL, was able to be used to some extent for practical use. On the other hand, the exclusion limit molecular weight and the adsorption amount of LDL,
Examining the relationship between this and the adsorption of proteins other than LDL (so-called non-specific adsorption), we found that as the exclusion limit molecular weight increases, the amount of LDL adsorbed increases;
It was found that this increase reaches a plateau when the exclusion limit exceeds 10 million, while adsorption of proteins other than LDL, such as IGG and IGM, becomes noticeable. Furthermore, when the exclusion limit molecular weight exceeds 100 million, the amount of immobilized ligand decreases, resulting in a decrease in the amount of LDL adsorbed, and nonspecific adsorption cannot be ignored. Therefore, the preferred exclusion limit molecular weight of the carrier used in the present invention is 100
The number is 10,000 to 100 million, most preferably 3 million to 70 million. Next, regarding the porous structure of the carrier, total porosity is preferable to surface porosity, and it is preferable that the pore volume is 20% or more. The shape of the carrier can be selected from any shape such as granules, fibers, membranes, and hollow fibers. When a particulate carrier is used, the particle size is preferably 1 μm or more and 5000 μm or less. Furthermore, it is advantageous if a functional group that can be used in an immobilization reaction or a functional group that can be easily activated is present on the surface of the carrier. Representative examples of these functional groups include amino groups, carboxyl groups, hydroxyl groups, thiol groups, acid anhydride groups, succinylimide groups, chlorine groups, aldehyde groups, amide groups, and epoxy groups. Typical examples of polymer hard gels suitable for the present invention include styrene-divinylbenzene copolymer,
Synthetic polymers such as cross-linked polyvinyl alcohol, cross-linked polyacrylate, cross-linked vinyl ether-maleic anhydride copolymer, cross-linked styrene-maleic anhydride copolymer, cross-linked polyamide, and hard porous materials such as porous cellulose gel; Examples include, but are not limited to, those whose surfaces are coated with polysaccharides, synthetic polymers, etc. These polymer hard gels may be used alone or in combination of two or more. Representative examples of polyanionic compounds with affinity for lipoproteins suitable for use in the present invention include heparin, dextran sulfate, chondroitin sulfate,
Chondroitin polysulfate, heparanic acid, keratan sulfate, heparitin sulfate, xylan sulfate, caronine sulfate, cellulose sulfate, chitin sulfate, chitosan sulfate, pectin sulfate, inulin sulfate, alginate sulfate, glycogen sulfate, polylactose sulfate, carrageenan sulfate, starch sulfate, Examples include sulfated polysaccharides such as polyglucose sulfate, laminarin sulfate, galactan sulfate, levan sulfate, and mepesulfate, phosphotungstic acid, polysulfated anethole, polyvinyl alcohol sulfate, and polyphosphoric acid. The most preferred examples include heparin, dextran sulfate, and chondroitin polysulfate. Compounds (ligands) that have affinity for lipoproteins
Various known methods can be used for immobilizing on a carrier. i.e. physical adsorption method,
These include ionic bonding methods, covalent bonding methods, etc. In order to use the adsorbent according to the present invention for treatment, it is important that the ligand does not detach during sterilization or treatment, so a strong covalent bonding method is preferable. It is desirable to form a bond in a cross-linked manner. Furthermore, a spacer may be introduced between the carrier and the ligand if necessary. The amount of immobilized ligand varies depending on the properties and activity of the ligand, but is preferably 0.02 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. More preferably, per ml of column volume
The amount is 0.5mg or more and 20mg or less. There are various ways in which the adsorbent according to the invention can be used therapeutically. The simplest method is to draw the patient's blood outside the body, store it in a blood bag, etc., mix it with the adsorbent (particles) of the present invention to remove LDL, and then pass it through a filter to remove the adsorbent (particles). There are ways to return the blood to the patient. Although this method does not require complicated equipment, it has the disadvantages that the amount of treatment per treatment is small, the treatment takes time, and the operation is complicated. The next method is to pack the adsorbent into a column and
It is installed in the extracorporeal circulation circuit and performs adsorption and removal online. Treatment methods include direct perfusion of whole blood and separation of plasma from blood and then passing the plasma through a column. Although the adsorbent according to the invention can be used in either method, it is most suitable for on-line processing as mentioned above. When removing LDL using the adsorbent according to the present invention, it is possible to improve the removal efficiency and selectivity by adding polyvalent metal ions to the blood or plasma to be treated. Examples of polyvalent metal ions used for this purpose include alkaline earth metal ions such as calcium, magnesium, barium, and strontium, genus element ions such as aluminum, genus element ions such as manganese, and genus element ions such as cobalt. . The present invention will be explained in more detail with reference to Examples below. Reference example Agarose gel (Biogel A5m manufactured by Biorad, particle size
50-100 mesh), polymer hard gel (Toyo Pearl HW65 manufactured by Toyo Soda Kogyo Co., Ltd., particle size 50-100μ)
m, and Cellulofine GC-700 manufactured by Chitsuso Co., Ltd.
(particle size: 45 to 105 μm) were uniformly filled in each tube, water was flowed through the tubes using a peristaltic pump, and the relationship between flow rate and pressure loss was determined. The results are shown in Figure 1. According to the results, the flow rate of polymer hard gel increases almost in proportion to the increase in pressure, whereas the flow rate of agarose gel does not increase even if the pressure is increased due to compaction. Example 1 Toyopearl HW55, a cross-linked acrylate gel (fully porous hard gel) (exclusion limit molecular weight for globular proteins (hereinafter abbreviated as protein exclusion limit))
700000, particle size, 50-100μm), HW60 (protein exclusion limit 1000000, particle size 50-100μm), HW65
(Protein exclusion limit 5000000, particle size 50-100μm),
HW75 (protein exclusion limit 50000000, particle size 50~
100 μm) Add 6 ml of saturated NaOH aqueous solution and 15 ml of epichlorohydrin to each 10 ml and heat at 50°C with stirring.
After a time reaction, an epoxidized gel was obtained. 20 ml of concentrated ammonia water was added to this gel and stirred at 50°C for 2 hours to introduce amino groups. Next, 200 mg of heparin was dissolved in 10 ml of water and the pH was adjusted to 4.5, and then 3 ml of the above amino group-containing gel was added thereto. Add 200 mg of 1-ethyl-3-(dimethylaminopropyl)-carbodiimide to this to bring the pH to 4.5.
The mixture was added while maintaining the temperature at 4°C, and the mixture was shaken at 4°C for 24 hours. After the reaction, 2 molar salt solution, 0.5 molar salt solution,
A heparin-immobilized gel was obtained by washing with water. The amount of immobilized heparin was 2.2 mg/ml and 1.8 mg/ml, respectively.
ml, 1.4 mg/ml, and 0.8 mg/ml. Example 2 Hard cellulose porous material (fully porous hard gel), Cellulofine GC700 (manufactured by Chitsuso Co., Ltd., protein exclusion limit 400000, particle size 45 to 105 μm), Cellulofine A-2 (manufactured by Chitsuso Co., Ltd. (prototype) ), protein exclusion limit: 700,000, particle size: 45-105 μm), Cellulofine A-3 (manufactured by Chitsuso Co., Ltd. (prototype), protein exclusion limit: approximately 5,000,000, particle size: 45-105 μm), and Take 10g and add 20% to it
4 g of NaOH and 12 g of heptane were added, and 1 drop of nonionic surfactant TWEEN20 was added and stirred to disperse the gel. After stirring at 40°C for 2 hours, 5 g of epichlorohydrin was added thereto, and the mixture was stirred at 40°C for 2 hours. After standing still, the supernatant was discarded, and the gel was washed with water to obtain an epoxidized gel. To this, 15 ml of concentrated ammonia water was added and stirred at 40°C for 1.5 hours, and the contents were filtered off by suction and washed with water to obtain a cellulose gel into which amino groups had been introduced. Next, 200 mg of heparin was dissolved in 10 ml of water, and 3 ml of the above amino group-containing gel was added thereto to adjust the pH to 4.5. To this was added 200 mg of 1-ethyl-3-(dimethylaminopropyl)-carbodiimide while maintaining the pH at 4.5, and the mixture was shaken at 4°C for 24 hours. After the reaction was completed, the gel was washed with a 2 molar salt solution, a 0.5 molar salt solution, and water to obtain a heparin-immobilized gel. The amount of immobilized heparin was 2.5 mg/ml, 2.2 mg/ml, and
It was 1.8 mg/ml. Example 3 Chondroitin polysulfate-immobilized Toyopearl gel HW65 was obtained in the same manner as in Example 1, except that chondroitin polysulfate was used instead of heparin. The amount of immobilization is
It was 1.2 mg/ml. Example 4 Cellulofine A-3 0.5 mol NaIO ₄ in 4 ml
The mixture was made up to 10 ml, stirred at room temperature for 1 hour, and washed with water to introduce aldehyde groups. Next, add this gel to PH
The suspension was suspended in 10 ml of phosphate buffer (No. 8), 50 mg of diethylamine was added, stirred at room temperature for 20 hours, and separated. This was suspended in 10 ml of 1% NaBH ₄ solution and reduced for 15 minutes, then collected and washed to introduce amino groups. Then dextran sulfate 300mg 0.25mol _NaIO4
Dissolve in 10 ml of solution and stir at room temperature for 4 hours, then add 200 mg of ethylene glycol and stir for 1 hour. After adjusting the pH of this solution to 8, the amino group-containing gel was suspended and stirred for 24 hours. After the reaction was completed, the gel was collected and washed with water, suspended in 10 ml of 1% NaBH ₄ solution, reduced for 15 minutes, and washed with water to obtain a dextran sulfate-immobilized cellulose gel. The amount of immobilization is 0.5
It was mg/ml. Example 5 1 ml of each adsorbent synthesized in Examples 1 to 4 was placed in a test tube, and 3 ml of human plasma (containing 0.02M CaCl ₂ ) was added to this.
was added and stirred, and after standing at 20°C for 15 minutes, the cholesterol concentration and LDL (β-lipoprotein) amount of the supernatant were measured. The results are shown in Table 1.

【table】

【table】 [Brief explanation of the drawing]

FIG. 1 is a graph showing the relationship between flow rate and pressure loss using various gels in a reference example.

Claims (1)

[Claims] 1. Exclusion limit molecular weight of globular protein is 1 million or more.1
A lipoprotein adsorbent made by immobilizing a polyanionic compound that has an affinity for lipoproteins onto a porous polymer hard gel of less than 100 million yen. 2. The adsorbent according to claim 1, wherein the porous polymer hard gel is made of a synthetic polymer. 3. The adsorbent according to claim 1, wherein the porous polymer hard gel is a porous cellulose gel. 4. The adsorbent according to claim 1, wherein the polyanion compound is a sulfated polysaccharide. 5 Sulfated polysaccharides include heparin, dextran sulfate,
At least one selected from chondroitin polysulfate
The adsorbent according to claim 4, which is a species. 6. The adsorbent according to claim 1, wherein the amount of the polyanion compound immobilized is 0.02 mg or more and 100 mg or less per ml of column volume.