WO2012041761A2

WO2012041761A2 - Botulinum neurotoxin polypeptides exhibiting a prolonged activity

Info

Publication number: WO2012041761A2
Application number: PCT/EP2011/066504
Authority: WO
Inventors: Juergen Frevert
Original assignee: Merz Pharma Gmbh & Co. Kgaa
Priority date: 2010-09-28
Filing date: 2011-09-22
Publication date: 2012-04-05
Also published as: WO2012041761A3

Abstract

The present invention pertains to the field of modified neurotoxin polypeptides. In particular, it relates to a polynucleotide encoding a neurotoxin polypeptide comprising at least two neurotoxin light chains and a heavy chain. The neurotoxin polypeptide encoded by the polynucleotide exhibits, in an aspect, a prolonged duration of the biological effect in a subject. Moreover, contemplated are a vector and a host cell which comprise the polynucleotide as well as a neurotoxin polypeptide encoded by the aforementioned polynucleotide. Further, the present invention relates to a medicament comprising the polynucleotide, the vector or the neurotoxin polypeptide and its therapeutic and cosmetic applications.

Description

Botulinum neurotoxin polypeptides exhibiting a prolonged activity

[0001] The present invention pertains to the field of modified neurotoxin polypeptides. In particular, it relates to a polynucleotide encoding a neurotoxin polypeptide comprising at least two neurotoxin light chains and a heavy chain. The neurotoxin polypeptide encoded by the polynucleotide exhibits, in an aspect, a prolonged duration of the biological effect in a subject. Moreover, contemplated are a vector and a host cell which comprise the polynucleotide as well as a neurotoxin polypeptide encoded by the aforementioned polynucleotide. Further, the present invention relates to a medicament comprising the polynucleotide, the vector or the neurotoxin polypeptide and its therapeutic and cosmetic applications.

[0002] Clostridium botulinum and Clostridium tetani produce highly potent neurotoxins, i.e. botulinum toxins (BoNTs) and tetanus toxin (TeNT), respectively. These Clostridial neurotoxins specifically bind to neuronal cells and disrupt neurotransmitter release. Each toxin is synthesized as an inactive unprocessed approximately 150 kDa single-chain protein. The posttranslational processing involves formation of disulfide bridges and limited proteolysis (nicking) by bacterial protease(s). Active dichain neurotoxin consists of two chains, an N-terminal light chain of approx. 50 kDa and a heavy chain of approx. 100 kDa linked by a disulfide bond. Neurotoxins structurally consist of three domains, i.e. the catalytic light chain, the heavy chain encompassing the translocation domain (N-terminal half) and the receptor binding domain (C-terminal half), see Krieglstein 1990, Eur J Biochem 188, 39; Krieglstein 1991 , Eur J Biochem 202, 41 ; Krieglstein 1994, J Protein Chem 13, 49.

[0003] Clostridium botulinum secretes seven antigenically distinct serotypes designated A to G of the BoNTs. All serotypes together with the related TeNT secreted by Clostridium tetani are zinc (Zn²⁺)-dependent endoproteases that block synaptic exocytosis by cleaving SNARE proteins and, in particular, SNAP-25. BoNTs cause, inter alia, the flaccid muscular paralysis seen in botulism and tetanus, see Fischer 2007, PNAS 104, 0447.

[0004] Despite its toxic effects, BoNTs have been used as a therapeutic agents in a large number of diseases. BoNT serotype A (BoNT/A) was approved for human use in the United States in 1989 for the treatment of strabism, blepharospasm, and other disorders. It is commercially available as a protein preparation, for example, under the tradename BOTOX (Allergan !nc) under the tradename DYSPORT (Ipsen Ltd). For therapeutic application the complex is injected directly into the muscle to be treated. At physiological pH, the toxin is released from the protein complex and the desired pharmacological effect takes place. An improved BoNT/A preparation being free of complexing proteins is available under the tradename XEOMIN (Merz Pharmaceuticals GmbH). [0005] BoNTs, in principle, weaken voluntary muscle strength and are, therefore, effective therapeutic agents for the therapy of diseases such as strabism, focal dystonia, including cervical dystonia, and benign essential blepharospasm. They have been further shown to relief hemifacial spasm, and focal spasticity, and moreover, to be effective in a wide range of other indications, such as gastrointestinal disorders, hyperhidrosis, and cosmetic wrinkle correction, see Jost 2007, Drugs 67, 669.

[0006] However, the effect of BoNTs is only temporary, which is the reason why repeated administration of BoNTs may be required to maintain a therapeutic effect. Moreover, the more frequently these drugs are applied the higher will be the risk for an adverse immune response against the neurotoxin polypeptides applied. Furthermore, the production of these highly toxic polypeptides is cumbersome and needs special care with respect to safety issues and is, thus, expensive. In light of these drawbacks of conventional neurotoxin polypeptides as drugs, means for improving the duration of the biological effective of neurotoxins would be highly appreciated for the neurotoxin therapy.

[0007] Thus, the technical problem underlying the present invention could be seen as the provision of means and methods which comply with the aforementioned needs. This technical problem has been solved by the embodiments characterized in the claims and herein below.

[0008] The present invention relates to a polynucleotide encoding a neurotoxin polypeptide comprising at least two neurotoxin light chains and a heavy chain.

[0009] The term "polynucleotide" as used herein refers to single- or double-stranded DNA molecules as well as to RNA molecules. Encompassed by the said term is genomic DNA, cDNA, hnRNA, mRNA as well as all naturally occurring or artificially modified derivatives of such molecular species. The polynucleotide may be in an aspect a linear or circular molecule. Moreover, in addition to the nucleic acid sequences encoding the aforementioned neurotoxin polypeptide, a polynucleotide of the present invention may comprise additional sequences required for proper transcription and/or translation such as 5^'- or 3^'-UTR sequences. [0010] The polynucleotide of the present invention encodes a neurotoxin polypeptide comprising at least three chains, i.e. the at least two light chains and one heavy chain. It is envisaged that the at least two Sight chains are adjacent to each other while only one light chain is adjacent to the heavy chain.

[0011] In an aspect, at least one of the light chains is a modified light chain having a protease domain with reduced protease activity. In such an aspect, the order of the modified light chain and the (unmodified) light chain in the neurotoxin molecule, in an aspect, is N-terminus - modified light chain - unmodified light chain - heavy chain - C terminus. In another aspect the order is N-terminus - unmodified light chain - modified tight chain - heavy chain - C terminus. However, the heavy chain shall in all chain orders be located at the C-terminus. The polynucleotide, in an aspect, may even encode a neurotoxin polypeptide having more than three chains as set forth in detail below. The modified light chain which is comprised by the neurotoxin polypeptide encoded by the polynucleotide of the present invention has a protease domain with reduced protease activity as described elsewhere herein in detail. The neurotoxin polypeptide and, in particular, its light chain and heavy chain are derivable from one of the antigenically different serotypes of Botulinum Neurotoxins, i.e. BoNT/A, BoNT/B, BoNT/CI , BoNT/D, BoNT/E, BoNT/F, BoNT/G, or Tetanus Neurotoxin (TeNT). For details on the positions of the chains in neurotoxin polypeptides see Krieglstein 1990, Eur J Biochem 188, 39; Krieglstein 1991 , Eur J Biochem 202, 41 ; and Krieglstein 1994, J Protein Chem 13, 49.

[0012] In an aspect, said light and heavy chain of the neurotoxin polypeptide (prior to the modification of the modified light chain) are the light and heavy chain of a neurotoxin selected from the group consisting of: BoNT/A, BoNT/B, BoNT/C1 , BoNT/D, BoNT/E, BoNT/F, BoNT/G or TeNT. In another aspect, said light and heavy chain of the neurotoxin polypeptide (prior to the modification of the modified light chain) are encoded by a polynucleotide which comprises a nucleic acid sequence as shown in SEQ !D NO: 1 (BoNT/A), SEQ ID NO: 3 (BoNT/B), SEQ ID NO: 5 (BoNT/C1), SEQ ID NO: 7 (BoNT/D), SEQ ID NO: 9 (BoNT/E), SEQ ID NO: 1 1 (BoNT/F), SEQ ID NO: 13 (BoNT/G) or SEQ ID NO: 15 (TeNT). Moreover, said light and heavy chain of the neurotoxin polypeptide (prior to the modification of the modified light chain) are encoded, in an aspect, by a polynucleotide comprising a nucleic acid sequence encoding an amino acid sequence as shown in any one of SEQ ID NO: 2 (BoNT/A), SEQ ID NO: 4 (BoNT/B), SEQ ID NO: 6 (BoNT/CI), SEQ ID NO: 8 (BoNT/D), SEQ ID NO: 10 (BoNT/E), SEQ ID NO: 12 (BoNT/F), SEQ ID NO: 14 (BoNT/G) or SEQ ID NO: 16 (TeNT).

[0013] In another aspect, the said light and heavy chain of the neurotoxin polypeptide (prior to the modification of the modified light chain) are encoded by a polynucleotide which is a variant of the aforementioned polynucleotides comprising one or more nucleotide substitutions, deletions and/or additions which in still another aspect may result in an encoded amino acid having one or more amino acid substitutions, deletions and/or additions. Moreover, such a variant polynucleotide shall, in another aspect, comprise a nucieic acid sequence being at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the nucleic acid sequence as shown in any one of SEQ ID NOs: 1 , 3, 5, 7, 9, 11 , 13 or 15 or a nucleic acid sequence which encodes an amino acid sequence being at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence as shown in any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, or 16. The term "identical" as used herein refers to sequence identity characterized by determining the number of identical amino acids between two nucleic acid sequences or amino acid sequences wherein the sequences are aligned so that the highest order match is obtained. It can be calculated using published techniques or methods codified in computer programs such as, for example, BLASTP, BLASTN or FASTA (Altschul 1990, J Mol Biol 215, 403). The percent identity values are, in one aspect, calculated over the entire amino acid sequence. A series of programs based on a variety of algorithms is available to the skilled worker for comparing different sequences. In this context, the algorithms of Needleman and Wunsch or Smith and Waterman give particularly reliable results. To carry out the sequence alignments, the program PileUp (Higgins 1989, CABIOS 5, 151 ) or the programs Gap and BestFit (Needleman 1970, J Mol Biol 48; 443; Smith 1981 , Adv Appl Math 2, 482), which are part of the GCG software packet (Genetics Computer Group 1991 , 575 Science Drive, Madison, Wisconsin, USA 53711 ), may be used. The sequence identity values recited above in percent (%) are to be determined, in another aspect of the invention, using the program GAP over the entire sequence region with the following settings: Gap Weight: 50, Length Weight: 3, Average Match: 10.000 and Average Mismatch: 0.000, which, unless otherwise specified, shall always be used as standard settings for sequence alignments. In an aspect, each of the aforementioned variant polynucleotides (prior to the modification of the modified light chain) encodes a polypeptide retaining one or more and, in another aspect, all of the biological properties of the respective neurotoxin polypeptide, i.e. the Bo NT/A, BoNT/B, BoNT/CI , BoNT/D, BoNT/E, BoNT/F, BoNT/G or Tetanus Neurotoxin (TeNT). Those of skill in the art will appreciate that full biological activity is maintained only after proteolytic activation, even though it is conceivable that the unprocessed precursor can exert some biological functions or be partially active. "Biological properties" as used herein refers to (a) receptor binding, (b) internalization, (c) translocation across the endosomal membrane into the cytosol, and/or (d) endoproteolytic cleavage of proteins involved in synaptic vesicle membrane fusion. In vivo assays for assessing bioiogical activity include the mouse LD50 assay and the ex vivo mouse hemidiaphragm assay as described by Pearce et al. (Pearce 1994, Toxicol Appl Pharmacol 128: 69-77) and Dressier et al. (Dressier 2005, Mov Disord 20:1617- 1619, Keller 2006, Neuroscience 139: 629-637). The biological activity is commonly expressed in Mouse Units (MU). As used herein, 1 MU is the amount of neurotoxic component, which kills 50% of a specified mouse population after intraperitoneal injection, i.e. the mouse i.p. LD50. In a further aspect, the variant polynucleotides can encode Neurotoxins having improved or altered biological properties, e.g., they may comprise cleavage sites which are improved for enzyme recognition or may be improved for receptor binding or any other property specified above.

[0014] Neurotoxin polypeptides comprise an N-terminal light chain, an intermediate linker and a C-terminal heavy chain. The neurotoxins are translated as single chain precursor molecules and become proteolytically cleaved into a mature, biologically active dichain form during processing. Proteolytic cleavage occurs at the linker in a manner such that the linker is either cleaved once or is removed after cleavage at the N- and C-termini of the linker. The neurotoxin polypeptide encoded by the polynucleotide of the present invention comprises at least three chains, i.e. at least one unmodified or modified light chain, at least one (unmodified) light chain and a heavy chain. The said chains in the single chain precursor neurotoxin polypeptide encoded by the polynucleotide of the present invention are all separated, in an aspect, by appropriate linker sequences which can be cleaved once or can be removed by dual cleavage during processing of the neurotoxin polypeptide. In another aspect, the light chains are, however, not separated by a linker sequence. The resulting neurotoxin polypeptide is a mature biologicaiiy active polypeptide having tri- or more chains. An appropriate linker which can be introduced between the chains is, in an aspect, the endogenous linker of the neurotoxin polypeptide, i.e. the linker peptide of BoNT/A, BoNT/B, BoNT/CI , BoNT/D, BoNT/E, BoNT/F, BoNT/G or Tetanus Neurotoxin (TeNT). In another aspect, the linker may be a modified linker comprising artificial protease cleavage sites which facilitate the proteolytic processing into the mature neurotoxin polypeptide. Appropriate linkers can be generated and introduced into the polynucleotide of the present invention in between the chain coding nucleic acid sequences by conventional molecular biology techniques including PCR- based mutagenesis techniques.

[0015] The term "modified light chain" refers to a neurotoxin polypeptide light chain which is modified as to exhibit a reduced protease activity compared to an unmodified light chain. The modifications envisaged by the present invention encompass modifications to single amino acids or to a group of amino acids. Modifications include the substitution, addition or deletion of amino acids as well as chemical modifications which alter their biochemical properties. The modification(s) carried out in order to obtain a modified light chain in accordance with the present invention shall result in a modified light chain having a reduced protease activity including also the case where no protease activity can be detected at all.

[0016] In an aspect, the modifications are carried out at the protease domain of the light chain. A protease domain as used in accordance with the present invention refers to the amino acids which are required for the conferring the protease activity to the light chain of the neurotoxin polypeptide. Different amino acids of the light chain are involved in the proteolytic cleavage reaction.

[0017] The neurotoxin protease is a zinc-dependent enzyme. The zinc ion is coordinated by two histidines (H) in the light chain which are part of the protease domain. The protease domains of the neurotoxin polypeptides, thus, comprise conserved histidine motifs (Schiavo 1992, J Biol Chem 267(33): 23479-83). The conserved histidine motifs are shown in the following table: Table: Conserved histidine motifs in different neurotoxin polypeptides

[0018] Thus, in an aspect, the protease domain of the modified light chain also comprises the amino acid sequence motif HELXH (SEQ ID NO: 20). Since the zinc ion is required for the proteolytic cleavage reaction, it will be understood that replacing the histidines which coordinate the said zinc ion will affect the protease function significantly. Thus, in the modified light chain of the neurotoxin polypeptide encoded by the polynucleotide of the invention the aforementioned amino acid sequence motif has, in an aspect, a sequence modification selected from the group consisting of:

(i) substitution of either the first (amino acid 1 of SEQ ID NO: 20) or the second (amino acid 5 of SEQ ID No: 20) histidine (H);

(ii) substitution of both histidines (H);

(iii) substitution of either the first or the second histidine (H) and the glutamic acid (E); and

(iv) substitution of the first and the second histidine (H) and the glutamic acid (E).

[0019] Other amino acid of the protease domain are also essential for the proteolytic reaction. Such essential amino acids can be identified by the skilled artisan by applying standard mutagenesis techniques and testing of the protease domain mutants in in vitro activity tests for SNAP-25 cleavage or in in vivo neurotoxicity assays. Among said essential amino acids, R363 and Y366 have been identified in BoNT/A which are both located in the active core of the light chain protease (Ahmed 2008, Protein J. 27(3): 151- 162). Therefore, in the modified light chain of the neurotoxin polypeptide encoded by the polynucleotide of the invention, in an aspect, said protease domain has a sequence modification at an amino acid position corresponding to position R363 and/or Y366 in the BoNT/A amino acid sequence.

[0020] It will be understood that the modifications of the modified light chain referred to above may either occur as single modifications, i.e. as single amino acid substitution, or may be combined. Due to the combination of different modifications, modified light chains having protease domains exhibiting a graded reduced protease activity can be generated. Accordingly, a polynucleotide of the invention may in one aspect encode a neurotoxin polypeptide comprising a modified light chain with a moderately reduced protease activity and, in another aspect, a neurotoxin polypeptide comprising a modified light chain with no protease activity. Reduced protease activity, in an aspect, refers to significantly reduced protease activity in comparison to a corresponding wildtype neurotoxin polypeptide, i.e. a neurotoxin polypeptide having one unmodified light and one unmodified heavy chain. Suitable tests for measuring the activity are known in the art as described elsewhere herein. No protease activity as used herein means no detectable protease activity in a suitable test referred to herein for measuring the protease activity.

[0021] in an aspect, a combination of different modifications which results in an essentially inactivated protease domain has the following amino acid substitutions: E224Q, R363A and Y366F. The amino acid positions which are modified are indicated for BoNT/A. Corresponding positions in other neurotoxin polypeptides can be identified by the skilled artisan based on an amino acid sequence alignment of the amino acids of the light chains of BoNT/A and the other neurotoxin polypeptides.

[0022] In another aspect, the polynucleotide encodes a neurotoxin polypeptide comprising more than one, i.e. two, three, four, five, six, seven, nine, ten or more, modified light chains, more than one, i.e. two, three, four, five, six, seven, nine, ten or more, light chains and a heavy chain. Moreover, in another aspect, if two or more light chains are comprised in a neurotoxin polypeptide encoded by the polynucleotide of the present invention, the said modified fight chains can be identical or differ with respect to the modifications. The duration of the biological effect of the neurotoxin polypeptide can be modulated by the balance of the number of available iight chains with protease activity and the Iight chains without or with reduced protease activity, i.e. the modified light chains. In principle, the more the number of modified Iight chains is with respect to the unmodified iight chains, the more prolonged the duration of the biological effect of the neurotoxin polypeptide shall be in a subject. [0023] In an aspect of the polynucleotide of the present invention, the said neurotoxin polypeptide exhibits a prolonged duration of the biological effect in a subject.

[0024] In a further aspect of the polynucleotide of the present invention, the said neurotoxin polypeptide comprises at least one more modified light chain. In an aspect, it comprises, thus, two modified light chains, one light chain and one heavy chain or three modified light chains, two light chains and one heavy chain.

[0025] Advantageously, it has been found in the studies underlying the present invention that the neurotoxin polypeptide encoded by the polynucleotide of the present invention does upon internalization into a neuron not more inhibit secretion of vesicles containing the neurotoxin receptor in their plasma membranes as the wiidtype neurotoxin because he wiidtype neurotoxin polypeptide inhibits also its own uptake. Moreover, the neurotoxin polypeptide encoded by the polynucleotide of the present invention has stability within the cytoplasm which is comparable to the stability of wiidtype neurotoxin polypeptide. Accordingly, the present invention provides for means which allow increasing the duration of the neurotoxin biological effect in a cell by increasing the amount of neurotoxin polypeptide, in particular its light chain, in a neuronal cell. This is achieved by the neurotoxin polypeptide encoded by the polynucleotide of the present invention which is capable of interrupting the self-inhibition of neurotoxin uptake and by maintaining the protein stability of the neurotoxin polypeptide at a wiidtype level. Thanks to the present invention, neurotoxin-based therapies become more efficient, cost-effective and safe because, due to the prolonged duration of the biological effect of the neurotoxin, less neurotoxin polypeptide must be administered to the patient. Thus, the costs for the therapy but also the risk of adverse immunological complications due to the generation of an anti-neurotoxin immune response in the patient can be reduced.

[0026] It is to be understood that the definitions and explanations of the terms made above apply mutatis mutandis for all aspects described in this specification in the following except as otherwise indicated.

[0027] The present invention contemplates a vector comprising the polynucleotide of the present invention. [0028] The term "vector", preferably, encompasses phage, plasmid, viral or retroviral vectors as well as artificial chromosomes, such as bacterial or yeast artificial chromosomes. Moreover, the term also relates to targeting constructs which allow for random or site- directed integration of the targeting construct into genomic DNA. Such target constructs, preferably, comprise DNA of sufficient length for either homologous or heterologous recombination as described in detail below. The vector encompassing the polynucleotides of the present invention, in an aspect, further comprises selectable markers for propagation and/or selection in a host. The vector may be incorporated into a host cell by various techniques well known in the art. For example, a plasmid vector can be introduced in a precipitate such as a calcium phosphate precipitate or rubidium chloride precipitate, or in a complex with a charged lipid or in carbon-based clusters, such as fullerens. Alternatively, a plasmid vector may be introduced by heat shock or electroporation techniques. Should the vector be a virus, it may be packaged in vitro using an appropriate packaging cell line prior to application to host cells. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host/cells. Moreover, in an aspect of the invention, the polynucleotide is operatively linked to expression control sequences allowing expression in prokaryotic or eukaryotic host cells or isolated fractions thereof in the said vector. Expression of the polynucleotide comprises transcription of the polynucleotide into a translatable mRNA. Regulatory elements ensuring expression in host cells are well known in the art. In an aspect, they comprise regulatory sequences ensuring initiation of transcription and/or poly-A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers. Possible regulatory elements permitting expression in prokaryotic host ceils comprise, e.g., the lac-, trp- or tac- promoter in E. co!i, and examples for regulatory elements permitting expression in eukaryotic host cells are the AOX1- or the GAL1- promoter in yeast or the CMV-, SV40-, RSV-promoter (Rous sarcoma virus), CMV-enhancer, SV40-enhancer or a globin intron in mammalian and other animal cells. Moreover, inducible expression control sequences may be used in an expression vector encompassed by the present invention. Such inducible vectors may comprise tet or lac operator sequences or sequences inducible by heat shock or other environmental factors. Suitable expression control sequences are well known in the art. Beside elements which are responsible for the initiation of transcription such regulatory elements may also comprise transcription termination signals, such as the SV40-poly-A site or the tk-poly-A site, downstream of the polynucleotide. In this context, suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pBluescript (Stratagene), pCDM8, pRc/CMV, pcDNAI , pcDNA3 (Invitrogen) or pSPORTI (Invitrogen). Preferably, said vector is an expression vector and a gene transfer or targeting vector. Expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno-associated virus, herpes viruses, or bovine papilloma virus, may be used for delivery of the polynucleotide or vector of the invention into a targeted cell population. Such approaches can also be used for gene therapy. Methods which are well known to those skilled in the art can be used to construct recombinant viral vectors; see, for example, the techniques described in Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1994). [0029] The present invention further relates to a host cell comprising the polynucleotide or the vector of the present invention. [0030] The term "host cell" as used herein encompasses prokaryotic and eukaryotic host cells. In an aspect the host cell is a bacterial cell and, in another aspect, a Firmicutes bacterial cell. In one aspect, the said bacterial host cell is an E.coli host cell. In another aspect, it is a Clostridium host cell. In a further aspect, the said Clostridium host cell is a Clostridium botulinum host cell, in even a further aspect, a cell of one of the aforementioned seven different serotypes of Clostridium botulinum. In yet another aspect, the bacteria! host cell is a Clostridium tetani host cell. In a further aspect, the host cell is a Bacillus host cell and in a particular aspect a Bacillus megaterium host cell. A eukaryotic host cell, in an aspect, is a cell of an animal cell line suitable for production of toxic proteins or a fungal host cell such as a yeast host cell.

[0031] The present invention also relates to a polypeptide encoded by the polynucleotide the present invention.

[0032] The term "polypeptide" as used herein encompasses isolated or essentially purified polypeptides being essentially free of other host cell polypeptides including the complexing proteins (HA70, HA17, HA33, or NTNH (NBP). The term, in another aspect, includes polypeptide preparations comprising the polypeptide of the present invention and other proteins in addition. Moreover, the term includes, in an aspect, chemically modified polypeptides. Such modifications may be artificial modifications or naturally occurring modifications. The polypeptide of the present invention shall have the biological properties referred to above. The polypeptide of the invention, in an aspect, can be manufactured by chemical synthesis or recombinant molecular biology techniques well known for the skilled artisan. In an aspect, such a method of manufacturing the polypeptide of the invention comprises (a) cu!turing the host cell of the present invention described elsewhere herein in more detail and (b) obtaining from the said host cell the polypeptide of the present invention. In an aspect of this method, the polypeptide can be obtained by conventional purification techniques from a host cell lysate including affinity chromatography, ion exchange chromatography, size exclusion chromatography and/or preparative gel electrophoresis. Protease activity of the polypeptide of the present invention can be assayed by SNAP-25 cleavage in vitro or by an in vivo neurotoxicity assay.

[0033] Moreover, the present invention contemplates a composition comprising the polynucleotide, the vector, or the polypeptide of the present invention for use as a medicament. [0034] The term "composition" refers to any composition formulated in solid, liquid, aerosol (or gaseous) form. Said composition comprises the compound of the invention, i.e. the polynucleotide, vector or polypeptide, optionally together with suitable auxiliary compounds such as diluents or carriers or further ingredients. In this context, it is distinguished for the present invention between auxiliary compounds, i.e. compounds which do not contribute to the effects elicited by the compound of the present invention upon application of the composition for its desired purpose, and further ingredients, i.e. compounds which contribute a further effect or modulate the effect of the compound of the present invention. Suitable diluents and/or carriers depend on the purpose for which the composition is to be used and the other ingredients. The person skilled in the art can determine such suitable diluents and/or carriers without further ado. Examples of suitable carriers and/or diluents are disclosed elsewhere herein.

[0035] The composition referred to herein above shall be used as a medicament. The term "medicament" as used herein refers to a pharmaceutical composition containing polynucleotide, vector or polypeptide of the present invention as pharmaceutical active compound.

[0036] The pharmaceutical composition referred to above comprises the biologically compound and, in one aspect, one or more pharmaceutically acceptable carrier. The active compound can be present in liquid or lyophifized form dependent on its chemical nature. It will be understood that for nucleic acid based compounds such as the polynucleotide or the vector of the present invention other carriers will be required as for the polypeptide of the present invention. In an aspect, said the latter compound, i.e. the polypeptide of the present invention, can be present together with glycerol, protein stabilizers (e.g., human serum albumin (HAS)) or non-protein stabilizers.

[0037] The active compound of the pharmaceutical composition is, in one aspect, administered in conventional dosage forms prepared by combining the drug with standard pharmaceutical carriers according to conventional procedures. These procedures may involve mixing, granulating, and compression, or dissolving the ingredients as appropriate to the desired preparation. It will be appreciated that the form and character of the pharmaceutical acceptable carrier or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration, and other well-known variables.

[0038] The pharmaceutically acceptable carrier must be acceptable in the sense of being compatible with the other ingredients of the formulation and being not deleterious to the recipient thereof. The said carrier may include, in an aspect, a solid, a gel, or a liquid. Exemplary of solid carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like. Examples of liquid carriers are phosphate buffered saline solution, syrup, oil, water, emulsions, various types of wetting agents, and the like. Similarly, the pharmaceutically acceptable carrier may, in an aspect, also include time delay material well known to the art, such as glyceryl mono-stearate or glyceryl distearate alone or with a wax. Suitable carriers comprise those mentioned above and others well known in the art, see, e.g., Remington^'s Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania.

[0039] The active compound of the pharmaceutical composition may be present in a diluent. The diluent is selected so as not to affect the biological activity of the compound. Examples of diluents are distilled water, physiological saline, Ringer's solutions, dextrose solution, and Hank's solution.

[0040] In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers and the like.

[0041] A therapeutically effective dose refers to an amount of the compound to be used in a pharmaceutical composition of the present invention which prevents, ameliorates or treats the symptoms accompanying a disease or condition referred to in this specification. Therapeutic efficacy and toxicity of the compound can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50.

[0042] The dosage regimen will be determined by the attending physician and other clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Dosage recommendations shall be indicated in the prescribers or users instructions in order to anticipate dose adjustments depending on the considered recipient.

[0043] The pharmaceutical composition may be used for human or non-human therapy of various diseases or disorders referred to elsewhere herein in a therapeutically effective dose.

[0044] The pharmaceutical compositions and formulations referred to herein are administered at least once in order to treat or ameliorate or prevent a disease or condition recited in this specification. However, the said pharmaceutical compositions may be administered more than one time. The pharmaceutical composition is, in one aspect, administered topically. The conventionally used drug administration route is intramuscular, subcutaneous (near glands). However, depending on the nature and the mode of action of a compound the pharmaceutical composition may be administered by other routes as well.

[0045] The medicament according to the present invention may in a further aspect of the invention comprise drugs such as anti-inflammatory drugs in addition to the aforementioned active compounds. [0046] Further, the present invention pertains to a composition comprising the polynucleotide, the vector, or the polypeptide of the present invention to be used for treating and/or preventing a disease selected from the group consisting of: voluntary muscle strength, focal dystonia, including cervical, cranial dystonia, and benign essential blepharospasm, hemifacial spasm, and focal spasticity, gastrointestinal disorders, hyperhidrosis, and cosmetic wrinkle correction, Blepharospasm, oromandibular dystonia, jaw opening type, jaw closing type, bruxism, Meige syndrome, lingual dystonia, apraxia of eyelid, opening cervical dystonia, antecoilis, retrocollis, laterocollis, torticollis, pharyngeal dystonia, laryngeal dystonia, spasmodic dysphonia/adductor type, spasmodic dysphonia/abductor type, spasmodic dyspnea, limb dystonia, arm dystonia, task specific dystonia, writer's cramp, musician's cramps, golfer's cramp, leg dystonia, thigh adduction, thigh abduction knee flexion, knee extension, ankle flexion, ankle extension, equinovarus, deformity foot dystonia, striatal toe, toe flexion, toe extension, axial dystonia, pisa syndrome, belly dancer dystonia, segmental dystonia, hemidystonia, generalised dystonia, dystonia in lubag, dystonia in corticobasal degeneration, dystonia in lubag, tardive dystonia, dystonia in spinocerebellar ataxia, dystonia in Parkinson's disease, dystonia in Huntington's disease, dystonia in Hallervorden-Spatz disease, dopa- induced dyskinesias/dopa-induced dystonia, tardive dyskinesias/tardive dystonia, paroxysmal dyskinesias/dystonias, kinesiogenic non-kinesiogenic action-induced palatal myoclonus, myoclonus myokymia, rigidity, benign muscle cramps, hereditary chin trembling, paradoxic jaw muscle activity, hemimasticatory spasms, hypertrophic branchial myopathy, maseteric hypertrophy, tibialis anterior hypertrophy, nystagmus, oscillopsia supranuclear gaze palsy, epilepsia, partialis continua, planning of spasmodic torticollis operation, abductor vocal cord paralysis, recalcitant mutational dysphonia, upper oesophageal sphincter dysfunction, vocal fold granuloma, stuttering Gilles de la Tourette syndrome, middle ear myoclonus, protective larynx closure, postlaryngectomy, speech failure, protective ptosis, entropion sphincter Odii dysfunction, pseudoachalasia, nonachalsia, oesophageal motor disorders, vaginismus, postoperative immobilisation tremor, bladder dysfunction, detrusor sphincter dyssynergia, bladder sphincter spasm, hemifacial spasm, reinnervation dyskinesias, cosmetic use craw's feet, frowning facial asymmetries, mentalis dimples, stiff person syndrome, tetanus prostate hyperplasia, adipositas, treatment infantile cerebral palsy strabismus, mixed paralytic concomitant, after retinal detachment surgery, after cataract surgery, in aphakia myositic strabismus, myopathic strabismus, dissociated vertical deviation, as an adjunct to strabismus surgery, esotropia, exotropia, achalasia, anal fissures, exocrine gland hyperactivity, Frey syndrome, Crocodile Tears syndrome, hyperhidrosis, axillar palmar plantar rhinorrhea, relative hypersalivation in stroke, in Parkinsosn's, in amyotrophic lateral sclerosis, spastic conditions, in encephalitis and myelitis autoimmune processes, multiple sclerosis, transverse myelitis, Devic syndrome, viral infections, bacterial infections, parasitic infections, fungal infections, in hereditary spastic paraparesis postapoplectic syndrome hemispheric infarction, brainstem infarction, myeion infarction, in central nervous system trauma, hemispheric lesions, brainstem lesions, myeion lesion, in central nervous system hemorrhage, intracerebral hemorrhage, subarachnoidal hemorrhage, subdural hemorrhage, intraspinal hemorrhage, in neoplasias, hemispheric tumors, brainstem tumors, and myeion tumor. [0047] The present invention is also concerned with the provision of a method for the manufacture of a medicament comprising formulating a composition comprising the polynucleotide, the vector, or the polypeptide of the present invention in a pharmaceutically acceptable form. [0048] It is well known how the polynucleotide, the vector or the polypeptide of the present invention can be manufactured. Examples for the manufacture are described elsewhere herein in detail. The said polynucleotide, vector or polypeptide is subsequently formulated as a composition comprising the said polynucleotide, vector or polypeptide as active compound in a pharmaceutically acceptable form. As described elsewhere herein in more detail, in an aspect, a pharmaceutically acceptable carrier can be added. Moreover, in another aspect, the active compound can be provided in a suitable diluent. It is to be understood that the formulation of a pharmaceutical composition takes place under GMP standardized conditions or the like in order to ensure quality, pharmaceutical security, and effectiveness of the medicament.

[0049] Dependent on the desired medical indication for the medicament to be manufactured, further adaptations of the formulation may be required. The skilled artisan is, however, well aware of said adaptations and can adapt the formulation process without further ado for the desired indications, i.e. the diseases or disorders to be treated by the medicament.

[0050] In an aspect, the said medicament is to be applied for treating a disease selected from the group consisting of: voluntary muscle strength, focal dystonia, including cervical, cranial dystonia, and benign essential blepharospasm, hemifacial spasm, and focal spasticity, gastrointestinal disorders, hyperhidrosis, and cosmetic wrinkle correction, Blepharospasm, oromandibular dystonia, jaw opening type, jaw closing type, bruxism, Meige syndrome, lingual dystonia, apraxia of eyelid, opening cervical dystonia, antecollis, retrocollis, !aterocollis, torticollis, pharyngeal dystonia, laryngeal dystonia, spasmodic dysphonia/adductor type, spasmodic dysphonia/abductor type, spasmodic dyspnea, limb dystonia, arm dystonia, task specific dystonia, writer's cramp, musician's cramps, golfer's cramp, leg dystonia, thigh adduction, thigh abduction knee flexion, knee extension, ankie flexion, ankle extension, equinovarus, deformity foot dystonia, striatal toe, toe flexion, toe extension, axial dystonia, pisa syndrome, belly dancer dystonia, segmental dystonia, hemidystonia, generalised dystonia, dystonia in !ubag, dystonia in corticobasal degeneration, dystonia in lubag, tardive dystonia, dystonia in spinocerebellar ataxia, dystonia in Parkinson's disease, dystonia in Huntington's disease, dystonia in Hallervorden-Spatz disease, dopa-induced dyskinesias/dopa-induced dystonia, tardive dyskinesias/tardive dystonia, paroxysmal dyskinesias/dystonias, kinesiogenic non-kinesiogenic action-induced palatal myoclonus, myoclonus myokymia, rigidity, benign muscle cramps, hereditary chin trembling, paradoxic jaw muscle activity, hemimasticatory spasms, hypertrophic branchial myopathy, maseteric hypertrophy, tibialis anterior hypertrophy, nystagmus, osciilopsia supranuclear gaze palsy, epilepsia, partialis continua, planning of spasmodic torticollis operation, abductor vocal cord paralysis, recalcitant mutational dysphonia, upper oesophageal sphincter dysfunction, vocal fold granuloma, stuttering Gilles de la Tourette syndrome, middle ear myoclonus, protective larynx closure, postlaryngectomy, speech failure, protective ptosis, entropion sphincter Odii dysfunction, pseudoachalasia, nonachalsia, oesophageal motor disorders, vaginismus, postoperative immobiiisatton tremor, bladder dysfunction, detrusor sphincter dyssynergia, bladder sphincter spasm, hemifacial spasm, reinnervation dyskinesias, cosmetic use craw's feet, frowning facial asymmetries, mentalis dimples, stiff person syndrome, tetanus prostate hyperplasia, adipositas, treatment infantile cerebral palsy strabismus, mixed paralytic concomitant, after retinal detachment surgery, after cataract surgery, in aphakia myositic strabismus, myopathic strabismus, dissociated vertical deviation, as an adjunct to strabismus surgery, esotropia, exotropia, achalasia, anal fissures, exocrine gland hyperactivity, Frey syndrome, Crocodile Tears syndrome, hyperhidrosis, axillar palmar plantar rhinorrhea, relative hypersalivation in stroke, in Parkinsosn's, in amyotrophic lateral sclerosis, spastic conditions, in encephalitis and myelitis autoimmune processes, multiple sclerosis, transverse myelitis, Devic syndrome, viral infections, bacterial infections, parasitic infections, fungal infections, in hereditary spastic paraparesis postapoplectic syndrome hemispheric infarction, brainstem infarction, myelon infarction, in central nervous system trauma, hemispheric lesions, brainstem lesions, myelon lesion, in central nervous system hemorrhage, intracerebral hemorrhage, subarachnoidal hemorrhage, subdural hemorrhage, intraspinal hemorrhage, in neoplasias, hemispheric tumors, brainstem tumors, and myelon tumor. [0051] The invention also pertains to a composition as specified above, which is a cosmetic composition to be applied for cosmetic purposes in connection with the aforementioned diseases or disorders. The cosmetic composition can be formulated as described for a pharmaceutical composition above. Cosmetic compositions are, in an aspect, also to be applied intramuscular. In an even further aspect of the invention, cosmetic compositions comprising the compounds referred to above, i.e. the polynucleotide, vector or polypeptide of the present invention, can be formulated as an anti-wrinkle agent.

[0052] All references cited in this specification are herewith incorporated by reference with respect to their entire disclosure content and the disclosure content specifically mentioned in this specification.

FIGURES [0053] Figure 1 shows schematic structures of a botulinum neurotoxin construct with two (active) ligh chains.

EXAMPLES

Example 1 : Expression and purification of the holotoxin of botuiinum neurotoxin type A in E. coli

[0054] The nucleic acid sequence coding for full-length botulinum neurotoxin type A (BoNT/A) carrying a thrombin was inserted into a modified pET29c plasmid (Novagen) using Ndei and Bglll. Expression of recombinant BoNT/A carrying a Strep- and a His-tag at the C-terminus and a thrombin cleavage site was performed in E. coli BL21(DE3) (Novagen) after induction with 1 mM IPTG at 16 °C for 16 h. Cultures were harvested and rBoNT/A was isolated from the soluble lysate. Recombinant BoNT/A was purified by affinity chromatography on HisTrap™ FF Column (GE Healthcare, Freiburg) according to the information of the supplier. The neurotoxin was eluted with 300 mM imidazole. The eluate was further purifed on a ion exchange chromatography at pH=8.0 on SP- Sepharose FF (GE Healthcare, Freiburg) and in the final polishing step by size exclusion chromatography on Superdex 200 (GE Healthcare, Freiburg). The purified neurotoxin showed a single band in SDS-polyacrylamide gelelectrophoresis. The tags were cleaved off by incubation with thrombin (0,00811/ pg/neurotoxin for 24 hours at room temperature at pH=7.7. The neurotoxin could be activated by incubation with trypsin (100pg neurotoxin/0, 25pg trypsin) for 1 hr at 37°. After SDS polyacrylamide gelectrophoresis two bands were visible: the light chain (=50 kD) and the heavy chain (~ 100 kD). The recombinant neurotoxin showed a specific potency of 6.5 pg/LD50-unit. Example 2: Preparation of BoNT/A with an additional BoNT/A-light chain

[0055] The gene for the light chain of BoNT/A was inserted into the pET29c plasmid generated in example 2 with a linker consisting of three glycines between the two light chains. Expression of the protein construct was performed in E. coli BL21(DE3) (Novagen) after induction with 1 mM IPTG at 16 °C for 16 h as in Example 1. Cultures were harvested and rBoNT/A was isolated from the soluble lysate. The protein was purified as described in Example 1 and activated after removal of the tags by incubation with trypsin.

Example 3: Analyis of the duration of the paralytic effect [0056] The duration of the effect was analysed in the mouse voluntary running system described by Keller (Recovery from botulinum neurotoxin poisoning in vivo. (Keller JE, Neuroscience. 2006 139(2):629-37). Doses of 0.8 U, 0.5 U and 0.3 U of the activated fusion protein of Example 2 were injected into the gastrocnemius muscle of 8 mice per dose. 0.8 U of the wildtype BoNT/A were injected for comparison. The running activity was monitored in cages equipped with a running wheel for 40 days. The running activity decreased to 10% of the initial activity (100%) after two days. The recovery to 100% of the 0.8 U dose group of the fusion BoNT/A was markedly longer than the recovery of the 0.8 U wildtype group.

Claims

1. A polynucleotide encoding a neurotoxin polypeptide comprising at least two neurotoxin light chains and a heavy chain.

2. The polynucleotide of claim 1 , wherein at least one of said at least two light chains is a modified light chain having a protease domain with reduced protease activity.

3. The polynucleotide of claim 2, wherein said protease domain comprises the amino acid sequence motif HELXH (SEQ ID NO: 20).

4. The polynucleotide of claim 3, wherein said amino acid sequence motif has a sequence modification selected from the group consisting of:

(i) substitution of either the first or the second H;

(ii) substitution of both H;

(iii) substitution of either the first or the second H and the E; and

(iv) substitution of the first and the second H and the E.

5. The polynucleotide of any one of claims 1 to 4, wherein said neurotoxin is BoNT/A.

6. The polynucleotide of any one of claims 2 to 5 wherein said protease domain has a sequence modification at an amino acid position corresponding to position R363 and/or Y366 in the BoNT/A amino acid sequence.

7. The polynucleotide of any one of claims 1 to 6, wherein the said neurotoxin polypeptide exhibits a prolonged duration of the biological effect in a subject

8. A vector comprising the polynucleotide of any one of claims 1 to 7.

9. A host cell comprising the polynucleotide of any one of claims 1 to 7 or the vector of claim 8.

10. A polypeptide encoded by the polynucleotide of any one of claims 1 to 7.

11. A composition comprising the polynucleotide of any one of claims 1 to 7, the vector of claim 8, or the polypeptide of claim 10 as a medicament.

12. A composition comprising the polynucleotide of any one of claims 1 to 7, the vector of claim 8, or the polypeptide of claim 10 to be used for treating and/or preventing a disease selected from the group consisting of: voluntary muscle strength, focal dystonia, including cervical, crania! dystonia, and benign essentia! blepharospasm, hemifacial spasm, and focal spasticity, gastrointestinal disorders, hyperhidrosis, and cosmetic wrinkle correction, Blepharospasm, oromandibu!ar dystonia, jaw opening type, jaw closing type, bruxism, Meige syndrome, lingual dystonia, apraxia of eyelid, opening cervical dystonia, antecollis, retrocollis, iaterocollis, torticollis, pharyngeal dystonia, laryngeal dystonia, spasmodic dysphonia/adductor type, spasmodic dysphonia/abductor type, spasmodic dyspnea, limb dystonia, arm dystonia, task specific dystonia, writer's cramp, musician's cramps, golfer's cramp, leg dystonia, thigh adduction, thigh abduction knee flexion, knee extension, ankle flexion, ankle extension, equinovarus, deformity foot dystonia, striatal toe, toe flexion, toe extension, axial dystonia, pisa syndrome, belly dancer dystonia, segmental dystonia, hemidystonia, generalised dystonia, dystonia in lubag, dystonia in corticobasal degeneration, dystonia in lubag, tardive dystonia, dystonia in spinocerebellar ataxia, dystonia in Parkinson's disease, dystonia in Huntington's disease, dystonia in Hailervorden-Spatz disease, dopa-induced dyskinesias/dopa-induced dystonia, tardive dyskinesias/tardive dystonia, paroxysmal dyskinesias/dystonias, kinesiogenic non-kinesiogenic action-induced palatal myoclonus, myoclonus myokymia, rigidity, benign muscle cramps, hereditary chin trembling, paradoxic jaw muscle activity, hemimasticatory spasms, hypertrophic branchial myopathy, maseteric hypertrophy, tibialis anterior hypertrophy, nystagmus, oscillopsia supranuclear gaze palsy, epilepsia, partialis continua, planning of spasmodic torticollis operation, abductor vocal cord paralysis, recalcitant mutational dysphonia, upper oesophageal sphincter dysfunction, vocal fold granuloma, stuttering Gilles de la Tourette syndrome, middle ear myoclonus, protective larynx closure, postiaryngectomy, speech failure, protective ptosis, entropion sphincter Odii dysfunction, pseudoachaiasia, nonachalsia, oesophageal motor disorders, vaginismus, postoperative immobilisation tremor, bladder dysfunction, detrusor sphincter dyssynergia, bladder sphincter spasm, hemifacial spasm, reinnervation dyskinesias, cosmetic use craw's feet, frowning facial asymmetries, mentalis dimples, stiff person syndrome, tetanus prostate hyperplasia, adipositas, treatment infantile cerebral palsy strabismus, mixed paralytic concomitant, after retinal detachment surgery, after cataract surgery, in aphakia myositic strabismus, myopathic strabismus, dissociated vertical deviation, as an adjunct to strabismus surgery, esotropia, exotropia, achalasia, anal fissures, exocrine gland hyperactivity, Frey syndrome, Crocodile Tears syndrome, hyperhidrosis, axillar palmar plantar rhinorrhea, relative hypersalivation in stroke, in Parkinsosn's, in amyotrophic lateral sclerosis, spastic conditions, in encephalitis and myelitis autoimmune processes, multiple sclerosis, transverse myelitis, Devic syndrome, viral infections, bacterial infections, parasitic infections, fungal infections, in hereditary spastic paraparesis postapoplectic syndrome hemispheric infarction, brainstem infarction, myelon infarction, in central nervous system trauma, hemispheric lesions, brainstem lesions, myelon lesion, in central nervous system hemorrhage, intracerebral hemorrhage, subarachnoidal hemorrhage, subdural hemorrhage, intraspinal hemorrhage, in neoplasias, hemispheric tumors, brainstem tumors, and myelon tumor.

3. A method for the manufacture of a medicament comprising formulating a composition comprising the polynucleotide of any one of claims 1 to 7, the vector of claim 8, or the polypeptide of claim 10 in a pharmaceutically acceptable form.

4. The method of claim 13, wherein said medicament is to be applied for treating a disease selected from the group consisting of: voluntary muscle strength, focal dystonia, including cervical, cranial dystonia, and benign essential blepharospasm, hemifacial spasm, and focal spasticity, gastrointestinal disorders, hyperhidrosis, and cosmetic wrinkle correction, Blepharospasm, oromandibular dystonia, jaw opening type, jaw closing type, bruxism, Meige syndrome, lingual dystonia, apraxia of eyelid, opening cervical dystonia, antecollis, retrocollis, laterocollis, torticollis, pharyngeal dystonia, laryngeal dystonia, spasmodic dysphonia/adductor type, spasmodic dysphonia/abductor type, spasmodic dyspnea, limb dystonia, arm dystonia, task specific dystonia, writer's cramp, musician's cramps, golfer's cramp, leg dystonia, thigh adduction, thigh abduction knee flexion, knee extension, ankle flexion, ankle extension, equinovarus, deformity foot dystonia, striatal toe, toe flexion, toe extension, axial dystonia, pisa syndrome, belly dancer dystonia, segmental dystonia, hemidystonia, generalised dystonia, dystonia in lubag, dystonia in corticobasal degeneration, dystonia in iubag, tardive dystonia, dystonia in spinocerebellar ataxia, dystonia in Parkinson's disease, dystonia in Huntington's disease, dystonia in Hallervorden-Spatz disease, dopa-induced dyskinesias/dopa-induced dystonia, tardive dyskinesias/tardive dystonia, paroxysmal dyskinesias/dystonias, kinesiogenic non-kinesiogenic action-induced palatal myoclonus, myoclonus myokymia, rigidity, benign muscle cramps, hereditary chin trembling, paradoxic jaw muscle activity, hemimasticatory spasms, hypertrophic branchial myopathy, maseteric hypertrophy, tibialis anterior hypertrophy, nystagmus, osci!lopsia supranuclear gaze palsy, epilepsia, partialis continua, planning of spasmodic torticollis operation, abductor vocal cord paralysis, recalcitant mutational dysphonia, upper oesophageal sphincter dysfunction, vocal fold granuloma, stuttering Gilles de la Tourette syndrome, middle ear myoclonus, protective larynx closure, postlaryngectomy, speech failure, protective ptosis, entropion sphincter Odii dysfunction, pseudoachalasia, nonachalsia, oesophageal motor disorders, vaginismus, postoperative immobilisation tremor, bladder dysfunction, detrusor sphincter dyssynergia, bladder sphincter spasm, hemifacial spasm, reinnervation dyskinesias, cosmetic use craw's feet, frowning facial asymmetries, mentalis dimples, stiff person syndrome, tetanus prostate hyperplasia, adipositas, treatment infantile cerebral palsy strabismus, mixed paralytic concomitant, after retinal detachment surgery, after cataract surgery, in aphakia myositic strabismus, myopathic strabismus, dissociated vertical deviation, as an adjunct to strabismus surgery, esotropia, exotropia, achalasia, anal fissures, exocrine gland hyperactivity, Frey syndrome, Crocodile Tears syndrome, hyperhidrosis, axiilar palmar plantar rhinorrhea, relative hypersalivation in stroke, in Parkinsosn's, in amyotrophic lateral sclerosis, spastic conditions, in encephalitis and myelitis autoimmune processes, multiple sclerosis, transverse myelitis, Devic syndrome, viral infections, bacterial infections, parasitic infections, fungal infections, in hereditary spastic paraparesis postapoplectic syndrome hemispheric infarction, brainstem infarction, myelon infarction, in central nervous system trauma, hemispheric lesions, brainstem lesions, myelon lesion, in central nervous system hemorrhage, intracerebral hemorrhage, subarachnoidal hemorrhage, subdural hemorrhage, intraspinal hemorrhage, in neoplasias, hemispheric tumors, brainstem tumors, and myelon tumor.