JPH0616839B2 - Immunoglobulin light thiein adsorbent for extracorporeal circulation treatment - Google Patents

Description

TECHNICAL FIELD The present invention relates to an adsorbent for extracorporeal circulation treatment for removing immunoglobulin light chain (hereinafter referred to as L chain) contained in body fluid.

[Problems to be Solved by Conventional Techniques and Inventions] Amyloidosis is a β-fibrillar protein called amyloid substance deposited in blood vessels, organs and other tissues, resulting in organ failure such as heart and kidney, and heart stimulation. It is a disease that causes serious disorders such as conduction disorder, progressive dementia, cerebrovascular disorder, and neuropathy.

It is known that amyloidosis has primary, secondary, familial, senile, and other disease types, and its protein tissues differ depending on the disease type. Primary amyloid-cis is formed by a protein called AL, and the precursor corresponding to the deposited amyloid substance is L chain. However, so far no effective treatment, especially drug therapy, has been found for this disease. The L chain is a low molecular weight protein consisting of 200 amino acids with a molecular weight of 23,000. So far, membrane separation for L chain removal,
There is almost no adsorbent separation, and the amount removed is small,
It has problems such as low selectivity and difficulty in sterilization, and is not practical for therapeutic use.

On the other hand, in addition to primary amyloidosis, there are diseases associated with abnormal L chain production. Typical diseases are multiple myeloma (myeloma), macroglobulinemia, and malignant lymphoma, and the abnormal L chain appearing in these diseases is a monoclonal protein called Bence Jones protein (BJP). is there. BJP is usually excreted in urine,
At that time, it inhibits reabsorption of other proteins, especially albumin, and exhibits so-called myeloma renal symptoms. In addition, a large amount of BJP in the serum is often deposited in the heart, kidney, etc., leading to iroidosis. Therefore, an effective method for removing BJP in blood is desired, but as with primary amyloidosis, there is currently no practical method for removing it.

An object of the present invention is to solve the above problems and to provide an inexpensive adsorbent for extracorporeal circulation treatment, which can efficiently adsorb and remove a large amount of L chains.

[Means for Solving Problems] The present invention is an extracorporeal method in which a compound having an alkyl group having 4 or more carbon atoms is immobilized on a porous water-insoluble carrier having a spherical protein having an exclusion limit molecular weight of 10,000 or more and 600,000 or less. The present invention relates to an L chain adsorbent for circulation therapy.

[Examples] The adsorbent of the present invention is obtained by immobilizing a compound having an alkyl group having 4 or more carbon atoms on a porous insoluble carrier. As a result of investigating a compound having an alkyl group of various carbon numbers while fixing a compound effective for adsorbing an L chain, the result was 4 carbon atoms.
It has been revealed that the compound having one or more alkyl groups is effective for L chain adsorption, and the compound having an alkyl group having less than 4 carbon atoms exhibits almost no L chain adsorption ability.
From this result, it is considered that the adsorption of the L chain on the adsorbent of the present invention is due to the hydrophobic interaction with the compound serving as a ligand, and that the compound having an alkyl group having less than 4 carbon atoms has a hydrophobic property. It is considered that since it is too small, it does not exhibit L chain adsorption ability.

The alkyl group in the present invention may be a linear, branched or cyclic aliphatic hydrocarbon,
These may be saturated hydrocarbons or may have one or more unsaturated bonds.

The compound having an alkyl group having 4 or more carbon atoms in the present invention is a compound having the above alkyl group, for example, n-
Linear alcohols having 4 or more carbon atoms such as pentanol, n-hexanol, and n-heptanol, branched alcohols having 4 or more carbon atoms such as isooctanol, 1,8-octanediol, 9 -Alcohols containing 4 or more carbon atoms containing heteroatoms, unsaturated carbons, cyclic hydrocarbons (such as decenol, 4-cyclohexyl-n-butyl alcohol, etc. (excluding one alcohol group), or these alcohols) Examples of compounds include amines, thiols, carboxylic acids and their derivatives, halides, aldehydes, hydrazides, isocyanates, oxirane ring compounds such as glycidyl ethers, and compounds in which functional groups such as halogenated silanes are replaced. Such as monoalkyl ethers of glycols and monoalkyl ethers of dicarboxylic acids. As described above, compounds having an alkyl group bonded to a hetero atom in the compound are also included, but the present invention is not limited thereto.

It should be noted that these compounds may be used alone, or may be used by mixing arbitrary two or more kinds of compounds.

The water-insoluble carrier used in the present invention, cross-linked polyvinyl alcohol, cross-linked polyacrylate, synthetic polymers such as cross-linked polyacrylamide and crystalline cellulose, cross-linked cellulose, cross-linked agarose, an organic carrier consisting of a polysaccharide such as cross-linked dextran, Representative examples include organic-organic composite carriers obtained by combining these, but the present invention is not limited thereto. Further, the above carriers may be used alone or in any mixture of two or more kinds.

The first property required of the water-insoluble carrier used in the present invention is that it has a large number of pores of an appropriate size, that is, it is porous. The L-chain to be adsorbed by the adsorbent of the present invention is a protein having a molecular weight of 23,000 as described above. To efficiently adsorb this protein, the L-chain can penetrate into the pores with a certain degree of probability. It is preferable that protein invasion does not occur as much as possible. There are various methods for measuring the pore size, and the mercury porosimetry method is most often used, but it is often not applicable to the porous water-insoluble carrier used in the present invention.

In the case of the present invention, it is appropriate to use the exclusion limit molecular weight as a measure of the pore size. Exclusion limit molecular weight is a molecule that cannot enter (exclude) in the pores of gel permeation chromatography as described in the publications (for example, Hiroyuki Hatano, Toshihiko Hanai, Experimental High Performance Liquid Chromatograph, Kagaku Dojin). The molecular weight of the one with the smallest molecular weight. The exclusion molecular weight is generally globular protein,
Although dextran, polyethylene glycol, etc. have been well investigated, it is appropriate to use the value obtained by using the globular protein as the carrier used in the present invention.

As a result of examination using carriers having various exclusion limit molecular weights and compounds having various alkyl chain lengths, it was revealed that the exclusion range molecular weight is 10,000 or more and 600,000 or less in the range of the pore diameter suitable for L chain adsorption. became. That is, when a carrier having an exclusion limit molecular weight of less than 10,000 is used, the amount of L chain adsorbed and removed is small and cannot be used practically, and when it exceeds 600,000, even when a compound having a long alkyl chain is fixed. Adsorption of proteins other than L chain (mainly albumin) becomes large, and it is not practical in terms of selectivity. Therefore, the exclusion limit molecular weight of the carrier used in the present invention is preferably 10,000 or more and 600,000 or less,
More preferably, it is 20,000 or more and 300,000 or less.

Next, regarding the porous structure of the carrier, considering the adsorption capacity per unit volume of the adsorbent, total porosity is preferable to surface porosity, the pore volume is 20% or more, and the specific surface area is 1 m ² / g.
The above is preferable.

The shape of the carrier may be any shape such as granular shape, fibrous shape, film shape, hollow fiber shape.

Further, as a property of the carrier used in the present invention, a hydrophilic carrier is preferable because non-specific adsorption is relatively small and L chain adsorption selectivity is good.

The hydrophilic carrier as used herein refers to a carrier having a contact angle with water of 60 degrees or less when a compound constituting the carrier is formed into a flat plate. Examples of such a carrier include cellulose, polyvinyl alcohol, ethylene-vinyl acetate copolymer saponified product, polyacrylamide, polyacrylic acid, polyethacrylic acid, polymethyl methacrylate, polyacrylic acid grafted polyethylene, polyacrylamide grafted polyethylene, and glass. Typical examples of the carrier include the following, but the present invention is not limited thereto.

Furthermore, it is convenient that a functional group that can be used for the immobilization reaction of the ligand is present on the surface of the carrier. Typical examples of these functional groups include a hydroxyl group, an amino group, an aldehyde group, a carboxyl group, a thiol group, a silanol group, an amide group, an epoxy group, a halogen group, a succinylimide group and an acid anhydride group.

As the carrier used in the present invention, either a hard carrier or a soft carrier can be used, but in order to use it as an adsorbent for extracorporeal circulation treatment, it is packed in a column and clogged when passing a liquid. It is important that no mechanical strength occurs, and for that purpose, sufficient mechanical strength is required. Therefore,
The carrier used in the present invention is more preferably a hard carrier. The term "hard carrier" as used herein means, for example, in the case of a granular gel, as shown in the following reference example, the relationship between the pressure loss Δp and the flow rate when the gel is uniformly packed in a cylindrical column and an aqueous fluid is flown is 0.3 kg / cm. Up to ^{2 is a} linear relationship.

The adsorbent of the present invention is obtained by fixing a compound having an alkyl group having 4 or more carbon atoms on a porous water-insoluble carrier.
As the immobilization method, various known methods can be used without particular limitation. However, since the adsorbent of the present invention is used for extracorporeal circulation treatment, it is important for safety to suppress desorption and elution of ligand during sterilization or treatment, and for that purpose, it should be immobilized by a covalent bond method. Is most preferred.

There are various methods of using the adsorbent of the present invention for treatment.
The simplest method is to draw the patient's blood out of the body, store it in a blood bag, mix it with the adsorbent of the present invention to remove the L chain, and then remove the adsorbent through a filter to return the blood to the patient. There is a way. This method does not require a complicated device, but has the drawbacks that the amount of one treatment is small, the treatment takes time, and the operation becomes complicated. Another method is to fill the column with an adsorbent and incorporate it in an extracorporeal circulation circuit to perform adsorption removal online. Treatment methods include a method of directly perfusing whole blood and a method of separating plasma from blood and then passing the plasma through a column. The adsorbent of the present invention can be used in any method, but is most suitable for online processing as described above.

Next, the present invention will be described in more detail based on Reference Examples and Examples, but the present invention is not limited to these.

Reference Example Agarose gel (Biorado Biogel A-5m, particle size 50-100 mesh), vinyl polymer gel (Toyo Corporation) were attached to a glass cylindrical column (inner diameter 9 mm, column length 150 mm) equipped with filters with a pore size of 15 μm on both ends. Toyopearl HW-65 manufactured by Soda Industry Co., Ltd., particle size 50 to 100 μm) and cellulose gel (Cellulofine GC-700m manufactured by Chisso Corporation, particle size 45)
˜105 μm) was uniformly filled, and water was flown by a peristaltic pump to determine the relationship between the flow rate and the pressure loss Δp. The results are shown in FIG.

As shown in Fig. 1, the flow rate of Toyopearl HW-65 and Cellulofine GC-700m increases almost in proportion to the increase of pressure, whereas Biogel A-5m causes consolidation and increases the flow rate even if the pressure is increased. It can be seen that does not increase. In the present invention, as in the former case, a gel having a linear relationship between the pressure loss Δp and the flow rate up to 0.3 kg / cm ² is called a hard gel.

Example 1 Cellulofine GC-3 which is a cellulosic porous hard gel
00m (Chisso Corporation, exclusion limit molecular weight of globular protein 9
After water was added to 170 ml of 0.000) to make the total amount 340 ml, 90 ml of 2M sodium hydroxide was added to 40 ° C. To this, 31 ml of epichlorohydrin was added and reacted at 40 ° C. for 2 hours with stirring. After completion of the reaction, it was thoroughly washed with water to obtain an epoxidized gel.

To 10 ml of this epoxidized gel, 200 mg of dodecylamine was added, and the mixture was allowed to stand still at room temperature in a 50% (v / v) ethanol aqueous solution.
Reacted for days. After the completion of the reaction, the gel was thoroughly washed with a 50% (v / v) ethanol aqueous solution and water to obtain a dodecylamine-immobilized gel.

To 0.5 ml of this adsorbent, 3 ml of lgA myeloma patient plasma having a Bence Jones protein concentration of 200 μg / ml, which is an immunoglobulin L chain, was added and incubated at 37 ° C. for 2 hours. Measure the concentrations of BJP and albumin in the supernatant,
The amount of BJP and albumin adsorbed per ml of the adsorbent and the BJP adsorption rate were determined. The results are shown in Table 1.

Example 2 Example 1 except that the dodecylamine was changed to cetylamine.
A cetylamine-immobilized gel was obtained in the same manner as. Using this adsorbent, an adsorption experiment was conducted in exactly the same manner as in Example 1. The results are shown in Table 1.

Comparative Example 1 Same as Example 1 except that the carrier was changed to Cellulofine GC-700m (manufactured by Chisso Corp., exclusion limit molecular weight of globular protein is 400,000) which is a cellulosic porous hard gel, and dodecylamine was changed to ethylamine. Then, an ethylamine-immobilized gel was obtained. Using this adsorbent, an adsorption experiment was conducted in exactly the same manner as in Example 1. The results are shown in Table 1.

Example 3 An n-butylamine-immobilized gel was obtained in the same manner as in Comparative Example 1 except that ethylamine was changed to n-butylamine. Using this adsorbent, an adsorption experiment was conducted in exactly the same manner as in Example 1. The results are shown in Table 1.

Example 4 An n-octylamine-immobilized gel was obtained in the same manner as in Comparative Example 1 except that n-octylamine was used instead of ethylamine. Using this adsorbent, an adsorption experiment was conducted in exactly the same manner as in Example 1. The results are shown in Table 1.

Example 5 Comparative Example 1 except that ethylamine was changed to dodecylamine
A dodecylamine-immobilized gel was obtained in the same manner as in. Using this adsorbent, an adsorption experiment was conducted in exactly the same manner as in Example 1. The results are shown in Table 1.

Example 6 A cetylamine-immobilized gel was obtained in the same manner as in Comparative Example 1 except that ethylamine was changed to cetylamine. Using this adsorbent, an adsorption experiment was conducted in exactly the same manner as in Example 1. The results are shown in Table 1.

Example 7 Cellulofine GC which is a cellulosic porous gel as a carrier
200m (manufactured by Chisso Corp., molecular weight exclusion limit for globular proteins 12
A dodecylamine-immobilized gel was obtained in the same manner as in Example 1 except that the amount was changed to 0.00000). Example 1 using this adsorbent
An adsorption experiment was conducted in exactly the same manner as. The results are shown in Table 1.

Example 8 Example 7 except that cetylamine was used instead of dodecylamine.
A cetylamine-immobilized gel was obtained in the same manner as in. Using this adsorbent, an adsorption experiment was conducted in exactly the same manner as in Example 1. The results are shown in Table 1.

From the results in Table 1, it can be seen that when the adsorbent of the present invention is used, L chains are efficiently adsorbed, but albumin is hardly adsorbed.

[Advantages of the Invention] The adsorbent of the present invention is inexpensive and has the effect of being able to selectively and efficiently remove large amounts of L chains contained in a liquid.

[Brief description of drawings]

FIG. 1 is a graph showing the results of examining the relationship between flow velocity and pressure loss using three types of gels.

Claims (2)

[Claims] 1. The exclusion limit molecular weight of globular proteins is 10,000 or more 60.
An immunoglobulin light chain adsorbent for extracorporeal circulation treatment, which comprises immobilizing a compound having an alkyl group having 4 or more carbon atoms on a porous water-insoluble carrier of 10,000 or less. 2. The immunoglobulin light chain adsorbent for extracorporeal circulation treatment according to claim 1, wherein the porous water-insoluble carrier is a hard carrier.