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Therapeutic composition comprising a botulinum neurotoxin

a technology of botulinum neurotoxin and composition, which is applied in the direction of drug composition, peptide/protein ingredient, peptide, etc., can solve the problems of no approved product comprising one of the type b-g toxins on the market, antibody-positive patients no longer responding to the complex, and the botulinum toxin complex (irrespective of the complex). achieve the effect of inducing neutralizing antibodies

Inactive Publication Date: 2011-09-08
MERZ PHARMA GMBH & CO KGAA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0010]On the other hand, the novel pharmaceutical can be employed with particular advantage for patients who have never, or not for many years, been treated with botulinum neurotoxin, because their antibody titer is low or zero from the outset. The advantage of the present invention is then that the increase in the titer in these patients due to the treatment with the pure toxin according to the present invention is zero, or at the most very insignificant. In other words, the novel therapeutic composition can be administered over long periods without losing its effect.
[0011]The induction of antibodies during therapy with a Clostridium botulinum toxin is thus prevented by administering a pure neurotoxin in place of the high molecular weight toxic complexes. The neurotoxin which has been completely separated from the complex proteins is immediately bioavailable and can bind directly to the nerve endings of the motor endplates.SUMMARY OF THE INVENTION
[0012]What we therefore believe to be comprised by our invention may be summarized inter alia in the following words:
[0013]One aspect of the present invention thus relates to a pharmaceutical preparation which comprises at least one of the botulinum neurotoxins from Clostridium botulinum of types A, B, C, D, E, F or G (or a mixture of two or more of these neurotoxins), all the neurotoxins being free of the complexing proteins naturally present in the complex.
[0014]In a preferred embodiment, the pharmaceutical preparation is such that the induction of neutralizing antibodies in the patient by the neurotoxin or the mixture of neurotoxins is reduced by comparison with the complexes or is zero.
[0015]A further preferred embodiment provides a pharmaceutical preparation which comprises as neurotoxin or as mixture of neurotoxins a natural neurotoxin or a mixture of natural neurotoxins.

Problems solved by technology

However, at present there is no approved product comprising one of the type B-G toxins on the market.
The direct consequence is that antibody-positive patients no longer respond to the complex.
However, they might be treated with other toxin types, although none of them is approved, for therapy.
When the patient has been tested with all the toxin types and has formed antibodies against them, further administration of a botulinum toxin complex (irrespective of the type) no longer provides a remedy.
It often takes years for the antibody titer to fall significantly, so that these patients are not (cannot be) treated (with botulinum neurotoxin) for long periods.
The long residence time does not result in increased uptake by the target cells, however, since poisoned target cells are no longer able to take up toxin.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Isolation of the Pure Neurotoxin

[0023]The pure neurotoxin from Clostridium botulinum type A is obtained by a process based on the process of DasGupta & Sathyamoorthy. Clostridium botulinum type A is cultivated in a 20 I fermenter in a medium consisting of 2% proteose peptone, 1% yeast extract, 1% glucose and 0.05% sodium thioglycolate. After growth for 72 hours, the toxin is precipitated by adding 3 N H2SO4 (final pH=3.5). The precipitated and centrifuged biomass is extracted with 0.2 M sodium phosphate buffer pH 6.0.

[0024]After removal of the nucleic acids by precipitation with protamine sulfate, the toxin is precipitated by adding ammonium sulfate. The precipitate which has been solubilized and dialyzed against 50 mM sodium phosphate pH 6.0 is bound to a DEAE-Sephadex column at the same pH and detached with 150 mM NaCl. This is followed by a chromatography on a QAE-Sephadex column which has been equilibrated with a 50 mM tris / HCl buffer pH 7.9. The toxin is eluted via an NaCl grad...

example 2

Production of a Finished Pharmaceutical Containing Botulinum Neurotoxin

[0025]The purified neurotoxin from Example 1 is used to prepare a solution which comprises 200 mouse LD50 units, 10 mg of sucrose and 2 mg of human serum albumin per ml. The solution (0.5 ml) is dispensed into vials and freeze-dried. The lyophilizates are reconstituted with physiological saline, and the biological activity is determined. The vials comprise 100±30 LD50 units.

example 3

Isolation of Pure Neurotoxin B

[0026]Clostridium botulinum type B is cultivated in the same medium and under the same conditions as type A and is processed as far as the ammonium sulfate precipitation. This is again followed by a DEAE-Sephadex chromatography at pH 6.0. The fractions eluted from the column with 150 mM NaCl are combined and dialyzed against sodium phosphate pH 7.0, followed by a chromatography on QAE-Sephadex. The toxin-containing fractions are chromatographed further on a DEAE-Sephadex column at pH 8.5 (50 mM tris / HCl pH 8.5).

[0027]Finally, the high-purity botulinum toxin type B is obtained by a chromatography on hydroxyapatite equilibrated with 10 mM Na phosphate pH 8.0. The bound homogeneous toxin is eluted with 80 mM Na phosphate pH 8.0 and subsequently the biological activity is determined in the LD50 assay (2-4×107 LD50 units / mg of protein).

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Abstract

A pharmaceutical preparation comprising one of the botulinum neurotoxins from Clostridium botulinum of types A, B, C, D, E, F or G or a mixture of two or more of these neurotoxins, wherein the neurotoxin or the mixture of neurotoxins is free of the complexing proteins which naturally form the botulinum neurotoxin complexes together with the neurotoxins.

Description

FIELD OF THE INVENTION[0001]A pharmaceutical preparation comprising one of the botulinum neurotoxins from Clostridium botulinum of types A, B, C, D, E, F or G or a mixture of two or more of these neurotoxins, wherein the neurotoxin or the mixture of neurotoxins is free of the complexing proteins which naturally form the botulinum neurotoxin complexes together with the neurotoxins.BACKGROUND OF THE INVENTION[0002]The present invention relates to pharmaceutical preparations which comprise a botulinum neurotoxin from Clostridium botulinum, the neurotoxin being free of the complexing proteins naturally present in the complex. The direct consequence thereof is the realization, on which the present invention is based, that with the free neurotoxin, in contrast to the complex, there is only a distinctly reduced, or no, induction of neutralizing antibodies in the patient. The present invention further relates to the use of botulinum neurotoxins from Clostridium botulinum for producing a med...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/48A61P25/00C12N9/52A61K8/00A61K8/72A61K8/64A61K8/99A61K38/00A61K38/16A61P17/00A61P21/00A61P21/02A61P25/06A61P25/08A61P25/14A61P27/02A61P29/00A61Q15/00C07K16/12
CPCC07K16/1282A61K38/4893A61P17/00A61P17/16A61P21/00A61P21/02A61P25/00A61P25/02A61P25/04A61P25/06A61P25/08A61P25/14A61P27/02A61P29/00A61P43/00A61K38/16Y02A50/30
Inventor BIGALKE, HANSFREVERT, JURGEN
Owner MERZ PHARMA GMBH & CO KGAA
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