Treatment of pain and inflammatory disorders

Abstract

To provide a polypeptide for use in therapy, e.g., a polypeptide for use in treating pain or an inflammatory disorder.SOLUTION: The present invention is directed to a polypeptide for use in treating pain or an inflammatory disorder. Therein: the polypeptide comprises a clostridial neurotoxin light-chain (L-chain), a clostridial neurotoxin translocation domain (HN domain) and / or a clostridial neurotoxin receptor binding domain (HC domain); and, when the polypeptide comprises a clostridial neurotoxin L-chain, the L-chain is catalytically inactive. Also provided are corresponding methods of treatment and uses.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to polypeptides for use in therapy, for example, the use of polypeptides for the treatment of pain or inflammatory disorders. [Background technology]

[0002] Bacteria in the Clostridia genus produce highly potent and specific protein toxins that can poison neurons and other cells to which they are delivered. Examples of such clostridial neurotoxins include those produced by C. tetani (TeNT) and C. botulinum (BoNT) serotypes A-G and X (see WO 2018 / 009903 A2), as well as those produced by C. baratii and C. butyricum. Both tetanus and botulinum toxins act by inhibiting the function of affected neurons, specifically neurotransmitter release. Botulinum toxin acts at the neuromuscular junction and inhibits cholinergic transmission in the peripheral nervous system, while tetanus toxin acts in the central nervous system.

[0003] In nature, clostridial neurotoxins are synthesized as single-chain polypeptides, which are post-translationally modified by a proteolytic cleavage event to form two polypeptide chains linked together by a disulfide bond. Cleavage occurs at a specific cleavage site, often referred to as the activation site, which is located between cysteine ​​residues that provide the interchain disulfide bond. It is this two-chain form that is the active form of the toxin. The two chains are designated the heavy chain (H chain), with a molecular mass of approximately 100 kDa, and the light chain (L chain), with a molecular mass of approximately 50 kDa. The H chain is an N-terminal translocation component (H N domain) and a C-terminal targeting component (H C The cleavage site is located between the L chain and translocation domain components. C Following binding of the domains and internalization of the bound toxin into the cell via endosomes, H NThe domain translocates the L chain across the endosomal membrane into the cytosol, where the L chain provides the protease function (also known as a non-cytotoxic protease).

[0004] Non-cytotoxic proteases act by proteolytically cleaving intracellular transport proteins known as SNARE proteins (e.g., SNAP-25, VAMP, or syntaxin). The acronym SNARE is derived from the term soluble NSF adhesion receptor, where NSF stands for N-ethylmaleimide-sensitive factor. SNARE proteins are essential for intracellular vesicle fusion and therefore for the secretion of molecules from cells via vesicular transport. The protease function is a zinc-dependent endopeptidase activity and displays high substrate specificity for SNARE proteins. Thus, once delivered to the desired target cell, non-cytotoxic proteases can inhibit cellular secretion from the target cell. The light-chain proteases of clostridial neurotoxins are non-cytotoxic proteases that cleave SNARE proteins.

[0005] Given the ubiquitous nature of SNARE proteins, clostridial neurotoxins, such as botulinum toxin, have been successfully employed in a wide range of therapeutic applications.

[0006] Clostridial neurotoxins are some of the most potent toxins known. For example, botulinum neurotoxins have median lethal doses (LD50s) in mice ranging from 0.5 to 5 ng / kg, depending on the serotype. 50) value. Therefore, the use of these toxins is not without risk. Spread of the toxin from the site of administration to surrounding tissues or the systemic circulation is thought to be responsible for the undesirable side effects of clostridial neurotoxin treatment, which can be life-threatening in extreme cases. This can be a particular concern when clostridial neurotoxins are used at high doses, concentrations, and / or injection volumes. Adverse effects reported with commercial BoNT / A therapeutics include asthenia, generalized muscle weakness, diplopia, ptosis, dysphagia, dysphonia, dysphagia, urinary incontinence, and respiratory distress. Difficulty swallowing and breathing can be life-threatening. And deaths related to the spread of toxin effects have been reported. Summary of the Invention

[0007] The present invention overcomes one or more of the problems noted above.

[0008] The present inventors have discovered that catalytically inactive clostridial neurotoxins are effective in treating pain. This finding is particularly surprising because catalytic activity resulting in SNARE protein cleavage is believed to be the essential mechanism of action underlying clostridial neurotoxin therapy. Therefore, the polypeptides of the present invention avoid the toxic side effects associated with conventional catalytically active clostridial neurotoxin therapy and constitute safer (substantially non-toxic) therapeutics. Advantageously, the polypeptides of the present invention can be administered in larger amounts compared to conventional catalytically active clostridial neurotoxin therapeutics. Furthermore, the reduced toxicity of the polypeptides of the present invention allows for ease of manufacture and handling during the product lifecycle and eliminates the need for physicians to calculate complex (e.g., individualized) dosing regimens aimed at avoiding toxicity in subjects.

[0009] Equally surprising, the present inventors have found that catalytically inactive Clostridial neurotoxins are effective in treating inflammatory disorders. DETAILED DESCRIPTION OF THE INVENTION

[0010] In one embodiment, the present invention provides a polypeptide (e.g., an analgesic polypeptide) for use in treating pain, wherein the polypeptide comprises a clostridial neurotoxin light chain (L chain), a clostridial neurotoxin translocation domain (H chain), or a clostridial neurotoxin translocation domain (H chain). N domain), and / or Clostridial neurotoxin receptor binding domain (H C domain), wherein when the polypeptide comprises a Clostridial neurotoxin L chain, the L chain is catalytically inactive.

[0011] In a related embodiment, a method for treating pain is provided, the method comprising administering to a subject a polypeptide (e.g., an analgesic polypeptide), wherein the polypeptide comprises a clostridial neurotoxin light chain (L chain), a clostridial neurotoxin translocation domain (H chain), ... N domain), and / or Clostridial neurotoxin receptor binding domain (H C domain), wherein when the polypeptide comprises a Clostridial neurotoxin L chain, the L chain is catalytically inactive.

[0012] In another related aspect, the present invention provides the use of a polypeptide (e.g., an analgesic polypeptide) in the manufacture of a medicament for treating pain, wherein the polypeptide comprises a Clostridial neurotoxin light chain (L chain), a Clostridial neurotoxin translocation domain (H chain), ... N domain), and / or Clostridial neurotoxin receptor binding domain (H C domain), wherein when the polypeptide comprises a Clostridial neurotoxin L chain, the L chain is catalytically inactive.

[0013] The polypeptides of the present invention preferably have analgesic properties, in other words, the polypeptides of the present invention are preferably analgesic polypeptides.

[0014] Preferably, the polypeptides of the invention do not promote neuronal growth or neuronal repair to treat pain, in other words, preferably, the polypeptides do not treat pain by means of either promoting neuronal growth, promoting neuronal repair, or promoting neuronal growth and repair.

[0015] In one embodiment, the present invention provides a polypeptide for use in treating an inflammatory disorder, wherein the polypeptide comprises a clostridial neurotoxin light chain (L chain), a clostridial neurotoxin translocation domain (H chain), or a clostridial neurotoxin translocation domain (H chain). N domain), and / or Clostridial neurotoxin receptor binding domain (H C domain), wherein when the polypeptide comprises a Clostridial neurotoxin L chain, the L chain is catalytically inactive.

[0016] In a related aspect, a method for treating an inflammatory disorder is provided, the method comprising administering to a subject a polypeptide, wherein the polypeptide comprises a clostridial neurotoxin light chain (L chain), a clostridial neurotoxin translocation domain (H chain), ... N domain), and / or Clostridial neurotoxin receptor binding domain (H C domain), wherein when the polypeptide comprises a Clostridial neurotoxin L chain, the L chain is catalytically inactive.

[0017] In another related aspect, the present invention provides for the use of a polypeptide in the manufacture of a medicament for treating an inflammatory disorder, wherein the polypeptide comprises a Clostridial neurotoxin light chain (L chain), a Clostridial neurotoxin translocation domain (H chain), ... N domain), and / or Clostridial neurotoxin receptor binding domain (H C domain), wherein when the polypeptide comprises a Clostridial neurotoxin L chain, the L chain is catalytically inactive.

[0018] The polypeptides of the present invention may have anti-inflammatory properties, in other words, the polypeptides of the present invention may be anti-inflammatory polypeptides.

[0019] When the polypeptide is used to treat an inflammatory disorder as described herein, the polypeptide may comprise the L chain of botulinum neurotoxin serotype X (BoNT / X), the H chain of BoNT / X, or the L chain of botulinum neurotoxin serotype X (BoNT / X). N domain and / or H of BoNT / X C domain, where if the polypeptide comprises a clostridial neurotoxin L chain, the L chain is catalytically inactive. For example, the polypeptide may comprise a catalytically inactive BoNT / X light chain and translocation domain and a receptor binding domain (H) from a different (i.e., non-BoNT / X) clostridial neurotoxin. C The present invention may be a chimeric botulinum neurotoxin (BoNT) comprising a light chain and translocation domain of a catalytically inactive BoNT / X and a receptor binding domain (H domain) from a different (i.e., non-BoNT / X) clostridial neurotoxin. C domain) (preferably H of BoNT / B C Corresponding methods of treatment and use are also provided.

[0020] Preferably, the polypeptides of the invention do not promote neuronal growth or neuronal repair to treat an inflammatory condition, in other words, preferably, the polypeptides do not treat an inflammatory condition by means of either promoting neuronal growth, promoting neuronal repair, or promoting neuronal growth and repair.

[0021] The term "promoting neuronal growth and / or neuronal repair" encompasses increasing the rate of neuronal growth and / or neuronal repair. The term "neuronal growth and / or neuronal repair" encompasses the reconstruction of damaged neuronal circuits, thereby restoring activity and / or neuronal connections to a network or population of neurons. Thus, the term "neuronal repair" as used herein encompasses the repair of specific neurons and the repair of neuronal circuits. The term also encompasses neuronal plasticity. The term "neuronal plasticity" as used herein encompasses axonal sprouting, dendritic sprouting, neurogenesis (e.g., the generation of new neurons), maturation, differentiation, and / or synaptic plasticity (e.g., involving alterations in synaptic strength, activity, anatomy, and / or connectivity). The term "promoting neuronal growth and / or neuronal repair" also encompasses promoting the establishment of functional synapses (e.g., at or near the site of injury). As used herein, the term "neuron growth" encompasses growth of any part of a neuron, including axonal and / or dendrite growth. The term also encompasses increased neurite length, neurite number (e.g., number of neurites per cell), and / or increased length and / or number of projections from the cell body or cell membrane of a neuron, e.g., axonal growth and / or axonal sprouting of a neuron, e.g., a neuron in a subject. Such axonal growth can facilitate connections and / or chemical communication between neurons.

[0022] Preferably, the polypeptides of the present invention do not promote a neuroimmune response to treat pain or inflammatory disorders. In this context, a neuroimmune response encompasses a microglial response. Thus, in one embodiment, the polypeptides of the present invention do not promote a microglial response to treat pain or inflammatory conditions.

[0023] In a preferred embodiment, the pain is not pain associated with or caused by a brain disorder. In another preferred embodiment, the inflammatory disorder is not an inflammatory brain disorder. The term "brain disorder" as used in this context is interchangeable with "brain disease." "Brain disorder" as used in this context encompasses disorders originating inside or outside the brain, including disorders associated with attacks on the body that cause brain tissue damage. Examples of brain disorders encompassed in this context include any one (or more) of traumatic brain injury, cancer (e.g., brain tumor), infectious disease (e.g., encephalitis, meningitis, brain abscess, and encephalitis), stroke, neurodegenerative disorders (e.g., Alzheimer's disease, Parkinson's disease, Parkinson's disease-related disorders, motor neuron diseases (e.g., amyotrophic lateral sclerosis), prion diseases, Huntington's disease, spinocerebellar ataxia, ataxia, Hallervorden-Spatz disease, and frontotemporal lobar degeneration), cerebral aneurysm, multiple sclerosis, anoxic insult, toxic insult, and metabolic insult. Brain damage can be caused by traumatic brain injury, cancer, infectious diseases (e.g., encephalitis, meningitis, brain abscess, and encephalitis), stroke, neurodegenerative disorders (e.g., Alzheimer's disease, Parkinson's disease, Parkinson's-related disorders, motor neuron diseases (e.g., amyotrophic lateral sclerosis), prion diseases, Huntington's disease, spinocerebellar ataxia, ataxia, Hallervorden-Spatz disease, and frontotemporal lobar degeneration), cerebral aneurysm, multiple sclerosis, anoxic insult, toxic insult, and / or metabolic insult.

[0024] The active clostridial neurotoxin L chain has non-cytotoxic protease activity. Specifically, the active clostridial neurotoxin L chain has endopeptidase activity and can cleave proteins of the exocytic fusion apparatus in target cells. The proteins of the exocytic fusion apparatus are preferably SNARE proteins, such as SNAP25, synaptobrevin / VAMP, or syntaxin.

[0025] The term "catalytically inactive" as used herein with respect to a clostridial neurotoxin L chain means that the L chain exhibits substantially no non-cytotoxic protease activity. Preferably, the term "catalytically inactive" as used herein with respect to a clostridial neurotoxin L chain means that the L chain exhibits substantially no non-cytotoxic protease activity. In one embodiment, a catalytically inactive clostridial neurotoxin L chain does not cleave proteins of the exocytosis fusion apparatus in target cells. The term "substantially no non-cytotoxic protease activity" means that a clostridial neurotoxin L chain has less than 5% of the non-cytotoxic protease activity of a catalytically active clostridial neurotoxin L chain, for example, less than 2%, less than 1%, or preferably less than 0.1% of the non-cytotoxic protease activity of a catalytically active clostridial neurotoxin L chain. Non-cytotoxic protease activity can be determined in vitro by incubating a test Clostridial neurotoxin L chain with a SNARE protein and comparing the amount of SNARE protein cleaved by the test Clostridial neurotoxin L chain to the amount of SNARE protein cleaved by a catalytically active Clostridial neurotoxin L chain under the same conditions. Conventional techniques such as SDS-PAGE and Western blotting can be used to quantitate the amount of cleaved SNARE protein. A suitable in vitro assay is described in WO 2019 / 145577 A1, which is incorporated herein by reference.

[0026] Cell-based and in vivo assays can also be used to determine whether a clostridial neurotoxin comprising an L chain and a functional cell-binding and translocation domain has non-cytotoxic protease activity. Assays such as the digit abduction score (DAS) assay, the dorsal root ganglion (DRG) assay, the spinal cord neuron (SCN) assay, and the mouse phrenic nerve hemidiaphragm (PNHD) assay are routine in the art. Suitable assays for determining non-cytotoxic protease activity may be those described in Aoki KR, Toxicon 39: 1815-1820; 2001 or Donald et al. (2018), Pharmacol Res Perspect, e00446, 1-14, which are incorporated herein by reference.

[0027] A catalytically inactive L chain may have one or more mutations that inactivate the catalytic activity. Therefore, a catalytically active L chain (e.g., as described herein) may be modified to introduce one or more mutations that inactivate the catalytic activity of the L chain. For example, a catalytically inactive L chain may contain a mutation of an active site residue. The mutation may be a substitution or deletion. However, substitutions, particularly substitutions with chemically similar amino acids, are preferred. Glutamic acid may be substituted with glutamine, histidine may be substituted with tyrosine, arginine may be substituted with glutamine, and / or tyrosine may be substituted with phenylalanine. Alternatively, either residue may be substituted with alanine.

[0028] The catalytically inactive BoNT / A light chain may contain mutations at H223, E224, H227, E262, R363, and / or Y366, preferably at least E224 and H227. Preferably, the catalytically inactive BoNT / A light chain may contain a substitution at E224 with glutamine (E224Q) and a substitution at H227 with tyrosine (H227Y). Position numbering corresponds to the amino acid positions of SEQ ID NO:60 and can be determined by aligning the polypeptide with SEQ ID NO:60. Because the presence of a methionine residue at position 1 of SEQ ID NO:60 is optional, one skilled in the art would take the presence / absence of the methionine residue into account when determining the amino acid residue numbering. For example, if SEQ ID NO:60 includes a methionine, the position numbering would be as defined above (e.g., His223 would become His223 in SEQ ID NO:60). Alternatively, if methionine is not present in SEQ ID NO: 60, the amino acid residue numbering should be adjusted by -1 (e.g., His223 would be His222 in SEQ ID NO: 60). Similar considerations apply when methionine is present / absent at position 1 of the other polypeptide sequences described herein, and one of ordinary skill in the art would have no difficulty determining the correct amino acid residue numbering using routine techniques in the art.

[0029] The catalytically inactive BoNT / B light chain can contain mutations at E231 and / or H234, preferably at E231 and H234. Preferably, the catalytically inactive BoNT / B light chain contains a substitution at E231 with glutamine (E231Q) and a substitution at H234 with tyrosine (H234Y). The position numbering corresponds to the amino acid positions of SEQ ID NO: 52 and can be determined by aligning the polypeptide with SEQ ID NO: 52. Because the presence of a methionine residue at position 1 of SEQ ID NO: 52 is optional, one skilled in the art would take the presence / absence of the methionine residue into account when determining the amino acid residue numbering.

[0030] The catalytically inactive BoNT / C light chain can contain mutations at H229, E230, and / or H233, preferably at H229, E230, and H233. Preferably, the catalytically inactive BoNT / C light chain contains a substitution at H229 with glycine (H229G), a substitution at E230 with threonine (E230T), and a substitution at H233 with asparagine (H233N). The position numbering corresponds to the amino acid positions of SEQ ID NO: 53 and can be determined by aligning the polypeptide with SEQ ID NO: 53. Because the presence of a methionine residue at position 1 of SEQ ID NO: 53 is optional, one skilled in the art would take the presence / absence of the methionine residue into account when determining the amino acid residue numbering.

[0031] The catalytically inactive BoNT / D light chain can contain mutations at H229, E230, H233, and / or H236, preferably at least E230 and H236. Preferably, the catalytically inactive BoNT / D light chain contains at least a substitution at E230 with glutamine (E230Q) and a substitution at H236 with tyrosine (H236Y). The position numbering corresponds to the amino acid positions of SEQ ID NO: 54 and can be determined by aligning the polypeptide with SEQ ID NO: 54. Because the presence of a methionine residue at position 1 of SEQ ID NO: 54 is optional, one skilled in the art would take the presence / absence of the methionine residue into account when determining the amino acid residue numbering.

[0032] The catalytically inactive BoNT / E light chain can contain mutations at E213 and / or H216, preferably at E213 and H216. Preferably, the catalytically inactive BoNT / E light chain contains substitutions at E213 (E213Q) with glutamine and H216 (H216Y) with tyrosine. The position numbering corresponds to the amino acid positions of SEQ ID NO: 55 and can be determined by aligning the polypeptide with SEQ ID NO: 55. Because the presence of a methionine residue at position 1 of SEQ ID NO: 55 is optional, one skilled in the art would take the presence / absence of the methionine residue into account when determining the amino acid residue numbering.

[0033] The catalytically inactive BoNT / F light chain can contain mutations at E228 and / or H231, preferably at E228 and H231. Preferably, the catalytically inactive BoNT / F light chain contains substitutions at E228 (E228Q) with glutamine and H231 (H231Y) with tyrosine. The position numbering corresponds to the amino acid positions of SEQ ID NO: 56 and can be determined by aligning the polypeptide with SEQ ID NO: 56. Because the presence of a methionine residue at position 1 of SEQ ID NO: 56 is optional, one skilled in the art would take the presence / absence of the methionine residue into account when determining the amino acid residue numbering.

[0034] The catalytically inactive BoNT / G light chain can contain mutations at E231 and / or H234, preferably at E231 and H234. Preferably, the catalytically inactive BoNT / G light chain contains substitutions at E231 (E231Q) with glutamine and H234 (H234Y) with tyrosine. The position numbering corresponds to the amino acid positions of SEQ ID NO: 57 and can be determined by aligning the polypeptide with SEQ ID NO: 57. Because the presence of a methionine residue at position 1 of SEQ ID NO: 57 is optional, one skilled in the art would take the presence / absence of the methionine residue into account when determining the amino acid residue numbering.

[0035] The catalytically inactive BoNT / X light chain can contain mutations at E228 and / or H231, preferably at E228 and H231. Preferably, the catalytically inactive BoNT / X light chain contains substitutions at E228 with glutamine (E228Q) and H231 with tyrosine (H231Y). The position numbering corresponds to the amino acid positions of SEQ ID NO: 59 and can be determined by aligning the polypeptide with SEQ ID NO: 59. Because the presence of a methionine residue at position 1 of SEQ ID NO: 59 is optional, one skilled in the art would take the presence / absence of the methionine residue into account when determining the amino acid residue numbering.

[0036] The catalytically inactive TeNT L chain may contain mutations at E234, R372, and / or Y375, preferably at least R372 and Y375 (e.g., at E234, R372, and Y375). Preferably, the catalytically inactive TeNT L chain contains a substitution at R372 with glutamine or alanine (R372Q or R372A), more preferably with alanine, and a substitution at Y375 with phenylalanine (Y375F). The position numbering corresponds to the amino acid positions of SEQ ID NO: 58 and can be determined by aligning the polypeptide with SEQ ID NO: 58. Because the presence of a methionine residue at position 1 of SEQ ID NO: 58 is optional, one skilled in the art would take the presence / absence of the methionine residue into account when determining the amino acid residue numbering.

[0037] The polypeptides of the present invention may be full-length clostridial neurotoxins (wherein the L chain is catalytically inactive) or fragments of clostridial neurotoxins that lack non-cytotoxic protease activity (e.g., H N Domain and / or H C In other words, the polypeptides of the present invention do not have non-cytotoxic protease activity.

[0038] The term "clostridial neurotoxin" encompasses toxins produced by C. botulinum (botulinum neurotoxin serotypes A, B, C1, D, E, F, G, and X), C. tetani (tetanus neurotoxin), C. butyricum (botulinum neurotoxin serotype E), and C. baratii (botulinum neurotoxin serotype F). The reference BoNT / A sequence is set forth as SEQ ID NO:51. The reference BoNT / B sequence is set forth as SEQ ID NO:52. The reference BoNT / C sequence is set forth as SEQ ID NO:53. The reference BoNT / D sequence is set forth as SEQ ID NO:54. The reference BoNT / E sequence is set forth as SEQ ID NO:55. The reference BoNT / F sequence is set forth as SEQ ID NO:56. The reference BoNT / G sequence is set forth as SEQ ID NO:57. The reference TeNT sequence is set forth as SEQ ID NO:58. The reference BoNT / X sequence is set forth as SEQ ID NO:59. The term "clostridial neurotoxin" also encompasses newly discovered members of the botulinum neurotoxin protein family expressed by non-clostridial microorganisms, such as the toxin encoded by Enterococcus that shares closest sequence identity with BoNT / X, the toxin encoded by Weissella oryzae called BoNT / Wo (NCBI RefSeq: WP_027699549.1) that cleaves VAMP2 at W89-W90, the toxin encoded by Enterococcus faecium (GenBank: OTO22244.1) that cleaves VAMP2 and SNAP25, and the toxin encoded by Chryseobacterium pipero (NCBI Ref.Seq: WP_034687872.1).

[0039] Thus, the clostridial neurotoxin may be selected from BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, BoNT / X, and TeNT (tetanus neurotoxin). Preferably, the clostridial neurotoxin is a botulinum neurotoxin, such as a botulinum neurotoxin selected from BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, and BoNT / X. For example, clostridial neurotoxin H NThe domains are A, B, C1, D, E, F, G, X from BoNT or H from TeNT. N The L chain can be an L chain from BoNT A, B, C1, D, E, F, G, X, or TeNT, provided that the L chain is catalytically inactive (e.g., modified to render it catalytically inactive). More preferably, the clostridial neurotoxin is BoNT / A.

[0040] As discussed above, (full-length) clostridial neurotoxins are formed from two polypeptide chains, a heavy chain (H chain) with a molecular mass of approximately 100 kDa and a light chain (L chain) with a molecular mass of approximately 50 kDa. The H chain contains a C-terminal targeting component (receptor binding domain or H chain). C domain) and N-terminal translocation component (H N domains). Botulinum neurotoxins (BoNTs) are produced by C. botulinum in the form of large protein complexes consisting of BoNT itself complexed with several accessory proteins. There are currently eight different classes of botulinum neurotoxins, namely botulinum neurotoxin serotypes A, B, C1, D, E, F, G, and X, all of which share a similar structure and mode of action. Different BoNT serotypes can be distinguished based on inactivation by specific neutralizing antisera. This classification by serotype correlates with the percentage sequence identity at the amino acid level. Based on the percentage sequence identity of amino acids, BoNT proteins of a given serotype can be further divided into different subtypes.

[0041] Conventional (catalytically active) BoNTs are absorbed in the gastrointestinal tract and, after entering the systemic circulation, bind to the presynaptic membranes of cholinergic nerve terminals, preventing their release of the neurotransmitter acetylcholine. BoNT / B, BoNT / D, BoNT / F, and BoNT / G cleave synaptobrevin / vesicle-associated membrane protein (VAMP), while BoNT / C1, BoNT / A, and BoNT / E cleave 25 kDa synaptosomal-associated protein (SNAP-25), and BoNT / C1 cleaves syntaxin. BoNT / X has been found to cleave SNAP-25, VAMP1, VAMP2, VAMP3, VAMP4, VAMP5, Ykt6, and syntaxin 1. Tetanus toxin is produced by a single serotype by C. tetani. C. butyricum produces BoNT / E, and C. baratii produces BoNT / F.

[0042] In one embodiment, a polypeptide of the invention can be encoded by a nucleotide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49, provided that, when the polypeptide comprises a clostridial neurotoxin L chain, the L chain is catalytically inactive. In one embodiment, a polypeptide of the invention can be encoded by a nucleotide sequence having at least 80%, 90%, 95%, or 98% sequence identity to any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, or 49, provided that, when the polypeptide comprises a clostridial neurotoxin L chain, the L chain is catalytically inactive. Preferably, a polypeptide of the invention can be encoded by a nucleotide sequence comprising any one of SEQ ID NOs: 1, 7, 9, 11, 13, 15, 17, 21, 25, 29, 33, 37, 41, 43, 45, 47, or 49.

[0043] In one embodiment, a polypeptide of the invention may comprise a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 74, 75, or 76, provided that, when the polypeptide comprises a clostridial neurotoxin light chain, the light chain is catalytically inactive. In one embodiment, a polypeptide of the invention may comprise a polypeptide sequence having at least 80%, 90%, 95%, or 98% sequence identity to any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 74, 75, or 76, provided that, when the polypeptide comprises a clostridial neurotoxin light chain, the light chain is catalytically inactive. Preferably, a polypeptide of the present invention may comprise the polypeptide sequence of any one of SEQ ID NOs: 2, 8, 10, 12, 14, 16, 18, 22, 26, 30, 34, 38, 42, 44, 46, 48, 50, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 74, 75, or 76.

[0044] In one embodiment, a polypeptide of the invention may comprise a fragment of a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 2, 10, 12, 14, 16, 18, 26, 34, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70, provided that, when the polypeptide comprises a clostridial neurotoxin light chain, the light chain is catalytically inactive. In one embodiment, a polypeptide of the invention may comprise a fragment of a polypeptide sequence having at least 80%, 90%, 95%, or 98% sequence identity to any one of SEQ ID NOs: 2, 10, 12, 14, 16, 18, 26, 34, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70, provided that, when the polypeptide comprises a clostridial neurotoxin light chain, the light chain is catalytically inactive. Preferably, a polypeptide of the invention may comprise a fragment of a polypeptide sequence comprising any one of SEQ ID NOs: 2, 10, 12, 14, 16, 18, 26, 34, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70, provided that, when the polypeptide comprises a clostridial neurotoxin L chain, the L chain is catalytically inactive. Fragments may comprise a catalytically inactive L chain, H chain, or any of the foregoing SEQ ID NOs. N Domain, or H C It can be a domain.

[0045] Preferably, the polypeptide of the present invention comprises (or consists of) a catalytically inactive clostridial neurotoxin L chain. In this context, referring to a catalytically inactive clostridial neurotoxin also encompasses a fragment of the clostridial neurotoxin L chain. A fragment of the clostridial neurotoxin L chain may have ≦400, ≦350, ≦300, ≦250, ≦200, ≦150, ≦100, or ≦50 amino acid residues of the clostridial neurotoxin L chain. In one embodiment, a fragment of the clostridial neurotoxin L chain has at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, or 200 amino acid residues of the clostridial neurotoxin L chain. For example, a fragment of the clostridial neurotoxin L chain may have 20 to 400, 50 to 300, or 100 to 200 amino acid residues of the clostridial neurotoxin L chain. However, it is preferred that reference to a catalytically inactive Clostridial neurotoxin refers to a full-length catalytically inactive Clostridial neurotoxin L chain.

[0046] Examples of light chain reference sequences include: Botulinum type A neurotoxin: amino acid residues 1-448 Botulinum type B neurotoxin: amino acid residues 1-440 Botulinum type C1 neurotoxin: amino acid residues 1-441 Botulinum type D neurotoxin: amino acid residues 1-445 Botulinum type E neurotoxin: amino acid residues 1-422 Botulinum type F neurotoxin: amino acid residues 1-439 Botulinum type G neurotoxin: amino acid residues 1-441 Tetanus neurotoxin: amino acid residues 1-457

[0047] In the recently identified BoNT / X, the light chain is reported to correspond to amino acids 1-439 thereof, with the boundaries of the light chain potentially varying by approximately 25 amino acids (eg, 1-414 or 1-464).

[0048] The reference sequences identified above should be considered as a guide, as minor variations may occur according to serosubtype. As an example, US 2007 / 0166332 (hereby incorporated by reference in its entirety) cites minor different Clostridial sequences: Botulinum type A neurotoxin: amino acid residues M1-K448 Botulinum type B neurotoxin: amino acid residues M1-K441 Botulinum type C1 neurotoxin: amino acid residues M1-K449 Botulinum type D neurotoxin: amino acid residue M1-R445 Botulinum type E neurotoxin: amino acid residue M1-R422 Botulinum type F neurotoxin: amino acid residues M1-K439 Botulinum type G neurotoxin: amino acid residues M1-K446 Tetanus neurotoxin: amino acid residues M1-A457

[0049] Suitable clostridial neurotoxin light chains are described herein.

[0050] The clostridial neurotoxin L chain may comprise a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 6, 24, 32, 40, 74, or 76, provided that the L chain is catalytically inactive (e.g., the L chain is inactivated by modification). In one embodiment, the clostridial neurotoxin L chain comprises a polypeptide sequence having at least 80%, 90%, 95%, or 98% sequence identity to any one of SEQ ID NOs: 6, 24, 32, 40, 74, or 76, provided that the L chain is catalytically inactive (e.g., the L chain is inactivated by modification). Preferably, the clostridial neurotoxin L chain comprises (more preferably consists of) a polypeptide sequence comprising any one of SEQ ID NOs: 6, 24, 32, or 40, e.g., SEQ ID NO: 74 or 76, which has been modified to catalytically inactivate the L chain.

[0051] The clostridial neurotoxin L chain can be encoded by a nucleotide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 5, 23, 31, or 39, provided that the L chain is catalytically inactive (e.g., the L chain is inactivated by modification). In one embodiment, the clostridial neurotoxin L chain is encoded by a nucleotide sequence having at least 80%, 90%, 95%, or 98% sequence identity to any one of SEQ ID NOs: 5, 23, 31, or 39, provided that the L chain is catalytically inactive (e.g., the L chain is inactivated by modification). Preferably, the clostridial neurotoxin L chain is encoded by a nucleotide sequence comprising any one of SEQ ID NOs: 5, 23, 31, or 39, which has been modified to catalytically inactivate the encoded L chain.

[0052] Since the catalytic activity of the light chain was not required for efficacy in treating pain, it is plausible that a polypeptide without an L chain (or containing only a fragment of an L chain) could treat pain. For similar reasons, it is plausible that a polypeptide without an L chain (or containing only a fragment of an L chain) could treat inflammatory conditions. Therefore, in one embodiment, the polypeptide contains a Clostridial neurotoxin translocation domain (H N domain) and / or Clostridial neurotoxin receptor binding domain (H C In one embodiment, the polypeptide of the present invention may comprise a Clostridial neurotoxin translocation domain (H domain). N domain) and Clostridial neurotoxin receptor binding domain (H C domain) is not included.

[0053] In one embodiment, the polypeptide of the present invention comprises (or consists of) a clostridial neurotoxin heavy chain (H chain). The H chain comprises a clostridial neurotoxin translocation domain (H N domain) and receptor binding domain (H CIn this context, the term "clostridial neurotoxin H chain" also encompasses a fragment of the clostridial neurotoxin H chain. A fragment of the clostridial neurotoxin H chain may have ≦800, ≦700, ≦600, ≦500, ≦400, ≦350, ≦300, ≦250, ≦200, ≦150, ≦100, or ≦50 amino acid residues of the clostridial neurotoxin H chain. In one embodiment, a fragment of the clostridial neurotoxin H chain has at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, or 200 amino acid residues of the clostridial neurotoxin H chain. For example, a fragment of a clostridial neurotoxin H chain may have 20 to 800, 30 to 600, 40 to 400, 50 to 300, or 100 to 200 amino acid residues of the clostridial neurotoxin H chain. However, it is preferred that reference to an H chain refers to a full-length H chain.

[0054] In one embodiment, the polypeptide of the invention comprises a Clostridial neurotoxin translocation domain (H N A fragment of a clostridial neurotoxin translocation domain may comprise (or consist of) a clostridial neurotoxin translocation domain. In this context, a reference to a clostridial neurotoxin translocation domain also encompasses a fragment of the translocation domain. A fragment of a clostridial neurotoxin translocation domain may have ≦400, ≦350, ≦300, ≦250, ≦200, ≦150, ≦100, or ≦50 amino acid residues of the clostridial neurotoxin translocation domain. In one embodiment, a fragment of a clostridial neurotoxin translocation domain has at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, or 200 amino acid residues of the clostridial neurotoxin translocation domain. For example, a fragment of a clostridial neurotoxin translocation domain may have 20 to 400, 50 to 300, or 100 to 200 amino acid residues of the clostridial neurotoxin translocation domain. However, it is preferred that a reference to a translocation domain is a reference to the full-length translocation domain.

[0055] The translocation domain is a fragment of the H chain of a clostridial neurotoxin approximately equivalent to the amino-terminal half of the H chain, or a domain corresponding to that fragment on an intact H chain. C The function is H C It can be removed by deletion of the amino acid sequence (either at the level of DNA synthesis or post-synthesis, by treatment with nucleases or proteases). C The function can be inactivated by chemical or biological treatment, and therefore, in some embodiments, the heavy chain may be unable to bind to the binding site on the target cell to which the native clostridial neurotoxin (i.e., the holotoxin) binds.

[0056] Examples of suitable (reference) translocation domains include: Botulinum type A neurotoxin - amino acid residues (449-871) Botulinum type B neurotoxin - amino acid residues (441-858) Botulinum type C neurotoxin - amino acid residues (442-866) Botulinum type D neurotoxin - amino acid residues (446-862) Botulinum type E neurotoxin - amino acid residues (423-845) Botulinum type F neurotoxin - amino acid residues (440-864) Botulinum type G neurotoxin - amino acid residues (442-863) Tetanus neurotoxin - amino acid residues (458-879)

[0057] The reference sequences identified above should be considered as a guide, as minor variations may occur according to serosubtype. As an example, US 2007 / 0166332 (hereby incorporated by reference in its entirety) cites minor different Clostridial sequences: Botulinum type A neurotoxin - amino acid residues (A449-K871) Botulinum type B neurotoxin - amino acid residues (A442-S858) Botulinum type C neurotoxin - amino acid residues (T450-N866) Botulinum type D neurotoxin - amino acid residues (D446-N862) Botulinum type E neurotoxin - amino acid residues (K423-K845) Botulinum type F neurotoxin - amino acid residues (A440-K864) Botulinum type G neurotoxin - amino acid residues (S447-S863) Tetanus neurotoxin - amino acid residues (S458-V879)

[0058] In the context of the present invention, various clostridial neurotoxins H containing a translocation domain N The H region from the heavy chain of a clostridial neurotoxin may be useful in embodiments of the present invention. N The region is approximately 410-430 amino acids in length and contains the translocation domain. Studies have shown that H N This demonstrates that the entire length of the region is not necessary for the translocation activity of the translocation domain. Thus, aspects of this embodiment include clostridial neurotoxin H containing a translocation domain having a length of, for example, at least 350 amino acids, at least 375 amino acids, at least 400 amino acids, or at least 425 amino acids. N Other aspects of this embodiment include a Clostridial neurotoxin H that includes a translocation domain having a length of, for example, at most 350 amino acids, at most 375 amino acids, at most 400 amino acids, or at most 425 amino acids. N It may include a region.

[0059] For further details on the genetic basis of toxin production in Clostridium botulinum and C. tetani, see Henderson et al (1997), The Clostridia: Molecular Biology and Pathogenesis, Academic Press.

[0060] Term H N is a naturally occurring neurotoxin H N portions, and modified H having non-naturally occurring amino acid sequences and / or synthetic amino acid residues.N In one embodiment, the modified H N The moiety still demonstrates the translocation function referred to above.

[0061] In one embodiment, the polypeptide of the present invention comprises a Clostridial neurotoxin receptor binding domain (H C In this context, a clostridial neurotoxin receptor-binding domain (H domain) is used. C ) means that the Clostridial neurotoxin receptor binding domain (H C ) fragments of the Clostridial neurotoxin receptor binding domain (H C ) fragments of the Clostridial neurotoxin receptor binding domain (H C In one embodiment, the clostridial neurotoxin receptor binding domain (H) may have ≦350, ≦300, ≦250, ≦200, ≦150, ≦100, or ≦50 amino acid residues. C ) fragments of the Clostridial neurotoxin receptor binding domain (H C ), at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, or 200 amino acid residues of a Clostridial neurotoxin receptor binding domain (H C ) fragments of the Clostridial neurotoxin receptor binding domain (H C However, the cytosporin receptor-binding domain (H) may have 20 to 350, 50 to 300, or 100 to 200 amino acid residues. C ) means that the full-length Clostridial neurotoxin receptor binding domain (H C ) is preferable.

[0062] Clostridial neurotoxin receptor binding domain (H C ) Examples of reference sequences include: BoNT / A - N872-L1296 BoNT / B - E859-E1291 BoNT / C1 - N867-E1291 BoNT / D - S863-E1276 BoNT / E - R846-K1252 BoNT / F - K865-E1274 BoNT / G - N864-E1297 TeNT - I880-D1315

[0063] In the recently identified BoNT / X, H C The domain is reported to correspond to amino acids 893-1306, with the domain boundaries potentially varying by approximately 25 amino acids (eg, 868-1306 or 918-1306).

[0064] Clostridial neurotoxin heavy chain (e.g., H C The light chain fragment (domain portion) may further comprise a translocation facilitating domain (or the fragment may be a translocation facilitating domain fragment), which facilitates delivery of the light chain into the cytosol of a target cell and is described, for example, in WO 08 / 008803 and WO 08 / 008805, each of which is incorporated herein by reference.

[0065] For example, the translocation-facilitating domain is CN The neurotoxin may comprise a domain or a fragment or variant thereof. More particularly, the neurotoxin may comprise a clostridial neurotoxin H CN The translocation facilitating domain may have a length of at least 200 amino acids, at least 225 amino acids, at least 50 amino acids, or at least 275 amino acids. CN The translocation facilitating domain preferably has a length of at most 200 amino acids, at most 225 amino acids, at most 250 amino acids, or at most 275 amino acids. Specific (reference) examples include: Botulinum type A neurotoxin - amino acid residues (872-1110) Botulinum type B neurotoxin - amino acid residues (859-1097) Botulinum type C neurotoxin - amino acid residues (867-1111) Botulinum type D neurotoxin - amino acid residues (863-1098) Botulinum type E neurotoxin - amino acid residues (846-1085) Botulinum type F neurotoxin - amino acid residues (865-1105) Botulinum type G neurotoxin - amino acid residues (864-1105) Tetanus neurotoxin - amino acid residues (880-1127)

[0066] The above sequence positions may vary slightly depending on the serotype / subtype. CN Further examples of domains include: Botulinum type A neurotoxin - amino acid residues (874-1110) Botulinum type B neurotoxin - amino acid residues (861-1097) Botulinum type C neurotoxin - amino acid residues (869-1111) Botulinum type D neurotoxin - amino acid residues (865-1098) Botulinum type E neurotoxin - amino acid residues (848-1085) Botulinum type F neurotoxin - amino acid residues (867-1105) Botulinum type G neurotoxin - amino acid residues (866-1105) Tetanus neurotoxin - amino acid residues (882-1127)

[0067] Preferred Clostridial Neurotoxin H C Domains are described herein.

[0068] Clostridial neurotoxin H C The domain may comprise a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 8, 22, 30, 38, 42, 44, 46, 48, or 50. In one embodiment, the Clostridial neurotoxin H CThe domain comprises a polypeptide sequence having at least 80%, 90%, 95%, or 98% sequence identity to any one of SEQ ID NOs: 8, 22, 30, 38, 42, 44, 46, 48, or 50. Preferably, the Clostridial neurotoxin H C The domain comprises (more preferably consists of) a polypeptide sequence comprising any one of SEQ ID NOs: 8, 22, 30, 38, 42, 44, 46, 48, or 50.

[0069] Clostridial neurotoxin H C The domain can be encoded by a nucleotide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 7, 21, 29, 37, 41, 43, 45, 47, or 49. In one embodiment, the Clostridial neurotoxin H C The domain is one encoded by a nucleotide sequence having at least 80%, 90%, 95%, or 98% sequence identity to any one of SEQ ID NOs: 7, 21, 29, 37, 41, 43, 45, 47, or 49. Preferably, the Clostridial neurotoxin H C The domain is one encoded by a nucleotide sequence comprising any one of SEQ ID NOs: 7, 21, 29, 37, 41, 43, 45, 47, or 49.

[0070] Any of the facilitating domains described above can be combined with any of the previously described translocation domain peptides suitable for use in the present invention. Thus, by way of example, a non-clostridial facilitating domain can be combined with a non-clostridial translocation domain peptide or a clostridial translocation domain peptide. Alternatively, a clostridial neurotoxin H CN The translocation facilitating domain may be combined with a non-clostridial translocation domain peptide. Alternatively, a clostridial neurotoxin H CN The facilitating domain may be combined with a Clostridial translocation domain peptide. Examples of these include: Botulinum type A neurotoxin - amino acid residues (449-1110) Botulinum type B neurotoxin - amino acid residues (442-1097) Botulinum type C neurotoxin - amino acid residues (450-1111) Botulinum type D neurotoxin - amino acid residues (446-1098) Botulinum type E neurotoxin - amino acid residues (423-1085) Botulinum type F neurotoxin - amino acid residues (440-1105) Botulinum type G neurotoxin - amino acid residues (447-1105) Tetanus neurotoxin - amino acid residues (458-1127)

[0071] In some embodiments, the polypeptides of the invention comprise a functional H of a clostridial neurotoxin. C In one embodiment, the polypeptide preferably lacks the last 50 C-terminal amino acids of a clostridial neurotoxin holotoxin. In another embodiment, the polypeptide preferably lacks the last 100, preferably the last 150, more preferably the last 200, particularly preferably the last 250, and most preferably the last 300 C-terminal amino acid residues of a clostridial neurotoxin holotoxin. Alternatively, the polypeptide may lack the last 50 C-terminal amino acid residues of a clostridial neurotoxin holotoxin. C The binding activity can be abolished / reduced by mutagenesis - for example, referring to BoNT / A for convenience, the modification of one or two amino acid residues (W1266 to L and Y1267 to F) on the ganglioside binding pocket can abolish / reduce the binding activity of H C This causes the region to lose its receptor-binding function. Analogous mutations can be made in non-serotype A clostridial peptide components, such as constructs based on botulinum B (W1262 to L and Y1263 to F) or botulinum E (W1224 to L and Y1225 to F) with mutations. Other mutations in the active site, such as Y1267S in botulinum type A toxin and the corresponding highly conserved residues in other clostridial neurotoxins, can be made by replacing H Cachieve the same reduction in receptor binding activity. Details of this and other mutations are described in Rummel et al (2004) (Molecular Microbiol. 51: 631-634), which is incorporated herein by reference.

[0072] Naturally occurring clostridial neurotoxins H C The peptide contains approximately 400-440 amino acid residues and is divided into two functionally distinct domains of approximately 25 kDa each: the N-terminal region (usually H CN peptide or domain) and the C-terminal region (usually H CC This fact is confirmed by the following publications, each of which is incorporated herein by reference in its entirety: Umland TC (1997) Nat. Struct. Biol. 4: 788-792;Herreros J (2000) Biochem. J. 347: 199-204;Halpern J (1993) J. Biol. 104: 359-364; Lacey DB (1998) Nat. Struct. Biol. 5: 898-902; Knapp (1998) Am. Cryst. Assoc. Abstract Papers 25: 90; Swaminathan and Eswaramoorthy (2000) Nat. Struct. Biol. 7: 1751-1759; and Rummel A (2004) Mol. Microbiol. 51(3), 631-643. Furthermore, the C-terminal region (H CC It is well established that the heavy chain H is responsible for the binding of clostridial neurotoxins to their natural cellular receptors, i.e., to the nerve endings of the neuromuscular junction—a fact also confirmed by the above publications. Therefore, in this specification, we refer to the heavy chain H as a functional heavy chain H that is unable to bind to the cell surface receptor to which the natural clostridial neurotoxins bind. CThe term "clostridial heavy chain lacking a peptide (or domain)" means that the clostridial heavy chain is simply a functional H CC In other words, to reduce its natural binding ability to nerve endings at the neuromuscular junction, CC Peptide regions may be partially or entirely deleted or otherwise modified (eg, by conventional chemical or proteolytic treatment).

[0073] Therefore, in one embodiment, the Clostridial neurotoxin H of the present invention N The peptides consist of the C-terminal peptide portion (H CC ) and therefore lacks the H of natural clostridial neurotoxins. C For example, in one embodiment, a C-terminally extended Clostridium H N The peptide lacks the C-terminal 40, 60, 80, 100, 120, 140, 150, or 160 amino acid residues of the Clostridial neurotoxin heavy chain. N The peptides consist of the entire C-terminal peptide portion of the clostridial neurotoxin (H CC ) and therefore lacks the H of natural clostridial neurotoxins. C By way of example, in one embodiment, Clostridium H N The peptide lacks the C-terminal 165 amino acid residues, or the C-terminal 170 amino acid residues, or the C-terminal 175 amino acid residues, or the C-terminal 180 amino acid residues, or the C-terminal 185 amino acid residues, or the C-terminal 190 amino acid residues, or the C-terminal 195 amino acid residues of the Clostridial neurotoxin heavy chain. N The peptide is a Clostridium H selected from the group consisting of: CC Lacking a reference sequence: Botulinum type A neurotoxin - amino acid residues (Y1111-L1296) Botulinum type B neurotoxin - amino acid residues (Y1098-E1291) Botulinum type C neurotoxin - amino acid residues (Y1112-E1291) Botulinum type D neurotoxin - amino acid residues (Y1099-E1276) Botulinum type E neurotoxin - amino acid residues (Y1086-K1252) Botulinum type F neurotoxin - amino acid residues (Y1106-E1274) Botulinum type G neurotoxin - amino acid residues (Y1106-E1297) Tetanus neurotoxin - amino acid residues (Y1128-D1315).

[0074] The reference sequences identified above should be considered as a guide, as minor variations may occur according to serosubtype.

[0075] In other embodiments, H C Fragments of the domain may be any of the H CC It may comprise a peptide.

[0076] The polypeptide of the present invention comprises a catalytically inactive clostridial neurotoxin L chain and a clostridial neurotoxin translocation domain (H N domain) and / or Clostridial neurotoxin receptor binding domain (H C For example, the polypeptide may comprise a catalytically inactive Clostridial neurotoxin L chain and a Clostridial neurotoxin translocation domain (H N ).

[0077] Suitable polypeptides comprising a catalytically inactive Clostridial neurotoxin light chain and translocation domain are described herein.

[0078] A polypeptide comprising a clostridial neurotoxin L chain and a translocation domain may comprise a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 4, 20, 28, 36, or 75, provided that the L chain is catalytically inactive (e.g., the L chain has been inactivated by modification). In one embodiment, a polypeptide comprising a clostridial neurotoxin L chain and a translocation domain comprises a polypeptide sequence having at least 80%, 90%, 95%, or 98% sequence identity to any one of SEQ ID NOs: 4, 20, 28, 36, or 75, provided that the L chain is catalytically inactive (e.g., the L chain has been inactivated by modification). Preferably, a polypeptide comprising a clostridial neurotoxin L chain and a translocation domain comprises (more preferably consists of) a polypeptide sequence comprising any one of SEQ ID NOs: 4, 20, 28, 36, or 75, e.g., SEQ ID NO: 75, which has been modified to catalytically inactivate the L chain.

[0079] A polypeptide comprising (or consisting of) a clostridial neurotoxin L chain and a translocation domain can be encoded by a nucleotide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 3, 19, 27, or 35, provided that the L chain is catalytically inactive (e.g., the L chain has been inactivated by modification). In one embodiment, a polypeptide comprising (or consisting of) a clostridial neurotoxin L chain and a translocation domain is encoded by a nucleotide sequence having at least 80%, 90%, 95%, or 98% sequence identity to any one of SEQ ID NOs: 3, 19, 27, or 35, provided that the L chain is catalytically inactive (e.g., the L chain has been inactivated by modification). Preferably, a polypeptide comprising (or consisting of) a clostridial neurotoxin L chain and a translocation domain is encoded by a nucleotide sequence comprising any one of SEQ ID NOs: 3, 19, 27, or 35, which has been modified to catalytically inactivate the encoded L chain.

[0080] Preferably, the polypeptide comprises a catalytically inactive Clostridial neurotoxin L chain, a Clostridial neurotoxin translocation domain (H N domain), and Clostridial neurotoxin receptor binding domain (H C domain).

[0081] In one embodiment, the polypeptide of the present invention comprises a Clostridial neurotoxin receptor binding domain (H C ) or at least the C-terminal portion of the Clostridial neurotoxin receptor-binding domain (H CC ) in one embodiment. Therefore, in one embodiment, the polypeptide of the present invention does not contain the C-terminal portion (H CC Advantageously, such polypeptides lack endogenous Clostridial neurotoxin receptor binding ability and may therefore exhibit fewer off-target effects in subjects administered the polypeptide.

[0082] The polypeptides of the present invention comprise a catalytically inactive clostridial neurotoxin light chain (L chain), a clostridial neurotoxin translocation domain (H chain), and a clostridial neurotoxin translocation domain (H chain). N domain), and / or Clostridial neurotoxin receptor binding domain (H C For example, the polypeptide may consist essentially of a catalytically inactive Clostridial neurotoxin L chain and a Clostridial neurotoxin translocation domain (H N ) can consist essentially of

[0083] Preferably, the polypeptide comprises a catalytically inactive Clostridial neurotoxin L chain, a Clostridial neurotoxin translocation domain (H N domain), and Clostridial neurotoxin receptor binding domain (H C It essentially consists of a set of domains.

[0084] The term "consisting essentially of" as used in this context means that the polypeptide does not further comprise one or more amino acid residues that confer additional functionality to the polypeptide, for example, when administered to a subject. In other words, the polypeptide does not comprise a catalytically inactive clostridial neurotoxin light chain (L chain), a clostridial neurotoxin translocation domain (H chain), or a clostridial neurotoxin translocation domain (H chain). N domain), and / or Clostridial neurotoxin receptor binding domain (H C A polypeptide "consisting essentially of" a clostridial neurotoxin light chain (L chain), a clostridial neurotoxin translocation domain (H chain), or a clostridial neurotoxin light chain (L chain) is "essentially composed of" a clostridial neurotoxin light chain (L chain), a clostridial neurotoxin translocation domain (H chain), or a clostridial neurotoxin light chain (L chain). N domain), and / or Clostridial neurotoxin receptor binding domain (H C Although the one or more additional amino acid residues may be included in one of the polypeptides (e.g., one of the polypeptide domains), the one or more additional amino acid residues do not confer additional functionality to the polypeptide, e.g., when administered to a subject. Additional functionality may include any enzymatic activity, binding activity, and / or any biological activity.

[0085] The polypeptides of the present invention comprise a catalytically inactive clostridial neurotoxin light chain (L chain), a clostridial neurotoxin translocation domain (H chain), and a clostridial neurotoxin translocation domain (H chain). N domain), and / or Clostridial neurotoxin receptor binding domain (H C For example, the polypeptide may consist of a catalytically inactive Clostridial neurotoxin L chain and a Clostridial neurotoxin translocation domain (H N )

[0086] Preferably, the polypeptide comprises a catalytically inactive Clostridial neurotoxin L chain, a Clostridial neurotoxin translocation domain (H N domain), and Clostridial neurotoxin receptor binding domain (H C domain).

[0087] In one embodiment, a polypeptide can contain a non-clostridial neurotoxin sequence in addition to any clostridial neurotoxin sequence, so long as the non-clostridial neurotoxin sequence (the sequence that is not a clostridial neurotoxin) does not block the ability of the polypeptide to achieve its therapeutic effect (e.g., to treat pain). Preferably, the non-clostridial neurotoxin sequence does not have catalytic activity, e.g., enzymatic activity. In one embodiment, the polypeptide of the present invention does not contain a catalytically active domain (e.g., a non-clostridial catalytically active domain). In one embodiment, the non-clostridial sequence does not bind to a cellular receptor. In other words, in one embodiment, the non-clostridial sequence is not a ligand for a cellular receptor. The cellular receptor can be a proteinaceous cellular receptor, such as an integral membrane protein. Examples of cellular receptors can be found in the IUPHAR Guide to Pharmacology Database, version 2019.4, available at https: / / www.guidetopharmacology.org / download.jsp#db_reports. Non-clostridial neurotoxin sequences can include tags to aid in purification, such as His tags. In one embodiment, the polypeptides of the invention do not contain a tag or a site for adding a label, such as a sortase acceptor or donor site.

[0088] In a preferred embodiment, the polypeptide of the invention comprises a catalytically inactive clostridial neurotoxin L chain, H N Domain and / or H C The domains do not include additional therapeutic or diagnostic agents (e.g., nucleic acids, proteins, peptides, or small molecule therapeutic or diagnostic agents). For example, in one embodiment, the polypeptides may not include a covalently or non-covalently associated therapeutic or diagnostic agent. Thus, the polypeptides of the invention preferably do not function as a delivery vehicle for additional therapeutic or diagnostic agents.

[0089] The polypeptides of the present invention may comprise (or consist of) modified clostridial neurotoxins or derivatives thereof, including but not limited to those described below, or modified clostridial neurotoxin fragments or derivative fragments, provided that any L chains present are catalytically inactive. The modified clostridial neurotoxin or derivative (or modified clostridial neurotoxin fragments or derivative fragments) may contain one or more amino acids that are modified compared to the native (unmodified) form of the clostridial neurotoxin (or clostridial neurotoxin fragment), or may contain one or more inserted amino acids that are not present in the native (unmodified) form of the clostridial neurotoxin (or clostridial neurotoxin fragment). For example, the modified clostridial neurotoxin (or clostridial neurotoxin fragment) may have an altered amino acid sequence in one or more domains relative to the native (unmodified) clostridial neurotoxin sequence (or clostridial neurotoxin fragment). Such modifications may alter the functional aspects of the toxin (or toxin fragment). Thus, in one embodiment, the polypeptide of the present invention is a modified Clostridial neurotoxin, or a modified Clostridial neurotoxin derivative, or a Clostridial neurotoxin derivative, or a modified Clostridial neurotoxin fragment or derivative fragment (e.g., a catalytically inactive L chain, an H chain, or a modified Clostridial neurotoxin fragment or derivative fragment). N Domain and / or H C domain), provided that any L chains present are catalytically inactive.

[0090] The polypeptides of the present invention are modified clostridial neurotoxins or clostridial neurotoxin fragments (e.g., H) having one or more modifications in the amino acid sequence of the heavy chain. C domain) (e.g., modified H CThe modified heavy chain may comprise (or consist of) a clostridial neurotoxin fragment (H domain), wherein the modified heavy chain binds to a target neuronal cell with greater or less affinity than a native (unmodified) clostridial neurotoxin or clostridial neurotoxin fragment, provided that any L chain present is catalytically inactive. C Such modifications on the domain modulate binding to ganglioside receptors and / or protein receptors on target neurons. C This may involve modifying residues on the ganglioside binding site or protein (SV2 or synaptotagmin) binding site of the domain. Examples of such modified Clostridial neurotoxins are described in WO 2006 / 027207 and WO 2006 / 114308, both of which are incorporated herein by reference in their entirety.

[0091] The modified clostridial neurotoxin (or clostridial neurotoxin fragment) may comprise one or more modifications that increase the isoelectric point of the clostridial neurotoxin compared to an equivalent unmodified clostridial neurotoxin (or clostridial neurotoxin fragment) that lacks the one or more modifications, provided that any present L chain is catalytically inactive. Suitable modified clostridial neurotoxins (provided that any present L chain is modified to be catalytically inactive) are described below and in WO 2015 / 004461 A1 and WO 2016 / 110662 A1, which are incorporated herein by reference. Exemplary sequences include SEQ ID NOs: 42 and 62 described herein.

[0092] In one embodiment, the polypeptides of the present invention comprise a modified BoNT / A or a fragment thereof (e.g., the H of BoNT / A). Cdomain or fragment thereof). The modified BoNT / A or fragment thereof can contain a modification at one or more amino acid residue(s) selected from ASN886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, SER955, GLN991, GLU992, GLN995, ASN1006, ASN1025, ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, HIS1064, ASN1080, GLU1081, GLU1083, ASP1086, ASN1188, ASP1213, GLY1215, ASN1216, GLN1229, ASN1242, ASN1243, SER1274, and THR1277.

[0093] The alterations can be alterations relative to a catalytically inactive BoNT / A shown as SEQ ID NO:2, where the amino acid residue numbering is determined by alignment with SEQ ID NO:2. Because the presence of a methionine residue at position 1 of SEQ ID NO:2 (and SEQ ID NOs corresponding to the modified BoNT / A polypeptides or fragments thereof described herein) is optional, one of skill in the art would take the presence / absence of the methionine residue into account when determining the amino acid residue numbering. For example, if SEQ ID NO:2 includes a methionine, the position numbering would be as defined above (e.g., ASN886 would be ASN886 in SEQ ID NO:2). Alternatively, if a methionine is not present in SEQ ID NO:2, the amino acid residue numbering should be adjusted by -1 (e.g., ASN886 would become ASN885 in SEQ ID NO:2). Similar considerations apply when a methionine is present / absent at position 1 of the other polypeptide sequences described herein, and one of skill in the art would have no difficulty determining the correct amino acid residue numbering using routine techniques in the art.

[0094] The alignments described herein for determining amino acid residue numbering may be performed using any of the methods described herein for determining sequence homology and / or % sequence identity.

[0095] The amino acid residue(s) shown for the above modifications are surface-exposed amino acid residue(s).

[0096] The modified BoNT / A or fragment thereof can contain a modification in one or more amino acid residue(s) selected from ASN886, ASN930, ASN954, SER955, GLN991, ASN1025, ASN1026, ASN1052, ASN1188, ASP1213, GLY1215, ASN1216, GLN1229, ASN1242, ASN1243, SER1274, and THR1277.

[0097] The term "one or more amino acid residue(s)," when used in the context of a modified BoNT / A or a fragment thereof, preferably means at least 2, 3, 4, 5, 6, or 7 of the indicated amino acid residue(s). Thus, a modified BoNT / A or a fragment thereof can contain at least 2, 3, 4, 5, 6, or 7 (preferably 7) modifications to the indicated amino acid residue(s). A modified BoNT / A or a fragment thereof can contain 1 to 30, 3 to 20, or 5 to 10 amino acid modifications. More preferably, the term "one or more amino acid residue(s)," when used in the context of a modified BoNT / A or a fragment thereof, means all of the indicated amino acid residue(s).

[0098] Preferably, other than the one or more amino acid modification(s) at the indicated amino acid residue(s), the modified BoNT / A or fragment thereof does not contain any additional amino acid modifications when compared to SEQ ID NO:2.

[0099] The modification may be selected from the following: i. Substitution of an acidic, surface-exposed amino acid residue with a basic amino acid residue; ii. Substitution of acidic surface-exposed amino acid residues with uncharged amino acid residues; iii. Substitution of uncharged surface-exposed amino acid residues with basic amino acid residues; iv. insertion of a basic amino acid residue; and v. Deletion of acidic surface-exposed amino acid residues.

[0100] The modifications set forth above result in a modified BoNT / A or fragment thereof having an increased positive surface charge and an increased isoelectric point when compared to the corresponding unmodified BoNT / A or fragment thereof.

[0101] The isoelectric point (pI) is a specific property of a given protein. As is well known in the art, proteins are made up of specific sequences of amino acids (also referred to as amino acid residues when on a protein). Each amino acid in the standard set of 20 has a different side chain (or R group), meaning that each amino acid residue on a protein exhibits different chemical properties, such as charge and hydrophobicity. These properties can be affected by the surrounding chemical environment, such as temperature and pH. The overall chemical characteristics of a protein will depend on the sum of these various factors.

[0102] Certain amino acid residues (discussed in more detail below) possess ionizable side chains that can assume a charge depending on the surrounding pH. Whether such a side chain is charged at a given pH depends on the pKa of the ionizable moiety in question, where pKa is the negative logarithm of the acid dissociation constant (Ka) for a given proton from the conjugate base.

[0103] For example, acidic residues such as aspartic acid and glutamic acid have side chain carboxylic acid groups with pKa values ​​of approximately 4.1 (the exact pKa value may depend on temperature, ionic strength, and the microenvironment of the ionizable group). Therefore, these side chains display a negative charge at a pH of 7.4 (often referred to as "physiological pH"). At lower pH values, these side chains will become protonated and lose their charge.

[0104] Conversely, basic residues such as lysine and arginine have nitrogen-containing side chains with pKa values ​​of approximately 10-12. Thus, these side chains display a positive charge at a pH of 7.4. These side chains will become deprotonated and lose their charge at higher pH values.

[0105] Thus, the overall (net) charge of a protein molecule depends on the number of acidic and basic residues present on the protein (and their degree of surface exposure) and the surrounding pH. Changing the surrounding pH changes the overall charge on the protein. Thus, for each protein, there is a given pH at which the number of positive and negative charges is equal and the protein exhibits no overall net charge. This point is known as the isoelectric point (pI). Isoelectric point is a standard concept in protein biochemistry and those skilled in the art will be familiar with it.

[0106] Thus, the isoelectric point (pI) is defined as the pH value at which a protein exhibits a net charge of zero. An increase in pI means that a higher pH value is required for the protein to exhibit a net charge of zero. Therefore, an increase in pI represents an increase in the net positive charge of the protein at a given pH. Conversely, a decrease in pI means that a lower pH value is required for the protein to exhibit a net charge of zero. Therefore, a decrease in pI represents a decrease in the net positive charge of the protein at a given pH.

[0107] Methods for determining the pI of a protein are known in the art and will be familiar to those skilled in the art. For example, the pI of a protein can be calculated from the average pKa value of each amino acid present on the protein ("calculated pI"). Such calculations can be performed using computer programs known in the art, such as the Compute-pI / MW tool from ExPASy (https: / / web.expasy.org / compute_pi / ). This is a preferred method for calculating pI according to the present invention. Comparisons of pI values ​​between different molecules should be made using the same calculation technique / program.

[0108] Where appropriate, the calculated pI of a protein can be confirmed experimentally using the technique of isoelectric focusing ("observed pI"). This technique uses electrophoresis to separate proteins according to their pI. Isoelectric focusing is typically performed using a gel with an immobilized pH gradient. When an electric field is applied, the protein migrates through the pH gradient until it reaches a pH at which it has a net charge of zero. This point is the protein's pI. The results provided by isoelectric focusing are typically relatively low resolution in nature; therefore, the inventors believe that the results provided by calculated pI (as described above) are more appropriate to use.

[0109] In this specification, unless otherwise stated, "pI" means "calculated pI."

[0110] The pI of a protein can be increased or decreased by modulating the number of basic and / or acidic groups displayed on its surface. This can be achieved by modifying one or more amino acids of the protein. For example, an increase in pI can be provided by reducing the number of acidic residues or by increasing the number of basic residues.

[0111] A modified BoNT / A or fragment thereof of the present invention can have a pI value that is at least 0.2, 0.4, 0.5, or 1 pI unit higher than that of a catalytically inactive BoNT / A (e.g., SEQ ID NO: 2) or fragment thereof. Preferably, the modified BoNT / A or fragment thereof can have a pI of at least 6.6, e.g., at least 6.8.

[0112] The properties of the 20 common amino acids are shown in the table below: [Table A]

[0113] The following amino acids are considered charged amino acids: aspartic acid (negative), glutamic acid (negative), arginine (positive), and lysine (positive).

[0114] At a pH of 7.4, the side chains of aspartic acid (pKa 3.1) and glutamic acid (pKa 4.1) are negatively charged, while the side chains of arginine (pKa 12.5) and lysine (pKa 10.8) are positively charged. Aspartic acid and glutamic acid are called acidic amino acid residues. Arginine and lysine are called basic amino acid residues.

[0115] The following amino acids are considered uncharged polar (meaning they can participate in hydrogen bonding) amino acids: asparagine, glutamine, histidine, serine, threonine, tyrosine, cysteine, methionine, and tryptophan.

[0116] The following amino acids are considered uncharged hydrophobic amino acids: alanine, valine, leucine, isoleucine, phenylalanine, proline, and glycine.

[0117] In an amino acid insertion, an additional amino acid residue (one not normally present) is incorporated onto a BoNT / A polypeptide sequence or fragment thereof, thereby increasing the total number of amino acid residues on said sequence. In an amino acid deletion, an amino acid residue is removed from a Clostridial toxin amino acid sequence, thereby reducing the total number of amino acid residues on said sequence.

[0118] Preferably, the modification is a substitution, which advantageously maintains the same number of amino acid residues in the modified BoNT / A or fragment thereof. In an amino acid substitution, an amino acid residue that forms part of the BoNT / A polypeptide sequence or a fragment thereof is replaced with a different amino acid residue. The replacement amino acid residue can be one of the 20 standard amino acids described above. Alternatively, the replacement amino acid in the amino acid substitution can be a non-standard amino acid (an amino acid that is not part of the standard set of 20 described above). By way of example, the replacement amino acid can be a basic non-standard amino acid, such as L-ornithine, L-2-amino-3-guanidinopropionic acid, or the D-isomers of lysine, arginine, and ornithine. Methods for introducing non-standard amino acids into proteins are known in the art and include recombinant protein synthesis using the E. coli auxotrophic expression host.

[0119] In one embodiment, the substitution is selected from substitution of an acidic amino acid residue with a basic amino acid residue, substitution of an acidic amino acid residue with an uncharged amino acid residue, and substitution of an uncharged amino acid residue with a basic amino acid residue. In one embodiment, where the substitution is substitution of an acidic amino acid residue with an uncharged amino acid residue, the acidic amino acid residue is replaced by its corresponding uncharged amide amino acid residue (i.e., aspartic acid is replaced by asparagine, glutamic acid is replaced by glutamine).

[0120] Preferably, the basic amino acid residue is a lysine or arginine residue. In other words, the substitution is with lysine or arginine. Most preferably, the modification is with lysine.

[0121] Preferably, the modified BoNT / A or fragment thereof for use in the present invention is Clostridial toxin H CNThe modified BoNT / A or a fragment thereof preferably contains 4 to 40 amino acid modifications located in the domain. The modified BoNT / A or a fragment thereof also preferably has a pI of at least 6.6. The modified BoNT / A preferably contains at least four amino acid modifications selected from ASN886, ASN930, ASN954, SER955, GLN991, ASN1025, ASN1026, and ASN1052, where the modifications include amino acid substitutions with lysine or arginine residues. For example, the modified BoNT / A or a fragment thereof may contain at least five amino acid modifications selected from ASN886, ASN930, ASN954, SER955, GLN991, ASN1025, ASN1026, ASN1052, and GLN1229, where the modifications include amino acid substitutions with lysine or arginine residues.

[0122] Methods for modifying proteins by substitution, insertion, or deletion of amino acid residues are known in the art. For example, amino acid modifications can be introduced by modifying the DNA sequence encoding the polypeptide (e.g., encoding unmodified BoNT / A or a fragment thereof). This can be accomplished using standard molecular cloning techniques. For example, by site-directed mutagenesis, in which short strands of DNA (oligonucleotides) encoding the desired amino acid(s) are used to replace the original coding sequence using polymerase enzymes, or by inserting / deleting portions of a gene with various enzymes (e.g., ligases and restriction endonucleases). Alternatively, modified gene sequences can be chemically synthesized.

[0123] In one embodiment, a polypeptide for use in accordance with the invention comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 42 and / or a polypeptide sequence encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 41. In one embodiment, a polypeptide for use in accordance with the invention comprises a polypeptide sequence having at least 80%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 42. Preferably, a polypeptide for use in accordance with the invention comprises the polypeptide sequence set forth as SEQ ID NO: 42. In one embodiment, a polypeptide for use in accordance with the invention comprises a polypeptide sequence encoded by a nucleotide sequence having at least 80%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 41. Preferably, a polypeptide for use in accordance with the invention comprises the polypeptide sequence encoded by the nucleotide sequence set forth as SEQ ID NO: 41.

[0124] In one embodiment, a polypeptide for use in accordance with the invention comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 62. In one embodiment, a polypeptide for use in accordance with the invention comprises a polypeptide sequence having at least 80%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 62. Preferably, a polypeptide for use in accordance with the invention comprises (more preferably consists of) the polypeptide sequence set forth as SEQ ID NO: 62.

[0125] SEQ ID NO: 42 is an example of a modified BoNT / A fragment, and SEQ ID NO: 62 is an example of a catalytically inactive modified BoNT / A polypeptide. Such modified BoNT / A polypeptides and fragments are particularly preferred for use in the present invention. The polypeptides set forth as SEQ ID NOs: 42 and 62 have several amino acid modifications (e.g., substitutions) compared to wild-type BoNT / A, which increase the isoelectric point of the polypeptide. Without wishing to be bound by theory, it is believed that the increased net positive charge promotes electrostatic interactions between the polypeptide and anionic extracellular components, thereby promoting binding between the polypeptide and the cell surface, and therefore increasing retention at the site of administration and / or duration of action. Therefore, it is postulated that treatment with SEQ ID NOs: 42 and 62 will be improved compared to equivalent polypeptides lacking the modifications.

[0126] In one embodiment, a polypeptide comprising a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 42 or 62 and / or comprising a polypeptide sequence encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 41 comprises substitutions at one or more (preferably two or more, three or more, four or more, five or more, or six or more, more preferably all) of positions 930, 955, 991, 1026, 1052, 1229, and 886.

[0127] Preferably, a polypeptide comprising a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 42 or 62 and / or comprising a polypeptide sequence encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 41 comprises a lysine or arginine (more preferably, lysine) at one or more of positions 930, 955, 991, 1026, 1052, 1229, and 886. In one embodiment, the polypeptide comprises a lysine or arginine (more preferably, lysine) at at least two, three, four, five, six, or all of positions 930, 955, 991, 1026, 1052, 1229, and 886. Most preferably, the polypeptide comprises a lysine or arginine (more preferably, lysine) at all of positions 930, 955, 991, 1026, 1052, 1229, and 886.

[0128] In one embodiment, the Clostridial neurotoxin H for use in the present invention C The H domain of the modified BoNT / A C The modified BoNT / A domain includes one or more amino acid residues selected from Y1117, F1252, H1253, and L1278. For example, the modified BoNT / A H C The domain may include one or more (preferably two or more) of the following modifications: Y1117V, F1252Y, H1253K, and L1278F or L1278H.

[0129] In one embodiment, the H of the modified BoNT / A C The domain contains the modifications Y1117V and H1253K, or Y1117V, F1252Y, H1253K, and L1278F, or Y1117V, F1252Y, H1253K, and L1278H.

[0130] Preferably, the H of the modified BoNT / A C The domain contains the modifications Y1117V and H1253K, or Y1117V, F1252Y, H1253K, and L1278H.

[0131] The alterations may be alterations relative to catalytically inactive BoNT / A shown as SEQ ID NO:2, where the amino acid residue numbering is determined by alignment with SEQ ID NO:2. Because the presence of a methionine residue at position 1 of SEQ ID NO:2 is optional, one of skill in the art would take the presence / absence of the methionine residue into account when determining the amino acid residue numbering. For example, if SEQ ID NO:2 includes a methionine, the position numbering would be as defined above (e.g., Y1117 would align with Y1117 of SEQ ID NO:2). Alternatively, if a methionine is not present in SEQ ID NO:2, the amino acid residue numbering should be corrected by -1 (e.g., Y1117 would align with Y1116 of SEQ ID NO:2). Similar considerations apply when a methionine is present / absent at position 1 of the other polypeptide sequences described herein, and one of skill in the art would have no difficulty determining the correct amino acid residue numbering using routine techniques in the art.

[0132] Modified BoNT / A H C The domain can comprise a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 46, 48, or 50, except for the H domain of the modified BoNT / A. C The domain contains the modifications described above. In one embodiment, the H of the modified BoNT / A C The domain comprises a polypeptide sequence having at least 80%, 90%, 95%, or 98% sequence identity to any one of SEQ ID NOs: 46, 48, or 50, except that the H domain of the modified BoNT / A C The domain contains the modifications described above. In one embodiment, the H of the modified BoNT / A C The domain comprises a polypeptide sequence having at least 99% or 99.9% sequence identity to any one of SEQ ID NOs: 46, 48, or 50, except that the H domain of the modified BoNT / A C The domain contains the modifications described above. Preferably, the modified BoNT / A H domain CThe domain comprises (and more preferably consists of) a polypeptide sequence comprising any one of SEQ ID NOs: 46, 48, or 50.

[0133] Modified BoNT / A H C The domain can comprise a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 46 or 50, provided that the H domain of the modified BoNT / A C The domain contains the modifications described above. In one embodiment, the H of the modified BoNT / A C The domain comprises a polypeptide sequence having at least 80%, 90%, 95%, or 98% sequence identity to any one of SEQ ID NOs: 46 or 50, except that the H of the modified BoNT / A C The domain contains the modifications described above. In one embodiment, the H of the modified BoNT / A C The domain comprises a polypeptide sequence having at least 99% or 99.9% sequence identity to any one of SEQ ID NOs: 46 or 50, except that the H of the modified BoNT / A C The domain contains the modifications described above. Preferably, the modified BoNT / A H domain C The domain comprises (and more preferably consists of) a polypeptide sequence comprising any one of SEQ ID NOs: 46 or 50.

[0134] Modified BoNT / A H C The domain can be encoded by a nucleotide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 45, 47, or 49, except for the H domain of the modified BoNT / A. C The domain contains the modifications described above. In one embodiment, the H of the modified BoNT / A C The domain is encoded by a nucleotide sequence having at least 80%, 90%, 95%, or 98% sequence identity to any one of SEQ ID NOs: 45, 47, or 49, except that the H domain of the modified BoNT / A CThe domain contains the modifications described above. In one embodiment, the H of the modified BoNT / A C The domain is encoded by a nucleotide sequence having at least 99% or 99.9% sequence identity to any one of SEQ ID NOs: 45, 47, or 49, except for the H domain of the modified BoNT / A. C The domain contains the modifications described above. Preferably, the modified BoNT / A H domain C The domain is one encoded by any one of SEQ ID NOs: 45, 47, or 49.

[0135] Modified BoNT / A H C The domain can be encoded by a nucleotide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 45 or 49, provided that the H domain of the modified BoNT / A is not C The domain contains the modifications described above. In one embodiment, the H of the modified BoNT / A C The domain is encoded by a nucleotide sequence having at least 80%, 90%, 95%, or 98% sequence identity to any one of SEQ ID NOs: 45 or 49, except for the H domain of the modified BoNT / A. C The domain contains the modifications described above. In one embodiment, the H of the modified BoNT / A C The domain is encoded by a nucleotide sequence having at least 99% or 99.9% sequence identity to any one of SEQ ID NOs: 45 or 49, except for the H domain of the modified BoNT / A. C The domain contains the modifications described above. Preferably, the modified BoNT / A H domain C The domain is that encoded by any one of SEQ ID NOs: 45 or 49.

[0136] The polypeptides of the present invention may comprise (or consist of) hybrid or chimeric clostridial neurotoxins (or fragments of hybrid or chimeric clostridial neurotoxins), provided that the L chain, when present, is catalytically inactive. A hybrid clostridial neurotoxin comprises at least a portion of the light chain from one clostridial neurotoxin or its subtype and at least a portion of the heavy chain from another clostridial neurotoxin or clostridial neurotoxin subtype. In one embodiment, a hybrid clostridial neurotoxin may contain the entire light chain from one clostridial neurotoxin subtype and a heavy chain from another clostridial neurotoxin subtype, provided that the L chain, when present, is catalytically inactive. In another embodiment, a chimeric clostridial neurotoxin may contain a portion of the heavy chain (e.g., the binding domain) of one clostridial neurotoxin subtype, and another portion of the heavy chain may be from another clostridial neurotoxin subtype. Similarly or alternatively, the therapeutic agent component can comprise a light chain portion from a different clostridial neurotoxin, provided that the L chain, when present, is catalytically inactive. Such hybrid or chimeric clostridial neurotoxins are useful, for example, as a means of delivering the therapeutic benefits of such clostridial neurotoxins to patients who are immunologically resistant to a given clostridial neurotoxin subtype, to patients who may have lower-than-average concentrations of receptors for a given clostridial neurotoxin heavy chain-binding domain, or to patients who may have protease-resistant mutants of membrane or vesicular toxin substrates (e.g., SNAP-25, VAMP, and syntaxin). Hybrid and chimeric clostridial neurotoxins are described in U.S. Pat. No. 8,071,110, which publication is incorporated herein by reference in its entirety. Thus, in one embodiment, the polypeptide of the present invention is or comprises a hybrid clostridial neurotoxin or chimeric clostridial neurotoxin, provided that the L chain is catalytically inactive.

[0137] In a particularly preferred embodiment, the polypeptide of the invention comprises a catalytically inactive BoNT / A light chain and translocation domain (LH N domain) and the BoNT / B receptor binding domain (H C The chimeric clostridial neurotoxin may be a chimeric clostridial neurotoxin comprising (preferably consisting of) the L-chain (L-domain) or a portion thereof. Suitable chimeric and / or hybrid clostridial neurotoxins may be those taught in WO 2017 / 191315 A1, which is incorporated herein by reference, except that the L-chain is catalytically inactive (e.g., inactivated by modification). Preferred such sequences include SEQ ID NOs: 44 and 61.

[0138] Catalytically inactive BoNT / A LH N The domain covalently binds to the H C The chimeric BoNT / A may be linked to a domain. Such chimeric BoNT / A is also referred to herein as a "BoNT / AB" or a "BoNT / AB chimera."

[0139] LH N The C-terminal amino acid residue of the domain is the LH of BoNT / A. N and H C Separating Domains 3 10 It may correspond to the first amino acid residue of the helix. C The N-terminal amino acid residue of the domain is the LH of BoNT / B. N and H C Separating Domains 3 10 It may correspond to the second amino acid residue of the helix.

[0140] As used herein, "BoNT / A LH N and H C Separating Domains 3 10 The first amino acid residue of the helix is ​​the LH N and H C Separating Domains 3 10 It refers to the N-terminal residue of the helix.

[0141] As used herein, "BoNT / B LHN and H C Separating Domains 3 10 The second amino acid residue of the helix is N and H C Separating Domains 3 10 The following amino acid residues are the N-terminal residues of the helix:

[0142] "3 10 A "helix" is a type of secondary structure found on proteins and polypeptides, along with alpha helices, beta sheets, and reverse turns. 3 10 The amino acids on the helix are arranged in a right-handed helix, where each turn is completed by three residues and ten atoms separating the intermolecular hydrogen bonds between them. Each amino acid has ten atoms in the ring formed by making hydrogen bonds, corresponding to a 120° turn on the helix (i.e., the helix has three residues per turn) and a 2.0 Å (=0.2 nm) translation along the helix axis. Most importantly, the NH group of an amino acid forms a hydrogen bond with the C=O group of the amino acid three residues before it. This repeated i+3→i hydrogen bond sequence forms three 10 Define the helix. 3 10 Helices are a standard concept in structural biology and are familiar to those skilled in the art.

[0143] These three 10 The helix corresponds to the four residues that form the actual helix plus two cap (or connecting) residues, one on each end of these four residues. N and H C Separating Domains 3 10 The "helix" consists of these six residues.

[0144] By performing structural analysis and sequence alignment, LH N and H C Separating Domains 3 10 The three helices were identified. 10 The helix is ​​located at its N-terminus (i.e., LHN at the C-terminal end of the domain) by an α-helix, and at its C-terminus (i.e., H C At the N-terminal end of the domain, it is surrounded by β-strands. 10 The first (N-terminal) residue of the helix (the cap or connecting residue) also corresponds to the C-terminal residue of this α-helix.

[0145] LH N and H C Separating Domains 3 10 The helices can be determined for botulinum neurotoxins A1 and B1, for example, from publicly available crystal structures of botulinum neurotoxins, such as 3BTA (http: / / www.rcsb.org / pdb / explore / explore.do?structureId=3BTA) and 1EPW (http: / / www.rcsb.org / pdb / explore / explore.do?structureId=1EPW), respectively.

[0146] Publicly available in silico modeling and alignment tools also allow for the identification of LH on other neurotoxins. N and H C Separating Domains 3 10It can be used to determine the orientation of the helices. Examples include the homology modeling servers LOOPP (Learning, Observing and Outputting Protein Patterns, http: / / loopp.org), PHYRE (Protein Homology / analogY Recognition Engine, http: / / www.sbg.bio.ic.ac.uk / phyre2 / ), and Rosetta (https: / / www.rosettacommons.org / ), the protein superposition server SuperPose (http: / / wishart.biology.ualberta.ca / superpose / ), the alignment program Clustal Omega (http: / / www.clustal.org / omega / ), and several other tools / services listed in Internet Resources for Molecular and Cell Biologists (http: / / molbiol-tools.ca / ). In particular, the "H N / H CN The region around the junction is highly structurally conserved, making it an ideal region for overlapping different serotypes.

[0147] For example, the following methodology demonstrates the effectiveness of this 3-fold increase over other neurotoxins: 10 can be used to determine the sequence of the helix: 1. The structural homology modeling tool LOOP ( http: / / loopp.org ) was used to obtain predicted structures of other BoNT serotypes based on the BoNT / A1 crystal structure ( 3BTA.pdb ); 2.The resulting structure (pdb) file is H CN The N-terminal end of the domain and approximately 80 residues before it (these are H NIt encompasses only the structurally highly conserved "H" domain. N / H CN ” edited to preserve the area; 3. The protein superposition server SuperPose ( http: / / wishart.biology.ualberta.ca / superpose / ) was used to superimpose each serotype onto the 3BTA.pdb structure; 4. The overlapping pdb files are the H of BoNT / A1. C First 3 of the domain 10 The helices were scanned to locate them, and then corresponding residues in other serotypes were identified; 5. Other BoNT serotype sequences were aligned by Clustal Omega to check that the corresponding residues were correct.

[0148] LH determined by this method N , H C , and 3 10 An example of a helical domain is provided below: [Table B]

[0149] Using structural analysis and sequence alignment, LH N and H C Separating Domains 3 10 The β-strand following the helix is ​​a conserved structure in all botulinum and tetanus neurotoxins, and LH N and H C Separating Domains 3 10 It was found that when starting from the first residue of the helix, it begins at the eighth residue (eg, at residue 879 for BoNT / A1).

[0150] BoNT / AB chimera is a chimera derived from BoNT / B. C LH from BoNT / A covalently linked to the domain Ndomain (having a catalytically inactive light chain), - Here, LH N The C-terminal amino acid residue of the domain is H of BoNT / A. C corresponds to the eighth amino acid residue at the N-terminus of the β-strand located at the start (N-terminus) of the domain, - where H C The N-terminal amino acid residue of the domain is the H C It corresponds to the seventh amino acid residue at the N-terminus of the β-strand located at the start (N-terminus) of the domain.

[0151] BoNT / AB chimera is a chimera derived from BoNT / B. C LH from BoNT / A covalently linked to the domain N domain (having a catalytically inactive light chain), - Here, LH N The C-terminal amino acid residue of the domain is the LH of BoNT / A. N corresponds to the C-terminal amino acid residue of the α-helix located at the end (C-terminus) of the domain, - where H C The N-terminal amino acid residue of the domain is the LH of BoNT / B. N It corresponds to the amino acid residue immediately C-terminal to the C-terminal amino acid residue of the α-helix located at the end (C-terminus) of the domain.

[0152] The rationale for the design process of the BoNT / AB chimera was to ensure that the secondary structure was not disrupted, thereby striving to minimize any changes in the tertiary structure. Without wishing to be bound by theory, the 3 10 It is hypothesized that not blocking the four central amino acid residues of the helix ensures an optimal conformation for the chimeric neurotoxin.

[0153] Catalytically inactive LH from BoNT / A NThe domain may correspond to amino acid residues 1 to 872 of SEQ ID NO: 2 or 61, or a polypeptide sequence having at least 70% sequence identity thereto. Catalytically inactive LH from BoNT / A N The domain may correspond to amino acid residues 1 to 872 of SEQ ID NO: 2 or 61, or a polypeptide sequence having at least 80%, 90%, or 95% sequence identity thereto. Preferably, the catalytically inactive LH domain from BoNT / A. N The domain corresponds to amino acid residues 1 to 872 of SEQ ID NO:2 or 61.

[0154] H from BoNT / B C The domain may correspond to amino acid residues 860 to 1291 of SEQ ID NO: 52, or a polypeptide sequence having at least 70% sequence identity thereto. C The domain may correspond to amino acid residues 860 to 1291 of SEQ ID NO: 52, or a polypeptide sequence having at least 80%, 90%, or 95% sequence identity thereto. Preferably, the H domain from BoNT / B C The domain corresponds to amino acid residues 860 to 1291 of SEQ ID NO:52.

[0155] Preferably, catalytically inactive LH N The domain corresponds to amino acid residues 1 to 872 of BoNT / A (SEQ ID NO: 2 or 61), and C The domain corresponds to amino acid residues 860 to 1291 of BoNT / B (SEQ ID NO: 52).

[0156] Preferably, H of BoNT / B C The domain may further comprise at least one amino acid residue substitution, addition, or deletion. CC The BoNT / B neurotoxin contains a subdomain of the nucleotide sequence H, which has the effect of increasing the binding affinity of the BoNT / B neurotoxin to human Syt II compared to the native BoNT / B sequence. CCSuitable amino acid residue substitutions, additions, or deletions on the subdomains are disclosed in WO 2013 / 180799 and WO 2016 / 154534 (both of which are incorporated herein by reference).

[0157] BoNT / B H CC Suitable amino acid residue substitutions, additions, or deletions on the subdomains include substitution mutations selected from the group consisting of V1118M, Y1183M, E1191M, E1191I, E1191Q, E1191T, S1199Y, S1199F, S1199L, S1201V, E1191C, E1191V, E1191L, E1191Y, S1199W, S1199E, S1199H, W1178Y, W1178Q, W1178A, W1178S, Y1183C, Y1183P, and combinations thereof.

[0158] BoNT / B H CC Suitable amino acid residue substitutions, additions, or deletions on the subdomain further include combinations of two substitution mutations selected from the group consisting of E1191M and S1199L, E1191M and S1199Y, E1191M and S1199F, E1191Q and S1199L, E1191Q and S1199Y, E1191Q and S1199F, E1191M and S1199W, E1191M and W1178Q, E1191C and S1199W, E1191C and S1199Y, E1191C and W1178Q, E1191Q and S1199W, E1191V and S1199W, E1191V and S1199Y, or E1191V and W1178Q.

[0159] BoNT / B H CC Suitable amino acid residue substitutions, additions, or deletions on the subdomains also include the combination of the three substitution mutations: E1191M, S1199W, and W1178Q.

[0160] Preferably, H of BoNT / B CC Preferred amino acid residue substitutions, additions, or deletions on the subdomains include the combination of two substitution mutations: E1191M and S1199Y.

[0161] The alterations can be alterations relative to unaltered BoNT / B, set forth as SEQ ID NO:52, where the amino acid residue numbering is determined by alignment with SEQ ID NO:52. Because the presence of a methionine residue at position 1 of SEQ ID NO:52 is optional, one of skill in the art would take the presence / absence of the methionine residue into account when determining the amino acid residue numbering. For example, if SEQ ID NO:52 includes a methionine, the position numbering would be as defined above (e.g., E1191 would be E1191 in SEQ ID NO:52). Alternatively, if a methionine is not present in SEQ ID NO:52, the amino acid residue numbering should be adjusted by -1 (e.g., E1191 would become E1190 in SEQ ID NO:52). Similar considerations apply when a methionine is present / absent at position 1 of the other polypeptide sequences described herein, and one of skill in the art would have no difficulty determining the correct amino acid residue numbering using routine techniques in the art.

[0162] In one embodiment, a polypeptide for use in accordance with the invention comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 61. In one embodiment, a polypeptide for use in accordance with the invention comprises a polypeptide sequence having at least 80%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 61. Preferably, a polypeptide for use in accordance with the invention comprises (more preferably consists of) the polypeptide sequence set forth as SEQ ID NO: 61.

[0163] Chimeric and / or hybrid Clostridial neurotoxins for use in the present invention can contain a portion of a BoNT / A polypeptide and a portion of a BoNT / B polypeptide, examples of which include the polypeptide set forth herein as SEQ ID NO:44.

[0164] In one embodiment, a polypeptide for use in accordance with the invention comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 44 and / or a polypeptide sequence encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 43. In one embodiment, a polypeptide for use in accordance with the invention comprises a polypeptide sequence having at least 80%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 44. Preferably, a polypeptide for use in accordance with the invention comprises the polypeptide sequence set forth as SEQ ID NO: 44. In one embodiment, a polypeptide for use in accordance with the invention comprises a polypeptide sequence encoded by a nucleotide sequence having at least 80%, 90%, 95%, or 98% sequence identity to SEQ ID NO: 43. Preferably, a polypeptide for use in accordance with the invention comprises the polypeptide sequence encoded by the nucleotide sequence set forth as SEQ ID NO: 43.

[0165] A suitable chimeric Clostridial neurotoxin can include BoNT / FA, provided that any L chain present is catalytically inactive. Thus, a polypeptide of the invention can include BoNT / FA or a fragment thereof, provided that any L chain present is catalytically inactive. Catalytically inactive forms of BoNT / FA are set forth herein as SEQ ID NOs: 26 and 34. Suitable fragments of BoNT / FA are also set forth herein as SEQ ID NOs: 28, 30, and 32.

[0166] In another preferred embodiment, the polypeptide of the present invention comprises a catalytically inactive BoNT / X light chain and translocation domain (LH). N domain) and a receptor-binding domain (H) from a different (i.e., non-BoNT / X) clostridial neurotoxin. CThe chimeric and / or hybrid clostridial neurotoxins may be those taught in WO 2020 / 065336 A1, which is incorporated herein by reference, except that the L chain is catalytically inactive (e.g., inactivated by modification). Such preferred sequences include SEQ ID NOs: 63-70, as described herein.

[0167] The chimeric clostridial neurotoxin contains the catalytically inactive light chain and translocation domain (LH) of BoNT / X. N domain), and may include: (i) BoNT / A receptor binding domain (H C domain) or part thereof; or (ii) BoNT / B receptor binding domain (H C domain) or part thereof; or (iii) BoNT / C receptor binding domain (H C domain) or part thereof; or (iv) BoNT / D receptor binding domain (H C domain) or part thereof; or (v) BoNT / E receptor binding domain (H C domain) or part thereof; or (vi) BoNT / F receptor binding domain (H C domain) or part thereof; or (vii) BoNT / G receptor binding domain (H C domain) or part thereof; or (viii) TeNT receptor binding domain (H C domain) or part thereof.

[0168] In one embodiment, a receptor binding domain (H) from a different (i.e., non-BoNT / X) clostridial neurotoxin domain is used. C The domain) or portion thereof may bind to synaptotagmin I and / or II (SytI / II).

[0169] Preferably, the chimeric Clostridial neurotoxin comprises a catalytically inactive light chain and translocation domain (LH) of BoNT / X. N domain) and the receptor binding domain of BoNT / B (H C domain) or parts thereof.

[0170] The light chain and translocation domain (LH) of catalytically inactive BoNT / X N domain) and a receptor-binding domain (H) from a different (i.e., non-BoNT / X) clostridial neurotoxin. C A polypeptide comprising the light chain and translocation domain (LH domain) of catalytically inactive BoNT / X or a portion thereof can comprise a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 63-70. In one embodiment, the polypeptide comprising the light chain and translocation domain (LH domain) of catalytically inactive BoNT / X or a portion thereof can comprise a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 63-70. N domain) and a receptor-binding domain (H) from a different (i.e., non-BoNT / X) clostridial neurotoxin. C A polypeptide comprising the light chain and translocation domain (LH domain) of catalytically inactive BoNT / X or a portion thereof comprises a polypeptide sequence having at least 80%, 90%, 95%, or 98% sequence identity to any one of SEQ ID NOs: 63 to 70. Preferably, the polypeptide comprises the light chain and translocation domain (LH domain) of catalytically inactive BoNT / X or a portion thereof. N domain) and a receptor-binding domain (H) from a different (i.e., non-BoNT / X) clostridial neurotoxin. C The polypeptide comprising the nucleotide sequence (domain) or a part thereof is (more preferably consists of) any one of SEQ ID NOs: 63 to 70. Of these polypeptides, SEQ ID NOs: 63 to 66 are most preferred.

[0171] The light chain and translocation domain (LH) of catalytically inactive BoNT / X N domain) and a receptor-binding domain (H) from a different (i.e., non-BoNT / X) clostridial neurotoxin. CA polypeptide comprising the N-terminal amino acid sequence designated as MGS (SEQ ID NO: 63-66) or a portion thereof may comprise the following N-terminal amino acid sequence: MGS. Where SEQ ID NO: 63-66 contains said N-terminal amino acid sequence, said sequence is optional. In one embodiment, SEQ ID NO: 63-66 lacks the N-terminal amino acid sequence designated as MGS. In one embodiment, SEQ ID NO: 63-66 comprises the N-terminal amino acid sequence designated as MGS.

[0172] Catalytically inactive LH from BoNT / X N The domain may correspond to amino acid residues 1 to 899 of SEQ ID NO: 63, or a polypeptide sequence having at least 70% sequence identity thereto. Catalytically inactive LH from BoNT / X N The domain may correspond to amino acid residues 1 to 899 of SEQ ID NO: 63, or a polypeptide sequence having at least 80%, 90%, or 95% sequence identity thereto. Preferably, the catalytically inactive LH domain from BoNT / X is N The domain can correspond to amino acid residues 1 to 899 of SEQ ID NO:63.

[0173] Catalytically inactive LH from BoNT / X N The domain may correspond to amino acid residues 4 to 899 of SEQ ID NO: 63, or a polypeptide sequence having at least 70% sequence identity thereto. Catalytically inactive LH from BoNT / X N The domain may correspond to amino acid residues 4 to 899 of SEQ ID NO: 63, or a polypeptide sequence having at least 80%, 90%, or 95% sequence identity thereto. Preferably, the catalytically inactive LH domain from BoNT / X is N The domain can correspond to amino acid residues 4 to 899 of SEQ ID NO:63.

[0174] Catalytically inactive LH from BoNT / X N The domain may correspond to amino acid residues 1 to 866 of SEQ ID NO: 65, or a polypeptide sequence having at least 70% sequence identity thereto. Catalytically inactive LH from BoNT / X NThe domain may correspond to amino acid residues 1 to 866 of SEQ ID NO: 65, or a polypeptide sequence having at least 80%, 90%, or 95% sequence identity thereto. Preferably, the catalytically inactive LH domain from BoNT / X is N The domain can correspond to amino acid residues 1 to 866 of SEQ ID NO:65.

[0175] Catalytically inactive LH from BoNT / X N The domain may correspond to amino acid residues 4 to 866 of SEQ ID NO: 65, or a polypeptide sequence having at least 70% sequence identity thereto. Catalytically inactive LH from BoNT / X N The domain may correspond to amino acid residues 4 to 866 of SEQ ID NO: 65, or a polypeptide sequence having at least 80%, 90%, or 95% sequence identity thereto. Preferably, the catalytically inactive LH domain from BoNT / X is N The domain can correspond to amino acid residues 4 to 866 of SEQ ID NO:65.

[0176] H from BoNT / B C The domain may correspond to amino acid residues 860 to 1291 of SEQ ID NO: 52, or a polypeptide sequence having at least 70% sequence identity thereto. C The domain may correspond to amino acid residues 860 to 1291 of SEQ ID NO: 52, or a polypeptide sequence having at least 80%, 90%, or 95% sequence identity thereto. Preferably, the H domain from BoNT / B C The domain corresponds to amino acid residues 860 to 1291 of SEQ ID NO:52.

[0177] Preferably, H of BoNT / B C The domain may further comprise at least one amino acid residue substitution, addition, or deletion. CC The BoNT / B neurotoxin contains a subdomain of the nucleotide sequence H, which has the effect of increasing the binding affinity of the BoNT / B neurotoxin to human Syt II compared to the native BoNT / B sequence. CCSuitable amino acid residue substitutions, additions, or deletions on the subdomains are disclosed in WO 2013 / 180799 and WO 2016 / 154534 (both of which are incorporated herein by reference).

[0178] BoNT / B H CC Suitable amino acid residue substitutions, additions, or deletions on the subdomains include substitution mutations selected from the group consisting of V1118M, Y1183M, E1191M, E1191I, E1191Q, E1191T, S1199Y, S1199F, S1199L, S1201V, E1191C, E1191V, E1191L, E1191Y, S1199W, S1199E, S1199H, W1178Y, W1178Q, W1178A, W1178S, Y1183C, Y1183P, and combinations thereof.

[0179] BoNT / B H CC Suitable amino acid residue substitutions, additions, or deletions on the subdomain further include combinations of two substitution mutations selected from the group consisting of E1191M and S1199L, E1191M and S1199Y, E1191M and S1199F, E1191Q and S1199L, E1191Q and S1199Y, E1191Q and S1199F, E1191M and S1199W, E1191M and W1178Q, E1191C and S1199W, E1191C and S1199Y, E1191C and W1178Q, E1191Q and S1199W, E1191V and S1199W, E1191V and S1199Y, or E1191V and W1178Q.

[0180] BoNT / B H CC Suitable amino acid residue substitutions, additions, or deletions on the subdomains also include the combination of the three substitution mutations: E1191M, S1199W, and W1178Q.

[0181] Preferably, H of BoNT / B CC Preferred amino acid residue substitutions, additions, or deletions on the subdomains include the combination of two substitution mutations: E1191M and S1199Y.

[0182] The alterations can be alterations relative to unaltered BoNT / B, set forth as SEQ ID NO:52, where the amino acid residue numbering is determined by alignment with SEQ ID NO:52. Because the presence of a methionine residue at position 1 of SEQ ID NO:52 is optional, one of skill in the art would take the presence / absence of the methionine residue into account when determining the amino acid residue numbering. For example, if SEQ ID NO:52 includes a methionine, the position numbering would be as defined above (e.g., E1191 would be E1191 in SEQ ID NO:52). Alternatively, if a methionine is not present in SEQ ID NO:52, the amino acid residue numbering should be adjusted by -1 (e.g., E1191 would become E1190 in SEQ ID NO:52). Similar considerations apply when a methionine is present / absent at position 1 of the other polypeptide sequences described herein, and one of skill in the art would have no difficulty determining the correct amino acid residue numbering using routine techniques in the art.

[0183] H from BoNT / A C The domain may correspond to amino acid residues 873 to 1296 of SEQ ID NO: 60, or a polypeptide sequence having at least 70% sequence identity thereto. C The domain may correspond to amino acid residues 873 to 1296 of SEQ ID NO: 60, or a polypeptide sequence having at least 80%, 90%, or 95% sequence identity thereto. Preferably, the H domain from BoNT / B C The domain corresponds to amino acid residues 873 to 1296 of SEQ ID NO:60.

[0184] In one embodiment, when the polypeptide is for use in treating an inflammatory disorder, the polypeptide used comprises a catalytically inactive BoNT / X L chain, a BoNT / X translocation domain (H chain), or a BoNT / X translocation domain (H chain). N domain), and the receptor binding domain of BoNT / A (H CThus, in one embodiment, when the polypeptide is for use in treating an inflammatory disorder, the polypeptide does not contain a catalytically inactive BoNT / X L chain, a BoNT / X translocation domain (H domain). N domain), and (i) the receptor-binding domain of BoNT / B (H C domain), (ii) the receptor-binding domain of BoNT / D (H C domain), or (iii) receptor binding of BoNT / F (H C domain).

[0185] Similarly, in one embodiment, when the polypeptide is for use in treating pain, the polypeptide used comprises a catalytically inactive BoNT / X L chain, a BoNT / X translocation domain (H chain), or a BoNT / X translocation domain (H chain). N domain), and the receptor binding domain of BoNT / A (H C Therefore, in one embodiment, when the polypeptide is for use in treating pain, the polypeptide does not contain a catalytically inactive L chain of BoNT / X, a translocation domain (H chain) of BoNT / X, or a non-catalytically inactive L chain of BoNT / X. N domain), and (i) the receptor-binding domain of BoNT / B (H C domain), (ii) the receptor-binding domain of BoNT / D (H C domain), or (iii) receptor binding of BoNT / F (H C domain).

[0186] H from BoNT / D C The domain may correspond to amino acid residues 865 to 1276 of SEQ ID NO: 54, or a polypeptide sequence having at least 70% sequence identity thereto. C The domain may correspond to amino acid residues 865 to 1276 of SEQ ID NO: 54, or a polypeptide sequence having at least 80%, 90%, or 95% sequence identity thereto. Preferably, the H domain from BoNT / D C The domain corresponds to amino acid residues 865 to 1276 of SEQ ID NO:54.

[0187] H from BoNT / F C The domain can correspond to amino acid residues 866 to 1278 of SEQ ID NO: 56, or a polypeptide sequence having at least 70% sequence identity thereto. C The domain can correspond to amino acid residues 866 to 1278 of SEQ ID NO: 56, or a polypeptide sequence having at least 80%, 90%, or 95% sequence identity thereto. Preferably, the H domain from BoNT / F C The domain corresponds to amino acid residues 866 to 1278 of SEQ ID NO:56, with a histidine to lysine substitution at position 1241 (H1241K).

[0188] Preferably, the chimeric Clostridial neurotoxin comprises a catalytically inactive light chain and translocation domain (LH) of BoNT / X. N domain) as well as receptor-binding domains (H) from different (i.e., non-BoNT / X) clostridial neurotoxins. C domain) or a part thereof and Cys-(Xaa) a -Ile-Asp / Glu-Gly-Arg-(Yaa) b -Cys (SEQ ID NO: 71), where a=1 to 10 and b=4 to 15. SEQ ID NO: 71 is an activation loop consensus sequence based on the BoNT / C1 activation loop.

[0189] Additionally, any polypeptide of the present invention may have a Cys-(Xaa) a -Ile-Asp / Glu-Gly-Arg-(Yaa) b -Cys (SEQ ID NO: 71), where a=1 to 10 and b=4 to 15. The activation loop may suitably comprise a Clostridial neurotoxin L chain and / or translocation domain (H) present on the polypeptides described herein. N Any activation loop sequence present in either the ATP domain may be replaced.

[0190] Xaa or Yaa, when used in the context of SEQ ID NO: 71, can be any amino acid. The number of amino acids at positions Xaa and Yaa is indicated by the letters "a" and "b," respectively. In one embodiment, "a" and "b" can be any integer that allows proteolytic cleavage of the activation loop to yield an active dichain clostridial neurotoxin. In one embodiment, "a" is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, "b" is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In one embodiment, "a" is ≦12, ≦11, ≦10, ≦9, ≦8, ≦7, ≦6, ≦5, or ≦4. In one embodiment, "b" is ≦20, ≦19, ≦18, ≦17, ≦16, ≦15, ≦14, ≦13, ≦12, ≦11, ≦10, or ≦9.

[0191] In one embodiment, "a" is 1 to 12, for example, 1 to 10. Preferably, "a" is 1 to 7, for example, 2 to 4. More preferably, "a" is 3. In one embodiment, "b" is 1 to 20, for example, 4 to 15. Preferably, "b" is 6 to 10. More preferably, "b" is 8.

[0192] It is not intended that Xaa or Yaa be limited to only one type of amino acid. Thus, one or more residues present at position Xaa can be independently selected from the standard amino acids aspartic acid, glutamic acid, arginine, lysine, histidine, asparagine, glutamine, serine, threonine, tyrosine, methionine, tryptophan, cysteine, alanine, glycine, valine, leucine, isoleucine, proline, and phenylalanine. One or more residues present at position Yaa can be independently selected from the standard amino acids aspartic acid, glutamic acid, arginine, lysine, histidine, asparagine, glutamine, serine, threonine, tyrosine, methionine, tryptophan, cysteine, alanine, glycine, valine, leucine, isoleucine, proline, and phenylalanine. Preferably, the amino acid at position Yaa (more preferably, immediately C-terminal to the Arg residue of SEQ ID NO: 71) is not proline.

[0193] Alternatively / in addition, one or more residues present at positions Xaa or Yaa can be independently selected from non-standard amino acids (amino acids that are not part of the standard set of 20 described above). By way of example, non-standard amino acids can include 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline, α-methylserine, trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allothreonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, L-ornithine, L-2-amino-3-guanidinopropionic acid, or D-isomers of lysine, arginine, and / or ornithine, and 4-fluorophenylalanine. Methods for incorporating non-standard amino acids onto proteins are known in the art and include recombinant protein synthesis using E. coli auxotrophic expression hosts.

[0194] The sequence Ile-Asp / Glu-Gly-Arg contained in SEQ ID NO: 71 refers to the site surprisingly found to be recognized by enterokinase (and factor Xa) in WO 2020 / 065336 A1. Said document describes a suitable method for cleaving at Ile-Asp / Glu-Gly-Arg, thereby generating a two-chain polypeptide. Preferably, the sequence is Ile-Asp-Gly-Arg, e.g., Cys-(Xaa) a -Ile-Asp-Gly-Arg-(Yaa) b -Cys. Enterokinase and factor Xa are believed to hydrolyze the peptide bond immediately C-terminal to Arg in SEQ ID NO:71 (i.e., the peptide bond between Arg and Yaa).

[0195] In one embodiment, the amino acid residue Xaa immediately N-terminal to He of SEQ ID NO:71 is an uncharged hydrophobic amino acid, preferably alanine. In some embodiments, "a" is at least 2, and Xaa includes at least an uncharged polar amino acid C-terminal and a charged basic amino acid immediately N-terminal thereto. The charged basic amino acid is preferably lysine. Thus, in embodiments where "a" is at least 2, Xaa can include at least Lys-Ala, where Ala is immediately N-terminal to He of SEQ ID NO:71.

[0196] In one embodiment, Xaa comprises or consists of the sequence HKA.

[0197] In one embodiment, the amino acid residue Yaa immediately C-terminal to Arg of SEQ ID NO:71 is an uncharged polar amino acid, preferably serine. In some embodiments, "b" is at least 2, and Yaa includes at least an N-terminal uncharged polar amino acid and an uncharged hydrophobic amino acid immediately C-terminal thereto. The uncharged hydrophobic amino acid is preferably leucine. Thus, in embodiments where "b" is at least 2, Yaa can include at least Ser-Leu, where Ser is immediately C-terminal to Arg of SEQ ID NO:71.

[0198] In one embodiment, Yaa comprises or consists of the sequence SLYNKTLDC.

[0199] In some embodiments, the polypeptides herein comprise an activation loop having at least 70% sequence identity to SEQ ID NO: 72. In one embodiment, the polypeptides herein comprise an activation loop having at least 80%, 85%, or 90% sequence identity to SEQ ID NO: 72. Preferably, the polypeptides herein comprise an activation loop having at least 95% sequence identity to SEQ ID NO: 72. More preferably, the polypeptides herein comprise an activation loop having at least 99% sequence identity to SEQ ID NO: 72.

[0200] In particularly preferred embodiments, the polypeptides herein comprise an activation loop that comprises, and more preferably consists of, SEQ ID NO:72.

[0201] The activation loop can also be a variant of SEQ ID NO: 72, such as SEQ ID NO: 73, or a sequence having at least 70% sequence identity thereto. SEQ ID NO: 73 is a variant of SEQ ID NO: 72 in which the enterokinase recognition site IDGR is mutated to IEGR. In one embodiment, a polypeptide herein comprises an activation loop having at least 70% sequence identity to SEQ ID NO: 73. In one embodiment, a polypeptide herein comprises an activation loop having at least 80%, 85%, or 90% sequence identity to SEQ ID NO: 73. Preferably, a polypeptide herein comprises an activation loop having at least 95% sequence identity to SEQ ID NO: 73. More preferably, a polypeptide herein comprises an activation loop having at least 99% sequence identity to SEQ ID NO: 73.

[0202] In particularly preferred embodiments, the polypeptides herein comprise an activation loop that comprises, and more preferably consists of, SEQ ID NO:73.

[0203] In one embodiment, an activation loop described herein (e.g., SEQ ID NO: 71, 72, or 73) can be modified to include an additional or alternative protease site, such as the protease site set forth as SEQ ID NO: 77. An example of such a modified activation loop is set forth as SEQ ID NO: 78. Thus, in one embodiment, a polypeptide herein comprises an activation loop having at least 70% sequence identity to SEQ ID NO: 78. In one embodiment, a polypeptide herein comprises an activation loop having at least 80%, 85%, or 90% sequence identity to SEQ ID NO: 78. Preferably, a polypeptide herein comprises an activation loop having at least 95% sequence identity to SEQ ID NO: 78. More preferably, a polypeptide herein comprises an activation loop having at least 99% sequence identity to SEQ ID NO: 78. In a particularly preferred embodiment, a polypeptide herein comprises an activation loop comprising, or more preferably consisting of, SEQ ID NO: 78.

[0204] In one embodiment, the polypeptide of the present invention may comprise (or consist of) a retargeted Clostridial neurotoxin, provided that any L chains present are catalytically inactive. In a retargeted Clostridial neurotoxin, the Clostridial neurotoxin is modified to include an exogenous ligand known as a targeting moiety (TM). The TM is selected to provide binding specificity for the desired target cell, and as part of the retargeting process, the natural binding moiety of the Clostridial neurotoxin (e.g., H C Domain or H CCdomain) may be removed. Retargeting technologies are described, for example, in EP-B-0689459, WO 1994 / 021300, EP-B-0939818, US 6,461,617, US 7,192,596, WO 1998 / 007864, EP-B-0826051, US 5,989,545, US 6,395,513, US 6,962,703, WO 1996 / 033273, EP-B-0996468, US 7,052,702, WO 1999 / 017806, EP-B-1107794, US 6,632,440, WO 2000 / 010598, WO 2001 / 21213, WO 2006 / 059093, WO 2000 / 62814, WO 2000 / 04926, WO 1993 / 15766, WO 2000 / 61192, and WO 1999 / 58571, all of which are incorporated herein by reference in their entireties. Thus, in one embodiment, the polypeptides of the invention are retargeted clostridial neurotoxins, provided that any L chains present are catalytically inactive. The polypeptides of the invention are retargeted clostridial neurotoxins, provided that any L chains present are catalytically inactive. C It lacks the domain and may also lack any functionally equivalent TM.

[0205] In embodiments in which the polypeptides described herein have a tag (eg, a His tag) and / or a linker for purification, the tag and / or linker are optional.

[0206] The polypeptides of the present invention may be free of complexing proteins present in a naturally occurring Clostridial neurotoxin complex.

[0207] The polypeptides of the present invention can be produced using recombinant nucleic acid technology. Thus, in one embodiment, the polypeptide (as described above) is a recombinant polypeptide.

[0208] In one embodiment, a nucleic acid (e.g., DNA) comprising a nucleic acid sequence encoding a polypeptide is provided. In one embodiment, the nucleic acid sequence is prepared as part of a DNA vector comprising a promoter and a terminator. The nucleic acid sequence can be selected from any of the nucleic acid sequences described herein.

[0209] In a preferred embodiment, the vector has a promoter which may be selected from: Promoter / Inducing agent / Typical induction conditions Tac (hybrid) / IPTG / 0.2mM (0.05-2.0mM) AraBAD / L-arabinose / 0.2% (0.002~0.4%) T7-lac operator / IPTG / 0.2mM (0.05-2.0mM)

[0210] In another preferred embodiment, the vector has a promoter selected from: Promoter / Inducing agent / Typical induction conditions Tac (hybrid) / IPTG / 0.2mM (0.05-2.0mM) AraBAD / L-arabinose / 0.2% (0.002~0.4%) T7-lac operator / IPTG / 0.2mM (0.05-2.0mM) T5-lac operator / IPTG / 0.2mM (0.05-2.0mM)

[0211] Nucleic acid molecules can be produced using any suitable process known in the art. Thus, nucleic acid molecules can be produced using chemical synthesis techniques. Alternatively, nucleic acid molecules of the present invention can be produced using molecular biology techniques.

[0212] The DNA constructs of the present invention are preferably designed in silico and then synthesized by conventional DNA synthesis techniques.

[0213] The nucleic acid sequence information referred to above is optionally modified for codon bias according to the final host cell (eg, E. coli) expression system to be employed.

[0214] The terms "nucleotide sequence" and "nucleic acid" are used interchangeably herein. Preferably, the nucleotide sequence is a DNA sequence.

[0215] The polypeptides of the present invention (especially any Clostridial neurotoxin portion thereof) can exist as a single chain or as two chains. However, the polypeptides exist as two chains in which a catalytically inactive light chain is connected to a heavy chain (or a component thereof, e.g., H) via a disulfide bond. N Preferably, the domain is linked to the

[0216] The invention provides methods for producing a single-chain polypeptide having a catalytically inactive light chain and a heavy chain, the method comprising expressing a nucleic acid described herein in an expression host, lysing the host cells to provide a host cell homogenate containing the single-chain polypeptide, and isolating the single-chain polypeptide. In one embodiment, the invention provides a method for proteolytically processing a polypeptide described herein, the method comprising contacting the polypeptide with a protease that hydrolyzes a peptide bond on the activation loop of the polypeptide, thereby converting the (single-chain) polypeptide into the corresponding two-chain polypeptide (e.g., where the catalytically inactive light chain and heavy chain are linked together by a disulfide bond).

[0217] Thus, the present invention provides a di-chain polypeptide obtainable by the method of the present invention.

[0218] As used herein, a "subject" can be a mammal, such as a human or other mammal. Preferably, "subject" refers to a human subject.

[0219] As used herein, the term "disorder" also encompasses "disease." In one embodiment, a disorder is a disease.

[0220] As used herein, the term "treat" or "treating" encompasses prophylactic treatment (e.g., to prevent the occurrence of a disorder (e.g., pain)) and corrective treatment (treatment of a subject already suffering from a disorder (e.g., pain)). Preferably, as used herein, "treat" or "treating" refers to corrective treatment.

[0221] As used herein, the term "treat" or "treating" refers to a disorder (eg, pain) and / or its symptoms.

[0222] Thus, the polypeptides of the present invention can be administered to a subject in a therapeutically effective amount or a prophylactically effective amount. Preferably, the polypeptides of the present invention are administered to a subject in a therapeutically effective amount.

[0223] A "therapeutically effective amount" is any amount of a polypeptide that, when administered alone or in combination with another agent to a subject for treating said disorder (e.g., pain) (or a symptom thereof), is sufficient to effect such treatment of the disorder or symptom thereof.

[0224] A "prophylactically effective amount" is any amount of a polypeptide that, when administered to a subject alone or in combination with another agent, inhibits or delays the onset or recurrence of a disorder (e.g., pain) (or symptoms thereof). In some embodiments, a prophylactically effective amount prevents the onset or recurrence of a disorder (e.g., pain) altogether. "Inhibiting" onset means either reducing the likelihood of the onset of a disorder (e.g., where the disorder is pain) (or symptoms thereof), or preventing onset altogether.

[0225] The polypeptides of the present invention can be formulated in any suitable manner for administration to a subject, for example, as part of a pharmaceutical composition. Thus, in one aspect, the present invention provides a pharmaceutical composition comprising a polypeptide of the present invention and a pharmaceutically acceptable carrier, excipient, adjuvant, propellant, and / or salt.

[0226] The polypeptides of the invention may be formulated for oral, parenteral, continuous infusion, inhalation, or topical use. Compositions suitable for injection may be in the form of a solution, suspension, or emulsion, or as a dry powder that is dissolved or suspended in a suitable base prior to use.

[0227] In the case of polypeptides that are to be delivered locally, the polypeptides can be formulated as creams (eg, for topical application) or for subdermal injection.

[0228] Local delivery means may include aerosols or other sprays (e.g., nebulizers), whereby an aerosol formulation of the polypeptide enables delivery to the lungs and / or other nasal and / or bronchial or airway passages.

[0229] The polypeptides of the invention may be administered to the patient by intrathecal or epidural injection in the spinal column at the level of the spinal segment involved in the innervation of the affected organ.

[0230] The route of administration can be via laparoscopic and / or localized injection. In one embodiment, the polypeptide of the present invention is administered at or near the site to be treated, preferably at the site to be treated. For example, the polypeptide can be administered intrathecally or intraspinally. In one embodiment, the route of administration of the polypeptide of the present invention can be perineural, intraneural, intraspinal, and / or intrathecal.

[0231] In one embodiment, the polypeptides of the present invention can be administered peripherally. In one embodiment, the polypeptides can be administered intradermally, subcutaneously, or intramuscularly. Preferably, the polypeptides of the present invention are administered intradermally.

[0232] Dosage ranges for administration of the polypeptides of the present invention should be those that produce the desired therapeutic and / or prophylactic effect. It will be understood that the required dosage range will depend on the precise nature of the Clostridial neurotoxin or composition, the route of administration, the nature of the formulation, the subject's age, the nature, extent, or severity of the subject's condition, any contraindications, and the judgment of the attending physician. Variations in these dosage levels can be adjusted using standard empirical routines for optimization.

[0233] In one embodiment, the polypeptide is administered in a fixed dose. The fixed dose may range from 50 pg to 250 μg, preferably from 100 pg to 100 μg. In one embodiment, the fixed dose may be at least 50 pg, 100 pg, 500 pg, 1 ng, 50 ng, 100 ng, 500 ng, 1 μg, or 50 μg. The dose may be a single fixed dose.

[0234] In a preferred embodiment, the polypeptide may be administered in an amount greater than 250 μg. In one embodiment, the polypeptide of the present invention may be administered in an amount greater than 500 μg, 1 mg, 10 mg, 100 mg, 500 mg, 1 g, or 5 g. In one embodiment, the polypeptide of the present invention may be administered in an amount equal to or less than 10 g, 5 g, 1 g, 500 mg, 100 mg, 10 mg, or 1 mg. Preferably, the polypeptide of the present invention is administered in an amount of 251 μg to 10 g, 251 μg to 5 g, 251 μg to 1 g, 251 μg to 500 mg, 251 μg to 100 mg, 251 μg to 10 mg, or 251 μg to 1000 μg, e.g., 251 μg to 500 μg. In one embodiment, the polypeptide of the present invention is administered in an amount of 500 μg to 5 g, e.g., 1 mg to 1 g or 1 g to 3 g. This is made possible by the non-toxic (eg, substantially non-toxic) nature of the polypeptides of the invention.

[0235] Fluid dosage forms are typically prepared using the polypeptide and a pyrogen-free sterile base. The clostridial neurotoxin can be either dissolved or suspended in the base, depending on the base and concentration used. In preparing a solution, the polypeptide can be dissolved in the base, and the solution can be made isotonic by adding sodium chloride, if necessary, and sterilized by filtration through a sterile filter using aseptic techniques before filling and sealing into suitable sterile vials or ampoules. Alternatively, if the stability of the solution is sufficient, the solution in the sealed container can be sterilized by autoclaving. Advantageously, additives such as buffers, solubilizers, stabilizers, preservatives or bactericides, suspending or emulsifying agents, and / or local anesthetics can be dissolved in the base.

[0236] Dry powders, which are dissolved or suspended in a suitable base prior to use, can be prepared by filling pre-sterilized ingredients into sterile containers using aseptic technique in a sterile area. Alternatively, the ingredients can be dissolved in suitable containers using aseptic technique in a sterile area. The product is then freeze-dried and the container is aseptically sealed.

[0237] Parenteral suspensions suitable for the routes of administration described herein are prepared in substantially the same manner, except that the sterile components are suspended in a sterile vehicle instead of being dissolved, and sterilization cannot be accomplished by filtration. The components may be isolated in a sterile state, or alternatively, they may be sterilized after isolation, for example, by gamma irradiation.

[0238] Advantageously, a suspending agent, such as polyvinylpyrrolidone, is included in the composition(s) to facilitate uniform distribution of the components.

[0239] Administration according to the present invention may utilize a variety of delivery technologies, including microparticle encapsulation or high-pressure aerosol bombardment.

[0240] Polypeptides of the invention are preferably administered repeatedly (e.g., up to 5, 10, 15, or 20 times) as part of a treatment regimen. Repeated administration means at least two administrations, e.g., at least 5, 10, 15, or 20 administrations. Thus, in one embodiment, polypeptides of the invention can be administered more than once to treat a subject (e.g., to treat pain in a subject). This is particularly true for the treatment of chronic conditions, such as chronic pain, where ongoing treatment is typically necessary. In one embodiment, polypeptides of the invention can be administered weekly, twice a month, monthly, every two months, every six months, or yearly, preferably at least twice a year or yearly. In one embodiment, polypeptides of the invention are administered more than once over a 10-year, 5-year, 2-year, or 1-year period. Preferably, polypeptides of the invention are administered more than once over a 1-year period. Treatment can continue for at least 6 months, 1 year, 2 years, 3 years, 5 years, 10 years, 15 years, 20 years, 25 years, or 30 years.

[0241] The polypeptide comprises a catalytically inactive clostridial neurotoxin light chain, a H N Domain and / or H C Preferably, the domain is not administered with an additional therapeutic or diagnostic agent (e.g., a nucleic acid, protein, peptide, or small molecule therapeutic or diagnostic agent). For example, in one embodiment, the polypeptide is not administered with an additional analgesic and / or anti-inflammatory agent. In one embodiment, the polypeptide of the invention is not administered with a covalently linked therapeutic agent. In one embodiment, the polypeptide of the invention is not administered with a non-covalently linked therapeutic agent.

[0242] The polypeptides described herein can be used to treat subjects suffering from one or more types of pain. The pain can be chronic or acute pain. The pain can be one or more selected from four categories of pain: nociceptive pain, neuropathic pain, mixed pain, and pain of unknown origin. Nociceptive pain can be caused by a known noxious stimulus to a nociceptor (pain receptor) and can be somatic or visceral. Neuropathic pain can be pain initiated or caused by a primary lesion or dysfunction of the nervous system. Mixed pain can be a combination of nociceptive pain and neuropathic pain.

[0243] Examples of pain (eg, chronic pain) that may be treated by the present invention include neuropathic pain, inflammatory pain, headache pain, somatic pain, visceral pain, referred pain, allodynia, mixed pain, and post-operative pain.

[0244] The term "pain" as used herein means any unpleasant sensory experience usually associated with a physical disorder. The physical disorder may or may not be apparent to the clinician. Pain is of two types: chronic and acute. "Acute pain" is pain of short duration with sudden onset. One type of acute pain is cutaneous pain felt due to injury to the skin or other superficial tissue, such as caused by a cut or burn. Cutaneous nociceptors terminate just below the skin and produce sharp, localized pain of short duration due to the high concentration of nerve endings. "Chronic pain" is pain other than acute pain.

[0245] The polypeptides of the present invention can be used to treat pain caused by or otherwise associated with any of the following neuropathic pain conditions. "Neuropathic pain" refers to abnormal sensory input from the peripheral nervous system, the central nervous system, or both, that results in discomfort. Neuropathic pain symptoms can involve persistent, idiopathic pain, and allodynia (a painful response to a stimulus that is not normally painful), hyperalgesia (an exaggerated response to a painful stimulus that normally causes only mild discomfort, e.g., a pinprick), or hyperalgesia (a brief discomfort that becomes prolonged, severe pain). Neuropathic pain can be caused by any of the following: 1. Traumatic insult, such as a nerve compression injury (e.g., nerve crush, nerve stretch, nerve entrapment, or incomplete nerve transsection), spinal cord injury (e.g., spinal cord hemisection), limb amputation, contusion, inflammation (e.g., spinal cord inflammation), or surgery. 2. Ischemic events, including, for example, stroke and heart attack. 3. Infectious agents. 4. Exposure to toxic agents including, for example, drugs, alcohol, heavy metals (e.g., lead, arsenic, mercury), industrial agents (e.g., fumes from solvents, adhesives), or nitrous oxide. 5. Diseases including, for example, inflammatory disorders, neoplastic tumors, acquired immune deficiency syndrome (AIDS), Lyme disease, leprosy, metabolic diseases, peripheral neuropathies such as neuromas, mononeuropathies, or polyneuropathies.

[0246] Types of neuropathic pain include:

[0247] 1. Neuralgia

[0248] Neuralgia is pain that radiates along the course of one or more specific nerves, usually without any obvious pathological alteration of the nerve structure. The causes of neuralgia are varied. Chemical itching, inflammation, trauma (including surgery), compression by nearby structures (e.g., tumors), and infection can all lead to neuralgia. However, in many cases, the cause is unknown or unidentifiable. While neuralgia is most common in the elderly, it can occur at any age. Neuralgia includes, without limitation, trigeminal neuralgia, postherpetic neuralgia, postherpetic neuralgia, glossopharyngeal neuralgia, sciatica, and atypical facial pain.

[0249] Neuralgia is pain in the distribution of a nerve or nerves. Examples are trigeminal neuralgia, atypical facial pain, and postherpetic neuralgia (caused by shingles or herpes). The affected nerves are responsible for sensing touch, temperature, and pressure in the facial area from the jaw to the forehead. The disorder generally causes brief episodes of severe pain, usually less than two minutes and limited to one side of the face. The pain can be described in various ways, such as "stabbing," "sharp," "thunderous," "burning," and even "tingling." In atypical forms of TN, the pain can also manifest as severe or simply tingling and can last for an extended period of time. The pain associated with TN is recognized as one of the most severe pains that can be experienced.

[0250] Simple stimuli such as eating, talking, washing the face, or any light touch or sensation (even the sensation of a gentle breeze) can trigger an attack. Attacks can occur in clusters or as isolated attacks.

[0251] Symptoms include sharp, stabbing or constant burning pain in the same location for each episode, usually located somewhere on or near the surface of the body, pain in the path of a specific nerve, impaired function of the affected body part due to the pain or muscle weakness due to accompanying motor nerve damage, increased sensitivity or numbness of the skin in the affected skin area (similar to a feeling from a local anesthetic such as a novocaine injection), and any touch or pressure is interpreted as painful. Movement may also be painful.

[0252] Trigeminal neuralgia is the most common form of neuralgia. It affects the trigeminal nerve, the main sensory nerve of the face ("trigeminal" literally means "three origins" and refers to the branching of the nerve into three branches). This condition involves sudden and brief attacks of severe pain on the side of the face along the area supplied by the trigeminal nerve on that side. The pain attacks can be severe enough to cause facial distortion, classically referred to as painful tics. In some cases, trigeminal neuralgia is caused by a blood vessel or small tumor pressing on the nerve. Disorders such as multiple sclerosis (an inflammatory disease affecting the brain and spinal cord), certain forms of arthritis, and diabetes (high blood sugar) can also cause trigeminal neuralgia, although the cause is not always identified. In this condition, certain movements, such as chewing, speaking, swallowing, or touching certain areas of the face, can trigger spasms of severe pain.

[0253] A related but somewhat uncommon neuralgia affects the glossopharyngeal nerve, which provides sensation to the throat. Symptoms of this neuralgia are brief, shock-like episodes of pain localized to the throat.

[0254] Nerve pain can occur after infections such as Shingles, which is caused by a type of herpes virus called varicella-zoster. This nerve pain produces a constant, burning pain after the Shingles rash has healed. The pain worsens with movement or touch of the affected area. Not everyone diagnosed with Shingles progresses to experiencing postherpetic neuralgia, which can be even more painful than Shingles. The pain and sensitivity can last for months or even years. The pain is usually in the form of intolerable sensitivity to any touch, especially light touch. Postherpetic neuralgia is not limited to the face. It can occur anywhere on the body, but it usually occurs at the location of the Shingles rash. Depression is not uncommon due to pain and social isolation during the illness.

[0255] Postherpetic neuralgia can be debilitating long after the symptoms of the original herpes infection have disappeared. Other infectious diseases that can cause neuralgia are syphilis and Lyme disease.

[0256] Diabetes is another common cause of nerve pain. This very common medical problem affects approximately 1 in every 20 adult Americans. Diabetes damages the arterioles that supply circulation to nerves, leading to nerve fiber dysfunction and, in some cases, nerve loss. Diabetes can produce almost any nerve pain, including trigeminal neuralgia, carpal tunnel syndrome (pain and numbness in the hand and wrist), and dysesthesias femoralgia (numbness and pain in the thigh due to damage to the lateral femoral cutaneous nerve). Strict blood sugar control can prevent diabetic nerve damage and can accelerate recovery in subjects who do develop nerve pain.

[0257] Other medical conditions that may be related to nerve pain are chronic kidney failure and porphyria, an inherited disorder in which the body is unable to rid itself of certain substances produced after the normal breakdown of blood. Certain medications can also cause this problem.

[0258] 2. Deafferentation

[0259] Deafferentation refers to the loss of sensory input from a part of the body and can be caused by interruption of either peripheral sensory fibers or nerves from the central nervous system. Deafferentation pain syndromes include, without limitation, brain or spinal cord injury, post-stroke pain, phantom pain, paraplegia, brachial plexus avulsion injury, and lumbar radiculopathy.

[0260] 3. Complex Regional Pain Syndrome (CRPS)

[0261] CRPS is a chronic pain syndrome resulting from sympathetically maintained pain and occurs in two forms. CRPS1 currently replaces the term "reflex sympathetic dystrophy syndrome." It is a chronic nerve disorder that most often occurs in the arms or legs after minor or major injury. CRPS1 is associated with severe pain, changes in the nails, bones, and skin, and increased sensitivity to touch in the affected extremity. CRPS2 replaces the term causalgia and results from identified injury to the nerve. CRPS includes, without limitation, CRPSI (reflex sympathetic dystrophy) and CRPS II (causalgia).

[0262] 4. Neuropathy

[0263] Neuropathy is a functional or pathological change of the nerves, clinically characterized by abnormalities of sensory or motor neurons.

[0264] Central neuropathies are functional or pathological changes in the central nervous system.

[0265] Peripheral neuropathy is a functional or pathological change in one or more peripheral nerves. Peripheral nerves relay information from the central nervous system (brain and spinal cord) to muscles and other organs, and from the skin, joints, and other organs back to the brain. Peripheral neuropathy occurs when these nerves fail to transmit information to and from the brain and spinal cord, resulting in pain, loss of sensation, or an inability to control muscles. In some cases, failure of nerves controlling blood vessels, intestines, and other organs leads to abnormal blood pressure, digestive problems, and loss of other basic bodily processes. Risk factors for neuropathy include diabetes, heavy alcohol use, and exposure to certain chemicals and drugs. Some people have a genetic predisposition to neuropathy. Prolonged pressure on nerves is another risk factor for developing nerve damage. Pressure injuries can be caused by prolonged immobility (e.g., lengthy surgery or prolonged illness) or compression of the nerve by a cast, splint, brace, clutch, or other device. Polyneuropathy refers to a broad range of processes that usually affect both sides of the body equally. Symptoms depend on which type of nerve is affected. The three main types of nerves are sensory, motor, and autonomic. Neuropathy can affect any one or a combination of all three types of nerves. Symptoms also depend on whether the condition affects the entire body or just one nerve (as from an injury). The cause of chronic inflammatory polyneuropathy is an abnormal immune response. The specific antigens, immune processes, and triggering factors are variable and, in many cases, unknown. It can occur in association with other conditions, such as HIV, inflammatory bowel disease, systemic lupus erythematosus, chronic active hepatitis, and blood cell disorders.

[0266] Peripheral neuropathies can involve functional or pathological changes in a single nerve or group of nerves (mononeuropathy) or functional or pathological changes affecting multiple nerves (polyneuropathy).

[0267] Peripheral neuropathies can include: Genetic disorders Charcot-Marie-Tooth disease Friedreich's ataxia Systemic or metabolic disorders Diabetes (diabetic neuropathy) Dietary deficiencies (especially vitamin B12) Excessive alcohol use (alcoholic neuropathy) Uremia (from kidney failure) cancer infectious or inflammatory conditions AIDS hepatitis Colorado Tick Fever diphtheria Guillain-Barré syndrome HIV infection without development of AIDS Leprosy lime Polyarteritis nodosa Rheumatoid arthritis sarcoidosis Sjögren's syndrome syphilis Systemic lupus erythematosus Amyloid Exposure to toxic compounds Sniffing glue or other toxic compounds Nitrous oxide Industrial chemicals - especially solvents Heavy metals (lead, arsenic, mercury, etc.) Neuropathies secondary to drugs, such as analgesic-induced nephropathy Miscellaneous causes Ischemia (reduced oxygen / reduced blood flow) Long-term exposure to cold Polyneuropathy Polyneuropathy is a peripheral neuropathy involving loss of movement or sensation in an area caused by damage or destruction of multiple peripheral nerves. Polyneuropathic pain includes, but is not limited to, post-polio syndrome, post-mastectomy syndrome, diabetic neuropathy, alcoholic neuropathy, amyloid, toxins, AIDS, hypothyroidism, uremia, vitamin deficiency, chemotherapy-induced pain, 2',3'-dideoxycytidine (ddC) treatment, Guillain-Barré syndrome, or Fabry disease. B mononeuropathy A mononeuropathy is a peripheral neuropathy involving loss of movement or sensation in an area caused by damage or destruction of a single peripheral nerve or nerve group. Mononeuropathy is most often caused by damage to a localized area resulting from injury or trauma, but occasionally, a systemic disorder can cause isolated nerve damage (as in mononeuritis multiplex). Common causes are direct trauma, prolonged pressure on the nerve, and compression of the nerve by swelling or injury of nearby body structures. Damage involves destruction of the nerve's myelin sheath (covering) or part of the nerve cell (axon). This damage slows or prevents the conduction of impulses along the nerve. Mononeuropathy can involve any part of the body. Mononeuropathic pain includes, without limitation, sciatic nerve dysfunction, common peroneal nerve dysfunction, radial nerve dysfunction, ulnar nerve dysfunction, cranial nerve mononeuropathy VI, cranial nerve mononeuropathy VII, cranial nerve mononeuropathy III (compression type), cranial nerve mononeuropathy III (diabetic type), axillary nerve dysfunction, carpal tunnel syndrome, femoral nerve dysfunction, tibial nerve dysfunction, Bell's palsy, thoracic outlet syndrome, carpal tunnel syndrome, and 6th (abducens) nerve palsy. C. Generalized peripheral neuropathy Generalized peripheral neuropathies are symmetric and usually result from a variety of systemic diseases and disease processes that affect the peripheral nervous system throughout its entirety. They are further subdivided into several categories: i. Distal axonopathy is the result of some metabolic or toxic derangement of neurons. It can be caused by metabolic diseases, such as diabetes, renal failure, deficiency syndromes, such as malnutrition and alcoholism, or the effects of toxins or drugs. Distal axonopathy (also known as dying-back neuropathy) is a type of peripheral neuropathy resulting from some metabolic or toxic derangement of peripheral nervous system (PNS) neurons. It is the most common response of nerves to metabolic or toxic abnormalities and, therefore, can be caused by metabolic diseases, such as diabetes, renal failure, deficiency syndromes, such as malnutrition and alcoholism, or the effects of toxins or drugs. The most common cause of distal axonopathy is diabetes, and the most common distal axonopathy is diabetic neuropathy. ii. Myelinopathy is caused by a primary attack on myelin, resulting in acute failure of impulse conduction. The most common cause is acute inflammatory demyelinating polyneuropathy (AIDP, also known as Guillain-Barré syndrome), but other causes include chronic inflammatory demyelinating polyneuropathy (CIDP), genetic metabolic disorders (e.g., leukodystrophies), or toxins. Myelinopathy is caused by primary destruction of myelin or myelin-forming Schwann cells, which leaves axons intact but causes acute failure of impulse conduction. This demyelination slows or completely blocks conduction of electrical impulses along the nerve. The most common cause is acute inflammatory demyelinating polyneuropathy (AIDP, better known as Guillain-Barré syndrome), but other causes include chronic inflammatory demyelinating polyneuropathy (CIDP), genetic metabolic disorders (e.g., leukodystrophies or Charcot-Marie-Tooth disease), or toxins. iii. Neuropathies are the result of destruction of peripheral nervous system (PNS) neurons. They can be caused by motor neuron disease, sensory neuropathies (e.g., shingles), toxins, or autonomic dysfunction. Neurotoxins, such as the chemotherapy drug vincristine, can cause neuropathy. Neuropathy is dysfunction caused by damage to neurons in the peripheral nervous system (PNS), resulting in peripheral neuropathy. It can be caused by motor neuron disease, sensory neuropathy (e.g., shingles), toxic substances, or autonomic dysfunction. People with neuropathy can develop symptoms differently depending on the cause, the way it affects neurons, and the type of neurons most affected. iv. Focal entrapment neuropathy (e.g., carpal tunnel syndrome)

[0268] When the pain is neuropathic pain, in one embodiment, the polypeptide used is a catalytically inactive BoNT / X L chain, a BoNT / X translocation domain (H chain), or a BoNT / X translocation domain (H chain). N domain), and / or the receptor binding domain of BoNT / X (H C In one embodiment, when the pain is neuropathic pain, the polypeptide used optionally does not contain an H domain from a different (i.e., non-BoNT / X) clostridial neurotoxin. C domain (e.g., H from BoNT / B) C The catalytically inactive BoNT / X L chain and translocation domain (H domain) are combined with N Preferably, when the pain is neuropathic pain, the polypeptide used does not contain a catalytically inactive BoNT / X L chain and translocation domain (H domain). N domain) and BoNT / B H C Does not include the domain.

[0269] The polypeptides of the present invention may be used to treat pain caused by or otherwise associated with any of the following inflammatory conditions: Similarly, the polypeptides of the present invention may be used to treat one or more of the following inflammatory conditions:

[0270] A. Arthritic disorders Arthritic disorders include, for example, rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus (SLE), gouty arthritis, scleroderma, osteoarthritis, psoriatic arthritis, ankylosing spondylitis, Reiter's syndrome (reactive arthritis), adult Still's disease, arthritis from viral infections, arthritis from bacterial infections such as gonococcal arthritis and nongonococcal bacterial arthritis (septic arthritis), late-stage Lyme disease, tuberculous arthritis, and arthritis from fungal infections such as blastomycosis.

[0271] B. Autoimmune diseases Autoimmune diseases include, for example, Guillain-Barré syndrome, Hashimoto's thyroiditis, pernicious anemia, Addison's disease, type 1 diabetes, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, Reiter's syndrome, and Graves' disease.

[0272] C. Connective tissue disorders Connective tissue disorders include, for example, spondyloarthritis, dermatomyositis, and fibromyalgia.

[0273] D. Injury Inflammation caused by injury, including, for example, crushing, puncturing, or stretching of tissue or joints, can lead to chronic inflammatory pain.

[0274] E. Infection Inflammation caused by infections, including, for example, tuberculosis or interstitial keratitis, can cause chronic inflammatory pain.

[0275] F.Neuritis Neuritis is an inflammatory process that affects a nerve or group of nerves. Symptoms depend on the nerve involved but can include pain, paresthesias, paresis, or hypoesthesia (numbness). Examples include: a. Brachial neuritis b. Retrobulbar neuropathy, an inflammatory process that affects the part of the optic nerve immediately behind the eyeball. c. Optic neuropathy, an inflammatory process affecting the optic nerve causing sudden loss of vision in the affected eye. The cause of optic neuritis is unknown. Sudden inflammation of the optic nerve (the nerve connecting the eye and brain) leads to swelling and destruction of the myelin sheath. Inflammation can sometimes be the result of a viral infection, or it can be caused by an autoimmune disease such as multiple sclerosis. Risk factors relate to possible causes. d. Vestibular neuritis, a viral infection that causes an inflammatory process affecting the vestibular nerve.

[0276] G. Joint inflammation Joint inflammation, such as that caused by bursitis or tendonitis, for example, can cause chronic inflammatory pain.

[0277] H. Sunburn and / or UV-induced damage

[0278] The polypeptides of the present invention can be used to treat pain caused by or otherwise associated with any of the following headache conditions: Headache (medically known as cephalus) is a condition of mild to severe pain in the head. In some cases, pain in the neck or upper back can also be interpreted as a headache. It can indicate an underlying local or systemic disease or can be the disorder itself.

[0279] A. Muscular / Myogenic Headache Muscular / myogenic headaches appear to involve tightness or tension in the facial and neck muscles. They may radiate to the forehead. Tension headaches are the most common form of myogenic headache.

[0280] Tension headaches are conditions involving pain or discomfort in the head, scalp, or neck, usually associated with muscle tightness in these areas. Tension headaches result from contraction of the neck and scalp muscles. One cause of this muscle contraction is a response to stress, depression, or anxiety. Any activity that keeps the head in one position for a long period of time without movement can cause a headache. Such activities include typing or using a computer, fine manual work, and using a microscope. Sleeping in a cold room or with the neck in an abnormal position can also trigger this type of headache. Tension headaches include, without limitation, episodic tension headaches and chronic tension headaches.

[0281] B. Vascular headache The most common type of vascular headache is migraine. Other types of vascular headache include cluster headaches, which cause repeated episodes of intense pain, and headaches resulting from high blood pressure. 1. Migraine Migraine is a heterogeneous disorder that generally involves recurrent headaches. Migraines differ from other headaches because they occur in conjunction with other symptoms, such as nausea, vomiting, or sensitivity to light. In most individuals, throbbing pain is felt on only one side of the head. Clinical features, such as the type of aura, the presence of a premonition, or associated symptoms, such as dizziness, may be seen in subgroups of subjects with different underlying pathophysiological and genetic mechanisms. Migraine headaches include, but are not limited to, migraine without aura (common migraine), migraine with aura (classic migraine), menstrual migraine, migraine equivalents (acephalic headaches), complex migraine, abdominal migraine, and mixed tension-type migraine. 2. Cluster headaches Cluster headaches affect one side of the head (unilateral) and may be associated with watery eyes and stuffy nose. They occur in clusters, recurring at the same time each day for several weeks, then subside.

[0282] D. Hypertensive headache

[0283] E. Traction and inflammatory headaches Traction and inflammatory headaches are usually symptoms of other disorders ranging from stroke to sinus infection.

[0284] F. Hormonal headaches

[0285] G. Rebound headache Rebound headaches, also known as medication overuse headaches, occur when medications to relieve headaches are taken too frequently. Frequently, rebound headaches occur daily and can be very painful.

[0286] H. Chronic sinus headache Sinusitis is inflammation of the paranasal sinuses, either bacterial, fungal, viral, allergic, or autoimmune. Chronic sinusitis is one of the most common complications of the common cold. Symptoms include nasal congestion, facial pain, headache, fever, general malaise, thick green or yellow nasal discharge, and a feeling of facial "fullness" that worsens with bending forward. In a few cases, chronic maxillary sinusitis can also be caused by the spread of bacteria from a dental infection. Chronic hyperplastic eosinophilic sinusitis is a noninfectious form of chronic sinusitis.

[0287] I. Organic headache

[0288] J. Paroxysmal headache Ital headache is a headache associated with epileptic seizure activity.

[0289] The polypeptides of the present invention can be used to treat pain caused by or otherwise associated with any of the following somatic pain conditions: Somatic pain originates in ligaments, tendons, bones, blood vessels, and even the nerves themselves. It is detected by somatic nociceptors. The paucity of pain receptors in these areas produces a dull, poorly localized pain of longer duration than cutaneous pain. Examples include sprains and fractures. Additional examples include:

[0290] A. Excessive muscle tension Excessive muscle tension can be caused by, for example, a sprain or strain.

[0291] B. Repetitive motion disorder Repetitive motion injuries can result from overuse of the hands, wrists, elbows, shoulders, neck, back, hips, knees, feet, legs, or ankles.

[0292] C. Muscle disorders Muscle disorders that cause somatic pain include, for example, polymyositis, dermatomyositis, lupus, fibromyalgia, polymyalgia rheumatica, and rhabdomyolysis.

[0293] D. Myalgia Myalgia is muscle pain and is a symptom of many diseases and disorders. The most common cause of myalgia is either overuse or overstretching of a muscle or muscle group. Myalgia without a history of trauma is often caused by a viral infection. More long-term myalgia may indicate metabolic myopathy, some nutritional deficiencies, or chronic fatigue syndrome.

[0294] E. Infection Infections can cause somatic pain. Examples of such infections include, for example, muscle abscesses, trichinosis, influenza, Lyme disease, malaria, Rocky Mountain spotted fever, avian influenza, the common cold, community-acquired pneumonia, meningitis, monkeypox, severe acute respiratory syndrome, toxic shock syndrome, trichinosis, typhoid fever, and upper respiratory tract infections.

[0295] F. Drugs Drugs can cause somatic pain, including, for example, cocaine, statins for lowering cholesterol (e.g., atorvastatin, simvastatin, and lovastatin), and ACE inhibitors for lowering blood pressure (e.g., enalapril and captopril).

[0296] The polypeptides of the invention may be used to treat pain caused by or otherwise associated with any of the following visceral pain conditions: Visceral pain originates in a visceral organ or organ of the body. Visceral nociceptors are located within the organs and cavities of the body. The greater paucity of nociceptors in these areas produces pain that is usually more aching and of longer duration than somatic pain. Visceral pain is extremely difficult to localize, and various injuries to visceral tissues exhibit "referred" pain, where sensation is localized to an area completely unrelated to the site of injury. Examples of visceral pain include:

[0297] A. Functional visceral pain Functional visceral pain includes, for example, irritable bowel syndrome and chronic functional abdominal pain (CFAP), functional constipation and functional dyspepsia, non-cardiac chest pain (NCCP), and chronic abdominal pain.

[0298] B. Chronic gastrointestinal inflammation Chronic gastrointestinal inflammation includes, for example, gastritis, inflammatory bowel diseases such as Crohn's disease, ulcerative colitis, microscopic colitis, diverticulitis, and gastroenteritis, interstitial cystitis, intestinal ischemia, cholecystitis, appendicitis, gastroesophageal reflux disease, ulcers, nephrolithiasis, urinary tract infections, pancreatitis, and hernias.

[0299] C. Autoimmune pain Autoimmune pain includes, for example, sarcoidosis and vasculitis.

[0300] D. Organic visceral pain Organic visceral pain includes, for example, pain resulting from traumatic, inflammatory, or degenerative lesions of the intestinal tract or produced by tumor impingement on sensory innervation.

[0301] E. Procedurally induced visceral pain Treatment-induced visceral pain includes, for example, pain associated with chemotherapy treatment or pain associated with radiation treatment.

[0302] The polypeptides of the present invention may be used to treat pain caused by or otherwise associated with any of the following referred pain conditions:

[0303] Referred pain results from pain localized in an area other than the site of the painful stimulus. Often, referred pain occurs when a nerve is compressed or damaged at or near its origin. In this situation, the sensation of pain will generally be felt in the territory served by the nerve, despite the injury originating elsewhere. A common example occurs in a herniated disc, where a nerve root arising from the spinal cord is compressed by adjacent disc material. While pain can arise from the damaged disc itself, pain will also be felt in the area served by the compressed nerve (e.g., the thigh, knee, or foot). Relieving pressure on the nerve root can improve referred pain, provided no permanent nerve damage has occurred. Myocardial ischemia (loss of blood flow to part of the myocardial tissue) is perhaps the most well-known example of referred pain. The sensation may occur as a feeling of restriction in the upper chest or as pain in the left shoulder, arm, or even hand.

[0304] The polypeptides of the present invention may be used to treat post-operative pain.

[0305] Postoperative (e.g., post-surgery) pain is an unpleasant sensation resulting from a surgical procedure. Postoperative pain can be caused by the incision, the procedure itself, wound closure, and tissue damage from any forces applied during the procedure. Postoperative pain (e.g., post-operative pain) can also derive from factors incidental to the surgery. For example, a subject may suffer back pain due to the way the subject is positioned on the operating table, or chest pain may be caused by an incision in the chest. A sore throat can also occur after general anesthesia, as insertion of a breathing tube can cause itching. However, postoperative pain caused by cuts to the skin and muscles from the surgical incision is most common.

[0306] For example, surgery (or more specifically, a surgical incision) may represent a "noxious stimulus" that causes pain. Noxious stimuli, which can induce tissue damage, can activate the release of neurotransmitters from nociceptive afferent endings and neuropeptides, such as substance P and calcitonin gene-related peptide (CGRP), from sensory endings. Noxious information is then transmitted from the peripheral nervous system to the central nervous system, where pain is perceived by the individual.

[0307] Postoperative pain can be caused by a combination of inflammation and nerve tissue damage. For example, degranulation of activated mast cells in response to tissue injury can result in the release of various substances, including proteases, cytokines, serotonin, and the extracellular space. These substances can sensitize (activate at a lower threshold) primary afferent neurons, producing pain hypersensitivity. Because tissues are highly innervated, any region of the body is susceptible to nerve damage from surgery.

[0308] By surgery is meant a medical procedure involving the treatment of injury or disease in a subject, including the subjecting of a body part to incision (optionally removing or repairing a damaged body part). The level of invasiveness (e.g., the level of surgical incision required) can vary between types of surgery, but surgery having a level of invasiveness that causes pain to the subject once the surgery is completed is intended to be encompassed.

[0309] The surgery may involve an incision in the skin and / or fascia and / or muscle. Preferably, the surgery involves an incision in the skin.

[0310] Surgery is not limited to those that may be performed by a physician and also includes, for example, dental surgery. Non-limiting examples of surgery include appendectomy, breast tissue biopsy, breast augmentation or reduction, face lift, cholecystectomy, coronary artery bypass, debridement (e.g., of wounds, burns, or infections), skin grafts, organ transplants, and tonsillectomy.

[0311] Preferably, "postoperative" can refer to a time period beginning at most one day after surgery (e.g., post-surgery). In other words, the term "postoperative" can refer to a time period beginning no more than one day after surgery. For example, the term "postoperative" can refer to a time period beginning 1 to 20 hours after surgery, optionally 2 to 15 hours after surgery, optionally 5 to 10 hours after surgery. Such a time period can represent a time period beginning at a chronological boundary point when the analgesic effect of the surgical anesthesia administered to the subject tapers off (e.g., tapers off) and the subject therefore begins to perceive pain.

[0312] Furthermore, the term "post-operative" may be used interchangeably with the term "post-surgery," as "post-operative" is used herein to mean "surgery."

[0313] Similarly, the term "postoperative pain" can refer to pain perceived (or, more specifically, beginning to be perceived) during a period beginning up to one day after surgery (e.g., post-surgery). In other words, the term "postoperative pain" can refer to pain perceived by a subject during a period beginning no more than one day after surgery. For example, the term "postoperative pain" can refer to pain perceived during a period beginning 1-20 hours after surgery, optionally 2-15 hours after surgery, optionally 5-10 hours after surgery.

[0314] The time period can be 1 to 50 weeks after surgery, for example, 5 to 45 weeks, 10 to 40 weeks, or 10 to 35 weeks.

[0315] This is in contrast to the term "perioperative period," which can refer, for example, to the time at or around the time a subject is undergoing surgery (e.g., the time the subject is in the operating room), preferably beginning at least one hour before surgery and / or ending less than one hour after surgery.

[0316] The present invention addresses a wide range of pain conditions, including chronic pain conditions. In some embodiments, the polypeptides of the present invention are for treating cancer and non-cancer pain.

[0317] Preferably, the polypeptides of the present invention are used to treat neuropathic pain. Neuropathic pain can be acute or chronic. In one embodiment, the neuropathic pain is injury-induced neuropathic pain (injury-associated neuropathic pain). In one embodiment, the neuropathic pain is chemotherapy-induced neuropathic pain (chemotherapy-associated neuropathic pain).

[0318] Preferably, the polypeptide of the present invention is used to treat inflammatory pain. Inflammatory pain can be acute or chronic. In one embodiment, inflammatory pain can be caused by burns. For example, inflammatory pain can be caused by UV damage (e.g., UV-B damage).

[0319] Most preferably, the polypeptides of the present invention are used to treat bladder pain syndrome, phantom limb pain, or migraine pain. Bladder pain syndrome may be caused by or associated with interstitial cystitis.

[0320] Treating pain preferably means reducing pain, in other words, in one embodiment, administration of a polypeptide of the invention reduces pain in a subject.

[0321] More specifically, "reduced" or "reducing" (in terms of pain) preferably means that a lower level of pain is perceived by the subject after administration of a polypeptide of the invention (after administration) when compared to the level of pain perceived by the subject prior to administration (before administration). For example, the level of perceived pain may be reduced by at least 15%, 25%, 35%, 45%, 55%, 65%, 75%, 85%, or 95% after administration relative to before administration. For example, the level of perceived pain may be reduced by at least 75%, preferably at least 85%, and more preferably at least 95% after administration.

[0322] Various means for assessing pain perception are known to those skilled in the art. For example, assessment of mechanical allodynia (either static or dynamic) is routinely used in human pain research, as described in Pogatzki-Zahn et al. (Pain Rep. 2017 Mar; 2(2): e588), which is incorporated herein by reference.

[0323] Suitable (but not limited to) methods for assessing a subject's pain perception include numerical rating scale (NRS) scores, although those skilled in the art will be aware of other methods that may be used in addition or alternatively, such as sensory threshold, pain perception threshold, static mechanical allodynia, dynamic mechanical allodynia, temporal summation, pressure pain threshold, conditioned stimulus pain modulation, and temperature threshold.

[0324] Other non-limiting examples of pain perception measures include the change in SF-36 score from baseline at each scheduled time point, the amount of rescue medication taken during the study, and the time to first dose of rescue medication. These may be considered "exploratory" endpoints or pain perception assessment measures.

[0325] Therefore, in a preferred embodiment, after administration of a polypeptide of the present invention, pain perception may be assessed by one or more of: (a) Numeric Rating Scale (NRS), (b) Stimulus-evoked NRS, (c) Temperature of the painful area, (d) Size of the painful area, (e) Time to onset of analgesic effect, (f) Peak analgesic effect, (g) Time to peak analgesic effect, (h) Duration of analgesic effect, and (i) SF-36 Quality of Life.

[0326] Those skilled in the art are aware of such methods for assessing pain perception. For convenience, further description of the Numerical Rating Scale and the Quality of Life Questionnaire-Short Form-36 is provided below.

[0327] Numerical Rating Scale (NRS). Typically, pain perception according to the present invention uses a Numerical Rating Scale (NRS). The NRS is an 11-point scale for rating a subject's pain perception. Subjects are asked to respond with a number from 0 to 10 that best matches their pain intensity, with zero representing "no pain" and the upper limit of 10 representing "the worst pain possible."

[0328] NRS can be used to assess various aspects of pain, including idiopathic average pain, idiopathic worst pain, and idiopathic pain at the time of assessment. Idiopathic average pain is assessed by asking the subject to select the number that best describes the subject's average pain (e.g., perceived pain) over a period of time, for example, at least 6 hours, 12 hours, 24 hours, or at least 48 hours. Idiopathic worst pain is assessed by asking the subject to select the number that best describes the subject's pain at its worst during a given period of time, for example, at least the previous 6 hours, 12 hours, 24 hours, or the previous 48 hours. Idiopathic pain at the time of assessment is assessed by asking the subject to select the number that best describes the pain the subject is in at the time of assessment.

[0329] The NRS can also be used to assess a subject's pain perception in response to a variety of different stimuli. To assess pain perception in response to a stimulus, the subject will be exposed to stimuli of various natures applied to the painful area. The subject will be asked what their NRS score was at that time point before administration and after the stimulus.

[0330] Examples of stimuli used include (i) light touch (which can be assessed by measuring pain on the surface of the painful area on a radial spoke line after application of von Frey filaments as described herein), (ii) pressure (pressure pain threshold), which can be assessed by asking the subject to provide an NRS score as increasing pressure is applied using a pressure algometer, and (iii) temperature (which can be assessed by asking the subject for an NRS score for hot, cold, and heat stimuli using a thermode applied to the painful area).

[0331] Preferably, administration of a polypeptide of the invention reduces the subject's NRS score after administration (eg, from a score of ≧7 to a score of ≦6) when compared to the subject's NRS score before administration.

[0332] Quality of Life Questionnaire Short Form-36 (SF-36). The SF-36 quality of life questionnaire can be used to assess a subject's pain perception. The SF-36 is a 36-item survey of a subject's health as reported by the subject. The SF-36 consists of eight scale scores (vitality, physical functioning, bodily pain, general health perception, role physical functioning, role emotional functioning, role social functioning, and mental health). Each scale is directly converted to a 0-100 scale, assuming each question carries equal weight. The higher the score recorded on the SF-36, the less disability there is.

[0333] Relevant parameters commonly tested in clinical trials for the treatment of pain are known in the art and can be readily selected by those of skill in the art. Examples of such parameters include, but are not limited to, NRS, stimulus-evoked NRS, temperature of the painful area, size of the painful area, time to onset of analgesic effect, peak analgesic effect, time to peak analgesic effect, duration of analgesic effect, and / or the SF-36 Quality of Life scale described herein. Methods for assessing these parameters are also known in the art and can be performed by those of skill in the art using routine methods and procedures.

[0334] Preferably, administration of a polypeptide of the invention increases the subject's SF-36 score after administration (e.g., from a score of ≦50 to a score of ≧50) when compared to the subject's SF-36 score before administration.

[0335] The inflammatory disorder treated by the polypeptides of the present invention can be an inflammatory disorder of the nervous system, cardiovascular system, respiratory system, digestive system, integumentary system, musculoskeletal system, urinary system, reproductive system, endocrine system, or lymphatic system.

[0336] The inflammatory disorder of the nervous system can be one or more selected from the group consisting of central nervous system inflammation (e.g., encephalitis, myelitis, meningitis, or arachnoiditis), peripheral nervous system inflammation (e.g., neuritis), ocular inflammation (e.g., dacryoadenitis, scleritis, episcleritis, keratitis, retinitis, chorioretinitis, blepharitis, conjunctivitis, or uveitis), and ear inflammation (e.g., otitis externa, otitis media, otitis interna, and mastoiditis).

[0337] The inflammatory disorder of the cardiovascular system can be one or more selected from the group consisting of carditis (eg, endocarditis, myocarditis, or pericarditis) and vasculitis (eg, arteritis, phlebitis, or capillaritis).

[0338] The inflammatory disorder of the respiratory system can be one or more selected from the group consisting of an inflammatory disorder of the upper respiratory system (e.g., sinusitis, rhinitis, pharyngitis, or laryngitis), an inflammatory disorder of the lower respiratory system (e.g., tracheitis, bronchitis, bronchiolitis, pneumonitis, or pleuritis), and mediastinitis.

[0339] The inflammatory disorder of the digestive system can be one or more selected from the group consisting of inflammation of the mouth (e.g., stomatitis, gingivitis, gingivostomatitis, glossitis, tonsillitis, sialadenitis / parotitis, cheilitis, pulpitis, or jaw inflammation), inflammation of the gastrointestinal tract (e.g., esophagitis, gastritis, gastroenteritis, enteritis, colitis, small intestine / colitis, duodenitis, ileitis, appendicitis, or proctitis), and inflammation of the accessory digestive organs (e.g., hepatitis, ascending cholangitis, cholecystitis, pancreatitis, or peritonitis).

[0340] The inflammatory disorder of the integumentary system can be one or more selected from the group consisting of dermatitis (eg, folliculitis), cellulitis, and hidradenitis.

[0341] The inflammatory disorder of the musculoskeletal system can be one or more selected from the group consisting of arthritis, dermatomyositis, soft tissue inflammation (e.g., myositis, synovitis / tendonitis, bursitis, enthesitis, fasciitis, capsulitis, epicondylitis, tendonitis, or panniculitis), osteochondritis, osteitis / osteomyelitis, spondylitis, periostitis, and chondritis.

[0342] The inflammatory disorder of the urinary system can be one or more selected from the group consisting of nephritis (eg, glomerulonephritis or pyelonephritis), ureteritis, cystitis, and urethritis.

[0343] The inflammatory disorder of the reproductive system can be one or more selected from the group consisting of inflammation of the female reproductive system (e.g., oophoritis, salpingitis, endometritis, parametritis, cervicitis, vaginitis, vulvitis, or mastitis), inflammation of the male reproductive system (e.g., orchitis, epididymitis, prostatitis, seminal vesiculitis, balanitis, presitis, or balanoposthitis), and inflammation associated with pregnancy, childbirth, and / or the newborn (e.g., chorioamnionitis, funisitis, or omphalitis).

[0344] The inflammatory disorder of the endocrine system can be one or more selected from the group consisting of insulitis, hypophysitis, thyroiditis, parathyroiditis, and adrenalitis.

[0345] The inflammatory disorder of the lymphatic system can be one or more selected from the group consisting of lymphangitis and lymphadenitis.

[0346] Preferably, the inflammatory disorder is one or more selected from complex regional pain syndrome, endometriosis, rheumatoid arthritis, cystitis, and neuritis. The cystitis is preferably interstitial cystitis. The neuritis is preferably peripheral neuritis.

[0347] Embodiments of the present invention relating to various therapeutic uses are intended to apply equally to methods of treatment, and vice versa.

[0348] sequence homology

[0349] Any of a variety of sequence alignment methods can be used to determine percent identity, including, without limitation, global methods, local methods, and hybrid methods, such as segment approaches. Protocols for determining percent identity are routine procedures within the skill of the art. Global methods align multiple sequences from the beginning to the end of the molecule and determine the best alignment by summing the scores of individual residue pairs and by imposing gap penalties. Non-limiting methods include, for example: CLUSTAL-W, see e.g., Julie D. Thompson et al., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position-Specific Gap Penalties and Weight Matrix Choice, 22(22) Nucleic Acids Research 4673-4680 (1994); and iterative refinement methods, see e.g., Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein. Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments, 264(4) J. Mol. Biol. 823-838 (1996). Local methods align multiple sequences by identifying one or more conserved motifs shared by all of the input sequences. Non-limiting methods include, for example, the following:Match-box, see e.g., Eric Depiereux and Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8(5) CABIOS 501-509 (1992); Gibbs sampling, see e.g., C.E. Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131) Science 208-214 (1993); Align-M, see e.g., Ivo Van Walle et al., Align-M - A New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics:1428-1435 (2004).

[0350] Therefore, percent sequence identity is determined by conventional methods. See, e.g., Altschul et al., Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915-19, 1992. Briefly, as shown below, two amino acid sequences are aligned to optimize the alignment score using a gap open penalty of 10, a gap extension penalty of 1, and the "blosum62" scoring matrix of Henikoff and Henikoff (ibid.) (amino acids are indicated by standard single-letter codes). Preferably, this method is used to align a sequence with a SEQ ID NO: described herein to determine amino acid position numbering, as described herein.

[0351] The "percent sequence identity" between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by the sequences. Therefore, the percent identity can be calculated as the number of identical nucleotides / amino acids multiplied by 100 divided by the total number of nucleotides / amino acids. The calculation of percent sequence identity can also take into account the number of gaps and the length of each gap that need to be introduced to optimize the alignment of two or more sequences. The sequence comparison and determination of percent identity between two or more sequences can be performed using specific mathematical algorithms such as BLAST. Those skilled in the art will be familiar with this.

number

[0352] The percent identity is then calculated as: [total number of identical matches × 100] / [length of the longer sequence + number of gaps introduced in the longer sequence to align the two sequences]

[0353] Substantially homologous polypeptides are characterized as having one or more amino acid substitutions, deletions, or additions. These changes are preferably minor in nature, i.e., conservative amino acid substitutions (see below) and other substitutions that do not significantly affect the folding or activity of the polypeptide, typically small deletions of from 1 to about 30 amino acids, and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, small linker peptides of up to about 20-25 residues, or affinity tags.

[0354] Conservative amino acid substitutions Basicity: Arginine lysine histidine Acidic: glutamic acid Aspartic acid polarity: glutamine Asparagine Hydrophobicity: Leucine Isoleucine Balin Aromaticity: phenylalanine Tryptophan Tyrosine small: glycine Alanine Serine Threonine methionine

[0355] In addition to the 20 standard amino acids, non-standard amino acids (e.g., 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline, and α-methylserine) can be substituted for amino acid residues in the polypeptides of the invention. A limited number of non-conservative amino acids, amino acids not encoded by the genetic code, and unnatural amino acids can be substituted for polypeptide amino acid residues. The polypeptides of the invention can also include amino acid residues that do not occur in nature.

[0356] Non-naturally occurring amino acids include, without limitation, trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allothreonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine. Several methods for incorporating non-naturally occurring amino acid residues into proteins are known in the art. For example, an in vitro system can be employed in which nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylating tRNAs are known in the art. Transcription and translation of plasmids containing nonsense mutations are performed in a cell-free system containing E. coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. (See, e.g., Robertson et al., J. Am. Chem. Soc. 113: 2722, 1991; Ellman et al., Methods Enzymol. 202: 301, 1991; Chung et al., Science 259: 806-9, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA 90: 10145-9, 1993.) In a second method, translation is carried out in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNA (Turcatti et al., J. Biol. Chem. 271: 19991-8, 1996). In the third method, E. coli cells are cultured in the absence of the natural amino acid to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine).Non-naturally occurring amino acids are incorporated into polypeptides in place of their natural counterparts. See Koide et al., Biochem. 33: 7470-6, 1994. Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2: 395-403, 1993).

[0357] A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for amino acid residues in the polypeptides of the invention.

[0358] Essential amino acids on the polypeptides of the invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989). Sites of biological interaction can also be determined by physical analysis of structures determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, along with mutations of putative contact site amino acids. See, e.g., de Vos et al., Science 255: 306-12, 1992; Smith et al., J. Mol. Biol. 224: 899-904, 1992; Wlodaver et al., FEBS Lett. 309: 59-64, 1992. The identity of essential amino acids can also be inferred from analysis of homology with related components (e.g., translocation or protease components) of the polypeptides of the invention.

[0359] Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions on a polypeptide, selecting functional polypeptides, and then sequencing the mutagenized polypeptides to determine the spectrum of permissible substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30: 10832-7, 1991; Ladner et al., U.S. Pat. No. 5,223,409; Huse, WO 92 / 06204) and region-directed mutagenesis (Derbyshire et al., Gene 46: 145, 1986; Ner et al., DNA 7: 127, 1988).

[0360] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994) and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide those of ordinary skill in the art with a comprehensive dictionary of many of the terms used in this disclosure.

[0361] The present disclosure is not limited by the exemplary methods and materials disclosed herein; any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, each nucleic acid sequence is written left to right in 5' to 3' orientation. Amino acid sequences are written left to right in amino to carboxy orientation.

[0362] The headings provided herein are not limitations of the various aspects or embodiments of the disclosure.

[0363] Amino acids are referred to herein using the amino acid name, three-letter abbreviation, or one-letter abbreviation. As used herein, the term "protein" encompasses proteins, polypeptides, and peptides. As used herein, the term "amino acid sequence" is synonymous with the term "polypeptide" and / or the term "protein." In some cases, the term "amino acid sequence" is synonymous with the term "peptide." In some cases, the term "amino acid sequence" is synonymous with the term "enzyme." The terms "protein" and "polypeptide" are used interchangeably herein. In this disclosure and claims, conventional one-letter and three-letter codes for amino acid residues may be used. The three-letter codes for amino acids are as defined in accordance with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that a polypeptide can be encoded by more than one nucleotide sequence due to the degeneracy of the genetic code.

[0364] Other definitions of terms may appear herein. Before exemplary embodiments are described in more detail, it should be understood that the present disclosure is not limited to the specific embodiments described, and as such may vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to be limiting, since the scope of the present disclosure is defined only by the appended claims.

[0365] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limit of that range is also specifically disclosed, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise. Each smaller range between any claimed or intervening value within a claimed range and any other claimed or intervening value within that claimed range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range. Subject to any specifically excluded limit within the claimed range, each range in which either limit is included in the smaller range, neither is included, or both limits are included is also encompassed within the disclosure. Where a claimed range includes one or both of the limits, ranges excluding either or both of those included limits are also encompassed within the disclosure.

[0366] It should be pointed out that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a clostridial neurotoxin" includes a plurality of such candidate agents, reference to "the clostridial neurotoxin" includes reference to one or more clostridial neurotoxins and equivalents thereof known to those skilled in the art, and so forth.

[0367] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application, and nothing herein should be construed as an admission that such publications constitute prior art against the claims appended hereto.

[0368] Embodiments of the present invention will now be described, by way of example only, with reference to the following figures and examples. [Brief explanation of the drawings]

[0369] [Figure 1] FIG. 1 shows the % SNAP25 cleavage in human neuronal cells treated with BoNT / A or BoNT / A(0). [Figure 2] FIG. 2 shows the % SNAP25 cleavage in rat neuronal cells treated with BoNT / A or BoNT / A(0). [Figure 3] (A) The characteristic startle response of a mouse suspended by its tail. (B) The scoring system used in the digit abduction score (DAS) assay. [Figure 4](A) shows an experimental schematic of a study using the chronic constriction injury (CCI) model of chronic neuropathic pain in adult male Sprague-Dawley rats (220-250 g). Days (D) before and after administration of BoNT / A (60 pg / kg i.p.), BoNT / A (30 pg / kg intraplantar (i.p.)), BoNT / A (60 pg / kg i.p.), vehicle (gelatin phosphate buffered saline (GPB) i.p.—negative control), or gabapentin (100 mg / kg po—positive control) are shown. Administration occurred on day 0 (D0), and CCI surgery was performed on D-14. vF indicates that the von Frey test was performed on the indicated day. (B) Mechanical sensitivity (measured by von Frey test) of the ipsilateral paw (i.e., the paw administered with the control composition, BoNT / A, or BoNT / A(0)) over time is shown for animals administered as described in (A). (C) Mechanical sensitivity (measured by von Frey test) of the contralateral paw (i.e., the paw not administered with the control composition, BoNT / A, or BoNT / A(0)) over time is shown for animals administered as described in (A). (D) Change in body weight for rats administered as described in (A) over time. [Figure 5](A) An experimental schematic of a study using the acute oxaliplatin-induced neuropathic pain model in adult male Sprague-Dawley rats is presented. Oxaliplatin (10 mg / kg intraperitoneally (i.p.)) and BoNT / A (1000 pg / kg i.p.), BoNT / A (50 pg / kg i.p.), BoNT / A (100 pg / kg i.p.), BoNT / A (160 pg / kg i.p.), or vehicle (GPB i.p.—negative control) were administered on day 0 (D0). As an additional negative control for oxaliplatin treatment, a subset of rats received 5% glucose i.p. and GBP (i.p.). Duloxetine (100 mg / kg po—positive control) was administered 1 h before testing on D3. The number of days and hours after administration are indicated. PI indicates that the paw immersion (cold) test was performed on the day indicated. (B) The cold sensitivity (measured by paw immersion test) of the ipsilateral paw (i.e., the paw administered with the control composition, BoNT / A, or BoNT / A(0)) over time is shown for animals administered as described in (A). (C) The cold sensitivity (measured by paw immersion test) of the contralateral paw (i.e., the paw not administered with the control composition, BoNT / A, or BoNT / A(0)) over time is shown for animals administered as described in (A). [Figure 6](A) An experimental schematic of a study using a model of chronic oxaliplatin-induced neuropathic pain in adult male Sprague-Dawley rats (180-210 g) is presented. Oxaliplatin was administered on day -2 (D-2). BoNT / A(0) (100 pg / kg i.p.), BoNT / A (100 pg / kg i.p.), or vehicle (GPB i.p.—negative control) was administered on day 0 (D0). Pregabalin (30 mg / kg po—positive control) was administered on day 3. The days before and after administration are indicated. vF and CP indicate that von Frey and cold-plate tests (respectively) were performed on the indicated days. (B) Mechanical sensitivity (measured by von Frey test) of the ipsilateral paw (i.e., the paw administered with the control composition, BoNT / A, or BoNT / A(0)) over time for animals administered as described in (A). (C) Mechanical sensitivity (measured by von Frey test) of the contralateral paw (i.e., the paw not administered with the control composition, BoNT / A, or BoNT / A(0)) over time for animals administered as described in (A). (D) Thermal sensitivity (measured by cold plate test) over time for animals administered as described in (A). [Figure 7] (A) An experimental schematic of a study using a model of inflammatory pain induced by acute ultraviolet B (UV-B) burn injury in adult male Wistar rats (180-210 g) is presented. BoNT / A (0) (100 pg / kg i.p.), BoNT / A (100 pg / kg i.p.), or vehicle (GPB i.p.—negative control) was administered on day 0 (D0). Indomethacin (5 mg / kg po—positive control) was administered 1 h before the D3 readout. Rats were exposed to UV-B (500 mJ / cm2) on day 1 (D1). vF indicates that the von Frey test was performed on the indicated day. (B) Mechanical sensitivity (measured by the von Frey test) is shown for animals treated as described in (A). [Figure 8]Mechanical sensitivity (measured by the von Frey test) of mice administered vehicle, catalytically active chimeric BoNT / XB (0.3 ng / kg, n = 10), catalytically active chimeric BoNT / XB (30 ng / kg, n = 10), catalytically inactive chimeric BoNT / XB(0) (0.3 ng / kg, n = 10), catalytically inactive chimeric BoNT / XB(0) (30 ng / kg, n = 10), BoNT / A (160 pg / kg, n = 10), or indomethacin (10 mg / kg, n = 9) is shown in untreated animals (baseline), 2 days after BoNT or vehicle administration and before complete Freund's adjuvant (CFA) administration (day 0 CFA, day 2), and 1 day after CFA administration (day 1 CFA, day 3). **P<0.1, ***P<0.01 (Dunnett's multiple comparisons versus vehicle after repeated measures two-way ANOVA).

[0370] Sequence Listing

[0371] Where an initiating Met amino acid residue or corresponding initiation codon is shown in any of the following SEQ ID NOs, said residue / codon is optional.

[0372] SEQ ID NO:1 - Nucleotide sequence of recombinant catalytically inactive BoNT / A (rBoNT / A(0)) SEQ ID NO:2—Polypeptide sequence of rBoNT / A(0) SEQ ID NO:3-rLH N Nucleotide sequence of / A (light chain plus translocation domain only) SEQ ID NO:4-rLH N Polypeptide sequence of / A SEQ ID NO:5 - Nucleotide sequence of rL / A (light chain only) SEQ ID NO: 6 - Polypeptide sequence of rL / A SEQ ID NO:7-rH C Nucleotide sequence of / A SEQ ID NO:8-rH C Polypeptide sequence of / A SEQ ID NO:9—Nucleotide sequence of rBoNT / B(0) SEQ ID NO: 10—Polypeptide sequence of rBoNT / B(0) SEQ ID NO: 11—Nucleotide sequence of rBoNT / C(0) SEQ ID NO: 12—Polypeptide sequence of rBoNT / C(0) SEQ ID NO: 13—Nucleotide sequence of rBoNT / E(0) SEQ ID NO: 14—Polypeptide sequence of rBoNT / E(0) SEQ ID NO: 15—Nucleotide sequence of rBoNT / F(0) SEQ ID NO: 16—Polypeptide sequence of rBoNT / F(0) SEQ ID NO: 17—Nucleotide sequence of rBoNT / A(0) (His-tagged) SEQ ID NO: 18—Polypeptide sequence of rBoNT / A(0) (His-tagged) SEQ ID NO: 19-rLH N Nucleotide sequence of / A (His-tagged) SEQ ID NO: 20-rLH N Polypeptide sequence of / A (His-tagged) SEQ ID NO:21-rH C Nucleotide sequence of / A (His-tagged) SEQ ID NO:22-rH C Polypeptide sequence of / A (His-tagged) SEQ ID NO: 23—Nucleotide sequence of rLC / A (His-tagged) SEQ ID NO: 24—Polypeptide sequence of rLC / A (His-tagged) SEQ ID NO: 25—Nucleotide sequence of rBoNT / FA(0) (His-tagged) SEQ ID NO: 26—Polypeptide sequence of rBoNT / FA(0) (His-tagged) SEQ ID NO: 27-rLH N Nucleotide sequence of / FA (His-tagged) SEQ ID NO: 28-rLH N Polypeptide sequence of / FA (His tagged) SEQ ID NO:29-rH C Nucleotide sequence of / FA (His-tagged) SEQ ID NO: 30-rH C Polypeptide sequence of / FA (His tagged) SEQ ID NO: 31 - Nucleotide sequence of rLC / FA (His-tagged) SEQ ID NO: 32—Polypeptide sequence of rLC / FA (His-tagged) SEQ ID NO: 33—Nucleotide sequence of rBoNT / F(0) (His-tagged) SEQ ID NO: 34—Polypeptide sequence of rBoNT / F(0) (His-tagged) SEQ ID NO: 35-rLH N Nucleotide sequence of / F (His-tagged) SEQ ID NO: 36-rLH N Polypeptide sequence of / F (His tagged) SEQ ID NO: 37-rH C Nucleotide sequence of / F (His-tagged) SEQ ID NO: 38-rH C Polypeptide sequence of / F (His tagged) SEQ ID NO: 39 - Nucleotide sequence of rLC / F (His-tagged) SEQ ID NO: 40—Polypeptide sequence of rLC / F (His-tagged) SEQ ID NO: 41 - Cationic rH C Nucleotide sequence of / A (His-tagged) SEQ ID NO: 42 - Cationic rH C Polypeptide sequence of / A (His-tagged) SEQ ID NO: 43-rH C Nucleotide sequence of / AB (His-tagged) SEQ ID NO: 44-rH C Polypeptide sequence of / AB (His-tagged) SEQ ID NO: 45-rH C Nucleotide sequence of the / A mutant Y1117V H1253K (His-tagged) SEQ ID NO: 46-rH C Polypeptide sequence of / A mutant Y1117V H1253K (His-tagged) SEQ ID NO: 47-rH C Nucleotide sequence of the / A mutant Y1117V F1252Y H1253K L1278F (His-tagged) SEQ ID NO: 48-rH CPolypeptide sequence of / A mutant Y1117V F1252Y H1253K L1278F (His-tagged) SEQ ID NO: 49-rH C Nucleotide sequence of the / A mutant Y1117V F1252Y H1253K L1278H (His-tagged) SEQ ID NO:50-rH C Polypeptide sequence of / A mutant Y1117V F1252Y H1253K L1278H (His-tagged) SEQ ID NO: 51—Polypeptide sequence of BoNT / A—UniProt P10845 SEQ ID NO: 52—Polypeptide sequence of BoNT / B—UniProt P10844 SEQ ID NO: 53—Polypeptide sequence of BoNT / C—UniProt P18640 SEQ ID NO: 54—Polypeptide sequence of BoNT / D—UniProt P19321 SEQ ID NO: 55—Polypeptide sequence of BoNT / E—UniProt Q00496 SEQ ID NO: 56—Polypeptide sequence of BoNT / F—A7GBG3 from UniProt SEQ ID NO: 57—Polypeptide sequence of BoNT / G—UniProt Q60393 SEQ ID NO: 58 - Polypeptide sequence of TeNT - UniProt P04958 SEQ ID NO: 59—Polypeptide sequence of BoNT / X SEQ ID NO: 60—Polypeptide sequence of unmodified BoNT / A1 SEQ ID NO: 61—Polypeptide sequence of mrBoNT / AB(0) SEQ ID NO: 62—Polypeptide sequence of mrBoNT / A(0) SEQ ID NO: 63—Polypeptide sequence of BoNT / XB(0) (His-tagged) SEQ ID NO:64—Polypeptide sequence of BoNT / XB(0) SEQ ID NO: 65—Polypeptide sequence of BoNT / XB(0) mutant (His-tagged) SEQ ID NO: 66—Polypeptide sequence of BoNT / XB(0) mutant SEQ ID NO: 67—Polypeptide sequence of BoNT / XA(0) SEQ ID NO: 68—Polypeptide sequence of BoNT / XA(0) mutant SEQ ID NO: 69—Polypeptide sequence of BoNT / XD(0) SEQ ID NO:70—Polypeptide sequence of BoNT / XF(0) SEQ ID NO:71 - C1 activation loop consensus sequence SEQ ID NO: 72-C1 activation loop SEQ ID NO: 73-C1 activation loop mutant SEQ ID NO: 74—Polypeptide sequence of rLC / A(0) (His-tagged) SEQ ID NO:75-rLH N Polypeptide sequence of / A(0) (His-tagged) SEQ ID NO: 76—Polypeptide sequence of rLC / X(0) SEQ ID NO:77 - PreScission protease site SEQ ID NO: 78-C1 activation loop mutant 2

[0373] SEQ ID NO: 1 - nucleic acid sequence, rBoNT / A(0)

[0374] SEQ ID NO:2 - Polypeptide sequence, rBoNT / A(0)

[0375] SEQ ID NO: 3 - nucleic acid sequence, rLH N / A

[0376] SEQ ID NO: 4 - Polypeptide sequence, rLH N / A MEFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLT SIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAV TLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSG KFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVDGIITSKT KSDDDDKNKALNLQCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKY TMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIF SGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDD LSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTLEAHHHHHHHHHH

[0377] SEQ ID NO: 5 - nucleic acid sequence, rL / A

[0378] SEQ ID NO: 6 - Polypeptide sequence, rL / A MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTS IVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTL AHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFS VDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLGLEAHHHHHHHHHH

[0379] SEQ ID NO: 7 - nucleic acid sequence, rH C / A

[0380] SEQ ID NO:8 - Polypeptide sequence, rH C / A MHHHHHHKNIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENN SGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKEL NEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVK NKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL

[0381] SEQ ID NO: 9 - nucleic acid sequence, rBoNT / B(0)

[0382] SEQ ID NO: 10 - Polypeptide sequence, rBoNT / B(0)

[0383] SEQ ID NO: 11 - nucleic acid sequence, rBoNT / C(0)

[0384] SEQ ID NO: 12 - Polypeptide sequence, rBoNT / C(0)

[0385] SEQ ID NO: 13 - nucleic acid sequence, rBoNT / E(0)

[0386] SEQ ID NO: 14 - Polypeptide sequence, rBoNT / E(0)

[0387] SEQ ID NO: 15 - nucleic acid sequence, rBoNT / F(0)

[0388] SEQ ID NO: 16 - Polypeptide sequence, rBoNT / F(0)

[0389] SEQ ID NO: 17 - nucleic acid sequence, rBoNT / A(0) (His-tagged)

[0390] SEQ ID NO: 18 - Polypeptide sequence, rBoNT / A(0) (His-tagged)

[0391] SEQ ID NO: 19 - nucleic acid sequence, rLH N / A (His-tagged)

[0392] SEQ ID NO: 20 - Polypeptide sequence, rLH N / A (His-tagged) MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTS IVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVT LAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGK FSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKS LDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMF HYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGA VILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSS KLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTENLYFQGASHHHHHHHH

[0393] SEQ ID NO: 21 - nucleic acid sequence, rH C / A (His-tagged)

[0394] SEQ ID NO: 22 - Polypeptide sequence, rH C / A (His-tagged) MHHHHHHENLYFQGKNIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIIN CMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFD KELNEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVV KNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL

[0395] SEQ ID NO: 23 - nucleic acid sequence, rLC / A (His tagged)

[0396] SEQ ID NO: 24 - Polypeptide sequence, rLC / A (His tagged) MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTS IVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVT LAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGK FSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEENLYFQGASHHHHHHHH

[0397] SEQ ID NO: 25 - nucleic acid sequence, rBoNT / FA(0) (His-tagged)

[0398] SEQ ID NO: 26 - Polypeptide sequence, rBoNT / FA(0) (His-tagged)

[0399] SEQ ID NO: 27 - nucleic acid sequence, rLH N / FA (His-tagged)

[0400] SEQ ID NO: 28 - Polypeptide sequence, rLH N / FA (His-tagged) MPVVINSFNYDDPVNDNTIIYIRPPYYETSNTYFKAFQIMDNVWIIPERYRLGIDPSLFNPPVSLKAGSDGYFDPNYLSTNTEKNKYLQIMIKLFKRINSKPAGQILLE EIKNAIPYLGNSYTQEEQFTTNNRTVSFNVKLANGNIVQQMANLIIWGPGPDLTTNKTGGIIYSPYQSMEATPYKDGFGSIMTVEFSPEYATAFNDISIASHSPSLFIKD PALILMHELIHVLHGLYGTYITEYKITPNVVQSYMKVTKPITSAEFLTFGGRDRNIVPQSIQSQLYNKVLSDYKRIASRLNKVNTATALINIDEFKNLYEWKYQFAKDS NGVYSVDLNKFEQLYKKIYSFTEFNLAYEFKIKTRLGYLAENFGPFYLPNLLDDSIYTEVDGFNIGALSINYQGQNIGSDINSIKKLQGQGVVSRVVRLCSNSNTKNSLC ITVNNRDLFFIASQESYGENTINTYKEIDDTTTLDPSFEDILDKVILNFNEQVIPQMPNRNVSTDIQKDNYIPKYDYNRTDIIDSYEVGRNYNTFFYLNAQKFSPNESN ITLTSSFDTGLLEGSKVYTFFSSDFINNINKPVQALLFIEWVKQVIRDFTTEATKTSTVDKLKDISLVVPYIGLALNIGDEIYKQHFAEAVELVGAGLLLEFSPEFLIPT LLIFTIKGYLTGSIRDKDKIIKTLDNALNVRDQKWKELYRWVVSKWLTTINTQFNKRKEQMYKALKNQATAIKKIIENKYNNYTTDEKSKIDSSYNINEIERTLNEKINL AMKNIEQFITESSIAYLINIINNETIQKLKSYDDLVRRYLLGYIRNHSSILGNSVEELNSKVNNHLDNGIPFELSSYTNDSLLIRYFNKNYGEENLYFQGASHHHHHHHH

[0401] SEQ ID NO: 29 - nucleic acid sequence, rH C / FA (His-tagged)

[0402] SEQ ID NO: 30 - Polypeptide sequence, rH C / FA (His-tagged) MLKYNCILNIKYEMDRDKLVDSSGYRSRINIGTGVKFSEIDKNQVQLSNLESSKIEVILNNGVIYNSMYENFSTSFWIRIPKYFRNINNEYKIISCMQNNSGWEVSLNFSN MNSKIIWTLQDTEGIKKTVVFQYTQNINISDYINRWIFVTITNNRLSNSKIYINGRLINEESISDLGNIHASNNIMFKLDGCRDPHRYIWIKYFNLFDKELNKKEIKDLYDN QSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYLDVNNVGIRGYMYLKGPRGRIVTTNIYLNSTLYMGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAG VEKILSAVEIPDVGNLSQVVVMKSENDQGIRNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIGKASRTFGCSWEFIPVDDGWGESSLENLYFQGASHHHHHHHH

[0403] SEQ ID NO: 31 - nucleic acid sequence, rLC / FA (His tagged)

[0404] SEQ ID NO: 32 - Polypeptide sequence, rLC / FA (His tagged) MPVVINSFNYDDPVNDNTIIYIRPPYYETSNTYFKAFQIMDNVWIIPERYRLGIDPSLFNPPVSLKAGSDGYFDPNYLSTNTEKNKYLQIMIKLFKRINSKPAGQILLEEIK NAIPYLGNSYTQEEQFTTNNRTVSFNVKLANGNIVQQMANLIIWGPGPDLTTNKTGGIIYSPYQSMEATPYKDGFGSIMTVEFSPEYATAFNDISIASHSPSLFIKDPALIL MHELIHVLHGLYGTYITEYKITPNVVQSYMKVTKPITSAEFLTFGGRDRNIVPQSIQSQLYNKVLSDYKRIASRLNKVNTATALINIDEFKNLYEWKYQFAKDSNGVYSVDL NKFEQLYKKIYSFTEFNLAYEFKIKTRLGYLAENFGPFYLPNLLDDSIYTEVDGFNIGALSINYQGQNIGSDINSIKKLQGQGVVSRVVRLCSNSENLYFQGASHHHHHHHH

[0405] SEQ ID NO: 33 - nucleic acid sequence, rBoNT / F(0) (His-tagged)

[0406] SEQ ID NO: 34 - Polypeptide sequence, rBoNT / F(0) (His-tagged)

[0407] SEQ ID NO: 35 - nucleic acid sequence, rL H N / F (His-tagged)

[0408] SEQ ID NO: 36 - Polypeptide sequence, rL H N / F (His-tagged) MPVVINSFNYNDPVNDDTILYMQIPYEEKSKKYYKAFEIMRNVWIIPERNTIGTDPSDFDPPASLENGSSAYYDPNYLTTDAEKDRYLKTTIKLFKRINSNPAGEVLLQE ISYAKPYLGNEHTPINEFHPVTRTTSVNIKSSTNVKSSIILNLLVLGAGPDIFENSSYPVRKLMDSGGVYDPSNDGFGSINIVTFSPEYEYTFNDISGGYNSSTESFIAD PAISLAHELIHALHGLYGARGVTYKETIKVKQAPLMIAEKPIRLEEFLTFGQDLNIITSAMKEKIYNNLLANYEKIATRLSRVNSAPPEYDINEYKDYFQWKYGLDKNA DGSYTVNENKFNEIYKKLYSFTEIDLANKFKVKCRNTYFIKYGFLKVPNLLDDDIYTVSEGFNIGNLAVNNRGQNIKLNPKIIDSIPDKGLVEKIVKFCKSVIPRKGTKAP PRLCIRVNNRELFFVASESSYNENDINTPKEIDDTTNLNNNYRNNLDEVILDYNSETIPQISNQTLNTLVQDDSYVPRYDSNGTSEIEEHNVVDLNVFFYLHAQKVPEGE TNISLTSSIDTALSEESQVYTFFSSEFINTINKPVHAALFISWINQVIRDFTTEATQKSTFDKIADISLVVPYVGLALNIGNEVQKENFKEAFELLGAGILLEFVPELLI PTILVFTIKSFIGSSENKNKIIKAINNSLMERETKWKEIYSWIVSNWLTRINTQFNKRKEQMYQALQNQVDAIKTVIEYKYNNYTSDERNRLESEYNINNIREELNKKVS LAMENIERFITESSIFYLMKLINEAKVSKLREYDEGVKEYLLDYISEHRSILGNSVQELNDLVTSTLNNSIPFELSSYTNDKILILYFNKLYKKENLYFQGASHHHHHHHH

[0409] SEQ ID NO: 37 - nucleic acid sequence, rH C / F (His-tagged)

[0410] SEQ ID NO: 38 - Polypeptide sequence, rH C / F (His-tagged) MIKDNSILDMRYENNKFIDISGYGSNISINGDVYIYSTNRNQFGIYSSKPSEVNIAQNNDIIYNGRYQNFSISFWVRIPKYFNKVNLNNEYTIIDCIRNNNNSGWKIS LNYNKIIWTLQDTAGNNQKLVFNYTQMISISDYINKWIFVTITNNRLGNSRIYINGNLIDEKSISNLGDIHVSDNILFKIVGCNDTRYVGIRYFKVFDTELGKTEIET LYSDEPDPSILKDFWGNYLLYNKRYYLLNLLRTDKSITQNSNFLNINQQRGVYQKPNIFSNTRLYTGVEVIIRKNGSTDISNTDNFVRKNDLAYINVVDRDVEYRLYA DISIAKPEKIIKLIRTSNSNNSLGQIIVMDSIGNNCTMNFQNNNGGNIGLLGFHSNNLVASSWYYNNIRKNTSSNGCFWSFISKEHGWQENENLYFQGASHHHHHHHH

[0411] SEQ ID NO: 39 - nucleic acid sequence, rLC / F (His tagged)

[0412] SEQ ID NO: 40 - Polypeptide sequence, rLC / F (His tagged) MPVVINSFNYNDPVNDDTILYMQIPYEEKSKKYYKAFEIMRNVWIIPERNTIGTDPSDFDPPASLENGSSAYYDPNYLTTDAEKDRYLKTTIKLFKRINSNPAGEVLLQEIS YAKPYLGNEHTPINEFHPVTRTTSVNIKSSTNVKSSIILNLLVLGAGPDIFENSSYPVRKLMDSGGVYDPSNDGFGSINIVTFSPEYEYTFNDISGGYNSSTESFIADPAIS LAHELIHALHGLYGARGVTYKETIKVKQAPLMIAEKPIRLEEFLTFGQDLNIITSAMKEKIYNNLLANYEKIATRLSRVNSAPPEYDINEYKDYFQWKYGLDKNADGSYTV NENKFNEIYKKLYSFTEIDLANKFKVKCRNTYFIKYGFLKVPNLLDDDIYTVSEGFNIGNLAVNNRGQNIKLNPKIIDSIPDKGLVEKIVKFCKSENLYFQGASHHHHHHHH

[0413] SEQ ID NO: 41 - nucleic acid sequence, cationic rH C / A (His-tagged)

[0414] SEQ ID NO: 42 - Polypeptide sequence, cationic rH C / A (His-tagged) MIINTSILNLRYESKHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKKAIVYNSMYENFSTSFWIRIPKYFNKISLNNEYTIINCMENNSGWKVSLNY GEIIWTLQDTKEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNKSKIYINGRLIDQKPISNLGNIHASNKIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDN QSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQ AGVEKILSALEIPDVGNLSQVVVMKSKNDKGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPLKLAAALEHHHHHH

[0415] SEQ ID NO: 43 - nucleic acid sequence, rH C / AB (His-tagged)

[0416] SEQ ID NO: 44 - Polypeptide sequence, rH C / AB (His-tagged) MILNNIILNLRYKDNNLIDLSGYGAKVEVYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFFWIRIPKYKNDGIQNYIHNEYTIINCMKNNSGWKISIRGN RIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTITNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELSQSNIEERYKIQSYS EYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKYNQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFKKEEMK LFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESGIVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTEKLAAALEHHHHHH

[0417] SEQ ID NO: 45 - nucleic acid sequence, rH C / A mutant Y1117V H1253K (His-tagged)

[0418] SEQ ID NO: 46 - Polypeptide sequence, rH C / A mutant Y1117V H1253K (His-tagged) MIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVSLN YGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLY DNQSNSGILKDFWGDYLQYDKPYYMLNLVDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNA SQAGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFKQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPLHHHHHHHHHH

[0419] SEQ ID NO: 47 - nucleic acid sequence, rH C / A mutant Y1117V F1252Y H1253K L1278F (His-tagged)

[0420] SEQ ID NO: 48 - Polypeptide sequence, rH C / A mutant Y1117V F1252Y H1253K L1278F (His-tagged) MIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVSLN YGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLY DNQSNSGILKDFWGDYLQYDKPYYMLNLVDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNA SQAGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNNGDIGFIGYKQFNNIAKLVASNWYNRQIERSSRTFGCSWEFIPVDDGWGERPLHHHHHHHHHH

[0421] SEQ ID NO: 49 - nucleic acid sequence, rH C / A mutant Y1117V F1252Y H1253K L1278H (His-tagged)

[0422] SEQ ID NO: 50 - Polypeptide sequence, rH C / A mutant Y1117V F1252Y H1253K L1278H (His-tagged) MIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVSL NYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKD LYDNQSNSGILKDFWGDYLQYDKPYYMLNLVDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLA TNASQAGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNNGDIGFIGYKQFNNIAKLVASNWYNRQIERSSRTHGCSWEFIPVDDGWGERPLHHHHHH

[0423] SEQ ID NO: 51 - Polypeptide sequence, BoNT / A - UniProt P10845 MPFVNKQFNYKDPVNGVDIAYIKIPNVGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT GLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEE ITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNG KKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSG AVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAK VNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKA MININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDK VNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINI GSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNN EYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTIT NNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELN EKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPR GSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQA GVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAK LVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL

[0424] SEQ ID NO: 52 - Polypeptide sequence, BoNT / B - UniProt P10844

[0425] SEQ ID NO: 53 - Polypeptide sequence, BoNT / C - UniProt P18640

[0426] SEQ ID NO: 54 - Polypeptide sequence, BoNT / D - UniProt P19321

[0427] SEQ ID NO: 55 - Polypeptide sequence, BoNT / E - UniProt Q00496

[0428] SEQ ID NO: 56 - Polypeptide sequence, BoNT / F - UniProt A7GBG3

[0429] SEQ ID NO: 57 - Polypeptide sequence, BoNT / G - UniProt Q60393

[0430] SEQ ID NO: 58 - Polypeptide sequence, TeNT - UniProt P04958

[0431] SEQ ID NO: 59 - Polypeptide sequence, BoNT / X MKLEINKFNYNDPIDGINVITMRPPRHSDKINKGKGPFKAFQVIKNIWIVPERYNFTNNT NDLNIPSEPIMEADAIYNPNYLNTPSEKDEFLQGVIKVLERIKSKPEGEKLLELISSSIP LPLVSNGALTLSDNETIAYQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEG TLSEVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMHELVHVTHNLYGIS NRNFYYNFDTGKIETSRQQNSLIFEELLTFGGIDSKAISSLIIKKIIETAKNNYTTLISE RLNTVTVENDLLKYIKNKIPVQGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVR KHYLKERPIDPIYVNILDDNSYSTLEGFNISSQGSNDFQGQLLESSYFEKIESNALRAFI KICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTNVSYPCSLLNGCIEVENKDLFLISN KDSLNDINLSEEKIKPETTVFFKDKLPPQDITLSNYDFTEANSIPSISQQNILERNEELY EPIRNSLFEIKTIYVDKLTTFHFLEAQNIDESIDSSKIRVELTDSVDEALSNPNKVYSPF KNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVIDKSSDTLAIVPYIGPLLNI GNDIRHGDFVGAIELAGITALLEYVPEFTIPILVGLEVIGGELAREQVEAIVNNNALDKRD QKWAEVYNITKAQWWGTIHLQINTRLAHTYKALSRQANAIKMNMEFQLANYKGNIDDKAK IKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMIPKVQDNLKNFDLETKKTLDK FIKEKEDILGTNLSSSLRRKVSIRLNKNIAFDINDIPFSEFDDLINQYKNEIEDYEVLNL GAEDGKIKDLSGTTSDINIGSDIELADGRENKAIKIKGSENSTIKIAMNKYLRFSATDNF SISFWIKHPKPTNLLNNGIEYTLVENFNQRGWKISIQDSKLIWYLRDHNNSIKIVTPDYI AFNGWNLITITNNRSKGSIVYVNGSKIEEKDISSIWNTEVDDPIIFRLKNNRDTQAFTLL DQFSIYRKELNQNEVVKLYNYYFNSNYIRDIWGNPLQYNKKYYLQTQDKPGKGLIREYWS SFGYDYVILSDSKTITFPNNIRYGALYNGSKVLIKNSKKLDGLVRNKDFIQLEIDGYNMG ISADRFNEDTNYIGTTYGTTHDLTTDFEIIQRQEKYRNYCQLKTPYNIFHKSGLMSTETS KPTFHDYRDWVYSSAWYFQNYENLNLRKHTKTNWYFIPKDEGWDED

[0432] Sequence number 60 - Polypeptide sequence, unmodified BoNT / A1

[0433] SEQ ID NO: 61 - Polypeptide sequence, mrBoNT / AB(0)

[0434] SEQ ID NO: 62 - Polypeptide sequence, mrBoNT / A(0) MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDT FTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYS TDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELN LVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESL EVDTNPLLGAGKFATDPAVTLAHQLIYAGHRLYGIAINPNRVFKVNTNAY YEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYT EDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAAN FNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKAL NDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQ QYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTM FHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKD DFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALS KRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQ YNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSM IPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIP FQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESKHLIDLSRYASKINI GSKVNFDPIDKNQIQLFNLESSKIEVILKKAIVYNSMYENFSTSFWIRIP KYFNKISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTKEIKQRVVFK YSQMINISDYINRWIFVTITNNRLNKSKIYINGRLIDQKPISNLGNIHAS NKIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDF WGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYL NSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQA GVEKILSALEIPDVGNLSQVVVMKSKNDKGITNKCKMNLQDNNGNDIGFI GFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL

[0435] SEQ ID NO: 63 - Polypeptide sequence, BoNT / XB(0) (His tagged) MGSMKLEINKFNYNDPIDGINVITMRPPRH SDKINKGKGPFKAFQVIKNIWIVPERYNFT NNTNDLNIPSEPIMEADAIYNPNYLNTPSE KDEFLQGVIKVLERIKSKPEGEKLLELISS SIPLPLVSNGALTLSDNETIAYQENNNIVS NLQANLVIYGPGPDIANNATYGLYSTPISN GEGTLSEVSFSPFYLKPFDESYGNYRSLVN IVNKFVKREFAPDPASTLMHQLVYVTHNLY GISNRNFYYNFDTGKIETSRQQNSLIFEEL LTFGGIDSKAISSLIIKKIIETAKNNYTTL ISERLNTVTVENDLLKYIKNKIPVQGRLGN FKLDTAEFEKKLNTILFVLNESNLAQRFSI LVRKHYLKERPIDPIYVNILDDNSYSTLEG FNISSQGSNDFQGQLLESSYFEKIESNALR AFIKICHKAIDGRSLYNKTLDCIEVENKDL FLISNKDSLNDINLSEEKIKPETTVFFKDK LPPQDITLSNYDFTEANSIPSISQQNILER NEELYEPIRNSLFEIKTIYVDKLTTFHFLE AQNIDESIDSSKIRVELTDSVDEALSNPNK VYSPFKNMSNTINSIETGITSTYIFYQWLR SIVKDFSDETGKIDVIDKSSDTLAIVPYIG PLLNIGNDIRHGDFVGAIELAGITALLEYV PEFTIPILVGLEVIGGELAREQVEAIVNNA LDKRDQKWAEVYNITKAQWWGTIHLQINTR LAHTYKALSRQANAIKMNMEFQLANYKGNI DDKAKIKNAISETEILLNKSVEQAMKNTEK FMIKLSNSYLTKEMIPKVQDNLKNFDLETK KTLDKFIKEKEDILGTNLSSSLRRKVSIRL NKNIAFDINDIPFSEFDDLINQYKNEIEDY EVLNLGAEDGKIKDLSGTTSDINIGSDIEI LNNIILNLRYKDNNLIDLSGYGAKVEVYDG VELNDKNQFKLTSSANSKIRVTQNQNIIF SVFLDFSVSFFWIRIPKYKNDGIQNYIHNEY TIINCMKNNSGWKISIRGNRIIWTLIDING KTKSVFFEYNIREDISEYINRWFFVTITNN LNNAKIYINGKLESNTDIKDIREVIANGEI IFKLDGDIDRTQFIWMKYFSIFNTELSQSN IEERYKIQSYSEYLKDFWGNPLMYNKEYYM FNAGNKNSYIKLKKDSPVGEILTRSKYNQN SKYINYRDLYIGEKFIIRRKSNSQSINDDI VRKEDYIYLDFFNLNQEWRVYTYKYFKKEE MKLFLAPIYDSDEFYNTIQIKEYDEQPTYS CQLLFKKDEESTDEIGLIGIHRFYESGIVF EEYKDYFCISKWYLKEVKRKPYNLKLGCNW QFIPKDEGWTEHHHHHHHHHH

[0436] SEQ ID NO: 64 - Polypeptide sequence, BoNT / XB(0) MGSMKLEINKFNYNDPIDGINVITMRPPRH SDKINKGKGPFKAFQVIKNIWIVPERYNFT NNTNDLNIPSEPIMEADAIYNPNYLNTPSE KDEFLQGVIKVLERIKSKPEGEKLLELISS SIPLPLVSNGALTLSDNETIAYQENNNIVS NLQANLVIYGPGPDIANNATYGLYSTPISN GEGTLSEVSFSPFYLKPFDESYGNYRSLVN IVNKFVKREFAPDPASTLMHQLVYVTHNLY GISNRNFYYNFDTGKIETSRQQNSLIFEEL LTFGGIDSKAISSLIIKKIIETAKNNYTTL ISERLNTVTVENDLLKYIKNKIPVQGRLGN FKLDTAEFEKKLNTILFVLNESNLAQRFSI LVRKHYLKERPIDPIYVNILDDNSYSLEG FNISSQGSNDFQGQLLLESSYFEKIESNALR AFIKICHKAIDGRSLYNKTLDCIEVENKDL FLISNKDSLNDINLSEEKIKPETTVFFKDK LPPQDITLSNYDFTEANSIPSISQQNILER NEELYEPIRNSLFEIKTIYVDKLTTFHFLE AQNIDESIDSSKIRVELTDSVDEALSNPNK VYSPFKNMSNTINSIETGITSTYIFYQWLR SIVKDFSDETGKIDVIDKSSDTLAIVPYIG PLLNIGNDIRHGDFVGAIELAGITALLEYV PEFTIPILVGLEVIGGELAREQVEAIVNNA LDKRDQKWAEVYNITKAQWWGTIHLQINTR LAHTYKALSRQANAIKMNMEFQLANYKGNI DDKAKIKNAISETEILNLKSVEQAMKNTEK FMIKLSNSYLTKEMIPKVQDNLKNFDLETK KTLDKFIKEKEDILGTNLSSSLRRKVSIRL NKNIAFDINDIPFSEFDDLINQYKNEIEDY EVLNLGAEDGKIKDLSGTTSDINIGSDIEI LNNIILNLRYKDNNLIDLSGYGAKVYDG VELNDKNQFKLTSSANSKIRVTQNQNIIFN SVFLDFSVSFWIRIPKYKNDGIQNYIHNEY TIINCMKNNSGWKISIRGNRIIWTLIDING KTKSVFFEYNIREDISEYINRWFFVTITNN LNNAKIYINGKLESNTDIKDIREVIANGEI IFKLDGDIDRTQFIWMKYFSIFNTELSQSN IEERYKIQSYSEYLKDFWGNPLMYNKEYYM FNAGNKNSYIKLKKDSPVGEILTRSKYNQN SKYINYRDLYIGEKFIIRRKSNSQSINDDI VRKEDYIYLDFFNLNQEWRVYTYKYFKKEE MKLFLAPIYDSDEFYNTIQIKEYDEQPTYS CQLLFKKDEESTDEIGLIGIHRFYESGIVF EEYKDYFCISKWYLKEVKRKPYNLKLGCNW QFIPKDEGWTE

[0437] SEQ ID NO: 65 - Polypeptide sequence, BoNT / XB(0) mutant (His-tagged) MGSMKLEINKFNYNDPIDGINVITMRPPRH SDKINKGKGPFKAFQVIKNIWIVPERYNFT NNTNDLNIPSEPIMEADAIYNPNYLNTPSE KDEFLQGVIKVLERIKSKPEGEKLLELISS SIPLPLVSNGALTLSDNETIAYQENNNIVS NLQANLVIYGPGPDIANNATYGLYSTPISN GEGTLSEVSFSPFYLKPFDESYGNYRSLVN IVNKFVKREFAPDPASTLMHQLVYVTHNLY GISNRNFYYNFDTGKIETSRQQNSLIFEEL LTFGGIDSKAISSLIIKKIIETAKNNYTTL ISERLNTVTVENDLLKYIKNKIPVQGRLGN FKLDTAEFEKKLNTILFVLNESNLAQRFSI LVRKHYLKERPIDPIYVNILDDNSYSTLEG FNISSQGSNDFQGQLLESSYFEKIESNALR AFIKICHKAIDGRSLYNKTLDCIEVENKDL FLISNKDSLNDINLSEEKIKPETTVFFKDK LPPQDITLSNYDFTEANSIPSISQQNILER NEELYEPIRNSLFEIKTIYVDKLTTFHFLE AQNIDESIDSSKIRVELTDSVDEALSNPNK VYSPFKNMSNTINSIETGITSTYIFYQWLR SIVKDFSDETGKIDVIDKSSDTLAIVPYIG PLLNIGNDIRHGDFVGAIELAGITALLEYV PEFTIPILVGLEVIGGELAREQVEAIVNNA LDKRDQKWAEVYNITKAQWWGTIHLQINTR LAHTYKALSRQANAIKMNMEFQLANYKGNI DDKAKIKNAISETEILLNKSVEQAMKNTEK FMIKLSNSYLTKEMIPKVQDNLKNFDLETK KTLDKFIKEKEDILGTNLSSSLRRKVSIRL NKNIAFDINDIPFSEFDDLINQYKNEILNN IILNLRYKDNNLIDLSGYGAKVEVYDGVEL NDKNQFKLTSSANSKIRVTQNQNIIFNSVF LDFSVSFWIRIPKYKNDGIQNYIHNEYTII NCMKNNSGWKISIRGNRIIWTLIDINGKTK SVFFEYNIREDISEYINRWFFVTITNNLNN AKIYINGKLESNTDIKDIREVIANGEIIFK LDGDIDRTQFIWMKYFSIFNTELSQSNIEE RYKIQSYSEYLKDFWGNPLMYNKEYYMFNA GNKNSYIKLKKDSPVGEILTRSKYNQNSKY INYRDLYIGEKFIIRRKSNSQSINDDIVRK EDYIYLDFFNLNQEWRVYTYKYFKKEEMKL FLAPIYDSDEFYNTIQIKEYDEQPTYSCQL LFKKDEESTDEIGLIGIHRFYESGIVFEEY KDYFCISKWYLKEVKRKPYNLKLGCNWQFI PKDEGWTEHHHHHHHHHH

[0438] SEQ ID NO: 66 - Polypeptide sequence, BoNT / XB(0) mutant MGSMKLEINKFNYNDPIDGINVITMRPPRH SDKINKGKGPFKAFQVIKNIWIVPERYNFT NNTNDLNIPSEPIMEADAIYNPNYLNTPSE KDEFLQGVIKVLERIKSKPEGEKLLELISS SIPLPLVSNGALTLSDNETIAYQENNNIVS NLQANLVIYGPGPDIANNATYGLYSTPISN GEGTLSEVSFSPFYLKPFDESYGNYRSLVN IVNKFVKREFAPDPASTLMHQLVYVTHNLY GISNRNFYYNFDTGKIETSRQQNSLIFEEL LTFGGIDSKAISSLIIKKIIETAKNNYTTL ISERLNTVTVENDLLKYIKNKIPVQGRLGN FKLDTAEFEKKLNTILFVLNESNLAQRFSI LVRKHYLKERPIDPIYVNILDDNSYSTLEG FNISSQGSNDFQGQLLESSYFEKIESNALR AFIKICHKAIDGRSLYNKTLDCIEVENKDL FLISNKDSLNDINLSEEKIKPETTVFFKDK LPPQDITLSNYDFTEANSIPSISQQNILER NEELYEPIRNSLFEIKTIYVDKLTTFHFLE AQNIDESIDSSKIRVELTDSVDEALSNPNK VYSPFKNMSNTINSIETGITSTYIFYQWLR SIVKDFSDETGKIDVIDKSSDTLAIVPYIG PLLNIGNDIRHGDFVGAIELAGITALLEYV PEFTIPILVGLEVIGGELAREQVEAIVNNA LDKRDQKWAEVYNITKAQWWGTIHLQINTR LAHTYKALSRQANAIKMNMEFQLANYKGNI DDKAKIKNAISETEILLNKSVEQAMKNTEK FMIKLSNSYLTKEMIPKVQDNLKNFDLETK KTLDKFIKEKEDILGTNLSSSLRRKVSIRL NKNIAFDINDIPFSEFDDLINQYKNEILNN IILNLRYKDNNLIDLSGYGAKVEVYDGVEL NDKNQFKLTSSANSKIRVTQNQNIIFNSVF LDFSVSFWIRIPKYKNDGIQNYIHNEYTII NCMKNNSGWKISIRGNRIIWTLIDINGKTK SVFFEYNIREDISEYINRWFFVTITNNLNN AKIYINGKLESNTDIKDIREVIANGEIIFK LDGDIDRTQFIWMKYFSIFNTELSQSNIEE RYKIQSYSEYLKDFWGNPLMYNKEYYMFNA GNKNSYIKLKKDSPVGEILTRSKYNQNSKY INYRDLYIGEKFIIRRKSNSQSINDDIVRK EDYIYLDFFNLNQEWRVYTYKYFKKEEMKL FLAPIYDSDEFYNTIQIKEYDEQPTYSCQL LFKKDEESTDEIGLIGIHRFYESGIVFEEY KDYFCISKWYLKEVKRKPYNLKLGCNWQFI PKDEGWTE

[0439] SEQ ID NO: 67 - Polypeptide sequence, BoNT / XA(0) MGSMKLEINKFNYNDPIDGINVITMRPPRH SDKINKGKGPFKAFQVIKNIWIVPERYNFT NNTNDLNIPSEPIMEADAIYNPNYLNTPSE KDEFLQGVIKVLERIKSKPEGEKLLELISS SIPLPLVSNGALTLSDNETIAYQENNIVS NLQANLVIYGPGPDIANNATYGLYSTPISN GEGTLSEVSFSFYLKPFDESYGNIRSLVN IVNKFVKREFAPDPASTLMHQLVYVTHNLY GISNRNFYYNFDTGKIETSRQQNSLIFEEL LTFGGIDSKAIISSLIIKKIIETAKNYTTL ISERLNTVTVENDLLKYIKNKIPVQGRLGN FKLDTAEFEKKLNTILFVLNESNLAQRFSI LVRKHYLKERPIDPIYVNILDDNSYSLEG FNISSQGSNDFQGQLLLESSYFEKIESNALR AFIKICHKAIDGRSLEVLFQGPLYNKTLDC IEVENKDLFLISNKDSLNDINLSEEKIKPE TTVFFKDKLPPQDITLSNYDFTEANSIPSI SQQNILERNEELYEPIRNSLFEIKTIYVDK LTTFHFLEAQNIDESIDSKIRVELTDSVD EALSNPNKVYSPFKNMSNTINSIETGITST YIFYQWLRSIVKDFSDETGKIDVIDKSSDT LAIVPYIGPLLNIGNDIRHGDFVGAIELAG ITALLEYVPEFTIPILVGLEVIGGELAREQ VEAIVNNALDKRDQKWAEVYNITKAQWWGT IHLQINTRLAHTYKALSRQANAIKMNMEFQ LANYKGNIDDKAKIKNAISETEILNLKSVE QAMKNTEKFMIKLSNSYLTKEMIPKVQDNL KNFDLETKKTLDKFIKEKEDILGTNLSSSL RRKVSIRLNKNIAFDINDIPFSEFDDLINQ YKNEIEDYEVLNLGAEDGKIKDLSGTTSDI NIGSDIEIINTSILNLRYESNHLIDLSRYA SKINIGSKVNFDPIDKNQIQLFNLESSKIE VILKNAIVYNSMYENFSTSFWIRIPKYFNS ISLNNEYTIINCMENNSGWKVSLNYGEIIW TLQDTQEIKQRVVFKYSQMINISDYINRWI FVTITNNRLNNSKIYINGRLIDQKPISNLG NIHASNNIMFKLDGCRDTHRYIWIKYFNLF DKELNEKEIKDLYDNQSNSGILKDFWGDYL QYDKPYYMLNLYDPNKYVDVNNVGIRGYMY LKGPRGSVMTTNIYLNSSLYRGTKFIIKKY ASGNKDNIVRNNDRVYINVVVKNKEYRLAT NASQAGVEKILSALEIPDVGNLSQVVVMKS KNDQGITNKCKMNLQDNNGNDIGFIGFHQF NNIAKLVASNWYNRQIERSSRTLGCSWEFI PVDDGWGERPL

[0440] SEQ ID NO: 68 - Polypeptide sequence, BoNT / XA(0) mutant MGSMKLEINKFNYNDPIDGINVITMRPPRH SDKINKGKGPFKAFQVIKNIWIVPERYNFT NNTNDLNIPSEPIMEADAIYNPNYLNTPSE KDEFLQGVIKVLERIKSKPEGEKLLELISS SIPLPLVSNGALTLSDNETIAYQENNNIVS NLQANLVIYGPGPDIANNATYGLYSTPISN GEGTLSEVSFSPFYLKPFDESYGNYRSLVN IVNKFVKREFAPDPASTLMHQLVYVTHNLY GISNRNFYYNFDTGKIETSRQQNSLIFEEL LTFGGIDSKAISSLIIKKIIETAKNNYTTL ISERLNTVTVENDLLKYIKNKIPVQGRLGN FKLDTAEFEKKLNTILFVLNESNLAQRFSI LVRKHYLKERPIDPIYVNILDDNSYSTLEG FNISSQGSNDFQGQLLESSYFEKIESNALR AFIKICHKAIDGRSLEVLFQGPLYNKTLDC IEVENKDLFLISNKDSLNDINLSEEKIKPE TTVFFKDKLPPQDITLSNYDFTEANSIPSI SQQNILERNEELYEPIRNSLFEIKTIYVDK LTTFHFLEAQNIDESIDSSKIRVELTDSVD EALSNPNKVYSPFKNMSNTINSIETGITST YIFYQWLRSIVKDFSDETGKIDVIDKSSDT LAIVPYIGPLLNIGNDIRHGDFVGAIELAG ITALLEYVPEFTIPILVGLEVIGGELAREQ VEAIVNNALDKRDQKWAEVYNITKAQWWGT IHLQINTRLAHTYKALSRQANAIKMNMEFQ LANYKGNIDDKAKIKNAISETEILLNKSVE QAMKNTEKFMIKLSNSYLTKEMIPKVQDNL KNFDLETKKTLDKFIKEKEDILGTNLSSSL RRKVSIRLNKNIAFDINDIPFSEFDDLINQ YKNEIINTSILNLRYESNHLIDLSRYASKI NIGSKVNFDPIDKNQIQLFNLESSKIEVIL KNAIVYNSMYENFSTSFWIRIPKYFNSISL NNEYTIINCMENNSGWKVSLNYGEIIWTLQ DTQEIKQRVVFKYSQMINISDYINRWIFVT ITNNRLNNSKIYINGRLIDQKPISNLGNIH ASNNIMFKLDGCRDTHRYIWIKYFNLFDKE LNEKEIKDLYDNQSNSGILKDFWGDYLQYD KPYYMLNLYDPNKYVDVNNVGIRGYMYLKG PRGSVMTTNIYLNSSLYRGTKFIIKKYASG NKDNIVRNNDRVYINVVVKNKEYRLATNAS QAGVEKILSALEIPDVGNLSQVVVMKSKND QGITNKCKMNLQDNNGNDIGFIGFHQFNNI AKLVASNWYNRQIERSSRTLGCSWEFIPVD DGWGERPL

[0441] SEQ ID NO: 69 - Polypeptide sequence, BoNT / XD(0) MGSMKLEINKFNYNDPIDGINVITMRPPRH SDKINKGKGPFKAFQVIKNIWIVPERYNFT NNTNDLNIPSEPIMEADAIYNPNYLNTPSE KDEFLQGVIKVLERIKSKPEGEKLLELISS SIPLPLVSNGALTLSDNETIAYQENNNIVS NLQANLVIYGPGPDIANNATYGLYSTPISN GEGTLSEVSFSPFYLKPFDESYGNYRSLVN IVNKFVKREFAPDPASTLMHQLVYVTHNLY GISNRNFYYNFDTGKIETSRQQNSLIFEEL LTFGGIDSKAISSLIIKKIIETAKNNYTTL ISERLNTVTVENDLLKYIKNKIPVQGRLGN FKLDTAEFEKKLNTILFVLNESNLAQRFSI LVRKHYLKERPIDPIYVNILDDNSYSTLEG FNISSQGSNDFQGQLLESSYFEKIESNALR AFIKICHKAIDGRSLYNKTLDCIEVENKDL FLISNKDSLNDINLSEEKIKPETTVFFKDK LPPQDITLSNYDFTEANSIPSISQQNILER NEELYEPIRNSLFEIKTIYVDKLTTFHFLE AQNIDESIDSSKIRVELTDSVDEALSNPNK VYSPFKNMSNTINSIETGITSTYIFYQWLR SIVKDFSDETGKIDVIDKSSDTLAIVPYIG PLLNIGNDIRHGDFVGAIELAGITALLEYV PEFTIPILVGLEVIGGELAREQVEAIVNNA LDKRDQKWAEVYNITKAQWWGTIHLQINTR LAHTYKALSRQANAIKMNMEFQLANYKGNI DDKAKIKNAISETEILLNKSVEQAMKNTEK FMIKLSNSYLTKEMIPKVQDNLKNFDLETK KTLDKFIKEKEDILGTNLSSSLRRKVSIRL NKNIAFDINDIPFSEFDDLINQYKNEIEDY EVLNLGAEDGKIKDLSGTTSDINIGSDIEN DSKILSLQNKKNALVDTSGYNAEVRVGDNV QLNTIYTNDFKLSSSGDKIIVNLNNNILYS AIYENSSVSFWIKISKDLTNSHNEYTIINS IEQNSGWKLCIRNGNIEWILQDVNRKYKSL IFDYSESLSHTGYTNKWFFVTITNNIMGYM KLYINGELKQSQKIEDLDEVKLDKTIVFGI DENIDENQMLWIRDFNIFSKELSNEDINIV YEGQILRNVIKDYWGNPLKFDTEYYIINDN YIDRYIAPESNVLVLVQYPDRSKLYTGNPI TIKSVSDKNPYSRILNGDNIILHMLYNSRK YMIIRDTDTIYATQGGECSQNCVYALKLQS NLGNYGIGIFSIKNIVSKNKYCSQIFSSFR ENTMLLADIYKPWRFSFKNAYTPVAVTNYE TKLLSTSSFWKFISRDPGWVE

[0442] SEQ ID NO: 70 - Polypeptide sequence, BoNT / XF(0) MGSMKLEINKFNYNDPIDGINVITMRPPRH SDKINKGKGPFKAFQVIKNIWIVPERYNFT NNTNDLNIPSEPIMEADAIYNPNYLNTPSE KDEFLQGVIKVLERIKSKPEGEKLLELISS SIPLPLVSNGALTLSDNETIAYQENNNIVS NLQANLVIYGPGPDIANNATYGLYSTPISN GEGTLSEVSFSPFYLKPFDESYGNYRSLVN IVNKFVKREFAPDPASTLMHQLVYVTHNLY GISNRNFYYNFDTGKIETSRQQNSLIFEEL LTFGGIDSKAISSLIIKKIIETAKNNYTTL ISERLNTVTVENDLLKYIKNKIPVQGRLGN FKLDTAEFEKKLNTILFVLNESNLAQRFSI LVRKHYLKERPIDPIYVNILDDNSYSTLEG FNISSQGSNDFQGQLLESSYFEKIESNALR AFIKICHKAIDGRSLYNKTLDCIEVENKDL FLISNKDSLNDINLSEEKIKPETTVFFKDK LPPQDITLSNYDFTEANSIPSISQQNILER NEELYEPIRNSLFEIKTIYVDKLTTFHFLE AQNIDESIDSSKIRVELTDSVDEALSNPNK VYSPFKNMSNTINSIETGITSTYIFYQWLR SIVKDFSDETGKIDVIDKSSDTLAIVPYIG PLLNIGNDIRHGDFVGAIELAGITALLEYV PEFTIPILVGLEVIGGELAREQVEAIVNNA LDKRDQKWAEVYNITKAQWWGTIHLQINTR LAHTYKALSRQANAIKMNMEFQLANYKGNI DDKAKIKNAISETEILLNKSVEQAMKNTEK FMIKLSNSYLTKEMIPKVQDNLKNFDLETK KTLDKFIKEKEDILGTNLSSSLRRKVSIRL NKNIAFDINDIPFSEFDDLINQYKNEIEDY EVLNLGAEDGKIKDLLSGTTSDINIGSDIEI KDNSILDMRYENNKFIDISGYGSNISINGD VYIYSTNRNQFGIYSSKPSEVNIAQNNDII YNGRYQNFSISFWVRIPKYFNKVNLNNEYT IIDCIRNNNSGWKISLNYNKIIWTLQDTAG NNQKLVFNYTQMISISDYINKWIFVTITNN RLGNSRIYINGNLIDEKSISNLGDIHVSDN ILFKIVGCNDTRYVGIRYFKVFDTELGKTE IETLYSDEPDPSILKDFWGNYLLYNKRYYL LNLLRTDKSITQNSNFLNINQQRGVYQKPN IFSNTRLYTGVEVIIRKNGSTDISNTDNFV RKNDLAYINVVDRDVEYRLYADISIAKPEK IIKLIRTSNSNNSLGQIIVMDSIGNNCTMN FQNNNGGNIGLLGFKSNNLVASSWYYNNIR KNTSSNGCFWSFISKEHGWQEN

[0443] SEQ ID NO:71 - C1 activation loop consensus sequence Cys-(Xaa) a -Ile-Asp / Glu-Gly-Arg-(Yaa) b -Cys, where a = 1-10 and b = 4-15

[0444] SEQ ID NO:72 - C1 activation loop CHKAIDGRSLYNKTLDC

[0445] SEQ ID NO:73 - C1 activation loop mutant CHKAIEGRSLYNKTLDC

[0446] SEQ ID NO: 74 - Polypeptide sequence, rLC / A(0) (His tagged) MPFVNKQFNY KDPVNGVDIA YIKIPNAGQM QPVKAFKIHN KIWVIPERDT FTNPEEGDLN PPPEAKQVPV SYYDSTYLST DNEKDNYLKG VTKLFERIYS TDLGRMLLTS IVRGIPFWGG STIDTELKVI DTNCINVIQP DGSYRSEELN LVIIGPSADI IQFECKSFGH EVLNLTRNGY GSTQYIRFSP DFTFGFEESL EVDTNPLLGA GKFATDPAVT LAHQLIYAGH RLYGIAINPN RVFKVNTNAY YEMSGLEVSF EELRTFGGHD AKFIDSLQEN EFRLYYYNKF KDIASTLNKA KSIVGTTASL QYMKNVFKEK YLLSEDTSGK FSVDKLKFDK LYKMLTEIYT EDNFVKFFKV LNRKTYLNFD KAVFKINIVP KVNYTIYDGF NLRNTNLAAN FNGQNTEINN MNFTKLKNFT GLFEENLYFQ GASHHHHHHH H

[0447] SEQ ID NO: 75 - Polypeptide sequence, rLH N / A(0) (His tagged) MPFVNKQFNY KDPVNGVDIA YIKIPNAGQM QPVKAFKIHN KIWVIPERDT FTNPEEGDLN PPPEAKQVPV SYYDSTYLST DNEKDNYLKG VTKLFERIYS TDLGRMLLTS IVRGIPFWGG STIDTELKVI DTNCINVIQP DGSYRSEELN LVIIGPSADI IQFECKSFGH EVLNLTRNGY GSTQYIRFSP DFTFGFEESL EVDTNPLLGA GKFATDPAVT LAHQLIYAGH RLYGIAINPN RVFKVNTNAY YEMSGLEVSF EELRTFGGHD AKFIDSLQEN EFRLYYYNKF KDIASTLNKA KSIVGTTASL QYMKNVFKEK YLLSEDTSGK FSVDKLKFDK LYKMLTEIYT EDNFVKFFKV LNRKTYLNFD KAVFKINIVP KVNYTIYDGF NLRNTNLAAN FNGQNTEINN MNFTKLKNFT GLFEFYKLLC VRGIITSKTK SLDKGYNKAL NDLCIKVNNW DLFFSPSEDN FTNDLNKGEE ITSDTNIEAA EENISLDLIQ QYYLTFNFDN EPENISIENL SSDIIGQLEL MPNIERFPNG KKYELDKYTM FHYLRAQEFE HGKSRIALTN SVNEALLNPS RVYTFFSSDY VKKVNKATEA AMFLGWVEQL VYDFTDETSE VSTTDKIADI TIIIPYIGPA LNIGNMLYKD DFVGALIFSG AVILLEFIPE IAIPVLGTFA LVSYIANKVL TVQTIDNALS KRNEKWDEVY KYIVTNWLAK VNTQIDLIRK KMKEALENQA EATKAIINYQ YNQYTEEEKN NINFNIDDLS SKLNESINKA MININKFLNQ CSVSYLMNSM IPYGVKRLED FDASLKDALL KYIYDNRGTL IGQVDRLKDK VNNTLSTDIP FQLSKYVDNQ RLLSTENLYF QGASHHHHHHH HH

[0448] SEQ ID NO: 76 - Polypeptide sequence, rLC / X(0) MKLEINKFNY NDPIDGINVI TMRPPRHSDK INKGKGPFKA FQVIKNIWIV PERYNFTNNT NDLNIPSEPI MEADAIYNPN YLNTPSEKDE FLQGVIKVLE RIKSKPEGEK LLELISSSIP LPLVSNGALT LSDNETIAYQ ENNNIVSNLQ ANLVIYGPGP DIANNATYGL YSTPISNGEG TLSEVSFSPF YLKPFDESYG NYRSLVNIVN KFVKREFAPD PASTLMHQLV YVTHNLYGIS NRNFYYNFDT GKIETSRQQN SLIFEELLTF GGIDSKAISS LIIKKIIETA KNNYTTLISE RLNTVTVEND LLKYIKNKIP VQGRLGNFKL DTAEFEKKLN TILFVLNESN LAQRFSILVR KHYLKERPID PIYVNILDDN SYSTLEGFNI SSQGSNDFQG QLLESSYFEK IESNALRAFI KIAPRNGLLY NAIYRNSK

[0449] SEQ ID NO:77 - PreScission protease site LEVLFQGP

[0450] SEQ ID NO:78 - C1 activation loop mutant 2 CHKAIDGRSLEVLFQGPLYNKTLDC [Example]

[0451] Example 1

[0452] BoNT / A(0) is catalytically inactive in vitro and in vivo

[0453] The catalytic activity of BoNT / A(0) (SEQ ID NO: 2) was tested in an in vitro cell-based model, which measures the cleavage of the BoNT / A target SNARE protein SNAP25. Figure 1 shows that, in contrast to wild-type BoNT / A (SEQ ID NO: 60), BoNT / A(0) does not cleave SNAP25 in a human neuron assay. Figure 2 confirms this result in a rat neuron assay.

[0454] As a confirmation, an in vivo DAS assay was performed using BoNT / A and BoNT / A(0). The DAS assay was performed by injecting 20 μl of clostridial toxin formulated in gelatin phosphate buffer into the mouse gastrocnemius / soleus muscle complex, followed by assessment of digit abduction scores using the method of Aoki (Aoki KR, Toxicon 39: 1815-1820; 2001). In the DAS assay, mice were briefly suspended by their tails to elicit a characteristic startle response (Figure 3A), in which the mice extend their hind limbs and abduct their hind digits. After clostridial toxin injection, the variable degree of digit abduction was scored on a 5-point scale (0 = normal to 4 = maximal reduction in digit abduction—Figure 3B). This provides a functional measure of paralysis induced by neurotoxin activity at the neuromuscular junction. Additionally, changes in body weight were assessed in mice within 7 days of administration. This provides a measure of the toxicity and unwanted effects of toxin diffusion from the site of administration. The results are presented in Table 1 below. [Table 1]

[0455] The results confirm that BoNT / A(0) is catalytically inactive in vivo and does not produce any symptoms of toxicity. Therefore, BoNT / A(0) is a safe, substantially non-toxic therapeutic agent.

[0456] Example 2

[0457] Treatment of chronic neuropathic pain (chronic constriction injury (CCI) rat model) with catalytically inactive BoNT

[0458] Materials and Methods

[0459] Chronic constriction injury (CCI) was performed as previously described by Bennett and Xie (1988), Pain, 33(1):87-107. On day -14, adult male Sprague-Dawley rats (220-250 g) were anesthetized before a segment of the left sciatic nerve was exposed and four loose silk ligatures were placed over the nerve. On day 0 (DO), rats were injected with either BoNT / A (30 pg / kg), BoNT / A (60 pg / kg), BoNT / A (0) (60 pg / kg), or vehicle (GPB) administered by the intraplantar (i.p.) route (n = 10-11 / group). The positive control, gabapentin (100 mg / kg), was administered orally (po) (n = 8 / group). Animals treated with gabapentin were tested 1, 2, and 4 h after treatment. Animals treated with BoNT / A, BoNT / A(0), or vehicle were tested on days 3, 5, and 9. Animals were evaluated for mechanical sensitivity in the von Frey test.

[0460] result

[0461] The experiment showed that administration of catalytically inactive BoNT (BoNT / A(0)) reduced the mechanical sensitivity of the ipsilateral paw (Figure 4B). Furthermore, BoNT / A(0) was more effective at reducing mechanical sensitivity when compared with comparably dosed BoNT / A. At later time points, BoNT / A(0) was also more effective than gabapentin. To confirm that the reduced sensitivity was the result of BoNT / A(0) administration, the mechanical sensitivity of the contralateral paw was also tested. The results showed that the difference in mechanical sensitivity of the contralateral paw between conditions was not significant (Figure 4C). Furthermore, confirming the substantial non-toxicity of BoNT / A(0), the change in body weight over time was similar to that of rats administered vehicle or gabapentin (Figure 4D). This contrasts with the change observed in rats administered catalytically active BoNT / A, which was statistically significantly different on day 9.

[0462] In conclusion, catalytically inactive Clostridial neurotoxins are surprisingly capable of reducing pain (e.g., chronic neuropathic pain), thereby suggesting that such neurotoxins are suitable pain treatment agents.

[0463] Example 3

[0464] Treatment of acute neuropathic pain (oxaliplatin rat model) with catalytically inactive BoNT

[0465] Materials and Methods

[0466] An experimental model of oxaliplatin-induced peripheral sensory neuropathy was induced by intraperitoneal injection of oxaliplatin (Ling et al (2007), Pain, 128(3):225-234; Ling et al (2007), Toxicology, 20;234(3):176-84). On day 0, adult male Sprague-Dawley rats (100-133 g) received either a sham procedure (5% glucose) or an i.p. injection of oxaliplatin (10 mg / kg). Immediately afterward, sham-treated animals received an i.p. injection of vehicle, and oxaliplatin-treated animals received an i.p. injection of BoNT / A (0) (1000 pg / kg), BoNT / A (50 pg / kg), BoNT / A (100 pg / kg), BoNT / A (160 pg / kg), or vehicle (GPB; n = 10 / group). On D3, the positive control duloxetine (100 mg / kg) was administered by the po route. Animals were evaluated for temperature (cold) sensitivity on D3 and D5.

[0467] result

[0468] The experiment showed that administration of catalytically inactive BoNT (BoNT / A(0)) reduced the cold sensitivity of the ipsilateral paw (Fig. 5B). There was no difference in the thermal sensitivity of the contralateral paw between groups treated with BoNT / A, BoNT / A(0), or vehicle (Fig. 5C).

[0469] In conclusion, catalytically inactive clostridial neurotoxins are surprisingly able to reduce acute neuropathic pain, thereby suggesting that such neurotoxins have general application for the treatment of pain.

[0470] Example 4

[0471] Treatment of chronic neuropathic pain (oxaliplatin rat model) with catalytically inactive BoNT

[0472] Materials and Methods

[0473] On day -2 (D-2), adult male Sprague-Dawley rats (180-210 g) received an i.p. injection of oxaliplatin (10 mg / kg) (n = 11-12 / group) before being treated with BoNT / A (100 pg / kg), BoNT / A (0) (100 pg / kg), or vehicle (GPB) by the i.p. route on day 0. The positive control, pregabalin, was administered on day 3 (n = 12). Animals were tested for mechanical sensitivity (von Frey test) and thermal sensitivity (cold plate test) on days 3, 6, and 9.

[0474] result

[0475] The experiments showed that administration of catalytically inactive BoNT (BoNT / A(0)) reduced the mechanical sensitivity (Fig. 6B) and cold sensitivity (Fig. 6D) of the ipsilateral paw.

[0476] In conclusion, catalytically inactive clostridial neurotoxins are surprisingly able to reduce chronic neuropathic pain in different chemotherapy-induced pain models.

[0477] Example 5

[0478] Treatment of inflammatory pain (UV-B burn rat model) with catalytically inactive BoNT

[0479] Materials and Methods

[0480] In humans and rodent models, ultraviolet (UV) B radiation induces both mechanical and thermal hyperalgesia. Adult male Wistar rats (180-210 g) were administered BoNT / A (100 pg / kg), BoNT / A(0) (100 pg / kg), or vehicle (GPB; n = 12 / group) by i.p. injection. Twenty-four hours later, the plantar surface of the ipsilateral paw was exposed to ultraviolet B (UVB) radiation for approximately 5 min at a dose of 500 mJ / cm. 2Animals received a dose of 100 mg / kg / day. 48 hours after UVB and 72 hours after BoNT / A, BoNT / A(0), or vehicle injection, animals were tested for mechanical sensitivity in the von Frey test. An additional group of UVB-exposed animals was injected with the positive control indomethacin 48 hours later and tested in the von Frey test 1 hour after injection (n=12 / group).

[0481] result

[0482] The experiment showed that administering catalytically inactive BoNT (BoNT / A(0)) reduced mechanical sensitivity (Figure 7B). In conclusion, catalytically inactive Clostridial neurotoxins are surprisingly able to reduce inflammatory pain (e.g., acute inflammatory pain), thereby confirming that such neurotoxins have general application for the treatment of pain.

[0483] The surprising finding that a catalytically inactive Clostridial neurotoxin reduced inflammatory pain indicated that it would find utility in treating the underlying inflammatory condition (including, for example, treating at least one symptom of the inflammatory condition, i.e., the associated pain). Therefore, it was considered plausible that a catalytically inactive Clostridial neurotoxin could be used to treat inflammatory conditions.

[0484] Example 6

[0485] Treatment of inflammatory pain (CFA-induced inflammatory pain model) with catalytically inactive chimeric BoNT

[0486] Materials and Methods

[0487] Prior to BoNT or vehicle administration, paw withdrawal thresholds (PWT, g) of 70 adult male C57 / BL6 mice (22-26 g) were assessed using von Frey filaments with increasing force for three consecutive days. The average of the last two days was considered the baseline. On day 0, BoNT / XB (0.3 and 30 ng / kg), BoNT / XB(0) (0.3 and 30 ng / kg), BoNT / A (160 pg / kg), or vehicle (840 μl / kg) was injected intraplantarly into the left hind paw under gas anesthesia (n = 10 / group). On day 2, PWT was reassessed prior to CFA injection. Then, under isoflurane anesthesia, a fixed 20 μL volume of CFA (1.5 mg / mL) was injected into the same hind paw. On day 3 (day 1 post-CFA), animals assigned to the indomethacin group were orally dosed with indomethacin (10 mg / kg, n=9) 1 hour prior to PWT assessment.

[0488] result

[0489] The experiment was carried out using the catalytically inactive BoNT / X L chain and translocation domain (LH of BoNT / X). N ) and the BoNT / B receptor binding domain BoNT / X(H C This study demonstrated that a catalytically inactive chimeric BoNT (BoNT / XB(0)) containing the BoNT / XB domain (BoNT / XB(0)) was effective in treating inflammatory pain. More specifically, Figure 8 shows that mechanical sensitivity after CFA induction of inflammatory pain was reduced in mice administered catalytically active BoNT / XB and catalytically inactive BoNT / XB(0) at a dose of 30 ng / kg. The reduction in sensitivity was comparable to that of BoNT / A or the positive control indomethacin.

[0490] The surprising finding that BoNT / XB(0) reduced inflammatory pain indicated that it would find utility in treating the underlying inflammatory condition, including, for example, treating at least one symptom of the inflammatory condition, i.e., associated pain. It was therefore considered further evidence of the viability of catalytically inactive clostridial neurotoxins for use in treating inflammatory conditions.

[0491] Example 7

[0492] Treatment of atopic dermatitis with catalytically inactive chimeric BoNTs

[0493] Vehicle or BoNT / XB(0) (40 pg / mouse, 100 pg / mouse, or 400 pg / mouse) was administered subcutaneously to the mid-back of adult C57 / BL6 mice 1 day prior to calcipotriol exposure. Mice were then treated with calcipotriol for 5 consecutive days. At the end of the study, animals were euthanized, and dorsal skin was collected, fixed, and processed for histological analysis. Epidermal thickness was assessed after hematoxylin-eosin staining. Immunolabeling was performed to identify CD45+ cells.

[0494] The experiment was carried out using the catalytically inactive BoNT / X L chain and translocation domain (LH of BoNT / X). N ) and the BoNT / B receptor binding domain BoNT / X(H C This study demonstrates that a catalytically inactive chimeric BoNT (BoNT / XB(0)) containing the α- and β-actin domains is effective in treating atopic dermatitis, a model inflammatory condition. Results demonstrate an improvement in dermal thickness after administration of BoNT / XB(0). Dermal thickness, an indicator of fibrosis in the inflammatory response to calcipotriol, is shown to be statistically significantly reduced in BoNT / XB(0)-treated animals. Furthermore, the anti-inflammatory effect of BoNT / XB(0) is confirmed by a reduction in the number of CD45-positive cells in BoNT / XB(0)-treated animals (CD45 transmits activation signals in inflammatory cells).

[0495] It is therefore concluded that BoNT / XB(0) has anti-inflammatory properties and therefore finds utility in treating inflammatory disorders.

[0496] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.

[0497] item

[0498] [Item 1] A polypeptide for use in the treatment of pain, wherein the polypeptide comprises a Clostridial neurotoxin light chain (L chain), a Clostridial neurotoxin translocation domain (H chain), N domain), and / or Clostridial neurotoxin receptor binding domain (H C domain), wherein when said polypeptide comprises a Clostridial neurotoxin L chain, said L chain is catalytically inactive. [Item 2] A method for treating pain, the method comprising administering to a subject a polypeptide, wherein the polypeptide comprises a clostridial neurotoxin light chain (L chain), a clostridial neurotoxin translocation domain (H chain), or a clostridial neurotoxin translocation domain (H chain). N domain), and / or Clostridial neurotoxin receptor binding domain (H C domain), wherein when said polypeptide comprises a Clostridial neurotoxin L chain, said L chain is catalytically inactive. [Item 3] 1. Use of a polypeptide in the manufacture of a medicament for treating pain, wherein the polypeptide comprises a Clostridial neurotoxin light chain (L chain), a Clostridial neurotoxin translocation domain (H ... N domain), and / or Clostridial neurotoxin receptor binding domain (H C domain), wherein when the polypeptide comprises a Clostridial neurotoxin L chain, the L chain is catalytically inactive. [Item 4] Item 1. The polypeptide for use according to item 1, the method according to item 2, or the use according to item 3, wherein the polypeptide does not treat pain by promoting neuronal growth, promoting neuronal repair, or promoting neuronal growth and repair. [Item 5] A polypeptide for use in treating an inflammatory disorder, wherein the polypeptide comprises a Clostridial neurotoxin light chain (L chain), a Clostridial neurotoxin translocation domain (H ... translocation domain (H chain), a Clo N domain), and / or Clostridial neurotoxin receptor binding domain (H C domain), wherein when said polypeptide comprises a Clostridial neurotoxin L chain, said L chain is catalytically inactive. [Item 6] A method for treating an inflammatory disorder, the method comprising administering to a subject a polypeptide, wherein the polypeptide comprises a clostridial neurotoxin light chain (L chain), a clostridial neurotoxin translocation domain (H chain), or a clostridial neurotoxin translocation domain (L chain). N domain), and / or Clostridial neurotoxin receptor binding domain (H C domain), wherein when said polypeptide comprises a Clostridial neurotoxin L chain, said L chain is catalytically inactive. [Item 7] 1. Use of a polypeptide in the manufacture of a medicament for treating an inflammatory disorder, wherein the polypeptide comprises a clostridial neurotoxin light chain (L chain), a clostridial neurotoxin translocation domain (H chain), ... Ndomain), and / or Clostridial neurotoxin receptor binding domain (H C domain), wherein when the polypeptide comprises a Clostridial neurotoxin L chain, the L chain is catalytically inactive. [Item 8] Item 8. The polypeptide for use according to item 5, the method according to item 6, or the use according to item 7, wherein the polypeptide does not treat the inflammatory condition by promoting neuronal growth, promoting neuronal repair, or promoting neuronal growth and repair. [Item 9] 10. A polypeptide for use, method or use according to any one of the preceding items, wherein said polypeptide does not comprise an additional catalytically active domain. [Item 10] The polypeptide comprises the Clostridial neurotoxin L chain, H chain, N Domain and / or H C 10. A polypeptide for use, method or use according to any one of the preceding items, wherein the domain does not comprise an additional therapeutic or diagnostic agent (e.g. a covalently or non-covalently linked therapeutic or diagnostic agent). [Item 11] 10. A polypeptide for use, method or use according to any one of the preceding items, wherein the polypeptide is not administered (e.g. sequentially or subsequently) with a further therapeutic or diagnostic agent. [Item 12] 10. A polypeptide for use, method or use according to any one of the preceding items, wherein the polypeptide comprises a catalytically inactive Clostridial neurotoxin light chain. [Item 13] The polypeptides include clostridial neurotoxin L chain, H chain, N Domain, and H C 8. A polypeptide for use, method or use according to any one of the preceding items, comprising a domain, wherein the L chain is catalytically inactive. [Item 14] The polypeptide essentially comprises a Clostridial neurotoxin light chain (L chain), a Clostridial neurotoxin translocation domain (H chain),N domain), and / or Clostridial neurotoxin receptor binding domain (H C 13. The polypeptide for use, method or use according to any one of items 1 to 12, comprising a Clostridial neurotoxin L chain (domain), wherein when said polypeptide comprises or consists essentially of a Clostridial neurotoxin L chain, said L chain is catalytically inactive. [Item 15] The polypeptide essentially comprises a Clostridial neurotoxin light chain (L chain) and a Clostridial neurotoxin translocation domain (H chain). N 15. The polypeptide for use, method or use according to any one of items 1 to 12 or 14, comprising a L chain (L domain) and a L chain (L domain), wherein the L chain is catalytically inactive. [Item 16] The polypeptide essentially comprises a Clostridial neurotoxin light chain (L chain), a Clostridial neurotoxin translocation domain (H chain), N domain), and Clostridial neurotoxin receptor binding domain (H C 10. A polypeptide for use, method or use according to any one of the preceding items, comprising a L chain (L domain) and a L chain (L domain) of the polypeptide for use, method or use according to any one of the preceding items, wherein the L chain is catalytically inactive. [Item 17] The polypeptide comprises a clostridial neurotoxin light chain (L chain), a clostridial neurotoxin translocation domain (H chain), N domain), and / or Clostridial neurotoxin receptor binding domain (H C 15. The polypeptide for use, method or use according to any one of items 1 to 12 or 14, comprising a Clostridial neurotoxin L chain (domain), wherein when said polypeptide comprises or consists of a Clostridial neurotoxin L chain, said L chain is catalytically inactive. [Item 18] The polypeptide comprises a Clostridial neurotoxin light chain (L chain) and a Clostridial neurotoxin translocation domain (H chain). N 18. The polypeptide for use, method or use according to any one of items 1 to 12, 15 or 17, comprising a L chain (L domain) and a L chain (L domain), wherein the L chain is catalytically inactive. [Item 19] The polypeptide comprises a clostridial neurotoxin light chain (L chain), a clostridial neurotoxin translocation domain (H chain), N domain), and Clostridial neurotoxin receptor binding domain (H C 18. The polypeptide for use, method or use according to any one of items 1 to 13, 14, 16 or 17, comprising a L chain (L domain) and a L chain (L domain), wherein the L chain is catalytically inactive. [Item 20] The polypeptide is a clostridial neurotoxin H N Domain and H C 19. The polypeptide for use, method or use according to any one of items 1 to 12, 14, 15, 17 or 18, wherein the polypeptide does not comprise both domains. [Item 21] 8. A polypeptide for use, method or use according to any one of the preceding items, wherein the polypeptide further does not comprise a non-clostridial catalytic domain. [Item 22] 8. The polypeptide for use, method or use according to any one of the preceding items, wherein said pain is chronic pain. [Item 23] 22. The polypeptide for use, method or use according to any one of items 1 to 21, wherein the pain is acute pain. [Item 24] 8. The polypeptide for use, method or use according to any one of the preceding items, wherein said pain is inflammatory pain. [Item 25] 25. The polypeptide for use, method or use according to item 24, wherein the inflammatory pain is caused by or associated with sunburn, UV-induced damage, arthritic disorders, autoimmune diseases, connective tissue disorders, injury, infection, neuritis, joint inflammation or headache (preferably muscular / myogenic headache, vascular headache, hypertensive headache, hormonal headache, rebound headache, chronic sinus headache, organic headache or paroxysmal headache). [Item 26] 24. The polypeptide for use, method or use according to any one of items 1 to 23, wherein the pain is neuropathic pain. [Item 27] 27. The polypeptide for use, method or use according to item 26, wherein said neuropathic pain is (or is caused by or associated with) neuralgia, deafferentation, complex regional pain syndrome (CRPS), or neuropathy (e.g. central or peripheral neuropathy). [Item 28] 24. The polypeptide for use, method or use according to any one of items 1 to 23, wherein the pain is mixed pain. [Item 29] 24. The polypeptide for use, method or use according to any one of items 1 to 23, wherein the pain is allodynia. [Item 30] 24. The polypeptide for use, method or use according to any one of items 1 to 23, wherein the pain is visceral pain. [Item 31] 31. Polypeptide for use, method or use according to item 30, wherein the visceral pain is (or is caused by or associated with) functional visceral pain, chronic gastrointestinal inflammation, autoimmune pain, organic visceral pain or treatment-induced visceral pain. [Item 32] 24. The polypeptide for use, method or use according to any one of items 1 to 23, wherein the pain is headache pain (e.g. migraine). [Item 33] 33. Polypeptide for use, method or use according to item 32, wherein the pain is migraine pain. [Item 34] 33. The polypeptide for use, method or use according to item 32, wherein the headache pain is caused by or associated with muscular / myopathic headache, vascular headache, hypertensive headache, hormonal headache, rebound headache, chronic sinus headache, organic headache or paroxysmal headache. [Item 35] 24. The polypeptide for use, method or use according to any one of items 1 to 23, wherein the pain is post-operative pain. [Item 36] 24. The polypeptide for use, method or use according to any one of items 1 to 23, wherein the pain is referred pain. [Item 37] 24. The polypeptide for use, method or use according to any one of items 1 to 23, wherein the pain is somatic pain. [Item 38] 38. The polypeptide for use, method or use according to item 37, wherein the pain is somatic pain and is caused by or associated with excessive muscle tension, repetitive motion disorders, muscle disorders, myalgia, infection or drugs. [Item 39] 24. The polypeptide for use, method or use according to any one of items 1 to 23, wherein said pain is bladder pain syndrome, preferably said bladder pain caused by or associated with interstitial cystitis. [Item 40] 24. The polypeptide for use, method or use according to any one of items 1 to 23, wherein the pain is phantom limb pain. [Item 41] 22. The polypeptide for use, method or use according to any one of items 5 to 21, wherein the inflammatory disorder is one or more selected from cystitis, endometriosis, rheumatoid arthritis, complex regional pain syndrome, and neuritis. [Item 42] 42. The polypeptide for use, method or use according to item 41, wherein the cystitis is interstitial cystitis. [Item 43] 42. The polypeptide for use, method or use according to item 41, wherein the neuritis is peripheral neuritis. [Item 44] 10. The polypeptide for use, method or use according to any one of the preceding items, wherein a single dose of said polypeptide administered is greater than 250 μg. [Item 45] 10. The polypeptide for use, method or use according to any one of the preceding items, wherein a single dose of said polypeptide administered is between 251 μg and 10 g. [Item 46] 10. The polypeptide for use, method or use according to any one of the preceding items, wherein a single dose of said polypeptide administered is between 251 μg and 1 g. [Item 47] 10. The polypeptide for use, method or use according to any one of the preceding items, wherein a single dose of the polypeptide administered is between 251 and 1000 μg. [Item 48] 10. A polypeptide for use, method or use according to any one of the preceding items, wherein the polypeptide is administered repeatedly (e.g. as part of a pain treatment regimen). [Item 49] 10. The polypeptide for use, method or use according to any one of the preceding items, wherein the polypeptide is administered intradermally. [Item 50] 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 74, 75, or 76, with the proviso that if the polypeptide comprises a Clostridial neurotoxin light chain, the light chain is catalytically inactive. [Item 51] 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 74, 75, or 76, with the proviso that if the polypeptide comprises a Clostridial neurotoxin light chain, the light chain is catalytically inactive. [Item 52] 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 74, 75, or 76, with the proviso that if the polypeptide comprises a Clostridial neurotoxin light chain, the light chain is catalytically inactive. [Item 53] 3. The polypeptide for use, method or use of any one of the preceding items 2, wherein the polypeptide comprises the polypeptide sequence of any one of SEQ ID NOs: 2, 8, 10, 12, 14, 16, 18, 22, 26, 30, 34, 38, 42, 44, 46, 48, 50, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 74, 75 or 76. [Item 54] 8. The polypeptide for use, method or use according to any one of the preceding items, wherein the polypeptide is a catalytically inactive BoNT / A. [Item 55] 10. The polypeptide for use, method or use according to any one of the preceding items, wherein the polypeptide is a modified Clostridial neurotoxin, such as a chimeric Clostridial neurotoxin or a hybrid Clostridial neurotoxin, and preferably the polypeptide does not comprise a naturally occurring Clostridial neurotoxin H chain. [Item 56] The polypeptide is a functional H of a clostridial neurotoxin. CC Domain or H C 56. A polypeptide for use, method or use according to any one of items 1 to 53 or 55, lacking the domain. [Item 57] 57. The polypeptide for use, method or use according to any one of items 1 to 53 or 55 to 56, wherein the polypeptide is a retargeted Clostridial neurotoxin comprising a non-Clostridial targeting moiety (TM). [Item 58] The polypeptide is a functional H of a clostridial neurotoxin. C 57. A polypeptide for use, method or use according to any one of items 1 to 53 or 55 to 56, lacking the domain and also lacking any functionally equivalent exogenous ligand targeting moiety (TM). [Item 59] A polypeptide for use, method, or use according to any one of the preceding items, wherein the polypeptide is not expressed in cells of the subject, for example, wherein the use or method does not comprise expressing a nucleic acid encoding the polypeptide by cells of the subject. [Item 60] 10. The polypeptide for use, method or use according to any one of the preceding items, wherein the polypeptide further comprises one or more non-clostridial neurotoxin sequences. [Item 61] 61. The polypeptide, method or use for use according to item 60, wherein the one or more non-clostridial neurotoxin sequences do not bind to a cellular receptor. [Item 62] 62. Polypeptide for use, method or use according to item 60 or 61, wherein the one or more non-clostridial neurotoxin sequences do not comprise a ligand for a cellular receptor. [Item 63] The polypeptide comprises a catalytically inactive light chain and translocation domain of BoNT / A and a receptor binding domain (H) of BoNT / B. C 63. The polypeptide, method or use according to any one of items 1 to 53, 55 or 59 to 62, wherein the BoNT is a chimeric botulinum neurotoxin (BoNT) comprising a nucleotide sequence (domain) and a nucleotide sequence (domain). [Item 64] The polypeptide may comprise a modified BoNT / A H polypeptide comprising a modification at one or more amino acid residues selected from ASN886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, SER955, GLN991, GLU992, GLN995, ASN1006, ASN1025, ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, HIS1064, ASN1080, GLU1081, GLU1083, ASP1086, ASN1188, ASP1213, GLY1215, ASN1216, GLN1229, ASN1242, ASN1243, SER1274, and THR1277. C 63. A polypeptide, method or use for the purpose according to any one of items 1 to 55 or 59 to 62, comprising a domain: wherein said modification is selected from: i. Substitution of acidic surface-exposed amino acid residues with basic amino acid residues; ii. Substitution of acidic surface-exposed amino acid residues with uncharged amino acid residues; iii. Substitution of uncharged surface-exposed amino acid residues with basic amino acid residues; iv. insertion of a basic amino acid residue; and v. Deletion of acidic surface-exposed amino acid residues. [Item 65] The polypeptide comprises a catalytically inactive botulinum neurotoxin serotype X (BoNT / X) L chain, a BoNT / X H chain, and a N domain and / or H of BoNT / X C63. A polypeptide, method or use for a purpose according to any one of items 1 to 53 or 55 to 62, comprising a domain. [Item 66] The polypeptide comprises a catalytically inactive BoNT / X light chain and translocation domain and a receptor binding domain (H) from a different (i.e., non-BoNT / X) clostridial neurotoxin. C 66. The polypeptide, method or use for the use according to any one of items 1 to 53, 59 to 62 or 65, wherein the BoNT is a chimeric botulinum neurotoxin (BoNT) comprising a nucleotide sequence (domain) and a nucleotide sequence (domain). [Item 67] The polypeptide comprises a catalytically inactive BoNT / X light chain and translocation domain and a BoNT / B receptor binding domain (H C 67. The polypeptide, method or use according to any one of items 1 to 53, 59 to 62 or 65 to 66, wherein the BoNT is a chimeric botulinum neurotoxin (BoNT) comprising a nucleotide sequence (domain) and a nucleotide sequence (domain). [Item 68] 68. The polypeptide for use, method or use according to any one of items 65 to 67, wherein the pain is inflammatory pain. [Item 69] The polypeptide is Cys-(Xaa) a -Ile-Asp / Glu-Gly-Arg-(Yaa) b 8. A polypeptide for use, method or use according to any one of the preceding items, comprising -Cys (SEQ ID NO: 71), wherein a=1 to 10 and b=4 to 15. [Explanation of symbols]

[0499] [Figure 1] Cleavage: Cutting [Figure 2] cleavage: cutting [Figure 4A] Surgery: surgery Administration: [Figure 4B] Paw withdrawal threshold: Paw withdrawal threshold Ipsilateral paw: ipsilateral paw Time: Time days: days Day 0: Day 0 Dosing day: Dosing day 2 weeks post surgery: 2 weeks after surgery Vehicle: Base Gabapentin: [Figure 4C] Contralateral paw: Opposite paw [Figure 4D] Body weight change: Weight change % vs wk2: % vs wk2 Day 3: Day 3 Day 5: Day 5 Day 9: Day 9 Days after injection: Days after injection [Figure 5A] injection: injection Administration: [Figure 5B] Latency: Waiting time Time after treatment (day): Time after treatment (day) Pre-induction: Baseline: Baseline glucose: glucose Vehicle: Base Oxaliplatin: Oxaliplatin Duloxetine: Duloxetine Ipsilateral paw: ipsilateral paw [Figure 5C] Contralateral paw: Opposite paw [Figure 6] injection: injection Administration: [Figure 6B] Withdrawal threshold: Day -2: Day -2 Day 0: Day 0 Day 3: Day 6: Day 9: Baseline: Baseline Days after neurotoxin injection: Days after neurotoxin injection Vehicle: Base Pregabalin: Pregabalin Ipsilateral paw: ipsilateral paw [Figure 6C] Contralateral paw: Opposite paw [Figure 6D] Latency to paw withdrawal: Latency to paw withdrawal [Figure 7A] Administration: post-UVB: After UVB [Figure 7B] Paw withdrawal threshold: Paw withdrawal threshold Baseline: Baseline time post-UVB: Time after UVB Day 3: after neurotoxin injection: after neurotoxin injection Vehicle: Base Indomethacin: Indomethacin [Figure 8] Paw withdrawal threshold: Paw withdrawal threshold Baseline: Baseline Day 0: Day 0 Day 2: Day 1: Day 3: Diluent: Indomethacin: Indomethacin or Vehicle Injection: injection: injection

Claims

1. A pharmaceutical comprising a polypeptide for use in the treatment of pain, wherein the polypeptide comprises a Clostridial neurotoxin light chain (L chain), a Clostridial neurotoxin translocation domain (HN domain), and / or a Clostridial neurotoxin receptor binding domain (HC domain), and wherein when the polypeptide comprises a Clostridial neurotoxin L chain, the L chain is catalytically inactive.

2. The pharmaceutical of claim 1, wherein the polypeptide does not treat pain by either promoting neuronal growth, promoting neuronal repair, or promoting neuronal growth and repair, and / or the polypeptide does not contain an additional therapeutic agent (e.g., a therapeutic agent covalently or non-covalently linked) to the Clostridial neurotoxin L chain, HN domain, and / or HC domain.

3. A pharmaceutical comprising a polypeptide for use in treating an inflammatory disorder, wherein the polypeptide comprises a Clostridial neurotoxin light chain (L chain), a Clostridial neurotoxin translocation domain (HN domain), and / or a Clostridial neurotoxin receptor binding domain (HC domain), and wherein when the polypeptide comprises a Clostridial neurotoxin L chain, the L chain is catalytically inactive.

4. The pharmaceutical of claim 3, wherein the polypeptide does not treat the inflammatory condition by either promoting neuronal growth, promoting neuronal repair, or promoting neuronal growth and repair, and / or the polypeptide does not contain an additional therapeutic agent (e.g., a therapeutic agent covalently or non-covalently linked) to the Clostridial neurotoxin L chain, HN domain, and / or HC domain. (a) the polypeptide does not contain an additional catalytically active domain; (b) the polypeptide does not include an additional diagnostic agent (e.g., a diagnostic agent covalently or non-covalently linked) to the clostridial neurotoxin L chain, HN domain, and / or HC domain; (c) the polypeptide is not administered (e.g., sequentially or subsequently) with an additional therapeutic or diagnostic agent; (d) the polypeptide comprises a catalytically inactive Clostridial neurotoxin L chain; (e) the polypeptide comprises a Clostridial neurotoxin L chain, an HN domain, and an HC domain, wherein the L chain is catalytically inactive; (f) the polypeptide consists essentially of a Clostridial neurotoxin light chain (L chain), a Clostridial neurotoxin translocation domain (HN domain), and a Clostridial neurotoxin receptor binding domain (HC domain), wherein the L chain is catalytically inactive; (g) the polypeptide further does not contain a non-clostridial catalytic domain. (h) the polypeptide is not expressed in cells of the subject, e.g., where a nucleic acid encoding the polypeptide is not expressed by cells of the subject. (i) the polypeptide further comprises one or more non-clostridial neurotoxin sequences, preferably the one or more non-clostridial neurotoxin sequences do not bind to a cellular receptor, and / or the one or more non-clostridial neurotoxin sequences do not comprise a ligand for a cellular receptor, and / or (j) the polypeptide comprises Cys-(Xaa) a -Ile-Asp / Glu-Gly-Arg-(Yaa) b -Cys (SEQ ID NO: 71), where a=1 to 10 and b=4 to 15; The pharmaceutical composition according to any one of claims 1 to 4. (a) the polypeptide consists essentially of a Clostridial neurotoxin light chain (L chain), a Clostridial neurotoxin translocation domain (HN domain), and / or a Clostridial neurotoxin receptor binding domain (HC domain), wherein when the polypeptide comprises or consists essentially of a Clostridial neurotoxin L chain, the L chain is catalytically inactive. (b) the polypeptide consists essentially of a Clostridial neurotoxin light chain (L chain) and a Clostridial neurotoxin translocation domain (HN domain), wherein the L chain is catalytically inactive; (c) the polypeptide comprises a Clostridial neurotoxin light chain (L chain), a Clostridial neurotoxin translocation domain (HN domain), and / or a Clostridial neurotoxin receptor binding domain (HC domain), wherein when the polypeptide comprises or consists of a Clostridial neurotoxin L chain, the L chain is catalytically inactive; and / or (d) the polypeptide consists of a Clostridial neurotoxin light chain (L chain) and a Clostridial neurotoxin translocation domain (HN domain), wherein the L chain is catalytically inactive; The pharmaceutical composition according to any one of claims 1 to 5.

7. The pharmaceutical described in any one of claims 1 to 6, wherein the polypeptide consists of a Clostridial neurotoxin light chain (L chain), a Clostridial neurotoxin translocation domain (HN domain), and a Clostridial neurotoxin receptor binding domain (HC domain), wherein the L chain is catalytically inactive.

8. The pharmaceutical according to claim 1, wherein the polypeptide does not contain both the HN domain and the HC domain of a Clostridial neurotoxin.

9. The pharmaceutical composition described in any one of claims 1 to 8, wherein the pain is chronic pain or acute pain.

10. The pharmaceutical composition of any one of claims 1 to 9, wherein the pain is bladder pain, inflammatory pain, neuropathic pain, mixed pain, allodynia, visceral pain, headache pain (e.g., migraine and / or migraine pain), post-operative pain, referred pain, somatic pain, or phantom limb pain.

11. (a) the inflammatory pain is caused by or associated with sunburn, UV-induced damage, arthritic disorders, autoimmune diseases, connective tissue disorders, injury, infection, neuritis, joint inflammation, or headache (preferably muscular / myogenic headache, vascular headache, hypertensive headache, hormonal headache, rebound headache, chronic sinusitis headache, organic headache, or paroxysmal headache), (b) the neuropathic pain is (or is caused by or associated with) neuralgia, deafferentation, complex regional pain syndrome (CRPS), or neuropathy (e.g., central or peripheral neuropathy); (c) the visceral pain is (or is caused by or associated with) functional visceral pain, chronic gastrointestinal inflammation, autoimmune pain, organic visceral pain, or treatment-induced visceral pain; (d) the headache pain is caused by or associated with muscular / myopathic headache, vascular headache, hypertensive headache, hormonal headache, rebound headache, chronic sinusitis headache, organic headache, or paroxysmal headache, (e) the somatic pain is caused by or associated with excessive muscle tension, repetitive motion disorders, muscle disorders, myalgia, infection, or drugs; (f) the bladder pain is caused by or associated with interstitial cystitis; The pharmaceutical composition according to claim 10.

12. The pharmaceutical described in any one of claims 3 to 8, wherein the inflammatory disorder is one or more selected from cystitis, endometriosis, rheumatoid arthritis, complex regional pain syndrome, and neuritis, and preferably, the cystitis is interstitial cystitis or the neuritis is peripheral neuritis.

13. The method of claim 12, wherein the single dose of the polypeptide administered is greater than 250 μg. (b) the single dose of the polypeptide administered is between 251 μg and 10 g; (c) the single dose of the polypeptide administered is between 251 μg and 1 g; (d) the single dose of the polypeptide administered is between 251 and 1000 μg; (e) the polypeptide is administered repeatedly (e.g., as part of a pain treatment regimen); (f) the polypeptide is administered intradermally; (g) the polypeptide comprises a polypeptide sequence having at least 70%, 80%, or 90% sequence identity to any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 74, 75, or 76, provided that, when the polypeptide comprises a Clostridial neurotoxin light chain, the light chain is catalytically inactive. (h) the polypeptide comprises the polypeptide sequence of any one of SEQ ID NOs: 2, 8, 10, 12, 14, 16, 18, 22, 26, 30, 34, 38, 42, 44, 46, 48, 50, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 74, 75, or 76; (i) the polypeptide is a catalytically inactive BoNT / A, and / or (j) the polypeptide is a modified Clostridial neurotoxin, e.g., a chimeric Clostridial neurotoxin or a hybrid Clostridial neurotoxin, preferably, the polypeptide does not contain a naturally occurring Clostridial neurotoxin H chain; The pharmaceutical composition according to any one of claims 1 to 12. (a) the polypeptide lacks a functional HCC domain or HC domain of a Clostridial neurotoxin; (b) the polypeptide is a retargeted clostridial neurotoxin that contains a non-clostridial targeting moiety (TM); (c) the polypeptide lacks a functional HC domain of a Clostridial neurotoxin and also lacks any functionally equivalent exogenous ligand targeting moiety (TM); (d) the polypeptide is a chimeric botulinum neurotoxin (BoNT) that contains a catalytically inactive light chain and translocation domain of BoNT / A and a receptor binding domain (HC domain) of BoNT / B; (e) the polypeptide comprises a modified HC domain of BoNT / A comprising a modification at one or more amino acid residues selected from ASN886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, SER955, GLN991, GLU992, GLN995, ASN1006, ASN1025, ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, HIS1064, ASN1080, GLU1081, GLU1083, ASP1086, ASN1188, ASP1213, GLY1215, ASN1216, GLN1229, ASN1242, ASN1243, SER1274, and THR1277: wherein said modification is selected from: i. replacement of acidic surface exposed amino acid residues with basic amino acid residues; ii. replacement of acidic surface exposed amino acid residues with uncharged amino acid residues; iii. replacement of uncharged surface exposed amino acid residues with basic amino acid residues; iv. insertion of a basic amino acid residue; and v. Deletion of acidic surface-exposed amino acid residues; (f) the polypeptide comprises a catalytically inactive botulinum neurotoxin serotype X (BoNT / X) L chain, a BoNT / X HN domain, and / or a BoNT / X HC domain; (g) the polypeptide is a chimeric botulinum neurotoxin (BoNT) that contains a catalytically inactive BoNT / X light chain and translocation domain and a receptor binding domain (HC domain) from a different (i.e., non-BoNT / X) clostridial neurotoxin; or (h) the polypeptide is a chimeric botulinum neurotoxin (BoNT) comprising a catalytically inactive BoNT / X light chain and a translocation domain and a BoNT / B receptor binding domain (HC domain); The pharmaceutical composition according to any one of claims 1 to 13.

15. The pharmaceutical described in claim 14, wherein the pain is inflammatory pain.

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