Clostridial neurotoxins with altered persistency

A clostridia and neurotoxin technology, applied in neuromuscular system diseases, medical preparations containing active ingredients, peptide/protein ingredients, etc. incorrect, etc.

Inactive Publication Date: 2011-07-20
MERZ PHARMA GMBH & CO KGAA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far the authors have not been able to provide any evidence for this effect, and newer experiments show that the whole conjecture is incorrect
[0016] Furthermore, even though in some cases, the addition of the motif leads to membrane localization, this approach cannot be used to modify botulinum toxin type A
Because the light chain of native botulinum toxin type A is already localized to the intracellular membrane, additional tethering to the membrane provides no additional effect

Method used

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  • Clostridial neurotoxins with altered persistency

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0324] Example 1 Construction of expression plasmids

[0325] The DNA sequence of the heavy chain of botulinum toxin type A was amplified from the chromosomal DNA of botulinum type A by PCR (database No. AAA23262). A recognition sequence encoding thrombin was added at the 5' end of the sequence. A sequence encoding an affinity tag peptide chain (such as His-tag or Strep-tag) suitable for further purification is added at the 3' end of the sequence. Insert the DNA into the expression vector. The DNA sequences of the first and second light chains were also of type A serotype, and were amplified by PCR in the same manner from chromosomal DNA of botulinum type A. Then, the sequence of the light chain was introduced into the expression vector twice consecutively, upstream of the thrombin recognition sequence (TE). Therefore, the sequence shows the following coding structure: LC-LC-TE-HC-Tag.

Embodiment 2

[0326] Example 2 Preparation of fusion proteins in E. coli

[0327] The fusion protein was transfected into E. coli TG1. Induction was performed at 21°C for 4 hours. Then, the fusion protein was purified by StrepTactin-Sepharose (IBA GmbH, Göttingen) column chromatography according to the manufacturer's instructions. The fusion protein is then activated by immobilized thrombin (thrombin-sepharose), which cleaves the peptide bond between the heavy chain and the two light chains. The remaining protein subunits are linked only by disulfide bonds.

Embodiment 3

[0328] Example 3 Persistence assay (extensor digitorum brevis, EDB)

[0329] Subjects administered 4 units of (Merz Pharmaceuticals GmbH) to the right EDB and administered 4 units of modified botulinum toxin (botulinum toxin type A fusion protein conjugated with an additional botulinum toxin type A light chain). The "compound muscle action potential (CMAP)" was measured electrophysiologically every 30 days. After 90 days, the amplitude of CMAP in the right EDB decreased to about 40% (compared to the initial activity), while that in the left EDB decreased to about 70%. The CMAP of the left EDB reached 40% after 150 days.

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Abstract

The invention relates to a polypeptide comprising: (a) a HC-domain or fragment thereof of the neurotoxic component of a clostridial toxin; and (b) a first LC domain or fragment thereof of the neurotoxic component of a clostridial toxin; and (c) at least one further LC domain or fragment thereof of the neurotoxic component of a clostridial toxin wherein the first and the at least one further LC domain may be the same or different from each other, and wherein each of said fragments of said first and of said at least one further LC domain still exhibits proteolytic activity.

Description

technical field [0001] The present invention relates to Clostridal neurotoxins, such as botulinum neurotoxins, which have an altered protein structure compared to the corresponding wild-type neurotoxins. Said differences in protein structure lead, inter alia, to changes in the activity cycle, such as prolongation of activity or persistence. Background technique [0002] Chemodenervation is the application of drugs to prevent a nerve from stimulating its target tissue, such as a muscle, gland, or another nerve. For example, chemical denervation is performed with phenol, alcohol, or botulinum toxin. Chemical denervation is indicated for patients with localized spasms of one or two large muscles or multiple small muscles. It is used to reduce symptoms such as muscle cramps, pain, and hyperreflexia. [0003] Chemodenervating agents block neuromuscular transmission at the neuromuscular junction, resulting in paralysis or partial paralysis of the affected skeletal muscles. The...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/33
CPCC07K14/33C12N9/52A61P17/00A61P21/00A61P43/00A61P5/00A61K38/48C07K16/1282
Inventor 朱尔根·弗瑞沃特
Owner MERZ PHARMA GMBH & CO KGAA
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