HK1171246B - Neurotoxins exhibiting shortened biological activity - Google Patents

Description

Neurotoxins exhibiting shortened biological activity

The invention relates to the field of pharmacy. In particular, polynucleotides encoding neurotoxin polypeptides exhibiting a reduced duration of biological effect in a subject are contemplated, wherein the polypeptides comprise at least one degradation signal in the light chain, as well as vectors and host cells comprising the polynucleotides, polypeptides encoded thereby, and antibodies that specifically bind to the polypeptides. Furthermore, the invention relates to medicaments comprising said polynucleotides and polypeptides and specific therapeutic applications of the medicaments. Furthermore, the invention contemplates methods for the production of said polypeptides and medicaments.

Botulinum and tetanus produce highly potent neurotoxins, namely botulinum toxin (BoNTs) and tetanus toxin (TeNT), respectively. These Clostridial Neurotoxins (CNTs) specifically bind to neuronal cells and interfere with neurotransmitter release. Each toxin was synthesized as an inactive, unprocessed, single-chain protein of approximately 150 kDa. Post-translational processing involves the formation of disulfide bonds and limited proteolysis (cleavage) by bacterial proteases. The active neurotoxin consists of two chains, an N-terminal light chain of about 50kDa and a heavy chain of about 100kDa, linked by disulfide bonds. CNTs consist of three domains, the catalytic light chain, the heavy chain including the translocation domain (half of the N-terminus) and the receptor binding domain (half of the C-terminus), see Krieglstein1990, EurJBiochem188, 39; krieglstein1991, EurJBiochem202, 41; krieglstein1994, JProteinChem13, 49. Botulinum neurotoxin is synthesized in the form of a molecular complex comprising a 150kDa neurotoxin protein and associated non-toxic complex proteins. The size of the complex varies from 300kDa to 900kDa based on the clostridial strain and different neurotoxin serotypes. The complex proteins in these complexes stabilize the neurotoxin and protect it from degradation, see Chen1998, InfectImmun66 (6): 2420-2425.

Botulinum secretes seven antigenically distinct serotypes called botulinum neurotoxins (bonts) of types a to G. The relevant tetanus neurotoxin (TeNT) secreted by all serotypes and Clostridium tetani is Zn²⁺Endoprotease, said Zn²⁺Endoproteases block synaptic exocytosis by cleaving SNARE proteins, see Couesnon, 2006, Microbiology, 152, 759. CNTs cause the visible flaccid muscle paralysis in botulism, see Fischer2007, PNAS104, 10447.

Despite its toxic effects, botulinum toxin has been used as a therapeutic agent for many diseases or conditions. Botulinum toxin serotype a was approved in the united states for the treatment of strabismus, blepharospasm and other disorders in humans in 1989. It is commercially available as a botulinum toxin protein a complex, for example under the trade name botox (allergananc) or DYSPORT (Ipsenltd). For therapeutic applications, the complex is injected directly into the muscle to be treated. At physiological pH, the toxin is released from the protein complex and produces the desired pharmacological effect. An improved non-complex neurotoxin a polypeptide preparation is available under the trade name xeomin (merzpharmaceuticals gmbh). The effects of botulinum toxin are only temporary, which is why repeated administration of botulinum toxin is required to maintain the therapeutic effect.

Clostridial neurotoxins attenuate voluntary muscle strength and are effective therapeutic agents for strabismus, focal dystonia including cervical dystonia, and benign idiopathic blepharospasm. Clostridial neurotoxins have further been shown to alleviate unilateral facial spasms and focal spasms, and, in addition, to be effective for a variety of other indications such as gastrointestinal disorders, hyperhidrosis, and cosmetic wrinkle reduction, see Jost2007, Drugs67, 669.

However, weakening muscle strength and contraction is also desirable for medical conditions or diseases such as wound healing, fixation of bones and tendon rupture treatment, post-surgical fixation, in particular for hemorrhoidectomy, introduction of dental implants or hip replacement (endoprostheses), knee arthroplasty, ophthalmic surgery, acne or irritable bowel disease. Neurotoxins typically exhibit their biological effects over a period of time that is longer than is actually required for effective treatment of the disease or condition. However, prolonged muscle paralysis is detrimental or at least not preferred in the treatment of said medical conditions or diseases. However, neurotoxins which exhibit their biological effect only for the desired period of time are not available.

The technical problem underlying the present invention can therefore be seen as providing means and methods which meet the aforementioned needs. This technical problem is solved by the embodiments characterized in the claims and below.

Accordingly, the present invention relates to a polynucleotide encoding a neurotoxin polypeptide exhibiting a reduced duration of a biological effect in a subject, wherein said polypeptide comprises at least one degradation signal in the light chain.

The term "polynucleotide" as used herein refers to single-or double-stranded DNA molecules as well as RNA molecules. The term includes genomic DNA, cDNA, hnRNA, mRNA, and naturally occurring or artificially modified derivatives of all of these molecular species. In one aspect, the polynucleotide may be a linear or circular molecule. Furthermore, in addition to the nucleic acid sequence encoding the aforementioned neurotoxin polypeptide, the polynucleotides of the present invention may comprise additional sequences required for proper transcription and/or translation, such as 5 'or 3' UTR sequences. The polynucleotides of the present invention encode modified neurotoxin polypeptides derived from one of the antigenically distinct serotypes of botulinum neurotoxin, namely BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F, BoNT/G or tetanus neurotoxin (TeNT). In one aspect of the invention, the polynucleotide comprises the sequence set forth in seq id 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: 11(BoNT/F), SEQ ID NO: 13(BoNT/G) or seq id no: 15 (TeNT). Further, included in one aspect are polynucleotide sequences comprising a nucleic acid sequence encoding a polypeptide shown as seq id no: 2(BoNT/A), SEQ ID NO: 4(BoNT/B), SEQ ID NO: 6(BoNT/C1), 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). On the other hand, the polynucleotide is a variant of the aforementioned polynucleotide, which comprises one or more nucleotide substitutions, deletions and/or additions, and on the other hand, the variant may still result in the encoded amino acid having one or more amino acid substitutions, deletions and/or additions. Furthermore, in another aspect, the variant polynucleotides of the invention comprise a variant nucleic acid sequence that is identical to the sequence of seq id nos: 1. 3, 5, 7, 9, 11, 13, or 15, or a variant of a nucleic acid sequence encoding an amino acid sequence that is 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 set forth in any one of seq id nos: 2, 4, 6, 8, 10, 12, 14, or 16, exhibits 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% identity in amino acid sequence. The term "identity" as used herein refers to sequence identity, which is characterized as follows: determining the number of amino acids that are identical between two nucleic acid sequences or amino acid sequences, wherein the sequences are aligned to obtain the highest order match. Sequence identity can be calculated using published techniques or methods encoded in computer programs, such as BLASTP, BLASTN or FASTA (Altschul1990, JMolBiol215, 403). In one aspect, a percent identity value is calculated over the complete amino acid sequence. The skilled person can obtain a series of programs based on various algorithms for comparing different sequences. In this context, the algorithms of Needleman and Wunsch or Smith and Waterman provide particularly reliable results. Sequence alignments can be performed using the program PileUp (Higgins1989, CABIOS5, 151) which is part of the GCG software package (genetics computer group1991, SciensDrive, Madison, Wisconsin, USA53711) or the programs Gap and BestFit (Needleman1970, JMolBiol 48; 443; Smith1981, AdvAppllMath 2, 482). In another aspect of the invention, the sequence identity values described above (in percent (%)) are determined using the program GAP over the entire sequence region using the following settings: empty bit weight: 50, length weight: 3, average matching: 10.000 and average mismatches: 0.000, which will always be used as a standard setting for sequence alignment, unless otherwise specifically noted.

In one aspect, each of the foregoing variant polynucleotides encodes a polypeptide that retains one or more or all of the biological properties of the respective neurotoxin polypeptide, i.e., BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F, BoNT/G or tetanus neurotoxin (TeNT). While it is contemplated that the unprocessed precursor may perform some biological function or be partially active, one skilled in the art will appreciate that it will retain full biological activity only after proteolytic activation. As used herein, "biological activity" refers to (a) receptor binding, (b) internalization, (c) translocation across the endosomal membrane into the cytoplasm, and/or (d) endoproteolytic cleavage of a protein involved in synaptic vesicle membrane fusion. In vivo assays for assessing biological activity include the mouse LD50 assay and the ex vivo mouse semi-membrane assay described by Pearce et al (Pearce1994, ToxicollApplPharmacol 128: 69-77) and Dressler et al (Dressler2005, MovDisord 20: 1617-1619, Keller2006, Neuroscience 139: 629-637). Biological activity is usually expressed in Mouse Units (MU). As used herein, 1MU is the amount of neurotoxin component that kills 50% of a particular population of mice, i.e., mouse i.p. ld50, after intraperitoneal injection. On the other hand, variant polynucleotides may encode neurotoxins with improved or altered biological properties, e.g. they may comprise cleavage sites which improve enzyme recognition or may improve receptor binding or any other property mentioned above.

In addition, one aspect includes a fusion polypeptide further comprising a detectable marker peptide or label. In one aspect, suitable tags are FLAG-tag, Myc-tag or His-tag, which also allow for more efficient purification of the tagged polypeptide. In one aspect, the detectable marker peptide includes a fluorescent protein such as GFP, BFP, and the like. In another aspect, the variant polynucleotide should encode a fusion neurotoxin polypeptide comprising a portion of at least two neurotoxin polypeptides of different serotypes, such as a fusion neurotoxin comprising the heavy chain of BoNT/A and the light chain of BoNT/E.

The neurotoxin polypeptide encoded by the polynucleotide of the present invention further comprises at least one degradation signal on its light chain. In one aspect of the invention, the light chain of the neurotoxin polypeptide encoded by a polynucleotide of the invention is obtained by modification of the light chain encoded by a polynucleotide comprising any of the aforementioned specific nucleic acid sequences or variants thereof as described above. Proteolytic cleavage of the precursor polypeptide (single chain polypeptide) produces the light chain of the neurotoxin polypeptide. The light chain is the N-terminal portion of the precursor polypeptide, which is obtained by proteolytic cleavage. In one aspect, the amino acid sequence of the light chain of the neurotoxin polypeptide referred to above can be deduced from the cleavage sites indicated in the table below.

The term "degradation signal" as used herein refers to a modification of the light chain of a neurotoxin polypeptide which results in increased degradation of the neurotoxin polypeptide by endogenous degradation pathways present in the organism to which the neurotoxin has been applied. In one aspect, the degradation pathway will be a proteasomal degradation pathway or a lysosomal degradation pathway. Alternatively, the degradation pathway may result in only partial degradation of the neurotoxin polypeptide, e.g., by one or more proteolytic cleavage steps. The degradation signal can be introduced into the light chain (i.e., localized (internal) within the light chain) or linked to the N-or C-terminus. The skilled person is well aware of suitable degradation signals and how to introduce or attach them to the light chain of the neuropeptide. Furthermore, by applying recombinant molecular biology techniques or chemical modifications, the skilled person can generate polynucleotides encoding such a neuropolypeptide with at least one degradation signal. For example, site-directed mutagenesis can be used to introduce the degradation signals mentioned below. Alternatively, the nucleic acid sequence of a polynucleotide comprising the coding sequence for the neurotoxin polypeptide and the envisaged degradation signal can be designed and subsequently the complete polynucleotide chemically synthesized.

In one aspect, the degradation signal is selected from:

a) at least one internally or terminally introduced PEST motif;

b) at least one internally or terminally introduced E3 ligase recognition motif;

c) an N-terminal oligolysine residue;

d) ubiquitin linked to the N-terminus;

e) the N-terminal proline is substituted by basic amino acid;

f) surface-displayed amino acid residues are substituted with lysine; and is

g) The substitution of the N-terminal proline with a basic amino acid is combined with the substitution of the surface displayed amino acid residue with lysine.

In one aspect, the E3 ligase recognition motif has a consensus sequence shown in the following table (where "X" may represent any of the naturally occurring amino acids):

PEST motifs are well known in the art as degradation signals (Rogers1986, Science 234: 364-. In one aspect, the PEST motif has the sequence set forth in rechstein 1996, TIBS 21: 267-271, the sequences disclosed in table 1 (incorporated herein by reference), which are useful in any of the following proteins: the flow rate of the gas to be treated is GCN4,fos, ornithine decarboxylase, Cactus, CLN2, CLN3 or NIMA.

The modified neurotoxin polypeptide encoded by the polynucleotide of the present invention exhibits a reduced duration of biological effect in a subject when administered as compared to the unmodified neurotoxin polypeptide. In one aspect, said biological effect observed in the subject causes muscle paralysis, i.e. a (reversible) inactivation of the muscle contractile capacity. In one aspect, the effect may be tested using a so-called mouse running assay (Keller2006, Neuroscience 139: 629) -637. The person skilled in the art can determine this biological effect without any special precautions. In one aspect, the reduced duration of the biological effect refers to a statistically significant reduced duration. One skilled in the art can determine whether the duration of the effect is statistically significantly reduced by applying standard statistical tests, such as determination of confidence intervals, determination of p-values, student's t-test, mann-whitney test, and the like. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99%. Preferred p-values are 0.1, 0.05, 0.01, 0.005 or 0.0001. Preferably, the probabilities envisioned by the present invention allow that diagnosis will be correct for at least 60%, at least 70%, at least 80%, or at least 90% of a given cohort or population of subjects. In one aspect, the reduced duration remains less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 30% or less than 20% of the normal duration, i.e., the duration observed for the unmodified neurotoxin polypeptide. In one aspect, the normal duration lasts about 4 months in the case of BoNT/A, 2 months in the case of BoNT/B, about 3 to 4 months in the case of BoNT/C, or about 4 weeks in the case of BoNT/E (Foran, JBiol. chem.278 (2): 1363-. It will be appreciated that the duration of the effect depends on the influence of the individual in the subject such as genetic background, age, lifestyle, etc. Thus, an approximate duration as referred to herein refers to the duration of each neurotoxin polypeptide indicated above (e.g., 4 months for BoNT/A or 4 weeks for BoNT/E) having a standard deviation of 25% or less, 20% or less, 15% or less, 10% or less, 5% or less.

Advantageously, it has been found in accordance with the present invention that neurotoxin polypeptides can be modified to exhibit a shortened biological effect in a subject to which they are administered. In principle, the introduction of a degradation signal into or linked to the light chain of the neurotoxin polypeptide can achieve this object, since the persistence of the light chain is found to be correlated with the duration of the biological effect. For a variety of therapeutic applications where it is desirable to inactivate neural activity, such as muscle paralysis, to promote wound healing, the shortened duration of the biological effect elicited by the neurotoxin polypeptide is beneficial.

The present invention contemplates vectors comprising the polynucleotides of the invention.

The term "vector" preferably includes phage, plasmid, viral or retroviral vectors, as well as artificial chromosomes, such as bacterial or yeast artificial chromosomes. Furthermore, the term also relates to targeting constructs that allow random or site-directed integration of the targeting construct into genomic DNA. Preferably, such a targeting construct comprises DNA of sufficient length for homologous or heterologous recombination as detailed below. In one aspect, a vector comprising a polynucleotide of the invention further comprises a selectable marker for propagation and/or selection in a host. The vector may be incorporated into a host cell by a variety of techniques known in the art. For example, the plasmid vector may be introduced into a precipitate, such as a calcium phosphate precipitate or a rubidium chloride precipitate, or into a complex with charged lipids, or into a carbon-based aggregate, such as fullerene. Alternatively, the plasmid vector may be introduced by heat shock or electroporation techniques. If the vector is a virus, the virus is packaged in vitro using a suitable packaging cell line prior to application to the host cell. Retroviral vectors may be replication competent or replication defective. In the latter case, propagation of the virus generally occurs only in the complementing host/cell. Furthermore, in one aspect of the invention, the polynucleotide is operably linked to an expression control sequence, allowing expression of said vector in a prokaryotic or eukaryotic host cell or an isolated fraction thereof. Expression of the polynucleotide includes transcription of the polynucleotide into translatable mRNA. Regulatory elements that ensure expression in host cells are well known in the art. In one aspect, they comprise regulatory sequences which ensure transcription initiation and/or poly-A signals which ensure transcription termination and transcript stabilization. Other regulatory elements may include transcriptional and translational enhancers. Possible regulatory elements which allow expression in prokaryotic host cells include, for example, the lac-, trp-or tac-promoter in E.coli, and examples of regulatory elements which allow expression in eukaryotic host cells are the AOX 1-or GAL 1-promoter in yeast or the CMV-, SV40-, RSV-promoter (Rous sarcoma virus), CMV-enhancer, SV 40-enhancer or globin intron in mammalian and other animal cells. In addition, inducible expression control sequences may be used in the expression vectors encompassed by the present invention. These inducible vectors may contain tet or lac operator sequences or sequences that are induced by heat shock or other environmental factors. Suitable expression control sequences are well known in the art. In addition to the elements responsible for transcription initiation, such regulatory elements may also comprise a transcription termination signal located downstream of the polynucleotide, such as the SV 40-poly-A site or the tk-poly-A site. In this context, suitable expression vectors are known in the art, such as the Okayama-berg cDNA expression vector pcDV1(Pharmacia), pBluescript (Stratagene), pCDM8, pRc/CMV, pcDNA1, pcDNA3(Invitrogen) or pSPORT1 (Invitrogen). Preferably, the vector is an expression vector and is a gene transfer or targeting vector. Expression vectors derived from viruses such as retroviruses, vaccinia viruses, adeno-associated viruses, herpes viruses, or bovine papilloma viruses may be used to deliver the polynucleotides or vectors of the invention to targeted cell populations. Recombinant viral vectors can be constructed using methods well known to those skilled in the art, see, e.g., techniques described in Sambrook, molecular cloning, 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).

Furthermore, the present invention relates to a host cell comprising a polynucleotide or a vector of the present invention.

The term "host cell" as used herein includes prokaryotic and eukaryotic host cells. The host cell is a bacterial cell on the one hand and a bacterial cell of the phylum firmicutes on the other hand. In one aspect, the bacterial cell is an escherichia coli host cell. In another aspect, it is a Clostridium host cell. In another aspect, the Clostridium host cell is a Clostridium botulinum host cell, and in even another aspect, is a cell of one of the seven different serotypes of Clostridium botulinum mentioned above. In another aspect, the bacterial host cell is a clostridium tetani host cell. In yet another aspect, the host cell is a Bacillus host cell and particularly a Bacillus megaterium host cell. In one aspect, the eukaryotic host cell is a cell of an animal cell line suitable for the production of toxic proteins or a fungal host cell such as a yeast host cell.

The invention also includes polypeptides encoded by the polynucleotides of the invention.

The term "polypeptide" as used herein includes isolated or substantially purified polypeptides that are substantially free of other polypeptides, including complex proteins of host cells (HA70, HA17, HA33, or NTNH (NBP) or polypeptide preparations that include additional other proteins.

Furthermore, the present invention relates to antibodies that specifically bind to the polypeptides of the present invention.

Antibodies against the polypeptides of the invention can be prepared by well-known methods using the polypeptides purified according to the invention or suitable fragments derived therefrom as antigens. Fragments suitable as antigens may be identified by antigenic determination algorithms well known in the art. These fragments may be obtained by proteolytic digestion of the polypeptides of the invention or may be synthetic peptides. In one aspect, the antibody of the invention is a monoclonal antibody, a polyclonal antibody, a single chain antibody, a human or humanized antibody, or a primatized antibody, chimeric antibody or fragment thereof. The antibodies encompassed by the present invention are also bispecific antibodies, synthetic antibodies, antibody fragments such as Fab, Fv or scFv fragments and the like, or chemically modified derivatives of any of these antibodies. The antibodies of the invention should bind specifically to the polypeptides of the invention (i.e., not cross-react with other polypeptides or peptides). In particular toAlternatively, the antibody should not cross-react with the unmodified neurotoxin polypeptide. Specific binding can be tested using a variety of well known techniques. Antibodies or fragments thereof can be obtained using methods such as those described in Harlow and Lane "Antibodies, laboratory Manual", CSHPress, ColdSpringHarbor, 1988. Using beiEt al (1975, Nature256(1975), 495) or Galfr (Galfr 1981, meth. Enzymol.73(1981)) can produce monoclonal antibodies by techniques initially described which involve the fusion of mouse myeloma cells with splenocytes derived from a mammal that has been immunized with an antigen, i.e., a polypeptide of the invention or an immunogenic fragment thereof. Antibodies can be used, for example, for immunoprecipitation and immunolocalization of the polypeptides of the invention, as well as for monitoring the presence of said polypeptides, for example in recombinant organisms, and for the identification of compounds that interact with the proteins according to the invention. For example, surface plasmon resonance used in BIAcore systems can be used to increase the efficiency of phage antibodies that bind to the protein epitopes of the invention (Schier1996, Humananibodieses hybrids 7, 97-105; Malmborg1995, J.Immunol. methods183, 7-13).

The polynucleotides or polypeptides of the invention are generally useful as medicaments.

In one aspect, the term "drug" as used herein refers to a pharmaceutical composition comprising a biologically active neurotoxin polypeptide or a polynucleotide encoding the polypeptide as a pharmaceutically active compound. The medicament may be for use in the treatment of a variety of diseases or conditions in a human or animal in a therapeutically effective dose. The medicament may be formulated using a variety of techniques, depending on the intended purpose of use. Different aspects of the medicament according to the invention are specified below.

In one aspect, the medicament comprises a biologically active neurotoxin polypeptide of the invention and one or more pharmaceutically acceptable carriers as a pharmaceutical composition. A pharmaceutically acceptable carrier must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient of the formulation. The pharmaceutically acceptable carrier used may include a solid, gel or liquid. Examples of solid carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, stearic acid and the like. Examples of liquid carriers are glycerol, phosphate buffered saline solution, water, emulsions, various wetting agents, and the like. Suitable carriers include those mentioned above and others well known in the art, see, for example, Remington's pharmaceutical sciences, mack publishing company, Easton, Pennsylvania. In particular, if the active ingredient of the medicament is a polynucleotide of the present invention, it will be appreciated that the vector may also be a virus or retrovirus suitable for gene therapy.

In one aspect, the drug is dissolved in a diluent prior to administration. The diluent is also selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water or physiological saline. In addition, the pharmaceutical composition or formulation may also include other carriers or non-toxic, non-therapeutic, non-immunogenic stabilizers and the like. Thus, in one aspect, the neurotoxin polypeptide of the present invention can be present in a liquid or lyophilized form. In one aspect, it may be present with glycerol, a protein stabilizer (HAS) or a non-protein stabilizer such as polyvinylpyrrolidone (PVP), hyaluronic acid, or free amino acids. In one aspect, suitable non-protein stabilizing agents are disclosed in WO2005/007185 or WO 2006/020208.

In another aspect, the drug will be provided in the form of a solution comprising the neurotoxin polypeptide. Furthermore, the solution may also comprise the above-mentioned carriers or stabilizers. In one aspect, stable liquid formulations of neurotoxin polypeptides may be provided, as disclosed in US7,211,261.

In one aspect, the pharmaceutical composition may be administered topically. Drugs are usually administered intramuscularly or subcutaneously (close to the gland), depending on the intended medical indication. However, depending on the nature of the compound and the mode of action, the pharmaceutical composition may also be administered by other routes.

A therapeutically effective dose refers to an amount of a neurotoxin polypeptide or polynucleotide of the present invention that prevents, ameliorates or treats the conditions or symptoms associated with the disease referred to in this specification. Therapeutic efficacy and toxicity of compounds can be determined by standard pharmaceutical protocols 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/ED 50. In one aspect, the medicament of the invention will contain the recommended dosage in the instruction of the prescriber or user so as to be adjusted according to the expected dosage for the individual subject.

The medicaments referred to herein have been developed for at least one administration in order to treat or ameliorate or prevent the diseases or conditions listed in the present specification. However, the drug may be administered more than once.

In another aspect of the invention, in addition to the biologically active neurotoxin polypeptide, the medicament according to the invention may comprise a pharmaceutical product which is added to the pharmaceutical composition during formulation of the medicament.

Furthermore, the present invention relates to the use of a polynucleotide or polypeptide of the invention for the preparation of a medicament for the treatment of wound healing, bone fixation and tendon rupture treatment, post-surgical fixation, in particular for hemorrhoidectomy, introduction of dental implants or hip arthroplasty (endoprostheses), knee arthroplasty, ophthalmic surgery, acne or irritable bowel disease.

The aforementioned medical conditions or disease-related symptoms are well known to those skilled in the art and are described in standard textbooks of medicine such as Stedman or pschrembl.

Furthermore, the present invention also relates to the use of a polynucleotide or polypeptide of the invention for the preparation of a diagnostic medicament for determining whether a subject is susceptible to neurotoxin therapy.

The above-mentioned diagnostic drug is the above-mentioned neurotoxin polypeptide drug. However, the drug may be applied over a period of time and in a dosage regimen that allows only determining whether the subject is responsive to the neurotoxin polypeptide or determining a suitable dosage regimen. Because the above neurotoxin polypeptide-although also having therapeutic potential-is in this respect mainly used for diagnostic purposes rather than therapeutic or improvement purposes, the drug comprising it is referred to as "diagnostic drug". Thus, such time-limited pre-screening with the modified neurotoxin polypeptides of the invention will aid in screening subjects susceptible to treatment with unmodified neurotoxin and in determining appropriate dosages. Because the duration of the biological effect caused by the modified neurotoxin of the present invention is reduced, the potential side effects of treatment based on unmodified neurotoxins, which generally last longer, can be reduced.

The present invention includes a method of making a neurotoxin polypeptide encoded by a polynucleotide of the present invention comprising the steps of:

a) culturing a host cell of the invention under conditions that allow expression of a neurotoxin polypeptide encoded by a polynucleotide of the invention, and

b) obtaining a neurotoxin polypeptide encoded by a polynucleotide of the present invention from a culture of host cells of a).

In one aspect, the polypeptide may be obtained from the culture by all conventional purification techniques including affinity chromatography, size exclusion chromatography, High Pressure Liquid Chromatography (HPLC), and precipitation techniques including antibody precipitation. In addition, in one aspect, the neurotoxin polypeptide obtained by the method of the invention may be free of complexing proteins. In another aspect, the neurotoxin polypeptide can be obtained in the form of a complex further comprising a neurotoxin polypeptide complexing protein. Further, in one aspect, obtaining as used herein includes activation of the neurotoxin polypeptide. This can be achieved by proteolytic cleavage of the (single-chain) neurotoxin polypeptide precursor either intracellularly by an endogenous or exogenous (e.g. recombinantly expressed) protease or extracellularly contacting the neurotoxin polypeptide with a protease under conditions allowing cleavage, as before, during or after the aforementioned purification.

Furthermore, contemplated according to the present invention are methods of preparing a medicament comprising the steps of the aforementioned methods of the invention and the further step of formulating a neurotoxin polypeptide encoded by a polynucleotide of the invention as a medicament.

It is understood that such a process for the preparation of a medicament is carried out according to GMP standards for pharmaceuticals to ensure quality, pharmaceutical safety and efficacy of the medicament. Suitable formulations of the medicament are described elsewhere in this specification. However, it is well within the skill of the art how such formulations can be prepared.

The present invention also includes a method of preparing a cosmetic composition comprising the steps of the method of the invention and the further step of formulating the neurotoxin polypeptide as a cosmetic composition.

The "cosmetic composition" as used herein may be formulated as described for the pharmaceutical composition above. In one aspect, as such, it is contemplated that the compounds of the present invention are used in substantially pure form. However, impurities are not pharmaceutically critical. Alternatively, the cosmetic composition may be applied intramuscularly. In another aspect, the cosmetic composition comprising neurotoxin can be formulated as an anti-wrinkle agent.

The invention also relates to such cosmetic compositions and to the use of the polynucleotides or polypeptides of the invention for the preparation of cosmetic compositions to be used as anti-wrinkle agents.

All references cited in this specification are hereby incorporated by reference in their entirety as well as the disclosure specifically mentioned in this specification.

Claims (13)

1. A polynucleotide encoding a neurotoxin polypeptide which exhibits a reduced duration of a biological effect in a subject, wherein said polypeptide comprises at least one degradation signal in the light chain, wherein said degradation signal is selected from the group consisting of:

a) at least one internally or terminally introduced PEST motif;

b) at least one internally or terminally introduced E3 ligase recognition motif.

2. The polynucleotide of claim 1, wherein said biological effect causes muscle paralysis in the subject.

3. The polynucleotide of claim 1, wherein said shortened duration lasts less than 4, 3, or 2 weeks.

4. The polynucleotide of any one of claims 1 to 3, wherein the light chain of said polypeptide is obtained by modifying a light chain encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of:

a) a nucleic acid sequence having the nucleotide sequence shown in seq id No. 1, 3, 5, 7, 9, 11, 13 or 15; and

b) a nucleic acid sequence having at least 98% identity to the nucleic acid sequence of a).

5. A vector comprising the polynucleotide of any one of claims 1 to 4.

6. A host cell comprising the polynucleotide of any one of claims 1 to 4 or the vector of claim 5.

7. The host cell of claim 6, wherein the cell is an E.coli cell or a Clostridium or Bacillus cell.

8. A polypeptide encoded by the polynucleotide of any one of claims 1 to 4.

9. A polynucleotide according to any one of claims 1 to 4 for use as a medicament.

10. A polypeptide according to claim 8 for use as a medicament.

11. Use of a polynucleotide according to any one of claims 1 to 4 or a polypeptide according to claim 8 in the manufacture of a medicament for the treatment of wound healing, bone fixation and tendon rupture treatment, post-surgical fixation, hemorrhoidectomy, introduction of dental implants or hip replacement, knee replacement, ophthalmic surgery, acne or irritable bowel disease.

12. A method of making a neurotoxin polypeptide encoded by a polynucleotide of any one of claims 1 to 4, comprising the steps of:

a) culturing the host cell of claim 6 under conditions which allow expression of the neurotoxin polypeptide encoded by the polynucleotide of any one of claims 1 to 4, and

b) obtaining a neurotoxin polypeptide encoded by a polynucleotide according to any one of claims 1 to 4 from a bacterial culture of a).

13. A method of preparing a medicament comprising the steps of the method of claim 12 and the further step of formulating a neurotoxin polypeptide encoded by a polynucleotide of any one of claims 1 to 4 as a medicament.