JPH04244231A - Adsorbing body suppressing activation of complement - Google Patents

Abstract

PURPOSE:To provide an adsorbing body developing effect capable of performing extracorporeal circulation treatment while holding high selectivity and adsorption quantity without almost generating the activation of the complement when beta2-microglobulin considered to be a substance causing a carpal canal syndrome is adsorbed and removed from blood. CONSTITUTION:A beta2-microglobulin adsorbing body for extracorporeal circulation treatment is obtained by fixing a ligand having a 4 or more C alkyl group to a porous water-insoluble carrier in an amount of 8-60mumol per 1ml of the carrier.

Description

[Detailed description of the invention]

0001

[Field of Industrial Application] The present invention is directed to β2-
β2 for extracorporeal circulation treatment to remove microglobulin
- Concerning microglobulin adsorbents.

0002

BACKGROUND OF THE INVENTION In recent years, a disease called carpal tunnel syndrome has frequently occurred in patients who have undergone artificial dialysis for a long period of time. Carpal tunnel syndrome is a disease in which the median nerve is compressed in the carpal tunnel region, resulting in symptoms of median nerve paralysis. A β-fibrillar protein called amyloid substance is deposited in the affected area of such a patient, and the precursor protein corresponding to this amyloid substance is β2, a low molecular weight protein consisting of amino acids present in the patient's blood.
- It has been shown to be a microglobulin.

[0003]Therefore, attempts have been made to separate such β2-microglobulin by using membranes that selectively separate components in blood or plasma depending on their sizes; At the same time, other useful proteins may be removed or β
2- Since there are drawbacks such as a small amount of microglobulin removed, β2-
A method that can remove microglobulin is desired.

[0004] Other methods for removing β2-microglobulin, which have not been attempted much at present, include, for example, removing β2-microglobulin using an immunoadsorbent in which anti-β2-microglobulin antibodies are immobilized on a carrier. A method of adsorption removal is being considered. Although these adsorbents exhibit high selectivity for β2-microglobulin, the ligands such as anti-β2-microglobulin antibodies are expensive, and the adsorbents have disadvantages such as poor storage stability and difficulty in sterilization. Therefore, it is not practical as a therapeutic adsorbent.

Furthermore, since these adsorbents directly contact blood or plasma components to adsorb and remove β2-microglobulin from the blood or plasma, and then return the blood to the patient's body, they have other useful properties. In addition to adsorption of blood components, activation of leukocytes, platelets, complement, etc. must also be considered. It is said that components in blood are less likely to be adsorbed to something with a hydrophilic surface, so a hydrophilic surface is advantageous. Hydrophilic surfaces are also advantageous for activation of leukocytes and platelets. However, cellulose-based carriers, which are hydrophilic hard carriers and are ideal for extracorporeal circulation therapy of blood or plasma, are known to cause activation of the complement system (P.
．． B. Sin) et al., Biochem. J., Vol. 193, p. 115 (1981)).

[0006] Complement is a heat-labile factor that exists in fresh serum and reacts nonspecifically with immune complexes, and is composed of nine component proteins: the first component, the second component, and the ninth component. Each component is activated in turn, and in the reactions from the first component to the fifth component, the polypeptide chain is cleaved, changing from a precursor to an active form. Some of the fragments generated in this way include C3a and C5a, which are called anaphylatoxins.
There are some substances that have strong biological activity, such as , and there is also a theory that activation of complement is a cause of carpal tunnel syndrome, so activation of complement must be avoided as much as possible.

[0007] The present inventors therefore discovered that they were able to adsorb and remove a large amount of β2-microglobulin, and further
As a result of extensive research into an adsorbent that has a high adsorption rate and amount of microglobulin, has excellent adsorption selectivity, and can suppress the activation of blood components, especially complement, we have discovered that hydrophobic ligands We found that by fixing the amount of β2-microglobulin on a carrier within a certain range, it was possible to suppress the activation of blood components, especially complement, while maintaining high adsorption amount and rate of β2-microglobulin, as well as high adsorption selectivity. From the beginning, the present invention was completed.

[0008]

[Means for Solving the Problems] That is, the present invention provides an adsorbent comprising a porous water-insoluble carrier immobilized with a ligand having an alkyl group having 4 or more carbon atoms, the amount of the ligand being 8
The present invention relates to a β2-microglobulin adsorbent for extracorporeal circulation therapy, characterized in that it is a carrier of ~60 μmol/ml.

[0009]

[Example] The β2-microglobulin adsorbent for extracorporeal circulation treatment of the present invention has a ligand having an alkyl group having 4 or more carbon atoms fixed on a porous water-insoluble carrier.

Porous water-insoluble carriers used in the present invention include, for example, inorganic carriers such as glass beads and silica gel, synthetic polymer compounds such as cross-linked polyvinyl alcohol, cross-linked polyacrylate, cross-linked polyacrylamide, and cross-linked polystyrene, and crystalline cellulose. Typical examples include organic carriers made of polysaccharides such as crosslinked cellulose, crosslinked agarose, and crosslinked dextrin, and composite carriers such as organic-organic and organic-inorganic that can be obtained by combining these. The porous water-insoluble carrier used in the present invention may be hydrophilic or hydrophobic, but hydrophilic carriers have relatively low nonspecific adsorption and have good adsorption selectivity for β2-microglobulin. preferable. Here, the hydrophilic carrier 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. Examples of such carriers include cellulose, cellulose derivatives, cross-linked polyvinyl alcohol, saponified ethylene-vinyl acetate copolymers, cross-linked polyacrylamide, cross-linked polyacrylic acid, cross-linked polymethacrylic acid, polymethyl methacrylate, and grafted polyacrylamide. Typical examples include carriers made of polyethylene and glass, but cellulose and cellulose derivatives have relatively high mechanical strength and are tough, so they are less likely to be destroyed or produce fine powder during operations such as stirring. When packed in a column, body fluids do not become compacted or clogged even when flowing at high flow rates, making it possible to flow at high flow rates.
In addition, the pore structure is not easily changed by high-pressure steam sterilization, etc. ■It is hydrophilic, and there are many hydroxyl groups that can be used for binding of ligands, and there is little non-specific adsorption. ■It is relatively strong even when the pore volume is increased. Because of its large capacity, it has an adsorption capacity comparable to that of soft gels, and has excellent blood compatibility compared to synthetic polymers, making it one of the most suitable carriers for use in the present invention.

[0011] The cellulose mentioned above refers to so-called natural cellulose or regenerated cellulose. For example, natural cellulose includes defatted cotton fibers, hemp fibers,
Typical examples include pulp obtained by removing lignin, hemicellulose, etc. from wood, and purified cellulose obtained by further refining the pulp, but are not limited to these. Note that the regenerated cellulose refers to cellulose obtained by forming cellulose into an easily moldable cellulose derivative, and then regenerating the cellulose by hydrolysis or the like.

[0012] The above-mentioned cellulose derivatives include, for example, those in which part or all of the hydroxyl groups of cellulose have been esterified or etherified, and those in which part of the hydroxyl groups of cellulose have been esterified and some have been etherified. , derived from cellulose.

Specific examples of cellulose in which some or all of the hydroxyl groups are esterified include cellulose acetate, cellulose propionate, cellulose butyrate, nitrocellulose, cellulose sulfate, cellulose phosphate, cellulose acetate butyrate, and cellulose nitrate. , dithiocarboxylic acid ester of cellulose (viscose rayon)
Examples include, but are not limited to, these.

Specific examples of cellulose in which part or all of the cellulose is etherified include methylcellulose, ethylcellulose, benzylcellulose, triethylcellulose, cyanethylcellulose, carboxymethylcellulose, carboxyethylcellulose, oxyethylcellulose, etc. It is not limited to.

Among the cellulose materials constituting the cellulose particles, purified cellulose has few impurities and
It is preferable because it leaves less undissolved matter when dissolved, and since cellulose esters such as cellulose acetate and cellulose propionate are soluble in a wide variety of solvents, the range of selection of various manufacturing conditions during particle production is wide. The distribution coefficient of the particles, the thickness of the skin layer, the size of the pores in the internal network structure, etc. can be easily adjusted, making it possible to manufacture desired cellulose-based particles, and furthermore, by hydrolysis, regenerated cellulose can be easily produced. It is more preferred because it can be made into particles.

By the way, the porous water-insoluble carrier used in the present invention may be either a hard carrier or a soft carrier, but in order to prevent clogging when filling a column and passing liquid through it, etc. Since sufficient mechanical strength is required, a hard carrier is more preferable. For example, in the case of a granular gel, the hard carrier used here means that when the 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 linear up to 0.3 kg/cm2. refer to something.

[0017] Note that it is advantageous if a functional group that can be used for the immobilization reaction of the ligand is present on the surface of the adsorbent. Typical examples of these functional groups include hydroxyl group,
Amino group, aldehyde group, carboxyl group, thiol group, silanol group, amide group, epoxy group, halogen group,
Examples include a succinylimide group and an acid anhydride group.

[0018] A ligand having an alkyl group having 4 or more carbon atoms is immobilized on the porous water-insoluble carrier used in the present invention, and various known methods can be used for immobilization without any particular restriction. It can be used for. However, since the adsorbent of the present invention is used for extracorporeal circulation treatment,
It is important for safety to suppress as much as possible the detachment and elution of the ligand during sterilization or treatment, and for this purpose, it is most preferable to immobilize the ligand by a covalent bonding method.

Examples of immobilization methods include the following. 1) A method of introducing long-chain fatty acids through ester bonds. 2) A method in which a halogenated long-chain alkyl is reacted with a cellulose alkoxide and introduced through an ether bond. 3) A method in which long-chain alkylamine is reacted with cellulose activated with epichlorohydrin and introduced through an amino bond.

The reason why a ligand having an alkyl group having 4 or more carbon atoms is used in the present invention is that when such a ligand is used, the ability to adsorb β2-microglobulin is greatly improved. A ligand having an alkyl group having less than 4 carbon atoms has low hydrophobicity and does not exhibit a satisfactory ability to adsorb β2-microglobulin.

The alkyl group may be a linear, branched or cyclic aliphatic hydrocarbon, and even if these are saturated hydrocarbons, they may have one or more unsaturated bonds. There may be.

Specific examples of the alkyl group include:
n-butyl group, n-pentyl group, n-hexyl group, n-
Examples include octyl group, n-decyl group, n-dodecyl group, n-tetradecyl group, n-hexadecyl group, n-octadecyl group, i-butyl group, cyclohexyl group, oleyl group, etc. Among these, n-dodecyl group group, n-tetradecyl group, n-hexadecyl group, and n-octadecyl group are preferable, and n-hexadecyl group and n-octadecyl group are most preferable.

Specific examples of the ligand having an alkyl group having 4 or more carbon atoms include saturated or unsaturated hydrocarbons having the alkyl group, alcohols, amines, thiols, carboxylic acids and derivatives thereof, halides, Examples include oxirane ring-containing compounds such as aldehydes, hydrazides, isocyanates, and glycidyl ethers, and halogenated silanes.In addition to these, there are also monoalkyl ethers of glycols, monoalkyl esters of dicarboxylic acids, etc. Compounds with attached alkyl groups can be used. Note that each of these compounds may be used alone,
Further, two or more arbitrary types may be mixed and used.

The amount of the ligand to be applied to the porous water-insoluble carrier is 8 to 60% per ml of the porous water-insoluble carrier.
It is preferably 15 to 35 μmol, particularly 15 to 35 μmol. If the amount given is less than 8 μmol, not only will the amount of β2-microglobulin adsorbed be insufficient, but contact with blood will activate complement and generate large amounts of C3a and C5a. Therefore, when performing extracorporeal circulation using such an adsorbent, a large amount of adsorbent must be used in order to improve the therapeutic effect, which not only increases the amount of extracorporeally circulating blood or plasma, but also increases the amount of complement. The possibility of anaphylactic shock due to activation also increases. Furthermore, if the amount exceeds 60 μmol, platelet adhesion and activation tend to occur, increasing the possibility of blood coagulation within the column.

There are various ways in which the adsorbent of the present invention can be used therapeutically. The simplest method is, for example, 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 β2-microglobulin, and then pass it through a filter to remove the adsorbent. There are ways to return the drug to the patient. This method is preferred because it does not require complicated equipment. Another 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 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 either method. Most suitable for online processing.

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

Example 1 Cellulose-based porous hard gel (number average particle size 540 μm
All particle sizes are within ±5%, and the smallest size that does not fit into the surface pores has a molecular weight of 16,000)
Water was added to 170 ml to bring the total volume to 340 ml, and then 90 ml of 2M sodium hydroxide was added to bring the temperature to 40°C. 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.

10 mg of n-decylamine was added as a ligand to 10 ml of this epoxidized gel, and the mixture was allowed to react in ethanol for 8 hours while standing at room temperature. After the reaction was completed, the gel was thoroughly washed with ethanol and water to obtain an n-decylamine immobilized gel.

[0029] When the amount of ligand added to the obtained gel was measured by the following method, it was found that 8.4 μm per ml of gel.
It was ol.

(Amount of ligand provided) It is determined by ion-exchanging the bound amine with hydrochloric acid and back titrating with an aqueous sodium hydroxide solution.

(Amount of β2-microglobulin and albumin adsorbed) Dialysis patient serum with a β2-microglobulin concentration of 65 μg/ml was added to 0.4 ml of this gel.
4 ml was added and incubated at 37°C for 2 hours. The concentrations of β2-microglobulin and albumin in the supernatant were measured, and the amount of β2-microglobulin adsorbed per ml of adsorbent was found to be 158 μg.
/ml gel.

Next, the method and results of a blood compatibility test for this adsorbent will be described.

(Activation of complement) First, the method and results for measuring the activation of complement in this gel will be described. In addition, C3a and C, which are the causes of anaphylactic shock, will be discussed here.
Complement activation was evaluated focusing on 5a.

[0034] After sterilization in an autoclave (121°C, 2
After washing 0.4 ml of gel in a polystyrene centrifuge tube three times with physiological saline supplemented with 5 U/ml heparin, 4 ml of fresh human blood was added and incubated at 37°C for 30 min. After shaking at a speed of 10 times/min, the plasma concentrations of activated complement fragments C3a and C5a were measured, and they were each 2083 ng/ml.
, 17.8 ng/ml.

When only 4 ml of fresh human blood was added as a blank without gel, C3a and C5a were 727 ng/ml and 11.3 ng/ml, respectively. When the activation was investigated, C3a and C5a were each 5905 ng/ml.
, 63.1 ng/ml. Thus, in the adsorbent with a gel containing a ligand amount of 8 to 15 μmol/ml, C3 was activated to some extent, but C5 was not activated much.

(Platelet permeability) Next, the method and results for measuring platelet permeability will be described. Pack 1 ml of adsorbent into a column with an inner diameter of 8 mm, flow 100 ml of physiological saline containing 7 U/ml heparin, and then apply blood at a flow rate of 0.5 ml/mi.
When the platelet permeability was measured after flowing at n for 30 minutes, it was 80 to 93%.

[0037] In addition, the rate was 90 to 98% for the gel in which no ligand was introduced.

Example 2 An adsorbent was synthesized in the same manner as in Example 1 except that the reaction time was changed to 4 hours. The obtained adsorbent was subjected to the same experiment as in Example 1, and the results are shown below.

[0039] Ligand feeding amount: 3.1 μmol/ml
Activation of gel complement: C3a concentration 3570ng/ml
C5a concentration: 34.2 ng/ml As described above, both C3 and C5 are considerably activated in the adsorbent in which the amount of ligand provided is 8 μmol/ml or less of the gel.

[0040] Regarding the platelet permeability, it is 82 to 9.
5%, which is better than the adsorbent of Example 1.

Example 3 An adsorbent was synthesized in the same manner as in Example 1 except that the reaction time was 36 hours and the amount of n-decylamine added was 40 mg. The obtained adsorbent was subjected to the same experiment as in Example 1, and the results are shown below.

[0042] Ligand feeding amount: 56.3 μmol/ml
Gel complement activation: C3a concentration 758 ng/ml
C5a concentration 10.9 ng/ml As shown, almost no complement activation occurs,
It can be seen that the platelet permeability is considerably reduced, ranging from 48 to 63%.

Example 4 An adsorbent was synthesized in the same manner as in Example 1 except that the reaction time was 24 hours and the amount of n-decylamine added was 20 mg. The obtained adsorbent was subjected to the same experiment as in Example 1, and the results are shown below.

[0044] Ligand feeding amount: 23.3 μmol/ml
Gel β2 - Microglobulin adsorption amount: 360 μg
/ml gel albumin adsorption amount: 4 mg/ml Gel complement activation: C3a concentration 1102 ng/ml C5a concentration 12.9 ng/ml Platelet permeability: 65-80% Example 5 The reaction time of Example 1 was 24 hours, The adsorbent was synthesized in the same manner except that the amount of n-decylamine added was 30 mg. The obtained adsorbent was subjected to the same experiment as in Example 1, and the results are shown below.

[0045] Ligand feeding amount: 34.8 μmol/ml
Activation of gel complement: C3a concentration 830 ng/ml
C5a concentration: 11.5 ng/ml Platelet permeability: 58-76% Thus, the amount of ligand is 15-35 per ml of carrier.
When the amount was in the μmol range, the adsorption amount of β2-microglobulin, adsorption selectivity, and blood compatibility were all at good levels.

The above results are summarized in Tables 1 and 2.

[0047]

[Table 1]

[0048]

[Table 2]

As described above, the amount of ligand provided was 8 μmol.
/ml or less, complement activation cannot be ignored, and 56
Since the permeability of platelets deteriorates when the amount exceeds μmol/ml, the range of the amount of ligand to be administered was determined.

Reference Example 1 Synthesis was carried out in the same manner as in Example 4 except that ethylamine was used instead of decylamine. The obtained adsorbent was subjected to the same experiment as in Example 1, and the results are shown below.

[0051] Ligand feeding amount: 33.4 μmol/ml
Gel β2 - Microglobulin adsorption amount: 25μg/m
1 Gel As described above, when the alkyl chain length of the ligand is 4 or less, the amount of β2-microglobulin adsorbed becomes extremely small.

Example 6 Synthesis was carried out in the same manner as in Example 4 except that hexylamine was used instead of decylamine. The obtained adsorbent was subjected to the same experiment as in Example 1, and the results are shown below.

[0053] Ligand feeding amount: 30.8 μmol/ml
Gel β2 - Microglobulin adsorption amount: 138 μg
/ml gel complement activation: C3a concentration 1350ng
/ml C5a concentration 13.1 ng/ml Platelet permeability: 67-82% Example 7 Synthesis was carried out in the same manner as in Example 4 except that hexadecylamine was used instead of decylamine. The obtained adsorbent was subjected to the same experiment as in Example 1, and the results are shown below.

[0054] Ligand feeding amount: 21.5 μmol/ml
Gel β2 - Microglobulin adsorption amount: 380 μg
/ml gel complement activation: C3a concentration 1078ng
/mlC5a concentration 12.6ng/ml Platelet penetration rate: 66-80%

[0055]

Effects of the Invention Since the adsorbent of the present invention has a specific amount of hydrophobic ligand, it maintains high selectivity and adsorption amount when β2-microglobulin is adsorbed and removed from blood, and it also absorbs complement. This has the effect that extracorporeal circulation treatment can be performed with almost no activation of the .

Claims (2)

[Claims] 1. An adsorbent comprising a porous water-insoluble carrier immobilized with a ligand having an alkyl group having 4 or more carbon atoms, characterized in that the amount of the ligand provided to the carrier is 8 to 60 μmol per ml of the carrier. A β2-microglobulin adsorbent for extracorporeal circulation treatment. 2. The β2-microglobulin adsorbent for extracorporeal circulation treatment according to claim 1, wherein the porous water-insoluble carrier comprises cellulose or a cellulose derivative.