FR2697163A1 - Process for the manufacture of botulinum toxin type A, crystalline, as a medicament. - Google Patents

Abstract

According to the present invention, there is provided a process for the manufacture of a crystalline botulinum toxin type A, as a medicament in particular for the treatment of strabismus and blepharospasm, by obtaining a pure toxin, process according to which botulinum toxin type A is subjected to efficient elimination of nucleic acids without denaturation of the toxin using a nucleic acid elimination agent comprising a natural substance, such as Chitoxan without denaturation or deactivation of the toxin, with an improved yield in toxic activity.

Description

PROCESS FOR THE MANUFACTURE OF BOTULINUM TOXIN TYPE A,

  CRYSTALLINE, AS A MEDICINAL PRODUCT.

  The present invention relates to a process for the manufacture of botulinum toxin type A, crystalline, as a medicament and, more particularly, to a process for the manufacture of botulinum toxin type A, crystalline, from a substance natural, such as, for example, Chitoxan (trademark of products made by Dainippon Seiyaku Co) by using a

  nucleic acid removal agent.

  Botulinum toxin, as a typical example of neurotoxin from bacteria, is a proteotoxin produced by Clostridium botulinum Botulinum toxin is classified into eight serotypes A, B, Cl, C 2, D, E, F, G according to antigenicity Among them, type A toxin was the first to be purified and crystallized (Lamnna,

  C.O E Mc Elroy and H W Eklund: Science, 103: 613-614, 1946).

  Until now, the method for manufacturing botulinum toxin type A, crystalline, required complicated procedures carried out in the following order: precipitation with an acid (1), washing with water, extraction with chloride of calcium and acid precipitation (2), extraction with a phosphate buffer, precipitation with ethanol and extraction and deposit in an ammonium sulphate solution In addition, since the manufacturing operation is complicated, it requires the competence and know-how of qualified and experienced people, and it only gives a low recovery rate of the toxin, and one cannot hope to obtain the required quantity and the required effect thereof. more, although a method comprising the combination of, for example, protamine treatment, ion exchange chromatography and gel filtration, has also been adopted, the

  crystallization yield is not interesting.

  One of the problems with crystallizing botulinum toxin type A is how to get a toxin as an easily crystallizable macromolecule as much as possible and the other problem is how to allow the elimination of substances that inhibit crystallization, comprising mainly the nucleic acids contained in a culture solution, without denaturing the toxin The first problem can be solved by choosing a strain producing botulinum toxin type A having a molecular mass of 500,000 to 900,000, and the solution to the second problem depends on how nucleic acids can be removed in

mild conditions.

  The present invention has been accomplished to overcome the aforementioned drawbacks of the prior art, and its aim is to propose a process for the manufacture of botulinum toxin type A, crystalline, capable of improving the stability and the efficiency thereof. as a medicament in particular for the treatment of strabismus and

  blepharospasm, by obtaining a pure toxin.

  The present invention has been made on the basis of an improvement in the prior art as described above, and it is an objective of the latter to improve the stability and the effectiveness of said toxin as medicine especially for the treatment of strabismus

and blepharospasm.

  To achieve the above objective, in accordance with the present invention, botulinum toxin type A is crystallized without loss of toxicity using a nucleic acid elimination agent comprising a substance

natural, such as Chitoxan.

  First of all, among strains producing botulinum toxin type A, strains producing toxin L with a molecular mass of 500,000 and toxin LL with a molecular mass of 900,000 with a titer of high hemagglutination and presenting a high toxicity, are chosen The strains are cultivated in a medium of

  appropriate culture, for example, an NZ- culture medium

  amine, an acid is added to a culture filtrate to cause precipitation, and a phosphate buffer solution whose molar concentration is varied is used in place of calcium chloride to extract

  the toxic ingredient effectively from the precipitate.

  Then, the liquid extract is repeatedly subjected to p H control, overnight rest, centrifugation, dissolution in acetate buffer solution, modification of p H and centrifugation to obtain a supernatant clear A nucleic acid removal agent is added to the supernatant in the

most effective conditions.

  Until now, calcium and an alcohol were used

  in the usual way to remove nucleic acids.

  However, no remarkable results can be obtained even when conditions, such as the concentration and p H of the nucleic acid eliminating agent, are modified.In addition, protamine is known as an eliminating agent. nucleic acids, but it causes precipitation not only of nucleic acids but also of necessary proteins, although it has an excellent effect as an elimination agent. In addition, it causes a problem of difference in homogeneity between each. batches, which is a specific defect in natural products Then, a procedure for removing excess protamine from the resulting precipitate must still be added Furthermore, Chitoxan (trademark of products manufactured by Dainippon Seiyaku Co ) is a natural substance of high molecular mass, which is a natural substance obtained by deacetylation of chitin (poly-N-acyl-pD-glucosami ne) contained in external shells, for example of crabs and shrimps, and the products C-7 and C-9, obtained by reduction of its molecular mass, can effectively eliminate the nucleic acids without loss of the toxin When a salting out with ammonium sulfate is carried out after elimination of the nucleic acids by Chitoxan, the excess Chitoxan does not precipitate and, after dissolution of the toxin released in a phosphate buffer solution, ammonium sulfate is gradually added and we let it rest

  to easily crystallize the toxin.

  In the manufacturing process using Duff's method, the yield of botulinum toxin type A, crystalline, was as low as 1.4-3.1%, assuming the culture solution to represent 100%. could be increased up to about 5% by improving the extraction process, the toxic activity could be kept up to 10% by the process for removing nucleic acids according to the present invention using Chitoxan. In view of what has just been explained, in accordance with the present invention, the nucleic acids can be eliminated effectively during the production of botulinum toxin type A, crystalline, the rate of conservation of the toxic activity can to be improved and, as a result, a remarkable effect can be achieved as a medicament especially for the treatment of strabismus

and blepharospasm.

  Other characteristics and advantages of the present invention will appear on reading the following example.

Example

  (Culture) Clostridium botulinum, Hall strain, culture medium

  NZ-amine, immobile culture at 350 C for 4 days.

  (Precipitation with an acid-1) Addition of sulfuric acid: p H adjusted to 3.5 with H 2504 3 N, left to stand overnight (40 C) Elimination of the supernatant (siphon) Centrifugation Precipitate: Dissolved in distilled water, p H adjusted to 5.0 with 0.1 N Na Cl Centrifugation (Extraction) Precipitate: Addition of a 0.2 M phosphate buffer solution (p H 6.0) Centrifugation Supernatant Precipitate: Addition of '' a 0.1 M tris-H Cl buffer solution with which 0.5 M Na Cl is associated (p H 7.5) Centrifugation (Precipitation by an acid-2) Addition of H Cl: p H adjusted to 3 , 7 with H Cl 2 N, left to stand overnight

(4 C)

  Removal of the supernatant (siphon) Precipitated centrifugation: Dissolved in a 0.05 M acetate solution (p H 5.0) Buffer centrifugation (Treatment with C-9) Supernatant: 5% C-9 solution added up to 0.1% in one minute, with stirring for 10 minutes in a container containing ice Centrifugation Supernatant Addition of ammonium sulphate: Solid ammonium sulphate added to obtain a solution

saturated at 50%.

  Put to rest overnight at C. Centrifugation Precipitate Precipitate dissolved in a 0.03 M phosphate buffer solution at p H 6.8 Dialysis Dialysis with a 0.03 M phosphate buffer solution at p H 6.8 (Crystallization ) Addition of ammonium sulphate: Ammonium sulphate added until the solution shows a pearly color, provided that the final concentration does not exceed a concentration of 0.9 M. Crystal sedimentation: Left at 40 C (Preparation of the 5% C-9 solution) Distilled water was added to product C-9 (Kurimover, manufactured by Dainippon Seiyaku, Lot No K 143) in a 10 to 1 ratio Then, H Cl i N was added in a ratio of 2, and stirred with a stirrer for 2 hours The p H was adjusted to 4-6 with 1 N sodium hydroxide The solution was diluted with distilled water

  until forming a 5% solution.

  It is obvious that, in this invention, a wide range of different operating modes can be established on the basis of the invention, without departing from the spirit and the field of definition thereof. invention is also not limited by its modes

specific operating procedures.

Claims (4)

  1 Method for manufacturing a botulinum toxin type A, crystalline, as a medicament characterized in that the botulinum toxin type A is crystallized without loss of toxicity using an agent   elimination of nucleic acids.   2 Method according to claim 1, characterized in that the nucleic acid elimination agent comprises a natural substance obtained by deacetylation chitin.   3 A method of manufacturing a crystalline botulinum toxin type A, as a medicament, characterized in that it comprises the steps of: culturing a strain of bacteria producing the botulinum toxin type A; precipitation of an filtrate from this culture with an acid; extraction of the precipitate with a phosphate buffer solution; adjustment of the p H of the liquid extract; resting the adjusted solution overnight; separation by centrifugation of the adjusted solution dissolution of the precipitate separated by an acetate buffer solution; modification of the p H value of the solution obtained; separation by centrifugation of the modified solution; and addition to the supernatant of a nucleic acids.   4 Method according to claim 3, characterized in that the nucleic acid elimination agent comprises a natural substance obtained by deacetylation chitin.