JPS59156431A - Adsorbent - Google Patents

Abstract

PURPOSE:To selectively adsorb and remove lipoprotein, by immobilizing a polyanion compound by an inorg. porous substance with an average pore size of 700-4,000Angstrom . CONSTITUTION:A polyanion compound having affinity to lipoprotein such as heparin, dextrane sulfate, chondroitin sulfate, chondroitin polysulfate, heparanic acid or keratanic acid is immobilized by an inorg. porous substance having an averate pore size of 700-4,000Angstrom , a void volume of 20% and a specific surface area of 3m<2>/g or more such as silica gel, alumina, porous glass, silica-alumina, hydroxyapatite or calcium silicate according to a physical adsorbing method, an ion bonding method or a covalent bonding method. The amount polyanion compound to be immobilized is pref. 0.5-20mg per milli-liter of a column volume.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an adsorbent for the removal of harmful components in blood. More specifically, the present invention relates to an adsorbent that selectively adsorbs and removes lipoproteins, particularly low-density lipoproteins (LD'L), from blood, plasma, or serum.

Among 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 level is several times higher than the normal value, leading to hardening of the coronary arteries.

To treat this problem, dietary therapy and drug treatments such as propcol 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 blood exchange therapy, in which the patient's plasma is separated and replaced with normal plasma or replacement fluid containing albumin, etc., is currently almost the only effective treatment. It is. However, as is well known, blood stool exchange therapy requires (1) the use of expensive fresh dJ or blood stool preparations; (2) There is a risk of infection with hepatitis virus, 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 disadvantage that the antigens and antibodies used are difficult and expensive to obtain. It has some disadvantages. Furthermore, adsorbents based on the principle of affinity chromatography, in which compounds having an affinity for harmful components (so-called ligands) are immobilized, have also been attempted. The ligand used for this is cheap and relatively selective compared to antigens and antibodies, but since the carrier is a soft gel such as agarose, it has a sufficient flow rate when packed in a column. was difficult to obtain. In other words, if an adsorbent of Cholera is to be used for blood and blood cancer flow therapy (so-called plasmapheresis) using an extracorporeal circulation circuit that has been developed in recent years, it is necessary to use a special column shape to obtain a high flow rate. There are many problems, such as the need to devise a new method and the need to prepare a spare column because clogging often occurs, and the situation has not reached a point where stable treatment can be performed.

Although it is obvious that a carrier with high mechanical strength should be used to improve the flow characteristics of the adsorbent, it is said that the use of these carriers lowers the adsorption capacity compared to soft gels such as 7 Gallone. .

On the other hand, it is known that polyanionic compounds such as sulfated polysaccharides have an affinity for riboproteins and form precipitates in the coexistence of metal ions (for example, M-Bur
nstej-n and H,R,5cho conn1-c
k.

Adv-j-n Lj-pidRes-, 11,67,
(1973) and is used for clinical analysis. However, when attempting to remove LDL from a patient's blood using this method, it is difficult to remove LDL from the patient's blood by at least 0.005% of the blood volume being treated.
It was necessary to add a polyanion compound of 0.02M or more and a metal ion of 0.02M or more, and it was necessary to separate the resulting precipitate by a method such as centrifugation, which was a complicated and highly dangerous operation and was practically impossible to apply. .

As a result of extensive research, the present inventors found that by using an inorganic porous material with a specific structure and fixing a polyanion compound that has an affinity for riboproteins, it is possible to achieve a method that is inexpensive, has good flow characteristics, and has high removal ability. The present invention was achieved by obtaining a lipoprotein adsorbent with excellent properties.

That is, in the present invention, the average pore diameter is 700X or more and 4ooo
This is a riboprotein adsorbent formed by immobilizing a polyanion compound having an affinity for riboproteins on an inorganic porous material having a size of Z or less.

The present invention will be explained in detail below.

The carrier used in the present invention has (1) pressure resistance, (
2) It is required to have pores with a relatively large diameter,
Inorganic porous materials are the most suitable carrier.

Inorganic porous materials do not swell with water and maintain sufficient mechanical strength in water. Therefore, unlike soft gels such as agarose, there is less risk of clogging, and the pressure loss is also small. It also has the advantage that the gel is less likely to be deformed or swelled by pressure or osmotic pressure, and the column volume is constant.

Next, regarding the size of the pores, first of all, it is necessary that the LDL to be removed can freely enter the pores. LDL is a huge particle with a molecular weight of at least 1,000,000 or more and a particle diameter of about 2,001. In order for these giant particles to freely and with a high probability of entering the pores, it is thought that the larger the pore diameter, the better.However, as the pore diameter increases, the surface area and pore volume generally decrease. descend. Therefore, an optimum pore size exists.

There are various methods for measuring the pore diameter, but in the present invention, the mercury intrusion method was adopted.

As a result of studies using inorganic porous materials with various pore sizes, the present inventors found that good LDL adsorption ability was obtained when using an inorganic porous material with an average pore size of 700X or more and 4000X or less; As described above, it has been found that when a porous body having an average pore diameter of 3000 or less is used, the highest LDL adsorption ability is exhibited.

Next, regarding the porous structure of the carrier, total porosity is preferable to surface porosity, and it is desirable that the pore volume is 20% or more. Further, it is desirable that the specific surface area is 3 m2711 or more.

The shape of the carrier can be selected from any shape such as granules, fibers, membranes, and holofibers. When using a particulate carrier, the particle size is preferably 1 μm or more and 5000 μm or less.

Typical examples of inorganic porous materials suitable for the present invention include, but are not limited to, silica gel, alumina, porous glass, silica alumina, hydroxyapatite, calcium silicate, zirconia, and zeolite 1. Furthermore, those whose surfaces are coated with polysaccharides, synthetic polymers, etc. may also be used. These inorganic porous bodies may be used alone or in combination of two or more types.

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, gratan sulfate, heparitin sulfate, xylan sulfate, caronine sulfate, and cellulose. Sulfuric acid, chitin sulfate, chitosan sulfate, pectin sulfate, inulin sulfate, alginate sulfate, glycogen sulfate, Vorilac, Thone sulfate,
carrageenan sulfate, starch sulfate, polyglucose sulfate,
Examples include, but are not limited to, sulfated polysaccharides such as laminarin sulfate, galactan sulfate, levan sulfate, and mebesulfate, phosphotungstic acid, polysulfated anethole, polyvinyl alcohol sulfate, and polyphosphoric acid.

Various known methods can be used to immobilize a compound (ligand) that has affinity for lipoproteins on a carrier. These include physical adsorption methods, ionic bonding methods, covalent bonding methods, etc. When using the adsorbent of 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, and even if an ionic bonding method is used, the ligand is It is desirable to form a bond in a cross-linked manner.

1. If necessary, a spacer may be introduced between the carrier and the ligand.

The amount of immobilized ligand varies depending on the properties and activity of the ligand, but in order to obtain a significant amount of riboprotein adsorption, it is preferably 0.02 m9 or more per 1 ml column volume.
Also, considering economic efficiency, it is 100m! i+ or less is desirable. More preferably 05 m/ml column volume
2 or more and 20m1i1 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 and store it in a blood bag, etc., mix it with the adsorbent of the present invention to remove LDL, then pass it through a filter to remove the adsorbent, and transfer the blood to the patient. There is a way to get it back. 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, incorporate it into an extracorporeal circulation circuit, and perform online adsorption and removal. Treatment methods include a method in which whole blood is directly passed through the tumor, and a method in which blood is separated from the blood and then the plasma is passed through a column. The adsorbent according to the invention can be used in either method, but 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 blood sample to be treated. Polyvalent metal ions used for this purpose include alkaline earth metal ions such as calcium, magnesium, barium, nitrogen, trontium, etc.
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 inches) equipped with filters with a pore size of 5 μm at both ends was coated with porous glass (Wako Tsumugi Co., Ltd.) as a representative example of the inorganic porous material.
FPG 2000. As a typical example of soft gel, agarose gel (Bj, ora) is a representative example of soft gel.
Manufactured by d company; Bj-ogel, A5M.

(particle size: 50 to 100 mesh) were uniformly filled in each container, water was flowed through each container using a veristatic pump, and the relationship between flow rate and pressure loss was determined. The results are shown in Figure 1.

In contrast to inorganic porous materials whose pressure increases, soft-kels cause compaction and do not increase flow rate even if the pressure increases.

Example 1 Porous glass FPG2000 (average pore diameter 19soA.

(Specific surface area: 1.3 m2/f/, particle size: 80-120 mesh) was heated in dilute nitric acid for 6 hours, washed with water, dried, and filtered at 500°C for a period of time. This was refluxed for 3 hours in a 10% toluene solution of r-aminopropyltriethoxysilane and washed with methanol to obtain γ-aminopyrobylated galanuo.

Next, dissolve 200μ of heparin in 10cc of water and adjust the pH
After adjusting to 4.5, 2FI γ-aminopropylated glass was added thereto. 200 m2 of 1-ethyl-O ÷ dimethylaminopropyl ÷ carbodiimide was added while maintaining the pH at 4.5, and the mixture was shaken at 4'C for 24 hours. After the reaction was completed, the glass was washed with a 2M salt solution, a 0.5M salt solution, and water to obtain a heparin-immobilized porous glass. Immobilized heparin was 12 μ/ml.

Example 2 Porous glass FPG2000 was replaced with FPG700 (average pore diameter 7 ooffi, specific surface area 37 m2/g + particle size 80~
120 mesh), FPG 100 o, (average pore size 1
o9;

Specific surface area 6.8 m2/fj, particle size 80-120 mesh), porous silica (MERK Li, chrosph
er5i4000.

Heparin was immobilized in the same manner as in Example 1, except that the average pore size was 400X and the particle size was 10 μm.

The amount of heparin immobilized was 5. ．． 2, 2.2.0
．． 8゜0.5 m? / 6 in ml.

Example 6 Chondroitin polysulfate-immobilized FPG2 was produced in the same manner as in Example 1 except that heparin was changed to chondroitin polysulfate.
I got 000. The amount of immobilized chondroitin polysulfate was 1.0 μ/g.

Example 4 800 ml of dextran sulfate was dissolved in 10 ml of 0.25 mol Na-04 solution and stirred at room temperature for 4 hours, then 200 ml of ethylene glycol was added and stirred for 1 hour. After adjusting the pH of this solution to 8, 4 ml of γ-aminopropylated F, PG2000 obtained in the same manner as in Example 1 was added and shaken for 24 hours.

After the reaction was completed, the gel was collected in a furnace, washed with water, and added with 1% NaBH.
4 solution 101R1 and reduced for 15 minutes, filtered and washed with water to obtain dextran sulfate-immobilized FPG2000.

The amount of immobilization was o, stq/g/.

Example 5 1 ml of each of the adsorbents synthesized in Examples 1 to 4 was placed in a test tube, human blood is zt (containing 0.02 M of 0a01 weight) was added thereto, stirred, and allowed to stand still for 15 minutes at 20°C. After incubation, the cholesterol, I, and DL concentrations of the supernatant were measured. The results are shown in Table 1.

[Brief explanation of the drawing]

Figure 1 shows a glass cylindrical column filled with porous glass FPG2000 and a soft gel (B
5 is a graph showing the relationship between the flow rate of water and the pressure loss for each case filled with J-Ogel A 5 m ). Patent applicant Makoto Asano, patent attorney representing Kanekabuchi Chemical Industry Co., Ltd. jc O,C:zd A! ;7n O,8/,θ

Claims (1)

[Scope of Claims] 1. A lipoprotein adsorbent comprising a polyanionic compound having an affinity for lipoproteins fixed to an inorganic porous material having an average pore diameter of 700X or more and 4000A or less. 2. The adsorbent according to claim 1, wherein the inorganic porous material is at least one selected from the group consisting of porous glass, porous silica gel, and porous alumina. The adsorbent according to claim 1, wherein the polyanion compound is a sulfated polysaccharide. 4. The adsorbent according to claim 3, wherein the sulfated polysaccharide is at least one selected from heparin, dequinutran sulfate, and chondroitin polysulfate. 5. The adsorbent according to claim 1, which uses an inorganic porous material having a specific surface area of 3 m2/g or more. 6 The amount of immobilized polyanion compound is column volume 1πt
The adsorbent according to claim 1, wherein the adsorbent has a p O,'02■ or more and 1oom9 or less.