JPH0516901B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to an adsorbent for extracorporeal circulation therapy for removing immunoglobulin light chains (hereinafter referred to as "L chains") contained in body fluids. [Prior art and problems to be solved by the invention] Amyloidosis is a phenomenon in which β-fibrillar proteins called amyloid substances are deposited in serum, organs, and other tissues, leading to organ failure in the heart, kidneys, etc., 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 formed by a protein called AL, and the precursor corresponding to the deposited amyloid substance is considered to be an L chain. however,
So far, no effective treatment, especially drug therapy, has been found for this disease. The L chain is a monomeric low molecular weight protein consisting of 200 amino acids with a molecular weight of 23,000. On the other hand, in addition to primary amyloidosis, there are diseases accompanied by abnormal L chain production. Typical diseases are multiple myeloma, macroglobulin plasma, and malignant lymphoma, and the abnormal L chain that appears in these diseases is a clonal protein called benzyjones protein (hereinafter referred to as BJP). Although BJP is normally excreted in the urine, it inhibits the reabsorption of other proteins, especially albumin, resulting in the so-called kidney symptoms of myeloma. Also, a large amount of serum
In many cases, BJP is deposited in the heart, kidneys, etc., leading to amyloidosis. Therefore, there is a desire for an effective method for removing blood BJP, but as with primary amyloidosis, there is currently no practical method for removing it. [Means for Solving the Problems] The present invention provides an L chain for extracorporeal circulation treatment, which is formed by immobilizing a compound having a 1ogP (P is a partition coefficient in an octanol-water system) value of 2.50 or more on a porous water-insoluble carrier. Regarding adsorbents. [Example] The adsorbent of the present invention is formed by immobilizing a compound having a 1ogP value of 2.50 or more on a porous water-insoluble carrier. The 1ogP value is a parameter for the hydrophobicity of a compound, and is the distribution coefficient P in a typical octanol-water system.
The method of finding is as follows. First, dissolve the compound in octanol (or water), add an equal amount of water (or octanol), and use a Griffin flask shaker.
Shaker) (manufactured by Griffin & George Ltd.) for 30
Shake for a minute. Thereafter, centrifugation is performed at 2000 rpm for 1 to 2 hours, and the concentration of the compound in the octanol layer and the aqueous layer is measured by various methods such as spectroscopy or GLC, and is determined by the following formula. P=Coct/Cw Coct: Concentration of the compound in the octanol layer Cw: Concentration of the compound in the water layer Many researchers have so far measured the 1ogP value of various compounds, but these actual values are It has been organized by C. Hansch et al.
THEIR USES; Chemical Reviews), vol. 71,
525, 1971). Regarding compounds for which actual measured values are not known, RFRekker has published his book "THE HYDROPHOBIC FRAGMENTAL CONSTANT".
FRAGMENTAL CONSTANT), Elsevier Sci. Publishing Company Amsterdam (Elsevier Sci.Pub.
The value (Σf) calculated using the hydrophobic fragment constant f shown in (1977)) can be used as a reference. hydrophobic fragment constant f
is a value indicating the hydrophobicity of various fragments determined by statistical processing based on a large number of 1ogP actual measurements, and the sum of the f values of each fragment that makes up the compound almost matches the 1ogP value. do. In searching for compounds that are effective in adsorbing L chains, we fixed and studied compounds with various 1ogP values, and found that
It was found that compounds with a 1ogP value of 2.50 or more are effective in adsorbing L chains, and compounds with a 1ogP value of less than 2.50 show almost no L chain adsorption ability. For example, when an alkylamine is immobilized, the alkylamine is n-
It was found that when changing from hexylamine (1ogP=2.06) to n-octylamine (1ogP=2.90), the L chain adsorption capacity increased dramatically during this period. From these results, it is thought that the adsorption of the L chain to the adsorbent of the present invention is due to the hydrophobic interaction between the L chain and the atomic group introduced onto the carrier by immobilizing a compound with a 1ogP value of 2.50 or more. It is considered that compounds with a 1ogP value of less than 2.50 do not exhibit L chain adsorption ability because their hydrophobicity is too small. In the present invention, as a compound to be immobilized on a porous water-insoluble carrier, any compound having a 1ogP 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, if the hydrophobicity of the atomic group fixed on the carrier due to elimination becomes smaller than Σf=2.50, it is not suitable as a compound for use in the present invention, considering the gist of the present invention. One typical example of this 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 immobilized on the carrier is C ₆ H ₅ CO-, and the Σf of this atomic group is 1 or less. Whether such a compound is suitable as a compound for use in the present invention may be determined by whether the 1ogP value of the compound in which the leaving group is replaced with hydrogen is 2.50 or more. Among compounds with a 1ogP value of 2.50 or higher, oxirane ring-containing compounds such as alcohols, amines, thiols, carboxylic acids and their derivatives, halides, aldehydes, hydrazides, isocyanates, glycidyl ethers, and halogenated silanes are suitable for use in carriers. Compounds having a functional group that can be used for bonding are preferred. Representative examples of such compounds include n-heptylamine, n-octylamine, decylamine, dodecylamine, hexadecylamine, octadecylamine, 2-aminooctene, naphthylamine, phenyl-n-propylamine, diphenylmethylamine, etc. amines, n-heptyl 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, dotecanoic acid, stearic acid, arachidonic acid, oleic acid, diphenylacetic acid, phenylpropionic acid, etc. carboxylic acids and carboxylic acid derivatives such as these acid halides, esters, amides,
Halides such as octyl chloride, octyl bromide, decyl chloride, dodecyl chloride, thiols such as octanethiol and dodecanethiol, halogenated silanes such as n-octyltrichlorosilane and octadecyltrichlorosilane, n-octylaldehyde, n- Examples include aldehydes such as capric aldehyde and dodecyl aldehyde. Furthermore, in addition to these, substituents in which the hydrogen atom of the hydrocarbon moiety of the above-mentioned exemplary compounds contain a heteroatom such as halogen, nitrogen, oxygen, and sulfur;
Among compounds substituted with other alkyl groups, etc.
Compounds with a 1ogP value of 2.50 or higher, as described in the above-mentioned review by C. Hansch et al.
Chemical Reviews (PARTITION)
COEFFICIENTS AND THEIR USES;
Chemical Reviews), Vol. 71, p. 525, 1971, pages 555 to 613.
Compounds having a logP of 2.50 or more can be used, but the present invention is not limited to these. Note that these compounds may be used alone, or any two or more types may be combined, and further, they may be used in combination with a compound having a 1ogP value of less than 2.50. Examples of water-insoluble carriers used in the present invention include inorganic carriers such as glass beads and silica gel, synthetic polymers such as cross-linked polyvinyl alcohol, cross-linked polyacrylate, cross-linked polyacrylamide, and cross-linked polystyrene, crystalline cellulose, cross-linked cellulose, cross-linked agarose, and cross-linked Typical examples include organic carriers made of polysaccharides such as dextran, and composite carriers such as organic-organic and organic-inorganic that can be obtained by combining these.Among these, hydrophilic carriers are particularly susceptible to non-specific adsorption. It is preferable because it is relatively small and the 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 made 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, etc., but porous cellulose gel (1) has relatively high mechanical strength and is tough, so it cannot be destroyed or generate fine powder by operations such as stirring; If the body fluid is packed in a column, 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 there are many hydroxyl groups that can be used for binding of ligands, resulting in less non-specific adsorption; (3) Comparatively, even if the pore volume is increased, It has excellent properties such as its high adsorption capacity, which is comparable to that of soft gels, and (4) its safety compared to synthetic polymer gels.
It is one of the most suitable carriers for use in the present invention. 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, but other It is preferable that protein invasion be 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. Exclusion limit molecular weight is a molecular weight that cannot enter the pores (excluded ) 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, it has become clear that the range of pore diameter suitable for adsorption of L 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 superficial porosity, the pore volume is 20% or more, and the specific surface area is 3 m ² / It is preferable that it is more than g. 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.
Representative examples of these functional groups include hydroxyl group, amino group, aldehyde group, carboxyl group, thiol group, silanol group, amide group, epoxy group, halogen group, succinylimide group, and acid anhydride group. Next, the carrier used in the present invention is a hard carrier,
Any soft carrier can be used, but in order to use it as an adsorbent for extracorporeal circulation therapy, it is important that it does not cause clogging when filling the column and passing liquid through it. 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 passed through it, the relationship between pressure loss ΔP and flow rate is linear up to 0.3 Kg/cm ² . The adsorbent of the present invention is obtained by immobilizing a compound with a 1ogP value of 2.50 or more on a porous water-insoluble carrier,
As the immobilization method, various known methods can be used 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,
The disadvantages are that the amount of 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 (Biogel A-5m manufactured by Biorade,
(particle size: 50 to 100 mesh), vinyl polymer gel (Toyo Pearl HW-65 manufactured by Toyo Soda Kogyo Co., Ltd., particle size:
50-100μm) and cellulose gel (Cellulofine GC-700m manufactured by Chitsuso Co., Ltd., particle size 45-105μm)
were filled uniformly, water was flowed through each tube using a peristaltic pump, and the relationship between flow rate and pressure loss Δp was determined. The results are shown in FIG. As shown in Figure 1, the flow rate of Toyopearl HW-65 and Cellulofine GC-700m increases almost in proportion to the increase in pressure, while the flow rate of Biogel A-
It can be seen that 5 m causes consolidation and the flow rate does not increase even if the pressure is increased. In the present invention, as in the former case, the relationship between pressure loss Δp and flow rate is 0.3.
A substance that has a linear relationship up to Kg/cm ² is called a hard gel. Example 1 Water was added to 170 ml of Cellulofine GCL-300m (manufactured by Chitsuso Co., Ltd., exclusion limit molecular weight of globular protein 90,000), which is a cellulose-based porous hard gel, and the total volume was 340 ml.
After that, add 90ml of 2M sodium hydroxide and add 40ml of 2M sodium hydroxide.
℃. 31 ml of epichlorohydrin was added to this, 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. 200 mg of dodecylamine (Σf=5.10) was added to 10 ml of this epoxidized gel, and the mixture was allowed to react in a 50% (v/v) ethanol aqueous solution at 45° C. for 6 days.
After completion of the reaction, 50% (v/v) ethanol aqueous solution,
Ethanol, 50% (v/v) ethanol aqueous solution,
The gel was thoroughly washed with water to obtain a dodecylamine-immobilized gel. 1gA of benzodiones protein, which is an immunoglobulin L chain, was added to 0.5ml of this adsorbent at a concentration of 200μg/ml.
3 ml of myeloma patient plasma was added and incubated at 37°C for 2 hours. The concentrations of BJP and albumin in the supernatant were measured, and the amount of adsorption of BJP and albumin per ml of adsorbent 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 cetylamine (Σf=7.22) was used instead of dodecylamine. Example 1 using this adsorbent
An adsorption experiment was conducted in exactly the same manner. Comparative Example 1 The carrier was changed to Cellulofine GC-700m (manufactured by Chitsuso Co., Ltd., exclusion limit molecular weight for globular proteins: 400000), which is a cellulose-based porous hard gel, and dodecylamine was changed to ethylamine (1ogP = -0.13). An ethylamine-immobilized gel was obtained in the same manner as in Example 1. 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 (1ogP=
n-
A butylamine immobilized gel was obtained. 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 (1ogP=
2.90) in the same manner as Comparative Example 1 except that n-
An octylamine immobilized gel was obtained. 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 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 ethylamine was replaced with cetylamine (Σf=7.22). An adsorption experiment was conducted in exactly the same manner as in Example 1 using this adsorbent. Results first
Shown in the table.

【table】

[Table] From the results in Table 1, when using the adsorbent of the present invention,
It can be seen that although BJP is efficiently adsorbed, albumin is hardly adsorbed. [Effects of the Invention] The adsorbent of the present invention is inexpensive and has the advantage of being able to efficiently adsorb and remove BJP contained in body fluids.

[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.

Claims (1)

[Scope of Claims] 1. Immunoglobulin L for extracorporeal circulation therapy, comprising a porous water-insoluble carrier immobilized with a compound having a 1ogP (P is partition coefficient in an octanol-water system) value of 2.50 or more.
Chain adsorbent. 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 globular proteins of 10,000 to 600,000. 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.