JPS59102436A - Adsorbent body - Google Patents

Abstract

PURPOSE:To provide a body for selectively adsorbing and removing lipoprotein in blood by immobilizing a polyanion compd. having affinity to lipoprotein to a specific porous polymer hard gell. CONSTITUTION:A material prepd. by immobilizing a polyanion compd. consisting of heparin, sulfated polysaccharide such as dextran sulfuric acid, chondroitin polysulfate or the like and having high affinity to lipoprotein on a porous cellulose gel having 1,000,000-100,000,000 limit mol. wt. for removal of spherical protein, such as a synthetic high polymer such as a styrene-divinyl benzene copolymer, crosslinked PVA or the like and porous cellulose gel, at 0.02-100mg for each 1ml of column volume is used as an adsorbent body for selectively adsorbing and removing the lipoprotein (LDL) in the human blood, plasma or serum. The LDL which is a cause for arteriosclerosis is adsorbed and removed selectively at a low cost and said adsorbent body is used for the treatment of the hyperlipemia.

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 that selectively adsorbs and removes 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. Particularly, in cases of flat cholesterol such as familial hyperlipidemia, the LDL value is several times higher than the normal value, which causes hardening of the coronary arteries. For this treatment, dietary therapy and drug therapy such as globucol and cholestyramine have been 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. . However, as is well known, plasma exchange therapy requires the use of: l) 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. 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 (so-called ligands) that have an affinity for harmful components 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. Met. In other words, if these adsorbents are to be used in blood or plasma perfusion therapy using the extracorporeal circulation circuit that has been developed in recent years (so-called Plasma 7 Elesis, etc.), special ingenuity is required in the column shape in order to obtain a cylindrical flow rate. In addition, there are many problems such as the need to prepare a spare column due to frequent clogging, and the situation has not been reached in which stable treatment can be performed.In order to improve the flow characteristics of the adsorbent, Although it is obvious that a carrier with high mechanical strength should be used, it is known that when these carriers are used, the adsorption capacity is lower than that of soft gels such as agarose.

On the other hand, it is known that polyanionic compounds such as monosulfated polysaccharides have affinity with lipoproteins and form precipitates in the coexistence of metal ions [for example, M, BurnsLein et al.
and H,R,5cholnick,Adv,
1nLipid, Res, It, 67 (19
73> ], used for clinical analysis, etc. However, if this method is used to remove LDL from the patient's blood, at least 0.05% of the blood 11c to be processed must be removed.
of a polyanion compound and metal ions of 0.02 M or more must be added, and the resulting precipitate must be separated by a method such as centrifugation, making the operation complicated and highly dangerous, making it virtually impossible to apply. It was impossible.

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, in the present invention, the exclusion limit molecular weight of globular proteins is 10
00,000 or more] porous polymer of excellent or less = 7-dogel,
This is a lipoprotein adsorbent made by immobilizing a polyanionic compound that has affinity for lipoproteins.

The present invention will be explained in detail below.

A carrier suitable for use in the present invention must: 1) be pressure resistant; 2) have pores with a relatively large diameter; Bolimano-Dogel 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, a substance having the above linear relationship up to at least 0.3 kyA is referred to as a no-dogel.

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 penetrate into the pores.

Next l/i: Even if LDL can enter the pores, unless the probability of entering the pores is high to a certain extent, its performance as an adsorbent will be low, i.e. partition between the mobile phase and stationary phase (inside the pores) It is considered that the larger the ratio (stationary phase concentration/mobile phase concentration) is, the more preferable it is. Therefore, it seems that the larger the hole 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 guideline for the pore diameter. The exclusion limit molecular weight is the molecular weight of molecules that cannot enter the pores (excluded) in gel permeation chromatography, as described in Hiroyuki Hatano and Toshihiko Hana, Experimental High Performance Liquid Chromatography, Kagaku Doujin. It refers to the molecular weight of the one with the smallest molecular weight. Phenomenologically, molecules with a molecular weight above the exclusion limit are eluted near the mobile phase volume Vo.
Exclusion limit molecular weight can be determined by examining the relationship with 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, globular proteins, dextran, borieterenoglycol, etc. have been well investigated, but lipoproteins have hardly been investigated. Therefore, it is appropriate to use p using the value obtained using the most similar globular protein (including viruses).

As a result of studies using various carriers with different exclusion limits, we found that, contrary to expectations, the exclusion limit molecular weight was smaller than the molecular weight of LDL1.
Even those with a resistance of about 1,000,000 ml show a certain degree of LDL absorption ability,
Also, the larger the pore size, the greater the capacity;
Rather, it has become clear that there is an optimal pore size range since proteins other than LDL are removed. In other words, when a carrier with an exclusion limit molecular weight of less than 1 million is used, the removal of LDL is too small to be practical, but even a carrier with an exclusion limit molecular weight of 1 million to several million, which is close to the molecular weight of LDL, can be put to practical use to some extent. A moist adsorbent was obtained. On the other hand, when examining the relationship between the exclusion limit molecular weight, the adsorption amount of LDL, and the adsorption of proteins other than LDL (so-called non-specific absorption layer), we find that as the exclusion limit molecular weight becomes thicker, the adsorption amount of LDL increases; This increase reaches a plateau when the exclusion limit exceeds 10 million, while proteins other than LDL, such as I
It was found that adsorption of GG, IGM, etc. became 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.
Non-specific adsorption can no longer be ignored. Therefore, the exclusion limit molecular weight of the phase used in the present invention is preferably from 1 million to 100 million, most preferably from 3 million to 70 million.

Regarding the porous structure of the next VC carrier, total porosity is preferable to surface porosity, and the pore volume is preferably 20% or more. 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 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 amine groups, carboxyl groups, hydroxyl groups, thiol groups, acid anhydride groups, succinylimide groups, chlorine groups, aldehyde groups, amide groups, and epoxy groups.

Representative examples of polymer hard gels suitable for the present invention include:
Synthetic polymers and porous materials such as styrene-divinylbene, Zen copolymer, cross-linked polyvinyl alcohol, cross-linked polyacrylate, cross-linked vinyl ether-maleic anhydride copolymer, cross-linked styrene-maleic anhydride copolymer, cross-linked polyamide, etc. Examples include, but are not limited to, hard porous bodies such as cellulose gel, and 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 riboproteins suitable for use in the present invention include hepa, phosphorus, texturan sulfate, chondroitin sulfate, chondroitin polysulfate, heparanic acid, keratanosulfate, hehalitene sulfate, xylan sulfate, caronine Sulfuric acid, cellulose sulfate, chitin chitosan sulfate, pectin acid, inulin sulfate, alginate sulfate, glycogen sulfate, polylactose sulfate, -
Lagenin sulfate, starch 7 sulfate, polyglucose sulfate,
Examples include sulfated polysaccharides such as laminarin sulfate, galactan sulfate, levan sulfate, and mepesulfate, phosphotungstic acid, polysulfated anethole, polyvinyl alcohol sulfate, and polyphosphoric acid. The most preferred examples include hevarino, dextran sulfate, and chondroitin polysulfate.

Various known methods can be used to immobilize a compound (ligand) that has affinity for lipoproteins on a carrier. That is, physical adsorption methods, 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 in order to obtain a significant amount of lipoprotein adsorption, it is preferably 0.02 mDa or more per 1 mβ column volume, and from economical considerations, it is 100 mFI or less. is desirable. More preferably, the column volume is 1. o per J, 5 my or more and 20 mf 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 out of the body and store it in a blood bag, etc., and then apply the adsorbent (particles) of the present invention to this.
There is a method of mixing blood to remove LDL, then passing it through a filter to remove adsorbents (particles), and returning 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 install it in an extracorporeal circulation circuit to perform online adsorption and removal. There are two processing methods: one in which whole blood is directly passed through the cancer, and the other in which plasma is separated from the blood and then passed 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. Polyvalent metal ions used for this purpose include alkaline earth metal ions such as calcium, magnesium, barium, and strontium;
Examples include ions of genus elements, ions of genus elements such as manganese, ions of genus elements such as cobalt, and the like.

The present invention will be explained in more detail with reference to Examples below.

Reference Example A glass cylindrical column (inner diameter 9 mm, column length 150 m+i) equipped with filters with a pore size of 15 μm at both ends was coated with agarose gel (Biorad Bioge#A 5 m).
1 particle size 50-100 mesh), polymer hard gel (
TOYOBAR HW65 manufactured by Toyo Soda Kogyo ■, particle size 5o~10
0μm1 and Chisso Cellulofine GC-700
, particle size: 45 to 105 μm) were uniformly filled in the tubes, water was flowed through the tubes using a peristaltic pump, and the relationship between flow rate and pressure loss was determined.

Results]! +1. According to this study, 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 by causing compaction.

Example 1 Toyovar HW55, a cross-linked acrylate gel (fully porous) (exclusion limit molecular weight of globular protein)
(hereinafter abbreviated as protein exclusion limit) 700.0 (10,
Particle size 50-100μm), )lW60 (protein exclusion limit 1,000.l[o, particle size 50-IQOpm), H
W65 (protein exclusion limit 5.000,000, particle size 50-100pm), HW75 (protein exclusion limit 50.0-00,00 (J, particle size 50-100 tt)
m) 6 tJ of saturated aqueous NaOH solution for each 10 ml.

Add 15 md of epichlorohydrin and heat to 50° while stirring.
The mixture was reacted at C for 2 hours to obtain an epoxidized gel. 20 mg of concentrated ammonia water was added to this gel and stirred at 50°C for 2 hours to introduce amino groups.

Next, dissolve niheparin 200 mF/p in the water of the tomb.
After adjusting to H4.5, 3-g of the above gel containing 7 amino groups was added thereto. This [1-ethyl-3-(dimethylaminopropyl)-carbodiimide 2004f] was added while keeping the pH at 4.5, and the mixture was incubated 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 is 2.2mf//ml each! , 1.8 m
f/ml, 1.4 mI/me.

It was 0.8 mf/ml.

Example 2 Hard cellulose porous material (fully porous hard gel λ Cellulofine GC700 (manufactured by Chisso ■, protein exclusion limit 4)
00,000, particle size 45-105 μm), Cellulofine A-2 (Chisso ■H (prototype), protein exclusion limit 700,000, particle size 45-105 μm), Cellulofine A-3 (Chisso (made by i5IO) (prototype), protein exclusion limit approximately 50,000,000, particle size 45-105
[μm] were sucked and allowed to take 1Of/l/I: 4y of 20% NaOH and 12y of Hegetane, and then 1 drop of nonionic surfactant TWEEN 20 was added and stirred to disperse the gel. . After stirring at 40°C for 2 hours, epichlorohydrin 5y 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. This l/il: I 5 m
Nine liters of concentrated ammonia water were stirred at 40° C. for 1.5 hours, and the contents were suction filtered and washed with water to obtain a cellulose gel into which amine groups had been introduced.

Next, dissolve Hepari/200 mf in rome water, add 3 ml of the above amino group-containing gel, and add
Adjusted to H4.5. To this was added 200 ml of 1-ethyl-3-(dimethylaminepropyl)-carboimide, f while maintaining the pH at 4.5K, and the mixture was incubated 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 fixed height is 2,5m9/, (1,2,
2 my/ml, 1.8 m'j/m (l).

Example 3 Chondroitin polysulfate-immobilized Toyopearl gel HW65 was obtained in the same manner as in Example 1 except that heparin was replaced with chondroitempo IJa acid. The amount of immobilization is 1.2 mf/
It was me.

Example 4 0.5 mol NaIO4 in Cellulophy 7A-34yJ'
Add 10. The mixture was stirred at room temperature for 1 hour, filtered and washed with water to introduce an aldehyde group. Next, this gel was suspended in 10 ml of pH 8 phosphate buffer, and 50 ml of diethylamine was added.
mf was added and stirred at room temperature for 20 hours to remove P. Add this to 1% NaBH4 solution 10. After suspension in J' and reduction for 15 minutes, the mixture was collected and washed to introduce an amine group.

Next, add 300 mf of dextran sulfate to 0.25 mol Na
IO4 solution 10. J [After dissolving and stirring at room temperature for 4 hours, add 200 mf of ethylene glycol and stir for 1 hour.

After adjusting the pH of this solution to 8VC, the above amino group-containing gel was suspended and stirred for 24 hours. After the reaction, the gel was collected and washed with water, suspended in 10 ml/C of 1% NaBH4 solution, exposed to light for 15 minutes, filtered and washed with water to obtain a dextran sulfate-immobilized cellulose gel. The amount of immobilization is 0.5 mf/
It was ml.

Example 5 Each of 14 adsorbents synthesized in Examples 1 to 4 was placed in a test tube,
This includes 3 ml of human plasma! (GaC (containing 120.02M) was added, stirred, and allowed to stand at 20°C for 15 minutes. The cholesterol concentration and LDL (β-lipoprotein) in the supernatant were
Children were measured. The results are shown in Table 1.

(↓The bottom is thick

[Brief explanation of drawings]

FIG. 1 is a graph showing the relationship between flow force and pressure loss using various gels in a reference example. Patent applicant Makoto Asano, agent of Kanebuchi Chemical Industry Co., Ltd., patent attorney - Procedural amendment (9 volumes, 1939), 20 accounts per month, Commissioner of the Japan Patent Office, Kazuo Wakasugi, Y, 1411, Indication of the case, 1982, Idera Kazari Application No. 212379 2. Title of the invention: 3. Relationship with the person making the amendment: Patent applicant: 2-2-4 Nakanoshima, Kita-ku, Osaka Mv “Shiwah>” H) Kanebuchi Chemical Industry 11, representative of the formula company, high school 1) Sho 4, agent 6, number of inventions increased by amendment 7, subject of amendment

Claims (6)

[Claims] (1) A lipoprotein adsorbent comprising a porous polymer hard gel having an exclusion limit molecular weight of 1,000,000 to 100,000,000 for globular proteins, and a polyanionic compound having an affinity for lipoproteins. (2) The adsorbent according to claim 1, wherein the porous polymer hard gel is made of a synthetic polymer. (3) Porous polymer hard gel is porous cellulose
The adsorbent according to claim 1, which is a gel. (4) The adsorbent according to claim 1, wherein the polyanionic compound is a sulfated polysaccharide. (5) The adsorbent according to claim 4, wherein the sulfated polysaccharide is at least one selected from heparin, dextran sulfate, and chondroitin polysulfate. (6) The adsorbent according to claim 1, wherein the amount of the polyanionic compound immobilized is 0.02 my or more and too my or less per 1 ml of column volume per hour.