JPH01265972A - Removing method of harmful component in humors - Google Patents

Abstract

PURPOSE:To make it possible to adsorb and remove harmful components in a humor selectively at a high flowing speed by contacting an adsorbing substance which is made by including a substance with the affinity to the objective substance to remove in a porous cellulose, to the humor. CONSTITUTION:By including a substance with the affinity to the objective substance to remove (called as legend hereafter) in a porous cellulose gel with the particle diameter within 1-5mu, such as acetylcellulose, in a common combination method or the like, an almost ideal adsorption substance can be obtained. Then, the adsorption substance and a humor are contacted by a means such as a method used for the external circulation treatment or the like. As the legand, not only the antigen-antibody reaction of the nucleic acid base or the like is utilized, but also a substance with a specific affinity to the substance to adsorb and remove a rheumatism factor such as iminogloburin can be used. In such a way, harmful components in humors can be removed selectively, safely, and efficiently.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for removing harmful components from body fluids. More specifically, the present invention relates to a method for adsorbing and removing harmful substances, viruses, harmful cells, etc. that appear in body fluids such as blood and plasma in immune diseases, inflammation such as nephritis and hepatitis, and viral diseases.

[Problems to be solved by conventional techniques and inventions] Extracorporeal circulation therapy, which originated from artificial dialysis, has developed greatly in recent years, and the number of diseases that can be treated is increasing. Along with this, there is a need for a means to more selectively remove harmful components that appear in body fluids and are thought to be closely related to the cause or progression of diseases. Separation force methods using membranes have been studied for this purpose, but such methods have the disadvantage of insufficient selectivity and large loss of body fluid components other than the substance to be removed.

Attempts have also been made to remove harmful components by adsorption, and so-called synthetic adsorbents, typified by activated carbon and Amberlite XAD, have been used mainly for liver diseases. However, the synthetic adsorbents have poor selectivity;
Furthermore, it has many drawbacks such as the inability to remove high molecular weight substances.

In order to further increase selectivity, attempts have been made to use so-called affinity adsorbents, in which a water-insoluble carrier holds a substance that has an affinity for the substance to be removed. However, many of these attempts have been based on repurposed carriers that are normally used for affinity stomatography.
It has become clear that it has various drawbacks that make it undesirable for use in extracorporeal circulation therapy.

That is, the carriers normally used for affinity chromatography are agarose, dextran, polyacrylamide, porous glass, porous silica, etc., but soft gels such as agarose, dextran, and polyacrylamide have (1) mechanical strength. Because it is weak, it is easily destroyed by operations such as stirring. (2) Because it has low pressure resistance, when body fluid is flowed at a high flow rate when packed in a column and used for extracorporeal circulation, it will cause compaction and the flow rate will not be stable. It has drawbacks such as clogging.

On the other hand, although inorganic porous materials such as porous glass have high pressure resistance, (1) they are easily destroyed by operations such as stirring and produce fine powder, and (2) components other than the target substance are adsorbed onto the carrier surface, so-called It has drawbacks such as non-specific adsorption which cannot be ignored.

Furthermore, polymer gels made of synthetic polymers have been proposed to overcome the drawback of slanting, but these drawbacks cannot be said to be fully overcome, and there are concerns about toxicity due to unreacted monomers, and adsorption capacity. It is also inferior to soft gel.

[Means for Solving Issue M] The present inventors have conducted intensive research to overcome the drawbacks such as slanting. As a result, the present inventors have injected a substance (hereinafter referred to as a ligand) that has an affinity for the substance to be removed into a porous cellulose gel. The present inventors have discovered that harmful components in body fluids can be selectively adsorbed and removed at a high flow rate by bringing an adsorbent holding body fluid into contact with body fluids, thereby completing the present invention.

In other words, the porous cellulose gel used in the present invention (1) has relatively high mechanical strength and is tough, so it is less likely to be destroyed or generate fine powder by operations such as stirring, and it can be packed into a column easily. (2) The gel is made of cellulose, so it does not become compacted or clogged even when body fluids are passed through it at high flow rates, so it is possible to flow at high flow rates. It is hydrophilic because it is composed of It has excellent features such as a comparable adsorption capacity and (4) higher safety than synthetic polymer gels.By retaining the ligand in the porous cellulose gel, it is ideal By bringing the adsorbent into contact with the body fluid, harmful components in the body fluid can be selectively adsorbed and removed at a high flow rate.

[Example] The porous cellulose gel used in the present invention is preferably a gel of cellulose itself, but may also be a gel of an esterified or etherified cellulose derivative, or a mixture of cellulose and the derivative. Examples of such cellulose derivatives include acetylcellulose, methylcellulose, ethylcellulose, and carboxymethylcellulose. Moreover, the shape of the gel particles is preferably spherical.

The porous cellulose gel can be produced by, for example, dissolving or swelling cellulose and/or cellulose derivatives using a solvent, then dispersing the cellulose and/or cellulose derivative in a different solvent that is immiscible with the used solvent to form spheres, and then gelling and regenerating the gel. It can be manufactured in

Cellulose and/or cellulose derivatives constituting the gel may be crosslinked. Typical examples of crosslinking agents include epichlorohydrin, dichlorohydrin, jepoxy compounds, dialdehyde compounds, diisocyanate compounds, cyanuric chloride, dialkoxyalkylsilane compounds, trialkoxyalkylsilane compounds, but are limited to these crosslinking agents. It's not like that.

The optimum pore diameter of the porous cellulose gel used in the present invention can be selected depending on the molecular weight and size of the substance to be removed. There are various methods for measuring pore diameter, and direct measurement methods include mercury porosimetry and electron microscopic observation, but these methods may not be applicable to water-containing particles. In such cases, the exclusion limit molecular weight can be used as a guideline for the pore diameter. What is the exclusion limit molecular weight (for example, Hiroyuki Hatano,
Written by Toshihiko Hana, Experimental High Performance Liquid Chromatograph, Chemistry Doujin)
As stated in et. Phenomenologically, molecules with molecular weights greater than the exclusion limit molecular weight are eluted near the volume of the mobile phase, so the exclusion limit molecular weight can be determined by examining the relationship with the elution volume using compounds of various molecular weights.

The exclusion limit molecular weight differs depending on the type of target compound, and the porous cellulose gel used in the present invention has an exclusion limit molecular weight (hereinafter referred to as exclusion limit molecular weight) of 5×1 when globular proteins and viruses are the substances to be removed.
The range is preferably from 0.03 to 1.times.10.sup.8. If the exclusion limit molecular weight exceeds IXLO8, it is not preferable because the retention of the ligand decreases, the adsorption amount of the substance to be removed decreases, and the strength of the gel decreases.

The pore volume of the porous cellulose gel can be determined based on the cellulose content. The cellulose content is expressed by the formula shown below.

Cellulose content (%)-X100 Vt-V.

(In the formula, W is the gel weight ffi (g), Vt is the volume of the gel-filled column (ml), and Vo is the amount of high molecular weight substance that cannot enter the gel pores passed through the gel-filled column. (If the high molecular weight substance elutes from the column, it is the elution volume (ml).) The porous cellulose gel used in the present invention preferably has a cellulose content of 2% or more and 60% or less, and less than 2%. If it is less than 60%, the strength of the gel decreases, and if it exceeds 60%, the pore volume becomes small, which is not preferable.

Generally, the smaller the particle size of the porous cellulose gel, the better from the viewpoint of adsorption capacity, but if the particle size becomes too small, the pressure loss will increase when packed in a column, etc., which is undesirable. Preferably, it is in the range of 5,000μ.

The following substances are representative examples of substances (ligands) that have an affinity for the substance to be removed used in the present invention.

To remove immune complexes, complement components such as C1, specific proteins such as protein A, antibodies against immune complexes, etc. can be used.

To remove autoantibodies that appear in autoimmune diseases, for example, to remove anti-nuclear antibodies and anti-DNA antibodies that appear in the blood in systemic lupus erythematosus, nucleobases, nucleosides, nucleotides, polynucleotides,
Furthermore, DNA, RNA, etc. can be used,
To remove anti-acetylcholine receptor antibodies that appear in myasthenia gravis, an acetylcholine receptor fraction component can be used.

In addition, antigens for each autoantibody can be used to remove autoantibodies.

Furthermore, in order to remove various harmful components that appear in the blood, antibodies against substances to be removed can be used. For example, antibodies against antigens on the surface of viruses can be used to remove hepatitis viruses, and anti-DNA antibodies can be used to remove DNA that appears in the blood in systemic lupus erythematosus. Anti-B for lymphocyte abnormalities
Lymphocytes can also be removed using cell antibodies, anti-pressor T cell bodies, and the like.

In addition to methods using antigen-antibody reactions as shown above,
A substance having a specific affinity for the adsorbed substance can be used. Typical examples include denatured or aggregated immunoglobulins for removing rheumatoid factors that appear in rheumatoid arthritis, γ-globulin or their fractionated components, amino acids such as tryptophan, and heparin for removing lipoproteins. Protein A for removing sulfated polysaccharides and immunoglobulins
1 hemoglobin for haptoglobin removal, lysine for plasminogen removal, immunoglobulin G1 for C19 removal, arginine for prekallikrein removal, cortisol for transcotin removal, hemin for hemovexin removal, endotoxin removal Examples include polymyxins. Furthermore, lectins such as concanavalin A1 conglutinin and phytohemagglutinin, nucleic acids, enzymes, substrates, coenzymes, etc. can also be used.

The substances to be removed and the ligands described above are merely representative examples, and the present invention is not limited to these. The substances to be removed include a variety of substances, ranging from those with a molecular ffi of less than 1000, such as bilirubin urate, to those with a molecular weight of several tens of millions, such as viruses. In addition, the porous cellulose gel of the carrier is usually determined according to the molecular weight and molecular size of the substance to be removed, but it is also influenced by the type of ligand, the shape of the molecule of the substance to be removed, etc. For those with several hundred, several hundred, or several thousand blades, it is appropriate to use those with an exclusion limit molecule of several thousand to several tens of blades, several thousand blades, or several thousand blades to 100 million blades, respectively. Further, the ligands may be used alone or in combination of two or more types.

Various known methods can be used to retain the ligand in the porous cellulose gel carrier. That is, physical methods, ionic bonding methods, covalent bonding methods, etc. can be mentioned. When using the removal method of the present invention for extracorporeal circulation treatment, it is important that the ligand does not detach, so a strong covalent bonding method is preferable, and even if other methods are used, measures to prevent detachment should be taken. is necessary.

Furthermore, a spacer may be introduced between the carrier and the ligand if necessary.

Various methods can be used for bringing the adsorbent into contact with body fluids. For example, an adsorbent is packed into a column equipped with a filter or mesh that allows body fluid components to pass through but not the adsorbent at the inlet and outlet, the column is installed in an extracorporeal circulation circuit, and blood, plasma, etc. are passed through the column. Although it is typically used for extracorporeal circulation therapy, it is not necessarily limited to such means.

The adsorbent can be sterilized by high-pressure steam as long as the ligand is not significantly denatured, and the sterilization process causes only slight changes in the shape and volume of the pores and particles.

Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited to these Examples.

Example 1 Cellulofine A-3 (Porous cellulose gel manufactured by Chisso ■, exclusion limit molecular weight 5X107, particle size 45-105
20% NaOH 4g, heptane 12 in loml
g1 Nonionic surfactant Tween 20
20) was added, 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. Add 15ml of concentrated ammonia water to the resulting gel,
The mixture was stirred at 0°C for 1.5 hours, and the contents were suction filtered 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 amino group-containing gel was added thereto to give a solution of pl+4.
5, then 200 mg of 1-ethyl-3-(dimethylaminopropyl)-carbodiimide was adjusted to pH 4.5.
The mixture was added while maintaining the temperature at 4°C, and was shaken for 24 hours at 4°C.

After the reaction was completed, the gel was washed with a 2M saline solution, a 0.5M saline solution, and water to obtain a cellulose gel retaining heparin. The amount of heparin retained was 1.8 mg/ml
Met. Furthermore, no breakage of the gel or generation of fine powder was observed during the operations up to this point.

Next, the obtained adsorbent has an inner diameter of 91III, 11 and a length of 47.
A column having an internal volume of approximately 3 ml was filled with the sample, and 18 ml of plasma from a hyperlipidemic patient was passed through the column at a rate of 0.3 ml/min. The pressure loss in the column was less than 1 g (15 m+s) throughout, and no clogging was observed.

By passing through the column, 65% of the total cholesterol in plasma was adsorbed, and total protein etc. decreased only slightly.

Example 2 The particle size of Cellulofine A-3 used in Example 1 was changed to 15
A heparin-retained cellulose gel was obtained in the same manner as in Example 1 except that the thickness was changed from 0 to 200μ. The amount of heparin retained was 1.5 mg/ml.

Fill the top gel into the same column as in Example 1, and add 18 ml of plasma from a hyperlipidemic patient (added with 200 U of heparin) to 0.2
Passed through the column at ml/min. The pressure drop in the column is 30
It was below mmHg, and the change was slight.

By passing through the column, 55% of the total cholesterol in plasma was adsorbed, and there was only a slight decrease in total protein and blood cells. Furthermore, there was little thrombus formation on the adsorbent surface.

Example 3 The heparin-retained gel obtained in Example 1 was sterilized using an autoclave at 120°C for 15 minutes, and the heparin-retained gel obtained in Example 1 was
18 ml of plasma from a hyperlipidemic patient was passed through the column at 0.3 ml/min. Both the pressure drop in the column and the adsorption of total cholesterol were the same as in Example 1.

Example 4 An epoxidized cellulose gel was produced in the same manner as in Example 1. Add 50ff1 of protein A to 10ml of the resulting gel.
After adjusting the pH to 9.5 and reacting at room temperature for 24 hours, the mixture was washed with a 2M saline solution, a 0.5M saline solution, and water. Then add monoethanolamine and add 1
The mixture was reacted for 6 hours to seal unreacted epoxy groups, and then washed with water to obtain a cellulose gel in which protein A was retained.

When the obtained adsorbent was packed into the same column as in Example 1 and 18 ml of human plasma was passed through the column at 0.3 ml/min, about 35% of globulin was adsorbed.

Note that the decrease in albumin was slight.

Example 5 20 ml of Cellulofine A-3 used in Example 1 was dispersed in water, and 6 g of cyanogen bromide was gradually added thereto while maintaining the PL at 11 to 12, followed by stirring for 10 minutes. The gel was broken off and washed with cold water and O, 1M NaHcO3 aqueous solution to obtain an activated gel. Then polymyxin sulfate 1
．． 5g to 0. After dissolving in 20 ml of IMNallCO3 aqueous solution and adding the top gel to this and shaking at 4°C for 24 hours, excess active groups were sealed using a monoethanolamine solution to form a cellulose gel with polymyxin retained. I got it.

1 ml of the resulting gel was packed into an oven column with an inner diameter of 7 mm, and endotoxin was added (approximately 100 μg/ml).
) When 5 ml of bovine blood was passed through the column at 0.3 ml/min, 60% of endotoxin was adsorbed.

Example 6 Cellulofine A-3 was mixed with Cellulofine A-2 (porous cellulose gel manufactured by Chisso Oki, exclusion limit molecule = 7xto
An epoxidized cellulose gel was produced in the same manner as in Example 1 except that the particle size was changed to 45 to 1105 AI.

0.10ml of human immunoglobulin was added to 10ml of the resulting gel.
After adjusting the pH to 9.0 and reacting at room temperature for 24 hours, the gel was separated and washed with a 2M saline solution, a 0.5M saline solution, and water. Next, unreacted epoxy groups were sealed using a monoethanolamine solution, and the cellulose gel in which immunoglobulin G was retained was removed.

Next, the obtained gel was packed into the same column as in Example 1,
When 18 ml of human plasma was passed through the column at 0.2 ml/min, about 30% of 019 was adsorbed.

[Effects of the Invention] According to the method of the present invention, harmful components in body fluids can be selectively removed. Furthermore, when the method of the present invention is applied to extracorporeal circulation therapy in which an adsorbent is packed into a column and body fluids are passed through at a high flow rate, the adsorbent is less likely to become compacted, clogged, or destroyed, so harmful substances can be removed safely and efficiently. can be removed.

Claims (1)

[Claims] 1. A method for removing harmful components in body fluids, which comprises bringing body fluids into contact with an adsorbent in which a porous cellulose gel holds a substance that has an affinity for the substance to be removed.