JP2730211B2 - Adsorbent for anti-acetylcholine receptor antibody - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to an adsorbent for an anti-acetylcholine sector antibody having a specific structure.

[Conventional technology and its problems] In patients with myasthenia gravis (MG), it has recently been found that an anti-acetylcholine receptor antibody (anti-AchR antibody) is specifically detected in plasma. Have been. Therefore, removal of anti-AchR antibody directly from the blood of a patient is considered to lead to improvement of symptoms, and plasma exchange therapy is evaluated as an effective method. However, plasma exchange therapy indiscriminately removes all of the plasma components, so that not only the necessary part of the plasma components is lost, but also plasma as a replenisher or lack of blood products, serum hepatitis or allergy Many problems such as complications have been pointed out.

Therefore, immunoadsorbents that specifically remove only anti-AchR antibodies from plasma have been developed recently, but their adsorption specificity and adsorption performance are not sufficient, and extracorporeal circulation using adsorbents for patient treatment has been developed. For performing blood purification therapy, it is desired that the AchR antibody can be removed with higher efficiency and that the adverse effect on blood is extremely small.

[Means for Solving the Problems] The present invention relates to an adsorbent for adsorbing and removing the anti-AchR antibody having a specific composition and structure from blood. That is, the present invention relates to a compound of the following formula [1] having two or more isocyanate groups in the molecule, a OCN-R-NCO [1], wherein R is An aliphatic chain having 1 to 12 carbon atoms or 6 carbon atoms
Represents up to 15 aromatic chains. A) an adsorbent for an anti-acetylcholine receptor antibody;

The polymer having a hydroxyl group used in the present invention may be any compound as long as it is water-insoluble, for example,
Examples thereof include agarose, cellulose, dextran, and polyethylene glycol. These may be used alone or as a mixture of two or more, but it is preferable to use a crosslinked one. The shape is not particularly limited, such as a particle shape, a fibrous shape, and a film shape. However, a particulate shape and a fibrous shape are preferable from the viewpoint of easy carrying reaction and handling. In the case of particles, the sphere diameter is 20 to 5000 μm, preferably 100 to 1000 μm from the viewpoints of clogging at the time of packing in a column and the adsorption speed of an adsorbate, but is not limited thereto.

As a compound having two or more isocyanate groups (—NCO) in the molecule, for example, the following formula: OCN—R—NCO (where R is an aliphatic chain having 1 to 12 carbon atoms or 6 to 6 carbon atoms) Fifteen
In addition to the bifunctional isocyanate represented by the formula (1), a polyfunctional isocyanate having three or more functional groups is exemplified.

Examples of the bifunctional isocyanate include hexamethylene diisocyanate, tetramethylene diisocyanate, o-tolidine isocyanate, tolylene diisocyanate, naphthylene-1,5-diisocyanate, and 4,4 ′.
-Diphenylmethane diisocyanate and the like,
The trifunctional isocyanate includes, for example, triphenylmethane-4,4 ', 4 "-triisocyanate. Among these compounds, hexamethylene diisocyanate has an adsorption activity of the obtained adsorbent. Is particularly preferred.

The adsorbent of the present invention can be produced, for example, as follows.

The water-insoluble polymer compound having a hydroxyl group is used in a dry state. When the polymer compound contains water, it is dried by ordinary means, but is used after repeated substitution treatment with an anhydrous organic solvent. The organic solvent used for substitution may be the same as the solvent usually used for the reaction, and examples thereof include dimethyl sulfoxide, dimethylacetamide, dimethylformamide, tetrahydrofuran, acetone, methyl ethyl ketone, and methyl isobutyl ketone.

The reaction is performed by suspending a water-insoluble polymer compound having a hydroxyl group in an organic solvent, adding a compound having two or more isocyanate groups in the molecule, and usually 10 to 200 ° C., preferably 3 to 3 ° C.
Perform at 0-110 ° C. with stirring. In this case, a hydroxyl group (-OH) present in the water-insoluble polymer compound having a hydroxyl group,
The proportion of the compound having two or more isocyanate groups in the molecule to the isocyanate group (-NCO) is not particularly limited, but is preferably -NCO / -OH> 1. After stirring for a predetermined time and the hydroxyl group and the isocyanate group react, the reaction product is repeatedly washed with a solvent in order to remove excess and unreacted isocyanate compound and other by-products. All reactions are carried out under the condition that no water enters the reaction system. After suction filtration of the reaction product, the reaction product is put into a reaction solvent, and the mixture is stirred for a while at room temperature, and the remaining unreacted product is dissolved in the solvent. The reaction is filtered off with suction, washed repeatedly with water and stored aseptically, usually in distilled water for injection.

 Hereinafter, the present invention will be described in more detail with reference to examples.

Production of adsorbent Cross-linked agarose (trade name: Sepharose CL-4B (Pharmacia)) was repeatedly washed with distilled water, filtered by suction, and sufficiently squeezed to obtain 10.0 g of wet cross-linked agarose. (0.77 g in an absolutely dry state) was added to 50 ml of dehydrated dimethyl sulfoxide (hereinafter referred to as “DMSO”), and the mixture was stirred for 2 hours at room temperature. In addition, the mixture was stirred at room temperature for 1 hour, and then the same operation of 30 ml (12.5 hours) and 30 ml (3 hours) was repeated, and finally, 20 ml of DMSO was added. As a result, 1.0 g of hexamethylene diisocyanate (hereinafter referred to as "HMDI") was added to the system, and the amount of dehydrated DMSO 10
The solution added to ml was charged and stirred at 100 ° C. for 2 hours to carry out a reaction. After removing the reaction solvent, 25 ml of fresh DMSO was charged and washed by stirring at room temperature for 1.5 hours. Thereafter, washing was performed in the same manner under the conditions of 25 ml (1.5 hours), 20 ml (1.5 hours), 20 ml (1.5 hours), 20 ml (11.5 hours), and 20 ml (2.5 hours). Titration analysis of the isocyanate group in the final washing solution (20 ml) revealed no isocyanate group. Thereafter, 30 ml of water was added to stop the reaction. The reaction was filtered off with suction and stirred overnight at room temperature in 20 ml of DMSO, then filtered again and washed repeatedly with large amounts of water. After suction filtration,
The sample was immersed in distilled water for injection and subjected to an adsorption test using an adsorbent whose water was squeezed out by suction filtration.

(Example 1 and Comparative Example 1) 0.5 ml (0.1 g dry weight) of the adsorbent produced by the above method
And 1.5 ml of plasma of a patient with myasthenia gravis of a high-value anti-AchR antibody were mixed with a vibrator, followed by incubation at 37 ° C. for 120 minutes. Shake every 30 minutes during this time. Thereafter, the supernatant was collected by centrifugation (3000 rpm, 15 min.), And the anti-AchR antibody was quantified by the IP method (Immunoprecipitaiton method; human IgG method). Table 1 shows the results. As a comparative example, an adsorption test was similarly performed using Sepharose CL-4B (manufactured by Pharmacia).

As is clear from Table 1, it can be seen that the composition has high adsorption performance even at a high concentration.

(Example 2 and Comparative Example 2) A column having an inner diameter of 40 mm and a length of 160 mm was packed with 200 ml of the adsorbent,
After 1 L and 2.5 L of plasma of a patient with myasthenia gravis, each of which had an anti-AchR antibody concentration, was passed through the column, the removal rate was measured in the same manner as in Example 1. Table 2 shows the results. As a comparative example, the same test was performed using Sepharose CL-4B.

As is evident from Table 2, when processing 1 liter, 120
Also, at a concentration of 500 pmol / ml, the removal rate was 100%. Also, the removal rate was as high as 83% and 61% in the treatment of 2.5 liters.

[Specific effects of the invention] If the adsorbent of the present invention is used for myasthenia gravis patients with high anti-AchR antibodies, the anti-AchR antibodies can be efficiently and selectively adsorbed and removed. In addition, the adsorbent of the present invention can be effectively used not only as an adsorbent for separating and purifying anti-AchR antibodies, but also used as a therapeutic device after being filled into an apparatus.

Claims (1)

(57) [Claims] 1. A water-insoluble polymer compound having a hydroxyl group,
A compound of the following formula [1] having two or more isocyanate groups in the molecule: OCN-R-NCO [1] (wherein R is an aliphatic chain having 1 to 12 carbon atoms or 6 carbon atoms)
Represents up to 15 aromatic chains. An anti-acetylcholine receptor antibody adsorbent obtained by binding