JPS6377457A - Immunoglobulin l chain adsorbing body for extracorporeal circulation remedy - Google Patents

Abstract

(57) [Abstract] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to immunoglobulin light chains (hereinafter referred to as
The present invention relates to an adsorbent for extracorporeal circulation treatment for removing L chains (referred to as L chains).

[Problems to be solved by conventional techniques and inventions Core amyloidosis is a phenomenon in which β-fibrillar proteins called amyloid substances are deposited in blood vessels, organs, and other tissues, leading to organ failure such as heart and kidney, and cardiac stimulation. It is a disease that causes serious disorders such as conduction disorders, progressive dementia, cerebrovascular disorders, and neurological disorders.

It is known that amyloidosis has disease types such as primary, secondary, familial, and senile, and its protein composition differs depending on the disease type. Primary amyloidosis is AL
The precursor substance corresponding to the deposited amyloid substance is said to be the L chain. However, so far no effective treatment, especially drug therapy, has been found for this disease. The L chain is a monomer with the molecule ff123. It is a low molecular weight protein consisting of 200 ooo amino acids.

On the other hand, in addition to primary amyloidosis, there are diseases accompanied by the production of normal chains. Typical diseases are multiple myeloma (myeloma), macroglobulin plasma, and malignant lymphoma, and the normal chain that appears in these diseases is a clonal protein called benzidigenes protein (hereinafter referred to as "BJP"). . Although the BJP is normally excreted,
At that time, it inhibits the reabsorption of other proteins, especially albumin,
It presents with so-called myeloma kidney symptoms.

Furthermore, a large amount of BJP in serum is deposited in the heart, kidneys, etc., often leading to amyloidosis. Because of this, blood BJ
Although an effective method for removing P is desired, as in the case of primary amyloidosis, there is currently no practical method for removing P.

[Means for Solving the Problems] The present invention provides a method for extracorporeal circulation therapy in which a compound having a 1ogP (P is a partition coefficient in an octanol-water system) value of 2.50 or more is immobilized on a porous water-insoluble carrier. This invention relates to an L chain adsorbent.

[Example] The adsorbent of the present invention is formed by immobilizing a compound having a logP value of 2.50 or more on a porous water-insoluble carrier.

The log P value becomes a parameter of the hydrophobicity of the compound,
The method of determining the distribution coefficient P in a typical octanol-water system is as follows. First, dissolve the compound in octatool (or water), add an equal amount of water (or octatool), and place it in a gritzin flask shaker.
(Griff'1n1'1ask 5haker) (
Gritzin & George Limited (Grl
(manufactured by Fl'in & George Ltd.) for 30 minutes. Thereafter, centrifugation is performed at 2000 rpm for 1 to 2 hours, and the concentration of the compound in the octatool layer and the aqueous layer is measured by various methods such as spectroscopic analysis or GLC, and is determined by the following formula.

P -Coct/Cv Coct: Compound concentration in the octanol layer Cv: Compound concentration in the aqueous layer
gP values have been actually measured, and these measured values have been organized by C. Hansch et al.
N C0EPFICIENTS ANDT! IEIRU
SES;Chemical Revlevs), 71
Vol. 525, 1971).

In addition, for compounds whose actual measured values are not known, R.
F. Rekker (R,P, Rekker) is the author (
[THE HYDROPHOBICPRAGME
NTAL C0N5TANT) J, El Sepia
Scientific Publishing Company
Amsterdam (Elsevier Sci, Pub,
Com,.

The value calculated using the hydrophobic fragment constant f shown in Am5terdaI11) (1977))
Σf) is a reference. The hydrophobic fragment constant f is a value indicating the hydrophobicity of various fragments determined through statistical processing based on a large number of actual measured OgP values, and is the sum of the f values of each fragment constituting a compound. almost coincides with the log P value.

In searching for compounds effective for L chain adsorption, various log
As a result of fixing and studying compounds with P values, logP
Compounds with a value of 2.50 or more are effective in adsorbing L chains, and l
It was found that compounds with an og P value of less than 2.50 showed almost no L chain adsorption ability. For example, when alkylamine is immobilized, n-hexylamine (l
ogP -2,08) to n-octylamine (log
P = 2.90), it was found that the L chain adsorption capacity increased dramatically during this period. From these results, the adsorption of the chain to the adsorbent of the present invention has a log P value of 2.5.
This is thought to be due to the hydrophobic interaction between the atomic group introduced onto the carrier by immobilization of zero or more compounds and the L chain.
Compounds with a log P value of less than 2650 are considered not to exhibit L chain adsorption ability because their hydrophobicity is too small.

In the present invention, as the compound to be immobilized on the porous water-insoluble carrier, any compound having a log P value of 2.50 or more can be used without any particular restriction. However, when a compound is bonded to a carrier by a chemical bonding method, a part of the compound often leaves, but if this leaving group contributes significantly to the hydrophobicity of the compound, in other words, The hydrophobicity of the atomic group fixed on the carrier by separation is Σf-2
, 50, the compound is unsuitable for use in the present invention in view of the gist of the present invention. One typical example is the case where benzoic acid isopentyl ester (Σf-4,15) is immobilized on a carrier having a hydroxyl group by transesterification. In this case, the atomic group actually fixed on the carrier is Ce Hs C
O-, and Σf of this atomic group is 1 or less. Whether such a compound is suitable as a compound for use in the present invention can be determined by determining the togp of the compound in which the leaving group is replaced with hydrogen.
The determination may be made based on whether the value is 2.50 or more.

Among compounds with an IogP value of 2.50 or more, alcohols, amines, thiols, carboxylic acids and their derivatives,
Preferred are compounds having a functional group that can be used for bonding to a carrier, such as oxirane ring-containing compounds such as halides, aldehydes, hydrazides, isocyanates, and glycidyl ethers, and halogenated silanes. Representative examples of such compounds include n-hebutylamine, n-octylamine, decylamine, dodecylamine, hexadecylamine, octadecylamine, 2-aminooctene, naphthylamine, phenyl-n-propylamine, diphenylmethylamine, etc. Amines, n-hebutyl alcohol, n-octyl alcohol, dodecyl alcohol,
hexadecyl alcohol, 1-octen-3-ol,
naphthol, diphenylmethanol, 4-phenyl-2
- Alcohols such as butanol, glycidyl ethers of these alcohols, n-octanoic acid, nonanoic acid, 2-nonenoic acid, decanoic acid, toticanic acid, stearic acid, arachidonic acid, oleic acid, diphenylacetic acid, phenylpropionic acid, etc. carboxylic acids and their acid halides, carboxylic acid derivatives such as esters and amides, halides such as octyl chloride, octyl bromide, decyl chloride, dodecyl chloride, thiols such as octanethiol and dodecanethiol, n-octyltri Examples include halogenated silanes such as chlorosilane and octadecyltrichlorosilane, and aldehydes such as n-octylaldehyde, n-capricaldehyde, and dodecylaldehyde. Furthermore, in addition to these, hydrogen atoms in the hydrocarbon moiety of the above-mentioned exemplified compounds include halogen, nitrogen,
Among compounds substituted with substituents containing heteroatoms such as oxygen and sulfur, and other alkyl groups, compounds with a logP value of 2.50 or more, the above-mentioned C.H.
Ansch) et al.'s review article ``Partition e Coefficients and There◆Uses; Chemical Reviews (PARTITION COEPPICIENTS A)
ND THEIRUSES.

Chemical Reviews), vol. 71, 52
Compounds having a log P of 2.50 or more as shown in the table on pages 555 to 813 of "Page 5, 1971" can be used, but the present invention is not limited to these. .

Incidentally, these compounds may be used alone or in combination of two or more of them, and furthermore, l
It may also be used in combination with compounds having an og P value of less than 2.50.

The water-insoluble carrier used in the present invention includes glass pieces,
Inorganic carriers such as silica gel, synthetic polymers such as cross-linked polyvinyl alcohol, cross-linked polyacrylate, cross-linked polyacrylamide, cross-linked polystyrene, and crystalline cellulose,
Typical examples include organic carriers made of polysaccharides such as cross-linked cellulose, cross-linked agarose, and cross-linked dextran, as well as composite carriers such as organic-organic and organic-inorganic that can be obtained by combining these.Among them, hydrophilic carriers is preferable because nonspecific adsorption is relatively small and L chain adsorption selectivity is good. The term "hydrophilic carrier" as used herein refers to a carrier whose contact angle with water is 60 degrees or less when the compound constituting the carrier is formed into a flat plate. Such carriers include cellulose, polyvinyl alcohol, saponified ethylene-vinyl acetate copolymer, polyacrylamide, polyacrylic acid, polymethacrylic acid, polymethyl methacrylate, polyacrylic acid-grafted polyethylene, polyacrylamide-grafted polyethylene, A typical example is a carrier made of glass or the like, but porous cellulose gels (1) have relatively high mechanical strength and are tough, so they can be destroyed or produce fine powder by operations such as stirring; If the column is filled with body fluids, it will not become compacted or clogged even if it is flowed at a high flow rate, making it possible to flow at a high flow rate.Also, the pore structure is not susceptible to changes due to autoclaving, etc. (2) Since the gel is composed of cellulose, it is hydrophilic and has many hydroxyl groups that can be used for binding of ligands, resulting in less non-specific adsorption; (3) The gel has a large pore volume. Because it has relatively high strength, it has superior adsorption capacity comparable to that of soft gels, and (4) it is safer than synthetic polymer gels. It is one of the carriers. The present invention is not limited to these. Note that the above-mentioned carriers may be used alone or in a mixture of two or more of them.

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. As mentioned above, the L chain to be adsorbed by the adsorbent of the present invention is a protein with a molecular weight of 23,000, and in order to efficiently adsorb this protein, the L chain can enter the pores with a certain degree of probability. It is preferable that the invasion of other proteins is prevented as much as possible.

There are various methods for measuring the pore diameter, and the mercury intrusion method is the most commonly used, but it is often not applicable to the porous water-insoluble carrier used in the present invention. In such cases, it is appropriate to use the exclusion limit molecular weight as a guideline for the pore diameter.

What is the exclusion limit molecular weight?As stated in books (for example, Hiroyuki Hatano and Toshihiko Hana, Experimental High Performance Liquid Chromatography, Kagaku Doujin), it is the molecular weight that 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. The exclusion limit molecular weight has generally been well investigated for globular proteins, dextran, polyethylene glycol, etc., but in the case of the carrier used in the present invention, it is appropriate to use the value obtained using globular proteins.

As a result of studies using carriers with various exclusion limit molecular weights, L
It has been revealed that the range of pore diameter suitable for adsorption of chains is an exclusion limit molecular weight of 10,000 to 600,000. In other words, if a carrier with an exclusion limit molecular weight of less than 10,000 is used, the amount of L chain adsorption and removal will be small, reducing its practicality, and if it exceeds 600,000, proteins other than L chains (mainly albumin) will be removed. adsorption becomes large, reducing its practicality in terms of selectivity. Therefore, the exclusion limit molecular weight of the carrier used in the present invention is preferably 10,000 to 600,000, more preferably 90,000 to 600,000.

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 3.
It is preferable that it is rd/g or more.

Further, the shape of the carrier can be selected from any shape such as granules, fibers, and hollow systems.

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

Next, as the carrier used in the present invention, both hard carriers and soft carriers can be used, but in order to use it as an adsorbent for extracorporeal circulation treatment, it is necessary to avoid clogging when filling a column and passing liquid. It is important that this does not occur, and for this purpose sufficient mechanical strength is required. Therefore, it is more preferable that the carrier used in the present invention is a hard carrier.

The hard carrier mentioned here is, for example, in the case of granular gel,
As shown in the reference example below, when gel is uniformly packed into a cylindrical column and an aqueous fluid is flowed, the relationship between the pressure loss ΔP and the flow rate is a linear relationship up to 0.3 kg/ClT12.

The adsorbent of the present invention can be obtained by immobilizing a compound having a logP value of 2.50 or more on a porous water-insoluble carrier, and various known methods can be used for the immobilization without any particular restrictions. . 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 the ligand as much as possible during sterilization or treatment. is most preferable.

There are various ways in which the adsorbent of the present 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, mix it with the adsorbent of the present invention to remove the L chain, remove the adsorbent through a filter, and return the blood to the patient. There is a way. 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.

In the next method, the adsorbent is packed into a column, installed in an extracorporeal circulation circuit, and adsorbed and removed 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 of the present invention can be used in either method, it is most suitable for on-line processing as described above.

Next, the adsorbent of the present invention will be explained in more detail based on Examples, but the present invention is not limited to these.

Reference Example Agarose gel (Blogel A-5 manufactured by Biorado) was placed on a glass cylindrical column (inner diameter 9 mm, column length 150 mm) equipped with filters with a pore diameter of I5 μm at both ends.
ms particle size 50 to 100 mesh), vinyl polymer gel (Toyo Pearl HW-65 manufactured by Toyo Soda Kogyo ■, particle size 50 to LOG side) and cellulose gel (Cellulofine GC-700m manufactured by Chisso ■, particle size 45) ~1010
5a was uniformly filled in each case, water was flowed through the tube using a peristaltic pump, and the relationship between the flow rate and the pressure loss Δp was determined. The results are shown in FIG.

As shown in Figure 1, while the flow rate of Toyopearl 8w-65 and Cellulofine GC-700a+ increases almost in proportion to the increase in pressure, BiogelA-5m causes consolidation and increases in flow rate even when the pressure increases. It can be seen that the flow rate does not increase. In the present invention, as in the former case, a gel in which the relationship between pressure loss Δp and flow rate is linear up to 0.3 kg/cm2 is referred to as a hard gel.

Example 1 Cellulofine GC, a cellulose-based porous hard gel
L-300+n (manufactured by Chisso ■, globular protein exclusion limit molecule ff190,000) Add water to 170ml to make a total volume of 340ml, then add 2M sodium hydroxide 9
0ml was added and the temperature was set at 40°C. To this was added 31 ml of epichlorohydrin, and the mixture was reacted at 40° C. for 2 hours with stirring. After the reaction was completed, it was thoroughly washed with water to obtain an epoxidized gel.

Dodecylamine (Σf
-5,10) Add 1g of 200ff, 50% (V/
V) Reaction was allowed to proceed in an aqueous ethanol solution at 45° C. for 6 days. After the reaction was completed, the gel was thoroughly washed in the following order: 50% (V/V) ethanol aqueous solution, ethanol, 50% (V/V) ethanol aqueous solution, and water to obtain a dodecylamine-immobilized gel.

3 ml of IgA myeloma patient plasma with a Pence Jones protein concentration of 200 μg/ml, which is an immunoglobulin L chain, was added to 0.5 ml of this adsorbent and incubated at 37° C. for 2 hours. The concentration of BJP and albumin in the supernatant was measured, and the concentration of BJP per ml of adsorbent was measured.
The adsorption amounts of P and albumin, and the adsorption rate of BJP were determined. The results are shown in Table 1.

Example 2 A cetylamine-immobilized gel was obtained in the same manner as in Example 1 except that dodecylamine was replaced with cetylamine (Σf-7.22). An adsorption experiment was conducted in exactly the same manner as in Example 1 using this adsorbent.

The results are shown in Table 1.

Comparative Example 1 The carrier was changed to Cellulofine GC-700m (manufactured by Chisso ■, exclusion limit molecular weight for globular proteins 400,000), which is a cellulose-based porous hard gel, and dodecylamine was changed to ethylamine (logP −-0,13). An ethylamine-immobilized gel was obtained in the same manner as in Example 1 except for the above. An adsorption experiment was conducted in exactly the same manner as in Example 1 using this adsorbent. The results are shown in Table 1.

Comparative Example 2 Ethylamine was replaced with n-butylamine (logP-0,9
An n-butylamine-immobilized gel was obtained in the same manner as in Comparative Example 1 except that 7) was changed. An adsorption experiment was conducted in exactly the same manner as in Example 1 using this adsorbent. The results are shown in Table 1.

Example 3 Ethylamine was converted to n-octylamine (logP-2,9
An n-octylamine-immobilized gel was obtained in the same manner as in Comparative Example 1 except that 0) was used. Example 1 using this adsorbent
An adsorption experiment was conducted in exactly the same manner. The results are shown in Table 1.

Example 4 A dodecylamine-immobilized gel was obtained in the same manner as in Comparative Example 1 except that ethylamine was replaced with dodecylamine (Σf-5,10). An adsorption experiment was conducted in exactly the same manner as in Example 1 using this adsorbent.

The results are shown in Table 1.

Example 5 A cetylamine-immobilized gel was obtained in the same manner as in Example 3, except that cetylamine (Σf-7.22) was used instead of ethylamine. An adsorption experiment was conducted in exactly the same manner as in Example 1 using this adsorbent.

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, BJP is efficiently adsorbed, but albumin is hardly adsorbed.

[Effects of the invention] The adsorbent of the present invention is inexpensive and absorbs B contained in body fluids.
This has the effect that JP can be efficiently adsorbed and removed.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the results of investigating the relationship between flow velocity and pressure loss using three types of gels. 21 Figure

Claims (1)

[Scope of Claims] 1. An immunoglobulin L chain adsorbent for extracorporeal circulation therapy, which is obtained by immobilizing a compound having a logP (P is a partition coefficient in an octanol-water system) value of 2.50 or more on a porous water-insoluble carrier. 2. The immunoglobulin L chain adsorbent for extracorporeal circulation therapy according to claim 1, wherein the porous water-insoluble carrier has an exclusion limit molecular weight of 10,000 to 600,000 for globular proteins. 3. The immunoglobulin L chain adsorbent for extracorporeal circulation treatment according to claim 1, wherein the porous water-insoluble carrier is a hydrophilic carrier. 4. The immunoglobulin L chain adsorbent for extracorporeal circulation treatment according to claim 1, wherein the porous water-insoluble carrier is a hard carrier.