Carrier for targeting nerve cells
The present invention relates to a transport protein which can be obtained by modifying the heavy chain of the neurotoxin formed by Clostridium botulinum wherein (i) the protein binds specifically to nerve cells with a higher or lower affinity as the native neurotoxin; (ii) the protein has an increased or reduced neurotoxicity compared to the native neurotoxin, the neurotoxicity being preferably determined in the hemidiaphragma assay; and/or (iii) the protein comprises a lower affinity against neutralizing antibodies compared to the native neurotoxin. The invention also relates to methods for producing the same and the use thereof in cosmetic and pharmaceutical compositions.
1. A transport protein, comprising a modified heavy chain of a Clostridium botulinum serotype B neurotoxin, wherein at least one amino acid in the positions valine 1118, tyrosine 1183, glutamate 1191, lysine 1192, glutamate 1245, and/or tyrosine 1256 of said heavy chain of the Clostridium botulinum serotype B neurotoxin is substituted by a naturally occurring amino acid, and wherein
the transport protein binds to the protein receptor of nerve cells with at least 15% higher or at least 15% lower affinity than a Clostridium botulinum serotype B native neurotoxin carboxy heavy chain.
2. The transport protein according to claim 1 , wherein the transport protein binds to synaptotagmin I or II.
3. The transport protein according to claim 1 , wherein the amino acid tyrosine is substituted in position 1183 by leucine.
4. The transport protein according to claim 1 , wherein the amino acid glutamate is substituted in position 1191 by leucine.
5. A composition comprising the transport protein according to claim 1 , further comprising an organic molecule, a peptide, or a protein, wherein said organic molecule, peptide, or protein is covalently bonded to the transport protein by a peptide bond, an ester bond, an ether bond, a sulphide bond, a disulphide bond or a carbon-carbon-bond, or wherein the protein and the transport protein are bonded covalently by an amino acid sequence.
6. The composition according to claim 5 , wherein the organic molecule is a virustatic, cytostatic, antibiotic or an immunoglobulin.
7. The composition according to claim 5 , wherein the protein is a protease.
8. The composition according to claim 7 , wherein the protease includes one or a plurality of light chains (LCs) of serotypes A, B, Cl, D, E, F and/or G of the Clostridium botulinum , neurotoxin.
9. The composition according to claim 7 , wherein the protease contains a protcolytically active fragment, derived from the light chain (LC) of a Clostridium botulinum neurotoxin selected from the group consisting of: the serotypes A, B, Cl, D, E, F and/or G of the Clostridium botulinum neurotoxin, and characterised in that it exhibits at least 0.01% of the proteolytic activity of the LC.
10. The composition according to claim 7 , wherein the protease specifically splits defined substrates within the cholinergic motor neurons.
11. The composition according to claim 10 , wherein the substrates are selected from proteins which are involved in the release of neurotransmitters in nerve cells and proteins which are capable of catalytic reactions within the nerve cell.
12. The composition according to claim 7 wherein the amino acid sequence that covalently bonds the transport protein and the protease is specifically recognized and split by an endopeptidase.
13. The composition according to claim 12 , wherein the amino acid sequence includes the sequence CXXXZKTKSLVPRGSKBXXC (SEQ ID NO:1), X being any desired amino acid and Z and B being selected independently of one another from alanine, valine, serine, threonine and glycine.
14. The composition according to claim 12 , wherein the protease and the transport protein are additionally bonded covalently by a disulphide bond; and
wherein, after splitting by the endopeptidase, the disulphide bond interlinks the protease and the transport protein, which, in turn, results in the formation of an active dual-chain protease.
15. A pharmaceutical composition containing the transport protein according to claim 1 as well as a pharmaceutically acceptable excipient, diluent and/or additive.
16. A method for treating a subject with a disorder or a disease for which a therapy with Botulinus neurotoxin is indicated, said method comprising administering to the subject an effective amount of pharmaceutical composition according to claim 15 .
17. The method according to claim 16 , wherein the disorder and the disease are selected from the group consisting of: hemi-facial spasm, spasmodic torticollis, blepharospasm, spasticities, dystonias, migraine, pain, disorders of the neck and lumbar vertebral column, strabism, hypersalivation, snoring, wound healing, and depressive disorders.
18. A cosmetic composition containing the transport protein according to claim 1 as well as, a cosmetically acceptable excipient, diluent and/or additive.
19. A method for treating the cosmetic indications hyperhidrosis and facial wrinkles, said method comprising administration of an effective amount of the cosmetic composition according to claim 18 .
20. The transport protein of claim 1 , wherein at least one amino acid in the positions valine 1118, tyrosine 1183, glutamate 1245, and/or tyrosine 1256 of the Botulinus neurotoxin serotype B protein sequences is substituted by a naturally occurring amino acid.
21. The transport protein according to claim 1 , wherein the amino acid tyrosine is substituted in position 1183 by arginine.