Treatment of pain & inflammatory disorders

Abstract

The present invention is directed to a polypeptide for use in treating pain or an inflammatory disorder, wherein the polypeptide comprises a clostridial neurotoxin light-chain (L-chain), a clostridial neurotoxin translocation domain (H Ndomain) and / or a clostridial neurotoxin receptor binding domain (H Cdomain), wherein when the polypeptide comprises a clostridial neurotoxin L-chain, the L-chain is catalytically inactive. Also provided are corresponding methods of treatment and uses.

Description

[Technical Field]

[0001] This invention relates to the use of polypeptides in therapeutics, for example, the use of polypeptides for treating pain or inflammatory disorders. [Previous Technology]

[0002] Bacteria in the genus *Clostridia* produce potent and specific protein toxins that can poison neurons and other cells to which they are delivered. Examples of such clostridia toxins include neurotoxins produced by serotypes AG and X of *C. tetani* (TeNT) and *C. botulinum* (BoNT) (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, particularly the release of neurotransmitters. Botulinum toxin acts at the neuromuscular junction and inhibits cholinergic transmission in the peripheral nervous system, while tetanus toxin acts on the central nervous system.

[0003] In nature, Clostridium neurotoxins are synthesized as single-chain polypeptides, which are formed through post-translational modification via protease cleavage, resulting in two polypeptide chains linked together by disulfide bonds. Cleavage occurs at a specific cleavage site, commonly referred to as the activation site, located between cysteine ​​residues that provide the inter-chain disulfide bond. This double-chain form is the active form of the toxin. These two chains are called the heavy chain (H chain), with a molecular weight of approximately 100 kDa; and the light chain (L chain), with a molecular weight of approximately 50 kDa. The H chain contains an N-terminal translocation component (HN domain) and a C-terminal targeting component (HC domain). The cleavage site is located between the L chain and the translocation component. After the HC domain binds to its target neuron and the bound toxin is internalized into the cell via the endosome, the HN domain transfers the L chain across the endosome membrane and into the cytoplasm. The L chain provides a protease function (also known as a non-cytotoxic protease).

[0004] Non-cytotoxic proteases act by 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 Attachment Receptor, where NSF stands for N-ethylmaleimide-Sensitive Factor. SNARE proteins are essential for intracellular vesicle fusion and therefore essential for the secretion of molecules transported from cells via vesicles. This protease functions as a zinc-dependent endopeptidase and exhibits high receptor specificity for SNARE proteins. Therefore, once delivered to the target cell, this non-cytotoxic protease can inhibit the cellular secretion of the target cell. The L-chain protease of Clostridium neurotoxin is a non-cytotoxic protease that cleaves SNARE proteins.

[0005] Given the ubiquitous nature of SNARE proteins, Clostridium neurotoxins such as botulinum toxin have been successfully used in a wide range of therapies.

[0006] Clostridium neurotoxins are known to have some of the most potent forms. For example, botulinum neurotoxin, depending on its serotype, has a median lethal dose (LD50) in mice ranging from 0.5 to 5 ng / kg. Therefore, the use of this toxin is not without risk. The diffusion of the toxin from the injection site into peripheral tissues or systemic circulation is a cause of undesirable side effects from clostridium neurotoxin treatment, and in extreme cases, can be life-threatening. This can be a particularly concerning issue when clostridium neurotoxins are used at high doses, concentrations, and / or injection volumes. Adverse reactions reported with commercially available BoNT / A therapy include asthenia, generalized muscle weakness, diplopia, ptosis, dysphagia, dysarthria, urinary incontinence, and respiratory distress. Dysphagia and respiratory distress can be life-threatening, and deaths have been reported in connection with the spread of the toxin's effects.

[0007] The present invention overcomes one or more of the above-mentioned problems. [Summary of the Invention]

[0008] [Invention Summary]

[0009] The inventors have discovered that catalytically inactive Clostridium difficile neurotoxins are effective in treating pain. This discovery is particularly surprising because it is believed that the catalytic activity leading to SNARE protein cleavage is a potential essential mechanism of action in Clostridium difficile neurotoxin therapy. Therefore, the peptides of the present invention avoid the toxic side effects associated with conventional catalytically active Clostridium difficile neurotoxin therapy and contribute to a safer (substantially non-toxic) treatment. Advantageously, the peptides of the present invention can be administered in larger doses compared to conventional catalytically active Clostridium difficile neurotoxin therapy. Furthermore, the reduced toxicity of the peptides of the present invention makes them easy to manufacture and handle throughout the product lifecycle and eliminates the need for physicians to perform complex (e.g., personalized) dosing regimen calculations to avoid toxicity in patients.

[0010] Equally surprising, the inventors have discovered that non-catalytically active Clostridium neurotoxins are effective in treating inflammatory disorders. [Simplified Explanation of the Diagram]

[0011] Specific embodiments of the present invention are described hereby by way of illustration only, with reference to the following figures and examples. Figure 1 shows the SNAP25 cleavage percentage in human neuronal cells administered BoNT / A or BoNT / A(0). Figure 2 shows the SNAP25 cleavage percentage in rat neuronal cells administered BoNT / A or BoNT / A(0). Figure 3(A) shows the characteristic startle response of a mouse suspended by its tail. (B) shows the score used for Digit Abduction Score (DAS) analysis. Figure 4(A) presents an experimental schematic diagram of a study using adult male Sprague-Dawley rats (220-250 g) in a chronic constriction injury (CCI) model of chronic neuropathic pain. Indicate the day before and after administration of BoNT / A(0) (60 pg / kg i.pl.), BoNT / A (30 pg / kg plantar (i.pl.)), BoNT / A (60 pg / kg i.pl.), mediator (gelatin phosphate buffer (GPB) i.pl. - negative control) or gabapentin (100 mg / kg po - positive control) (D). Administration occurred on day 0 (D0) and CCI surgery was performed on day -14. vF indicates the date of the indicated von Frey test. (B) shows the mechanosensitivity (measured by von Frey test) of the ipsilateral paw (i.e., the paw of the control group, BoNT / A, or BoNT / A(0) administered) over time in animals administered as described in (A). (C) Shows the mechanosensitivity (measured via von Frey test) of the contralateral paw (i.e., the paw not given the control group, BoNT / A, or BoNT / A(0)) over time in animals given as described in (A). (D) Shows the change in body weight of rats given as described in (A) over time. Figure 5(A) presents a schematic diagram of an experimental study of a pattern of acute oxaliplatin-induced neuropathic pain in adult male Sprague-Dawley rats. On day 0 (D0), patients were given oxaliplatin (10 mg / kg intraperitoneally (ip)) and BoNT / A(0) (1000 pg / kg i.pl.), BoNT / A (50 pg / kg i.pl.), BoNT / A (100 pg / kg i.pl.), BoNT / A (160 pg / kg i.pl.), or media (GPB i.pl. - negative control).As another negative control for oxaliplatin treatment, a subset of rats was administered 5% glucose ip and GBP (i.pl.). Duloxetine (100 mg / kg po - positive control) was administered 1 hour before the D3 test. Days and hours after administration are shown. PI indicates the paw immersion (cold) test performed on the indicated day. (B) Shows the cold sensitivity (measured by paw immersion test) of the ipsilateral paw (i.e., the paw given the control adult, BoNT / A, or BoNT / A(0)) over time in animals administered as described in (A). (C) Shows the cold sensitivity (measured by paw immersion test) of the contralateral paw (i.e., the paw not given the control adult, BoNT / A, or BoNT / A(0)) over time in animals administered as described in (A). Figure 6(A) shows a schematic diagram of the study of chronic oxaliplatin-induced neuropathic pain patterns using adult male Sprague-Dawley rats (180–210 g). Oxaliplatin was administered on day-2 (D-2). On day 0 (D0), BoNT / A(0) (100 pg / kg i.pl.), BoNT / A (100 pg / kg i.pl.), or mediator (GPB i.pl. - negative control) was administered. On day 3, pregabalin (30 mg / kg po. - positive control) was administered. Days before and after administration are shown. vF and CP indicate the von Frey and cold plate tests performed on the indicated days, respectively. (B) Mechanosensitivity of the ipsilateral paw (i.e., the paw given the control adult, BoNT / A, or BoNT / A(0)) over time as described in (A) (measured by the von Frey test). (C) Mechanosensitivity of the contralateral paw (i.e., the paw not given the control adult, BoNT / A, or BoNT / A(0)) over time as described in (A) (measured by the von Frey test). (D) Thermal sensitivity of the animal given the control adult (as described in (A)) over time (measured by the cold plate test). Figure 7(A) presents a schematic diagram of an experimental study using adult male Wistar rats (180–210 g) inducing inflammatory pain patterns from acute ultraviolet B (UV-B) sunburn. On day 0 (D0), rats were administered BoNT / A(0) (100 pg / kg i.pl.), BoNT / A (100 pg / kg i.pl.), or mediator (GPB i.pl. - negative control). One hour before the D3 readout, rats were administered indomethacin (5 mg / kg po - positive control). On day 1 (D1), rats were exposed to UV-B (500 mJ / cm2).vF indicates that the von Frey test was performed on the indicated date. (B) shows the mechanosensitivity of the animals administered as described in (A) (measured by the von Frey test). Figure 8 shows the mechanosensitivity of mice administered the media, catalytically active chimeric BoNT / XB (0.3 ng / kg, n=10), catalytically active chimeric BoNT / XB (30 ng / kg, n=10), non-catalytically active chimeric BoNT / XB(0) (0.3 ng / kg, n=10), non-catalytically active chimeric BoNT / XB(0) (30 ng / kg, n=10), BoNT / A (160 pg / kg, n=10), or indomethacin (10 mg / kg, n=9) (measured by the von Frey test). Sensitivity was demonstrated in untreated animals (baseline), 2 days after administration of BoNT or mediator, and before administration of Complete Freund's adjuvant (CFA) (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 comparison relative to mediator after repeated measures two-way ANOVA).

Implementation Method

[0012] [Detailed Description of the Invention]

[0013] Similarly, the present invention provides a polypeptide (e.g., analgesic polypeptide) for use in the treatment of pain, wherein the polypeptide comprises a Clostridium neurotoxin light chain (L chain), a Clostridium neurotoxin transfer domain (HN domain) and / or a Clostridium neurotoxin receptor binding domain (HC domain), wherein when the polypeptide comprises a Clostridium neurotoxin L chain, the L chain is non-catalytically active.

[0014] In a related state, a method for treating pain is provided, the method comprising administering a polypeptide (e.g., an analgesic polypeptide) to a subject, wherein the polypeptide comprises a Clostridium neurotoxin light chain (L chain), a Clostridium neurotoxin transfer domain (HN domain) and / or a Clostridium neurotoxin receptor binding domain (HC domain), wherein when the polypeptide comprises a Clostridium neurotoxin L chain, the L chain is non-catalytically active.

[0015] In another related embodiment, the present invention provides the use of a polypeptide (e.g., analgesic polypeptide) in the manufacture of a medicine for treating pain, wherein the polypeptide comprises a Clostridium neurotoxin light chain (L chain), a Clostridium neurotoxin transfer domain (HN domain) and / or a Clostridium neurotoxin receptor binding domain (HC domain), wherein when the polypeptide comprises a Clostridium neurotoxin L chain, the L chain is non-catalytically active.

[0016] The polypeptide of the present invention preferably has analgesic properties. In other words, the polypeptide of the present invention is preferably an analgesic polypeptide.

[0017] Preferably, the polypeptide of the present invention neither promotes neuronal growth nor repairs neurons to treat pain. In other words, preferably, the polypeptide does not treat pain by any of the following means: by promoting neuronal growth, by promoting neuronal repair, or by promoting both neuronal growth and repair.

[0018] Similarly, the present invention provides a polypeptide for use in treating inflammatory disorders, wherein the polypeptide comprises a Clostridium neurotoxin light chain (L chain), a Clostridium neurotoxin transfer domain (HN domain) and / or a Clostridium neurotoxin receptor binding domain (HC domain), wherein when the polypeptide comprises a Clostridium neurotoxin L chain, the L chain is non-catalytically active.

[0019] In a related state, a method for treating inflammatory disorders is provided, the method comprising administering a polypeptide to a subject, wherein the polypeptide comprises a Clostridium neurotoxin light chain (L chain), a Clostridium neurotoxin transfer domain (HN domain) and / or a Clostridium neurotoxin receptor binding domain (HC domain), wherein when the polypeptide comprises a Clostridium neurotoxin L chain, the L chain is non-catalytically active.

[0020] In another related embodiment, the present invention provides the use of a polypeptide in the manufacture of a medicine for treating inflammatory disorders, wherein the polypeptide comprises a Clostridium neurotoxin light chain (L chain), a Clostridium neurotoxin transfer domain (HN domain) and / or a Clostridium neurotoxin receptor binding domain (HC domain), wherein when the polypeptide comprises a Clostridium neurotoxin L chain, the L chain is non-catalytically active.

[0021] The polypeptide of the present invention may have anti-inflammatory properties. In other words, the polypeptide of the present invention may be an anti-inflammatory polypeptide.

[0022] As described herein, a polypeptide is used to treat inflammatory disorders. The polypeptide may comprise a botulinum neurotoxin serotype X (BoNT / X) L chain, a BoNT / X HN domain, and / or a BoNT / X HC domain, wherein when the polypeptide comprises a Clostridium neurotoxin L chain, the L chain is non-catalytically active. For example, the polypeptide may be a chimeric botulinum neurotoxin (BoNT) comprising a non-catalytically active BoNT / X light chain and a transfer domain, and a receptor-binding domain (HC domain) derived from a different (i.e., non-BoNT / X) Clostridium neurotoxin. Thus, in one embodiment, the present invention provides a polypeptide for use in treating inflammatory disorders, wherein the polypeptide comprises a non-catalytically active BoNT / X light chain and a transfer domain, and a receptor-binding domain (HC domain) derived from a different (i.e., non-BoNT / X) Clostridium neurotoxin (preferably a BoNT / B Hc domain). Corresponding treatment methods and uses are also provided.

[0023] Preferably, the polypeptide of the present invention neither promotes neuronal growth nor repairs neurons to treat inflammatory conditions. In other words, preferably, the polypeptide does not treat inflammatory conditions by any of the following means: by promoting neuronal growth, by promoting neuronal repair, or by promoting both neuronal growth and repair.

[0024] The term "promoting neuronal growth and / or neuronal repair" encompasses an increased 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 communication in a neuronal network or population. Therefore, the term "neuronal repair" as used herein encompasses the repair of a specific neuron and the repair of neuronal circuits. This term also encompasses neuronal plasticity. The term "neuronal plasticity" as used herein encompasses axonal budding, dendritic budding, neurogenesis (e.g., the generation of new neurons), maturation, differentiation, and / or synaptic plasticity (e.g., including changes 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., located at or near the site of injury). The term "neuronal growth" as used herein encompasses the growth of any part of a neuron, including the growth of axons and / or dendrites. This term encompasses an increase in neurite length, the number of neurites (e.g., the number of neurites per cell), and / or an increase in the length and / or number of protrusions from the cell body or cell membrane of a neuron, such as axonal growth and / or axonal budding, for example, in neurons within an object. This axonal growth can facilitate connections and / or chemical communication between neurons.

[0025] Preferably, the polypeptide of the present invention does not promote a neuroimmune response to treat pain or inflammatory disorders. In this context, a neuroimmune response encompasses the response of microglia. Therefore, in one specific embodiment, the polypeptide of the present invention does not promote a microglia response to treat pain or inflammatory conditions.

[0026] In one preferred embodiment, the pain is not related to 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 may be used interchangeably with "brain disease." "Brain disorder" as used in this context encompasses disorders originating from within or outside the brain, including disorders related to physical injury causing damage to brain tissue. Examples of brain disorders covered in this context include any one or more of the following: traumatic brain injury, cancer (e.g., brain tumor), 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 disease, Huntington's disease, spinocerebellar ataxia, motor disorders, Hallervorden-Spatz disease, and frontotemporal lobar degeneration), cerebral aneurysms, multiple sclerosis, hypoxic injury, toxic injury, and metabolic injury. Brain disorders 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), Prien's disease, Huntington's disease, spinocerebellar ataxia, motor ataxia, Hallewarden-Scholes disease, frontotemporal degeneration), cerebral aneurysms, multiple sclerosis, hypoxic injury, toxic injury, and / or metabolic injury.

[0027] The active Clostridium neurotoxin L-chain possesses non-cytotoxic protease activity. Specifically, the active Clostridium neurotoxin L-chain possesses endopeptidase activity and is capable of cleaving the protein of the exocytosis fusion construct in the target cell. The protein of the exocytosis fusion construct is preferably a SNARE protein, such as SNAP-25, synaptobrevin / VAMP, or a synaptic fusion protein.

[0028] The term "non-catalytically active" as used herein with respect to the Clostridium neurotoxin L-chain means that the L-chain substantially does not exhibit non-cytotoxic protease activity. Preferably, the term "non-catalytically active" as used herein with respect to the Clostridium neurotoxin L-chain means that the L-chain does not exhibit non-cytotoxic protease activity. In one specific embodiment, the non-catalytically active Clostridium neurotoxin L-chain is a protein that does not cleave the exocytosis fusion structure of the target cell. The term "substantially lacking non-cytotoxic protease activity" means that the Clostridium neurotoxin L-chain has less than 5% of the non-cytotoxic protease activity of the catalytically active Clostridium neurotoxin L-chain, for example, less than 2%, 1%, or preferably less than 0.1% of the non-cytotoxic protease activity of the catalytically active Clostridium neurotoxin L-chain. Non-cytotoxic protease activity can be determined in vitro by culturing the test Clostridium neurotoxin L chain with SNARE protein and comparing the amount of SNARE protein cleaved by the test Clostridium neurotoxin L chain with the amount cleaved by the catalytically active Clostridium neurotoxin L chain under the same conditions. The amount of cleaved SNARE protein can be quantified using conventional techniques such as SDS-PAGE and Western blotting. Suitable in vitro analyses are described in WO 2019 / 145577 A1, which is incorporated herein by reference.

[0029] Cell-based and in vivo assays can also be used to determine whether Clostridium neurotoxins containing L-chains and functional cell-binding and transfer domains possess non-cytotoxic protease activity. As routine analyses in this field include digit abduction score (DAS) analysis, dorsal root ganglion (DRG) analysis, spinal cord neuron (SCN) analysis, and mouse phrenic nerve hemidiaphragm (PNHD) analysis. Suitable analyses for determining non-cytotoxic protease activity may be those described in Aoki KR, Toxicon 39: 1815-1820; 2001 or Donalde et al. (2018), Pharmacol Res Perspect, e00446, 1-14, which are incorporated herein by reference.

[0030] The non-catalytically inactive L-chain may have one or more mutations that inactivate the catalytic activity. Thus, the 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, the non-catalytically inactive L-chain may contain mutations at the active site residues. The mutations may be substitutions or deletions, but substitutions are preferred, especially substitutions with chemically similar amino acids. Glutamic acid may be substituted with glutamic acid, histidine with tyrosine, arginine with glutamic acid, and / or tyrosine with phenylalanine. Alternatively, any residue may be substituted with alanine.

[0031] The non-catalytically inactive BoNT / AL chain may include mutations in H223, E224, H227, E262, R363, and / or Y366, preferably at least in E224 and H227. Preferably, the non-catalytically inactive BoNT / AL chain may include a glutamic acid substitution in E224 (E224Q) and a tyrosine substitution in H227 (H227Y). The position number corresponds to the amino acid position of SEQ ID NO: 60 and can be determined by comparing the polypeptide with SEQ ID NO: 60. Since the presence of the methionine residue at position 1 of SEQ ID NO: 60 is not essential, those skilled in the art will consider the presence / absence of the methionine residue when determining the amino acid residue number. For example, if SEQ ID NO: 60 includes methionine, the position numbering will be as defined above (e.g., His223 will be His223 of SEQ ID NO: 60). Alternatively, if the methionine is not present in SEQ ID NO: 60, the amino acid residue numbering should be modified by -1 (e.g., His223 will be His222 of SEQ ID NO: 60). Similar considerations apply when methionine at position 1 of other polypeptide sequences described herein is present / absent, and those skilled in the art will readily determine the correct amino acid residue numbering using conventional techniques of the art.

[0032] The non-catalytically inactive BoNT / BL chain may contain mutations at E231 and / or H234, preferably at E231 and H234. Preferably, the non-catalytically inactive BoNT / BL chain contains a glutamic acid substitution at E231 (E231Q) and a tyrosine substitution at H234 (H234Y). The position number corresponds to the amino acid position of SEQ ID NO: 52 and can be determined by comparing the polypeptide with SEQ ID NO: 52. Since the presence of the methionine residue at position 1 of SEQ ID NO: 52 is not essential, when determining the amino acid residue number, those skilled in the art will consider the presence / absence of the methionine residue.

[0033] The non-catalytically inactive BoNT / CL chain may include mutations in H229, E230, and / or H233, preferably H229, E230, and H233. More preferably, the non-catalytically inactive BoNT / CL chain includes a substitution of glycine in H229 (H229G), a substitution of threonine in E230 (E230T), and a substitution of aspartic acid in H233 (H233N). The position number corresponds to the amino acid position of SEQ ID NO: 53 and can be determined by comparing the polypeptide with SEQ ID NO: 53. Since the presence of the methionine residue at position 1 of SEQ ID NO: 53 is not essential, those skilled in the art will consider the presence / absence of the methionine residue when determining the amino acid residue number.

[0034] The non-catalytically inactive BoNT / DL chain may include mutations in H229, E230, H233, and / or H236, preferably at least in E230 and H236. Preferably, the non-catalytically inactive BoNT / DL chain includes at least a glutamic acid substitution at E230 (E230Q) and a tyrosine substitution at H236 (H236Y). The position number corresponds to the amino acid position of SEQ ID NO: 54 and can be determined by comparing the polypeptide with SEQ ID NO: 54. Since the presence of the methionine residue at position 1 of SEQ ID NO: 54 is not essential, those skilled in the art will consider the presence / absence of the methionine residue when determining the amino acid residue number.

[0035] The non-catalytically inactive BoNT / EL chain may include mutations in E213 and / or H216, preferably in E213 and H216. Preferably, the non-catalytically inactive BoNT / EL chain includes a glutamic acid substitution in E213 (E213Q) and a tyrosine substitution in H216 (H216Y). The position number corresponds to the amino acid position of SEQ ID NO: 55 and can be determined by comparing the polypeptide with SEQ ID NO: 55. Since the presence of the methionine residue at position 1 of SEQ ID NO: 55 is not essential, those skilled in the art will consider the presence / absence of the methionine residue when determining the amino acid residue number.

[0036] The non-catalytically inactive BoNT / FL chain may include mutations in E228 and / or H231, preferably in E228 and H231. Preferably, the non-catalytically inactive BoNT / FL chain includes a glutamic acid substitution in E228 (E228Q) and a tyrosine substitution in H231 (H231Y). The position number corresponds to the amino acid position of SEQ ID NO: 56 and can be determined by comparing the polypeptide with SEQ ID NO: 56. Since the presence of the methionine residue at position 1 of SEQ ID NO: 56 is not essential, those skilled in the art will consider the presence / absence of the methionine residue when determining the amino acid residue number.

[0037] The non-catalytically inactive BoNT / GL chain may include mutations in E231 and / or H234, preferably in E231 and H234. Preferably, the non-catalytically inactive BoNT / GL chain includes a glutamic acid substitution in E231 (E231Q) and a tyrosine substitution in H234 (H234Y). The position number corresponds to the amino acid position of SEQ ID NO: 57 and can be determined by comparing the polypeptide with SEQ ID NO: 57. Since the presence of the methionine residue at position 1 of SEQ ID NO: 57 is not essential, when determining the amino acid residue number, those skilled in the art will consider the presence / absence of the methionine residue.

[0038] The non-catalytically inactive BoNT / XL chain may include mutations in E228 and / or H231, preferably in E228 and H231. Preferably, the non-catalytically inactive BoNT / XL chain includes a glutamic acid substitution in E228 (E228Q) and a tyrosine substitution in H231 (H231Y). The position number corresponds to the amino acid position of SEQ ID NO: 59 and can be determined by comparing the polypeptide with SEQ ID NO: 59. Since the presence of the methionine residue at position 1 of SEQ ID NO: 59 is not essential, when determining the amino acid residue number, those skilled in the art will consider the presence / absence of the methionine residue.

[0039] The non-catalytically inactive TeNT L chain may include mutations in E234, R372, and / or Y375, preferably in at least R372 and Y375 (e.g., in E234, R372, and Y375). Preferably, the non-catalytically inactive TeNT L chain includes a substitution of glutamic acid or alanine in R372 (R372Q or R372A), more preferably alanine; and a substitution of phenylalanine in Y375 (Y375F). The position number corresponds to the amino acid position of SEQ ID NO: 58 and can be determined by comparing the polypeptide with SEQ ID NO: 58. Since the presence of the methionine residue at position 1 of SEQ ID NO: 58 is not essential, those skilled in the art will consider the presence / absence of the methionine residue when determining the amino acid residue number.

[0040] The polypeptide of the present invention may comprise a fragment of a full-length Clostridium neurotoxin (wherein the L chain is non-catalytically active) or a Clostridium neurotoxin fragment (e.g., an HN domain and / or an HC domain) that does not possess non-cytotoxic protease activity. In other words, the polypeptide of the present invention does not possess non-cytotoxic protease activity.

[0041] The term "clostridium neurotoxin" encompasses toxins produced by Clostridium botulinum (botulinum neurotoxin serotypes A, B, C1, D, E, F, G, and X), Clostridium tetani (tetanus neurotoxin), Clostridium butyricum (botulinum neurotoxin serotype E), and Clostridium pasteurellii (botulinum neurotoxin serotype F). Reference sequences for BoNT / A are shown in SEQ ID NO: 51. Reference sequences for BoNT / B are shown in SEQ ID NO: 52. Reference sequences for BoNT / C are shown in SEQ ID NO: 53. Reference sequences for BoNT / D are shown in SEQ ID NO: 54. Reference sequences for BoNT / E are shown in SEQ ID NO: 55. Reference sequences for BoNT / F are shown in SEQ ID NO: 56. Reference sequences for BoNT / G are shown in SEQ ID NO: 57. Reference sequences for TeNT are shown in SEQ ID NO: 58. Reference sequences for BoNT / X are shown in SEQ ID NO: 59. The term "clostridium neurotoxin" can also encompass newly discovered members of the botulinum neurotoxin protein family expressed by non-clostridium microorganisms, such as: a toxin encoded by Enterococcus, which has the closest sequence identity to the BoNT / X sequence; a toxin encoded by Weissella oryzae, called BoNT / Wo (NCBI Ref. Seq: WP_027699549.1), which cleaves VAMP2 at W89-W90; a toxin encoded by Enterococcus faecium (GenBank: OTO22244.1), which cleaves VAMP2 and SNAP25; and a toxin encoded by Chryseobacterium pipero (NCBI Ref. Seq: WP_034687872.1).

[0042] Thus, the Clostridium 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 Clostridium neurotoxin is a botulinum neurotoxin, such as selected from the following botulinum neurotoxins: BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, and BoNT / X. For example, the HN domain of the Clostridium neurotoxin may be an HN domain derived from BoNT A, B, Cl, D, E, F, G, X, or TeNT. Similarly, the L chain may be an L chain derived from BoNT A, B, Cl, D, E, F, G, X, or TeNT, provided that the L chain is non-catalytically inactive (e.g., modified to be non-catalytically inactive). More preferably, the Clostridium neurotoxin is BoNT / A.

[0043] As discussed above, (full-length) Clostridium neurotoxin is composed of two polypeptide chains: a heavy chain (H chain) with a molecular weight of approximately 100 kDa and a light chain (L chain) with a molecular weight of approximately 50 kDa. The H chain contains a C-terminal targeting component (receptor-binding domain or HC domain) and an N-terminal transfer component (HN domain). Botulinum neurotoxin (BoNT) is produced by Clostridium botulinum as a large protein complex, composed of BoNT itself and some accessory proteins. Currently, there are eight different types of botulinum neurotoxin, namely: botulinum neurotoxin serotypes A, B, C1, D, E, F, G, and X, all of which have similar structures and modes of action. Different BoNT serotypes can be distinguished based on the inactivation of specific neutralizing antisera, and this classification based on serotype is related to the percentage of sequence identity at the amino acid level. The BoNT protein of a specified serotype is further divided into different subtypes based on the percentage of amino acid sequence identity.

[0044] Conventional (catalytically active) BoNT is absorbed in the gastrointestinal tract and, upon entering systemic circulation, binds to the presynaptic membrane of cholinergic nerve endings, preventing the release of the neurotransmitter acetylcholine. BoNT / B, BoNT / D, BoNT / F, and BoNT / G cleave vesicle-associated membrane protein (VAMP); BoNT / C1, BoNT / A, and BoNT / E cleave the 25 kDa synaptosomal-associated protein (SNAP-25); and BoNT / C1 cleaves synaptic fusion protein. BoNT / X has been found to cleave SNAP-25, VAMP1, VAMP2, VAMP3, VAMP4, VAMP5, Ykt6, and synaptic fusion protein 1. Tetanus toxin is produced by Clostridium tetani in a single serotype. Clostridium butyricum produces BoNT / E, while Clostridium pasteurellum produces BoNT / F.

[0045] In one specific embodiment, the polypeptide of the present invention may be encoded by a nucleotide sequence having at least 70% sequence identity with any of the following 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 contains a Clostridium neurotoxin L-chain, the L-chain is non-catalytically active. In one specific embodiment, the polypeptide of the present invention may be encoded by a nucleotide sequence having at least 80%, 90%, 95%, or 98% sequence identity with any of the following 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 contains a Clostridium neurotoxin L-chain, the L-chain is non-catalytically active. Preferably, the polypeptide of the present invention may be encoded by a nucleotide sequence comprising any of the following SEQ ID NOs: 1, 7, 9, 11, 13, 15, 17, 21, 25, 29, 33, 37, 41, 43, 45, 47, or 49.

[0046] In one specific embodiment, the polypeptide of the present invention may comprise a polypeptide sequence having at least 70% sequence identity with any of the following 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 Clostridium neurotoxin L-chain, the L-chain is non-catalytically active. In one specific embodiment, the polypeptide of the present invention may comprise a polypeptide sequence having at least 80%, 90%, 95% or 98% sequence identity with any of the following 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 Clostridium neurotoxin L-chain, the L-chain is non-catalytically active. Preferably, the polypeptide of the present invention may comprise a polypeptide sequence of any of the following SEQ ID NO: 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.

[0047] In one specific embodiment, the polypeptide of the present invention may comprise a fragment of a polypeptide sequence having at least 70% sequence identity with any of the following 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 Clostridium neurotoxin L chain, the L chain is non-catalytically active. In one specific embodiment, the polypeptide of the present invention may comprise a fragment of a polypeptide sequence having at least 80%, 90%, 95% or 98% sequence identity with any of the following 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 Clostridium neurotoxin L-chain, the L-chain is non-catalytically active. Preferably, the polypeptide of the present invention may comprise a fragment containing a polypeptide sequence comprising any of the following 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 contains a Clostridium neurotoxin L-chain, the L-chain is non-catalytically active. The fragment may be the non-catalytically active L-chain, HN domain, or HC domain of the SEQ ID NO.

[0048] Preferably, the polypeptide of the present invention comprises (or consists of) a non-catalytically active Clostridium neurotoxin L chain. The reference herein to non-catalytically active Clostridium neurotoxin also encompasses fragments of the Clostridium neurotoxin L chain. Fragments of the Clostridium neurotoxin L chain may have ≤400, ≤350, ≤300, ≤250, ≤200, ≤150, ≤100, or ≤50 amino acid residues of the Clostridium neurotoxin L chain. In one specific embodiment, the fragment of the Clostridium neurotoxin L chain has at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, or 200 amino acid residues of the Clostridium neurotoxin L chain. For example, fragments of the L-chain of Clostridium neurotoxin may have 20-400, 50-300, or 100-200 amino acid residues of the L-chain of Clostridium neurotoxin. However, it is preferred that the reference to non-catalytically active Clostridium neurotoxin refers to the full-length non-catalytically active L-chain of Clostridium neurotoxin.

[0049] Examples of L-chain reference sequences include: Type A botulinum toxin neurotoxin: amino acid residues 1-448; Type B botulinum toxin neurotoxin: amino acid residues 1-440; Type C1 botulinum toxin neurotoxin: amino acid residues 1-441; Type D botulinum toxin neurotoxin: amino acid residues 1-445; Type E botulinum toxin neurotoxin: amino acid residues 1-422; Type F botulinum toxin neurotoxin: amino acid residues 1-439; Type G botulinum toxin neurotoxin: amino acid residues 1-441; Tetanus neurotoxin: amino acid residues 1-457.

[0050] For the recently identified BoNT / X, the L chain has been reported to correspond to its amino acid 1-439, where the L chain boundary may vary by about 25 amino acids (e.g., 1-414 or 1-464).

[0051] The reference sequences identified above should be considered as guidelines, as slight variations may occur depending on the subserotype. For example, US 2007 / 0166332 (incorporated here in its entirety by reference) cites the following different Clostridium difficile sequences: Type A botulinum neurotoxin: amino acid residues M1-K448; Type B botulinum neurotoxin: amino acid residues M1-K441; Type C1 botulinum neurotoxin: amino acid residues M1-K449; Type D botulinum neurotoxin: amino acid residues M1-R445; Type E botulinum neurotoxin: amino acid residues M1-R422; Type F botulinum neurotoxin: amino acid residues M1-K439; Type G botulinum neurotoxin: amino acid residues M1-K446; Tetanus neurotoxin: amino acid residues M1-A457.

[0052] Herein lies a suitable Clostridium neurotoxin L chain.

[0053] The Clostridium neurotoxin L chain may comprise a polypeptide sequence having at least 70% sequence identity with any of the following SEQ ID NOs: 6, 24, 32, 40, 74, or 76, provided that the L chain is non-catalytically active (e.g., the L chain has been modified to be inactive). In one embodiment, the Clostridium neurotoxin L chain comprises a polypeptide sequence having at least 80%, 90%, 95%, or 98% sequence identity with any of the following SEQ ID NOs: 6, 24, 32, 40, 74, or 76, provided that the L chain is non-catalytically active (e.g., the L chain has been modified to be inactive). Preferably, the Clostridium neurotoxin L chain comprises (more preferably constitutes) a polypeptide sequence comprising any of the following SEQ ID NOs: 6, 24, 32, or 40, which has been modified to make the L chain non-catalytically active, for example, SEQ ID NO: 74 or 76.

[0054] The Clostridium neurotoxin L-chain may be encoded by a nucleotide sequence having at least 70% sequence identity with any of the following SEQ ID NOs: 5, 23, 31, or 39, provided that the L-chain is non-catalytically active (e.g., the L-chain has been modified to be inactive). In one specific embodiment, the Clostridium neurotoxin L-chain is encoded by a nucleotide sequence having at least 80%, 90%, 95%, or 98% sequence identity with any of the following SEQ ID NOs: 5, 23, 31, or 39, provided that the L-chain is non-catalytically active (e.g., the L-chain has been modified to be inactive). Preferably, the Clostridium neurotoxin L-chain is encoded by a nucleotide sequence comprising any of the following SEQ ID NOs: 5, 23, 31, or 39, which has been modified to make the L-chain non-catalytically active.

[0055] Considering that the efficacy of pain treatment does not require the catalytic activity of light chains, it is credible that peptides not containing L chains (or containing only fragments of L chains) can treat pain. For similar reasons, it is credible that peptides not containing L chains (or containing only fragments of L chains) can treat inflammatory conditions. Therefore, in one embodiment, the peptide may contain a Clostridium neurotoxin transfer domain (HN domain) and / or a Clostridium neurotoxin receptor binding domain (HC domain). In one embodiment, the peptide of the present invention does not contain either the Clostridium neurotoxin transfer domain (HN domain) or the Clostridium neurotoxin receptor binding domain (HC domain).

[0056] In one specific embodiment, the polypeptide of the present invention comprises (or consists of) a Clostridium neurotoxin heavy chain (H chain). The H chain comprises a Clostridium neurotoxin transfer domain (HN domain) and a receptor-binding domain (HC domain). In this context, reference to the Clostridium neurotoxin H chain also encompasses fragments of the Clostridium neurotoxin H chain. Fragments of the Clostridium neurotoxin H chain may have ≤800, ≤700, ≤600, ≤500, ≤400, ≤350, ≤300, ≤250, ≤200, ≤150, ≤100, or ≤50 amino acid residues of the Clostridium neurotoxin H chain. In one specific embodiment, a fragment of the Clostridium neurotoxin H chain has at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, or 200 amino acid residues of the Clostridium neurotoxin H chain. For example, a fragment of the Clostridium neurotoxin H chain may have 20-800, 30-600, 40-400, 50-300, or 100-200 amino acid residues of the Clostridium neurotoxin H chain. However, it is preferred that the reference to H chain refers to the full-length H chain.

[0057] In one specific embodiment, the polypeptide of the present invention comprises (or is composed of) a Clostridium neurotoxin transfer domain (HN domain). In this context, reference to the Clostridium neurotoxin transfer domain also encompasses fragments of the transfer domain. Fragments of the Clostridium neurotoxin transfer domain may have ≤400, ≤350, ≤300, ≤250, ≤200, ≤150, ≤100, or ≤50 amino acid residues of the Clostridium neurotoxin transfer domain. In one specific embodiment, fragments of the Clostridium neurotoxin transfer domain have at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, or 200 amino acid residues of the Clostridium neurotoxin transfer domain. For example, fragments of the Clostridium neurotoxin transfer domain may have 20-400, 50-300, or 100-200 amino acid residues of the Clostridium neurotoxin transfer domain. However, it is preferred that the transfer domain refer to the full-length transfer domain.

[0058] The transfer domain is a fragment of the H chain of the Clostridium neurotoxin, approximately corresponding to half of the amino terminus of the H chain, or a domain corresponding to that fragment in the intact H chain. In one embodiment, the HC function of the H chain can be removed by deleting the HC amino acid sequence (at the DNA synthesis level or at the post-synthetic level by nuclease or protease treatment). Alternatively, the HC function can be deactivated by chemical or biological treatment. Thus, in some embodiments, the H chain can be a binding site that cannot bind to the target cell to which the natural Clostridium neurotoxin (i.e., the whole toxin) binds.

[0059] Examples of suitable (reference) transfer domains include: Botulinum toxin type A - amino acid residues (449-871), Botulinum toxin type B - amino acid residues (441-858), Botulinum toxin type C - amino acid residues (442-866), Botulinum toxin type D - amino acid residues (446-862), Botulinum toxin type E - amino acid residues (423-845), Botulinum toxin type F - amino acid residues (440-864), Botulinum toxin type G - amino acid residues (442-863), and Tetanus toxin - amino acid residues (458-879).

[0060] The reference sequence identified above should be considered as a guide, as slight variations may occur depending on the subserotype. For example, US 2007 / 0166332 (incorporated herein by reference) cites slightly different Clostridium sequences: Type A botulinum toxin neurotoxin - amino acid residues (A449-K871); Type B botulinum toxin neurotoxin - amino acid residues (A442-S858); Type C botulinum toxin neurotoxin - amino acid residues (T450-N866); Type D botulinum toxin neurotoxin - amino acid residues (D446-N862); Type E botulinum toxin neurotoxin - amino acid residues (K423-K845); Type F botulinum toxin neurotoxin - amino acid residues (A440-K864); Type G botulinum toxin neurotoxin - amino acid residues (S447-S863); Tetanus neurotoxin - amino acid residues (S458-V879).

[0061] In the context of this invention, various clostridium neurotoxin HN regions containing transfer domains can be used in the forms of this invention. The HN region from the heavy chain of a clostridium neurotoxin is about 410-430 amino acids long and contains a transfer domain. Studies have shown that the full length of the HN region from the heavy chain of a clostridium neurotoxin is not necessary for the transfer activity of the transfer domain. Thus, the form of this specific embodiment may include a clostridium neurotoxin HN region containing a transfer domain having, for example, the following lengths: at least 350 amino acids, at least 375 amino acids, at least 400 amino acids, and at least 425 amino acids. Other forms of this specific embodiment may include a clostridium neurotoxin HN region containing a transfer domain having, for example, the following lengths: up to 350 amino acids, up to 375 amino acids, up to 400 amino acids, and up to 425 amino acids.

[0062] For more detailed information on the genetic basis of botulinum and tetanus toxin production, see Henderson et al. (1997) in The Clostridia: Molecular Biology and Pathogenesis, Academic Press.

[0063] The term HN encompasses both naturally occurring neurotoxin HN moieties and modified HN moieties having amino acid sequences not found in nature and / or synthetic amino acid residues. In one specific embodiment, the modified HN moieties still exhibit the aforementioned transfer function.

[0064] In one specific embodiment, the polypeptide of the present invention comprises (or is composed of) a Clostridium neurotoxin receptor-binding domain (HC domain). In this context, reference to the Clostridium neurotoxin receptor-binding domain (HC) also encompasses fragments of the Clostridium neurotoxin receptor-binding domain (HC). Fragments of the Clostridium neurotoxin receptor-binding domain (HC) may have ≤350, ≤300, ≤250, ≤200, ≤150, ≤100, or ≤50 amino acid residues of the Clostridium neurotoxin receptor-binding domain (HC). In one specific embodiment, fragments of the Clostridium neurotoxin receptor-binding domain (HC) have at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, or 200 amino acid residues of the Clostridium neurotoxin receptor-binding domain (HC). For example, fragments of the Clostridium neurotoxin receptor-binding domain (HC) may have 20-350, 50-300, or 100-200 amino acid residues of the Clostridium neurotoxin receptor-binding domain (HC). However, it is preferred that the reference to the Clostridium neurotoxin receptor-binding domain (HC) refers to the full-length Clostridium neurotoxin receptor-binding domain (HC).

[0065] Examples of reference sequences for the neurotoxin receptor-binding domain (HC) of Clostridium difficile 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, and TeNT-I880-D1315.

[0066] For the recently identified BoNT / X, the HC domain has been reported to correspond to its amino acids 893-1306, where the boundary of the domain may vary by about 25 amino acids (e.g., 868-1306 or 918-1306).

[0067] The Clostridium neurotoxin H chain (e.g., the HC domain portion) may further include a transfer-promoting domain (or a fragment thereof may be a transfer-promoting domain fragment). This domain promotes the delivery of the L chain into the cytoplasm of the target cell, and has been described, for example, in WO 08 / 008803 and WO 08 / 008805, each of which is incorporated herein by reference.

[0068] For example, the transfer-promoting domain may comprise a Clostridium neurotoxin HCN domain or a fragment or variant thereof. More specifically, the Clostridium neurotoxin HCN transfer-promoting domain may have a length of at least 200 amino acids, at least 225 amino acids, at least 250 amino acids, or at least 275 amino acids. In this respect, the Clostridium neurotoxin HCN transfer-promoting 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: Type A botulinum toxin neurotoxin - amino acid residues (872-1110); Type B botulinum toxin neurotoxin - amino acid residues (859-1097); Type C botulinum toxin neurotoxin - amino acid residues (867-1111); Type D botulinum toxin neurotoxin - amino acid residues (863-1098); Type E botulinum toxin neurotoxin - amino acid residues (846-1085); Type F botulinum toxin neurotoxin - amino acid residues (865-1105); Type G botulinum toxin neurotoxin - amino acid residues (864-1105); Tetanus neurotoxin - amino acid residues (880-1127).

[0069] The above sequence positions may vary slightly depending on the serotype / subtype. Other suitable (reference) examples of the HCN domain of Clostridium neurotoxin include: Type A botulinum toxin neurotoxin - amino acid residues (874-1110) Type B botulinum toxin neurotoxin - amino acid residues (861-1097) Type C botulinum toxin neurotoxin - amino acid residues (869-1111) Type D botulinum toxin neurotoxin - amino acid residues (865-1098) Type E botulinum toxin neurotoxin - amino acid residues (848-1085) Type F botulinum toxin neurotoxin - amino acid residues (867-1105) Type G botulinum toxin neurotoxin - amino acid residues (866-1105) Tetanus neurotoxin - amino acid residues (882-1127)

[0070] Herein lies a suitable Clostridium neurotoxin HC domain.

[0071] The Clostridium neurotoxin HC domain may comprise a polypeptide sequence having at least 70% sequence identity with any of the following SEQ ID NOs: 8, 22, 30, 38, 42, 44, 46, 48, or 50. In one specific embodiment, the Clostridium neurotoxin HC domain comprises a polypeptide sequence having at least 80%, 90%, 95%, or 98% sequence identity with any of the following SEQ ID NOs: 8, 22, 30, 38, 42, 44, 46, 48, or 50. Preferably, the Clostridium neurotoxin HC domain comprises (more preferably constitutes) a polypeptide sequence comprising any of the following SEQ ID NOs: 8, 22, 30, 38, 42, 44, 46, 48, or 50.

[0072] The HC domain of the Clostridium neurotoxin may be encoded by a nucleotide sequence having at least 70% sequence identity with any of the following SEQ ID NOs: 7, 21, 29, 37, 41, 43, 45, 47, or 49. In one specific embodiment, the HC domain of the Clostridium neurotoxin is encoded by a nucleotide sequence having at least 80%, 90%, 95%, or 98% sequence identity with any of the following SEQ ID NOs: 7, 21, 29, 37, 41, 43, 45, 47, or 49. Preferably, the HC domain of the Clostridium neurotoxin is encoded by a nucleotide sequence comprising any of the following SEQ ID NOs: 7, 21, 29, 37, 41, 43, 45, 47, or 49.

[0073] Any of the above-described promoting domains may be combined with any of the aforementioned transfer domain peptides applicable to the present invention. For example, a non-Clostridium promoting domain may be combined with a non-Clostridium transfer domain peptide or with a Clostridium transfer domain peptide. Alternatively, a Clostridium neurotoxin HCN transfer promoting domain may be combined with a non-Clostridium transfer domain peptide. Alternatively, the HCN-promoting domain of Clostridium neurotoxin can be combined with the Clostridium transfer domain peptide, examples of which include: Botulinum toxin type A - amino acid residues (449-1110); Botulinum toxin type B - amino acid residues (442-1097); Botulinum toxin type C - amino acid residues (450-1111); Botulinum toxin type D - amino acid residues (446-1098); Botulinum toxin type E - amino acid residues (423-1085); Botulinum toxin type F - amino acid residues (440-1105); Botulinum toxin type G - amino acid residues (447-1105); Tetanus neurotoxin - amino acid residues (458-1127).

[0074] In some specific embodiments, the peptide of the present invention may lack the functional HC domain of Clostridium neurotoxin. In one specific embodiment, the polypeptide preferably lacks the last 50 amino acids at the C-terminus of the Clostridium neurotoxin holotoxin. In another specific embodiment, the polypeptide preferably lacks the last 100 amino acid residues at the C-terminus of the Clostridium neurotoxin holotoxin, more preferably the last 150, more preferably the last 200, particularly preferably the last 250, and most preferably the last 300. Alternatively, HC binding activity can be eliminated / reduced by mutagenesis—for example, for convenience, referring to BoNT / A, modification of one or two amino acid residues in the ganglioside binding pocket (W1266 to L and Y1267 to F) causes the HC region to lose its receptor binding function. Isofunctional mutations can be made into the peptide components of non-serum-type Clostridium A, for example, based on constructs of mutated Clostridium B (W1262 mutated to L and Y1263 mutated to F) or Clostridium E (W1224 mutated to L and Y1225 mutated to F). Other mutations at the active site achieve the same elimination of HC receptor binding activity, such as the highly preserved residues corresponding to Y1267S in Clostridium A botulinum toxin and other Clostridium neurotoxins. Details of this and other mutations are described in Rummel et al. (2004) (Molecular Microbiol. 51:631-634), which is incorporated herein by reference.

[0075] The HC peptide of natural Clostridium neurotoxin contains about 400-440 amino acid residues and is composed of two functionally different domains, each of which is about 25 kDa, namely the N-terminal region (usually referred to as HCN peptide or domain) and the C-terminal region (usually referred to as HCC peptide or domain). This fact is confirmed by the following published documents, 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. Chem. 268: 15, pp. 11188-11192; Rummel A (2007) PNAS 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, existing literature has sufficiently demonstrated that the C-terminal region (HCC), consisting of 160-200 amino acid residues at the C-terminus, is responsible for the binding of Clostridium neurotoxin to its natural cell receptors, i.e., to the nerve endings at the neuromuscular junction—a fact also confirmed by the aforementioned published literature. Thus, throughout this specification, the mention of a Clostridium heavy chain lacking a functional heavy chain HC peptide (or domain), preventing the heavy chain from binding to the cell surface receptors of the natural Clostridium neurotoxin, indicates that the Clostridium heavy chain lacks only the functional HCC peptide. In other words, the HCC peptide region can be partially or completely deleted, or modified in other ways (e.g., through conventional chemical or proteolytic treatment) to reduce its natural binding ability to nerve endings at the neuromuscular junction.

[0076] Thus, in one specific embodiment, the Clostridium neurotoxin HN peptide of the present invention lacks the C-terminal peptide portion (HCC) of the Clostridium neurotoxin, and therefore lacks the HC-binding function of the natural Clostridium neurotoxin. For example, in one specific embodiment, the C-terminal extended Clostridium HN peptide lacks 40, 60, 80, 100, 120, 140, 150, or 160 amino acid residues of the C-terminal of the Clostridium neurotoxin heavy chain. In another specific embodiment, the Clostridium HN peptide of the present invention lacks the complete C-terminal peptide portion (HCC) of the Clostridium neurotoxin, and therefore lacks the HC-binding function of the natural Clostridium neurotoxin. For example, in one specific embodiment, the Clostridium HN peptide lacks the following C-terminal amino acid residues: 165, 170, 180, 190, or 195 amino acid residues of the Clostridium neurotoxin heavy chain. For another example, the Clostridium HN peptide of the present invention lacks the Clostridium HCC reference sequence selected from the following groups: Type A botulinum neurotoxin amino acid residues (Y1111-L1296), Type B botulinum neurotoxin amino acid residues (Y1098-E1291), Type C botulinum neurotoxin amino acid residues (Y1112-E1291), Type D botulinum neurotoxin amino acid residues (Y1099-E1276), Type E botulinum neurotoxin amino acid residues (Y1086-K1252), Type F botulinum neurotoxin amino acid residues (Y1106-E1274), Type G botulinum neurotoxin amino acid residues (Y1106-E1297), and Tetanus neurotoxin amino acid residues (Y1128-D1315).

[0077] The reference sequence identified above should be considered as a guide, as slight variations may occur depending on the subserotype.

[0078] In other specific embodiments, the fragment of the HC domain may include the HCC peptide as described herein.

[0079] The polypeptide of the present invention may comprise a non-catalytically active Clostridium neurotoxin L-chain and a Clostridium neurotoxin transfer domain (HN domain) and / or a Clostridium neurotoxin receptor binding domain (HC domain). For example, the polypeptide may comprise a non-catalytically active Clostridium neurotoxin L-chain and a Clostridium neurotoxin transfer domain (HN).

[0080] Hereinafter describes a suitable polypeptide comprising a non-catalytically active Clostridium neurotoxin L chain and a transfer domain.

[0081] A polypeptide comprising a Clostridium neurotoxin L-chain and a transfer domain may comprise a polypeptide sequence having at least 70% sequence identity with any of the following SEQ ID NOs: 4, 20, 28, 36, or 75, provided that the L-chain is non-catalytically active (e.g., the L-chain has been modified and is not activated). A polypeptide comprising a Clostridium neurotoxin L-chain and a transfer domain may comprise a polypeptide sequence having at least 80%, 90%, 95%, or 98% sequence identity with any of the following SEQ ID NOs: 4, 20, 28, 36, or 75, provided that the L-chain is non-catalytically active (e.g., the L-chain has been modified and is not activated). Preferably, the polypeptide comprising the L chain and transfer domain of Clostridium neurotoxin comprises (more preferably constitutes) a polypeptide sequence comprising any of the following SEQ ID NOs: 4, 20, 28, 36 or 75, which has been modified to make the L chain non-catalytically active, such as SEQ ID NO: 75.

[0082] The polypeptide comprising (or composed of) the Clostridium neurotoxin L-chain and transfer domain may be encoded by a nucleotide sequence having at least 70% sequence identity with any of the following SEQ ID NOs: 3, 19, 27, or 35, provided that the L-chain is non-catalytically active (e.g., the L-chain has been modified and is not activated). In one specific embodiment, the polypeptide comprising (or composed of) the Clostridium neurotoxin L-chain and transfer domain is encoded by a nucleotide sequence having at least 80%, 90%, 95%, or 98% sequence identity with any of the following SEQ ID NOs: 3, 19, 27, or 35, provided that the L-chain is non-catalytically active (e.g., the L-chain has been modified and is not activated). Preferably, the polypeptide comprising (or composed of) the Clostridium neurotoxin L chain and transfer domain is encoded by a nucleotide sequence comprising any of the following SEQ ID NOs: 3, 19, 27 or 35, which has been modified to render the encoded L chain non-catalytically active.

[0083] Preferably, the polypeptide comprises a non-catalytically active Clostridium neurotoxin L chain, a Clostridium neurotoxin transfer domain (HN domain), and a Clostridium neurotoxin receptor binding domain (HC domain).

[0084] In one specific embodiment, the polypeptide of the present invention does not contain a Clostridium neurotoxin receptor-binding domain (HC) or at least the C-terminal portion of a Clostridium neurotoxin receptor-binding domain (HCC). Thus, in one specific embodiment, the polypeptide of the present invention lacks the C-terminal portion of the Clostridium neurotoxin receptor-binding domain (HCC). Advantageously, such a polypeptide lacks endogenous Clostridium neurotoxin receptor binding capacity, and therefore exhibits fewer off-target effects in subjects to which the polypeptide is administered.

[0085] The polypeptide of the present invention may be substantially composed of a non-catalytically active Clostridium neurotoxin light chain (L chain), a Clostridium neurotoxin transfer domain (HN domain), and / or a Clostridium neurotoxin receptor-binding domain (HC domain). For example, the polypeptide may be substantially composed of a non-catalytically active Clostridium neurotoxin L chain and a Clostridium neurotoxin transfer domain (HN).

[0086] Preferably, the polypeptide is essentially composed of a non-catalytically active Clostridium neurotoxin L chain, a Clostridium neurotoxin transfer domain (HN domain), and a Clostridium neurotoxin receptor binding domain (HC domain).

[0087] The term "substantially composed of" as used in this context means that the polypeptide does not contain one or more additional amino acid residues that confer additional functionality to the polypeptide, for example, when administered to a subject. In other words, a polypeptide "substantially composed of" a non-catalytically active Clostridium neurotoxin light chain (L chain), a Clostridium neurotoxin transfer domain (HN domain), and / or a Clostridium neurotoxin receptor-binding domain (HC domain) may contain one or more additional amino acid residues (for those non-catalytically active Clostridium neurotoxin light chains (L chain), Clostridium neurotoxin transfer domains (HN domains), and / or Clostridium neurotoxin receptor-binding domains (HC domains)) but these additional amino acid residues do not confer additional functionality to the polypeptide, for example, when administered to a subject. Additional functionality may include enzymatic activity, binding activity, and / or any discretionary physiological activity.

[0088] The polypeptide of the present invention may be composed of a non-catalytically active Clostridium neurotoxin light chain (L chain), a Clostridium neurotoxin transfer domain (HN domain), and / or a Clostridium neurotoxin receptor-binding domain (HC domain). For example, the polypeptide may be composed of a non-catalytically active Clostridium neurotoxin L chain and a Clostridium neurotoxin transfer domain (HN).

[0089] Preferably, the polypeptide is composed of a non-catalytically active Clostridium neurotoxin L chain, a Clostridium neurotoxin transfer domain (HN domain), and a Clostridium neurotoxin receptor binding domain (HC domain).

[0090] In one embodiment, in addition to any Clostridium neurotoxin sequence, the polypeptide may also contain a non-Clostridium neurotoxin sequence, provided that the non-Clostridium neurotoxin sequence does not interfere with the polypeptide's ability to achieve its therapeutic effect (e.g., pain relief). Preferably, the non-Clostridium neurotoxin sequence is not catalytically active, for example, enzyme-like. In one embodiment, the polypeptide of the present invention does not contain a catalytically active domain (e.g., a non-Clostridium catalytically active domain). In one embodiment, the non-Clostridium neurotoxin sequence is not bound to a cell receptor. In other words, in one embodiment, the non-Clostridium neurotoxin sequence is not a ligand for a cell receptor. The cell receptor may be a protein cell receptor, such as a membrane host protein. Examples of cell 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-Clostridium neurotoxin sequences may include tags that facilitate purification, such as His tags. In one specific embodiment, the polypeptides of the present invention do not contain sites for labeling or adding labels, such as sortase receptors or donor sites.

[0091] In a preferred embodiment, the polypeptide of the present invention does not contain therapeutic or diagnostic agents (e.g., nucleic acids, proteins, peptides, or small molecule therapeutic or diagnostic agents) except for the non-catalytically active L chain, HN domain, and / or HC domain of the Clostridium neurotoxin. For example, in one embodiment, the polypeptide may not contain covalently or non-covalently linked therapeutic or diagnostic agents. Thus, the polypeptide of the present invention is preferably not used as a delivery carrier for other therapeutic or diagnostic agents.

[0092] The polypeptides of the present invention may comprise (or consist of) modified Clostridium neurotoxins or derivatives thereof, or modified Clostridium neurotoxin fragments or derived fragments, including but not limited to those listed below, wherein any L-chain present is non-catalytically active. The modified Clostridium neurotoxins or derivatives (or modified Clostridium neurotoxin fragments or derived fragments) may contain one or more amino acids modified compared to the native (unmodified) form of the Clostridium neurotoxin (or Clostridium neurotoxin fragment); or may contain one or more inserted amino acids not present in the native (unmodified) form of the Clostridium neurotoxin (or Clostridium neurotoxin fragment). For example, relative to the native (unmodified) Clostridium neurotoxin sequence (or Clostridium neurotoxin fragment), the modified Clostridium neurotoxin (or Clostridium neurotoxin fragment) may have a modified amino acid sequence in one or more domains. Such modification can alter the functional aspects of the toxin (or toxin fragment). Thus, in one specific embodiment, the polypeptide of the present invention is or comprises a modified Clostridium neurotoxin, or a modified Clostridium neurotoxin derivative, or a Clostridium neurotoxin derivative, or a modified Clostridium neurotoxin fragment or derivative fragment (e.g., a non-catalytically active L-chain, HN domain, and / or HC domain), provided that any present L-chain is non-catalytically active.

[0093] The polypeptide of the present invention may comprise (or consist of) a modified Clostridium neurotoxin or Clostridium neurotoxin fragment (e.g., an HC domain) having one or more modifications to the amino acid sequence of the heavy chain (such as the modified HC domain), wherein the modified heavy chain binds to the target nerve cell with a binding affinity higher or lower than that of the natural (unmodified) Clostridium neurotoxin or Clostridium neurotoxin fragment, provided that any present L chain is non-catalytically active. Such modification of the HC domain may include modifying residues in the ganglioside binding site or protein (SV2 or synaptotagmin) binding site of the HC domain, thereby altering the binding to the ganglioside receptor and / or the protein receptor of the target nerve cell. Examples of such modified Clostridium neurotoxins are described in WO 2006 / 027207 and WO 2006 / 114308, both of which are incorporated herein by reference in their entirety.

[0094] Modified Clostridium neurotoxins (or Clostridium neurotoxin fragments) may contain one or more modifications that increase the isoelectric point of the Clostridium neurotoxin compared to an equivalent unmodified Clostridium neurotoxin (or Clostridium neurotoxin fragment) lacking the one or more modifications, provided that any present L-chain is non-catalytically inactive. Suitable modified Clostridium neurotoxins (provided that any present L-chain has been modified to be non-catalytically inactive) are described below and in WO 2015 / 004461 A1 and WO 2016 / 110662 A1, which are incorporated herein by reference. Example sequences include SEQ ID NOs: 42 and 62 described herein.

[0095] In one specific embodiment, the polypeptide of the present invention may comprise a modified BoNT / A or a fragment thereof (e.g., a BoNT / A HC domain or a fragment thereof). Modified BoNT / A or fragments thereof may be modifiers containing one or more amino acid residues selected from the following: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274, and THR 1277.

[0096] When compared with the non-catalytically active BoNT / A shown in SEQ ID NO: 2, this modification may be a modification in which the amino acid residue number is determined by comparison with SEQ ID NO: 2. Since the presence of the methionine residue at position 1 of SEQ ID NO: 2 (and the SEQ ID NO corresponding to the modified BoNT / A polypeptide or fragment thereof described herein) is not essential, when determining the amino acid residue number, those skilled in the art will consider the presence / absence of the methionine residue. For example, if SEQ ID NO: 2 includes methionine, the position number will be as defined above (e.g., ASN 886 will be ASN 886 of SEQ ID NO: 2). Alternatively, if the methionine is not present in SEQ ID NO: 2, the amino acid residue number should be modified by -1 (e.g., ASN 886 will be ASN 885 of SEQ ID NO: 2). When the methionine at position 1 of other polypeptide sequences described herein is present or absent, similar considerations apply, and those skilled in the art will readily determine the correct amino acid residue number using conventional techniques in this art.

[0097] Any of the methods described herein for determining sequence homology and / or sequence identity % may be used to perform the alignment described herein for determining amino acid residue numbers.

[0098] The above indicates that the amino acid residues used for modification are surface-exposed amino acid residues.

[0099] The modified BoNT / A or its fragments may contain modifications located on one or more amino acid residues selected from the following: ASN 886, ASN 930, ASN 954, SER 955, GLN 991, ASN 1025, ASN 1026, ASN 1052, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274 and THR 1277.

[0100] When used in the context of modified BoNT / A or fragments thereof, the term "one or more amino acid residues" preferably means at least 2, 3, 4, 5, 6, or 7 of the referred amino acid residues. Thus, the modified BoNT / A or fragments thereof may contain at least 2, 3, 4, 5, 6, or 7 (preferably 7) modifications located at the referred amino acid residues. The modified BoNT / A or fragments thereof may contain 1-30, 3-20, or 5-10 amino acid modifications. More preferably, when used in the context of modified BoNT / A or fragments thereof, the term "one or more amino acid residues" means all of the referred amino acid residues.

[0101] Preferably, apart from one or more amino acid modifications located at the indicated amino acid residues, the modified BoNT / A or fragment thereof does not contain any further amino acid modifications when compared with SEQ ID NO: 2.

[0102] The modification may be selected from: i. replacing an amino acid residue exposed on an acidic surface with a basic amino acid residue; ii. replacing an amino acid residue exposed on an acidic surface with a non-charged amino acid residue; iii. replacing a non-charged amino acid residue exposed on an acidic surface with a basic amino acid residue; iv. inserting a basic amino acid residue; and v. deleting an amino acid residue exposed on an acidic surface.

[0103] The modifications mentioned above result in the modified BoNT / A or its fragments having an increased positive surface charge and an improved isoelectric point when compared with the corresponding unmodified BoNT / A or its fragments.

[0104] The isoelectric point (pI) is a characteristic of a specified protein. As is well known in the art, proteins are composed of a specific sequence of amino acids (also referred to as amino acid residues in proteins). The amino acids in the twenty standard groups have different side chains (or R groups), meaning that each amino acid residue in 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 properties of a protein will depend on the sum of these various factors.

[0105] Certain amino acid residues (described in detail below) have ionizable side chains that can exhibit a charge depending on the surrounding pH. At a given pH, whether such a side chain is charged depends on the pKa of the relevant ionizable portion, where pKa is the negative logarithm of the acid dissociation constant (Ka) from a particular proton of the conjugate base.

[0106] For example, acidic residues such as aspartic acid and glutamic acid have side-chain carboxylic acid groups with a pKa value of approximately 4.1 (the exact pKa value can depend on temperature, ionic strength, and the microenvironment of ionizable groups). Thus, these side chains exhibit a negative charge at pH 7.4 (commonly referred to as "physiological pH"). At lower pH values, these side chains will become protonated and lose their charge.

[0107] Conversely, basic residues such as lysine and arginine have nitrogen-containing side chain groups with pKa values ​​of approximately 10-12. Therefore, these side chains exhibit a positive charge at pH 7.4. At higher pH values, these side chains will become deprotonated and lose their charge.

[0108] Therefore, the overall (net) charge of a protein molecule depends on the number of acidic and basic residues present in the protein (and their surface exposure) and the surrounding pH. Changing the surrounding pH changes the overall charge of the protein. Thus, for each protein, there is a specific pH at which the number of positive and negative charges is equal and the protein exhibits no total net charge. This point is known as the isoelectric point (pI). The isoelectric point is a standard concept in protein biochemistry and is well known to those skilled in the art.

[0109] Therefore, the isoelectric point (pI) is defined as the pH value at which a protein exhibits zero net charge. An increase in pI means that a higher pH value is required for a protein to exhibit zero net charge. Thus, 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 a protein to exhibit zero net charge. Thus, a decrease in pI represents a decrease in the net positive charge of the protein at a given pH.

[0110] In this technical field, methods for determining the pI of proteins are known and familiar to those skilled in the art. For example, the pI of a protein can be calculated from the average pKa value of the amino acids present in the protein ("calculated pI"). This calculation can be performed using computer programs known in this technical field, such as the Compute pI / MW Tool from ExPASy (https: / / web.expasy.org / compute_pi / ), which is a preferred method for calculating pI according to the present invention. Comparisons of pI values ​​between different molecules should be performed using the same calculation technique / program.

[0111] Where appropriate, the calculated pI of a protein can be experimentally confirmed using isoelectric focusing (“observed pI”). This technique uses electrophoresis to separate proteins based on their pI. Isoelectric focusing is generally performed using a gel with an immobilized pH gradient. When an electric field is applied, the protein moves across the pH gradient until it reaches its pH with zero net charge; this point is the protein's pI. The results provided by isoelectric focusing are generally of relatively low resolution, therefore the inventors believe that the results provided by calculated pI (as described above) are more suitable.

[0112] Unless otherwise stated, throughout this description, "pI" means "calculated pI".

[0113] The pI of a protein can be increased or decreased by altering the number of basic and / or acidic groups exposed 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.

[0114] The modified BoNT / A or its fragments of the present invention may have a pI value that is at least 0.2, 0.4, 0.5 or 1 pI unit higher than that of non-catalytically active BoNT / A (e.g., SEQ ID NO: 2) or its fragments. Preferably, the modified BoNT / A or its fragments may have a pI of at least 6.6, for example, at least 6.8.

[0115] The following table lists the properties of 20 standard amino acids: amino acids side chain Aspartic acid Asp D Charged (acidic) glutamic acid Glu E Charged (acidic) Arginine Arg R Charged (alkaline) lysine Lys K Charged (alkaline) histidine His H Uncharged (polarity) aspartic acid Asn N Uncharged (polarity) glutamic acid Gln Q Uncharged (polarity) serine Ser S Uncharged (polarity) threonine Thr T Uncharged (polarity) Tyrosine Tyr Y Uncharged (polarity) Methionine Met M Uncharged (polarity) tryptophan Trp W Uncharged (polarity) Cysteine Cys C Uncharged (polarity) alanine Ala A Non-electrostatic (hydrophobic) Glycine Gly G Non-electrostatic (hydrophobic) Valine Val V Non-electrostatic (hydrophobic) Leucine Leu L Non-electrostatic (hydrophobic) Isoleucine Ile I Non-electrostatic (hydrophobic) proline Pro P Non-electrostatic (hydrophobic) Phenylan Phe F Non-electrostatic (hydrophobic)

[0116] The following amino acids are considered to be charged amino acids: aspartic acid (negative), glutamic acid (negative), arginine (positive), and lysine (positive).

[0117] At pH 7.4, the side chains of aspartic acid (pKa 3.1) and glutamic acid (pKa 4.1) have negative charges, while the side chains of arginine (pKa 12.5) and lysine (pKa 10.8) have positive charges. Aspartic acid and glutamic acid are referred to as acidic amino acid residues. Arginine and lysine are referred to as basic amino acid residues.

[0118] The following amino acids are considered to be non-polar (meaning they can participate in hydrogen bonding): aspartic acid, glutamic acid, histidine, serine, threonine, tyrosine, cysteine, methionine, and tryptophan.

[0119] The following amino acids are considered to be non-electrically charged hydrophobic amino acids: alanine, valine, leucine, isoleucine, phenylalanine, proline, and glycine.

[0120] In the case of amino acid insertion, an additional amino acid residue (not normally present) is incorporated into the BoNT / A polypeptide sequence or a fragment thereof, thus increasing the total number of amino acid residues in the sequence. In the case of amino acid deletion, an amino acid residue is removed from the Clostridium toxin amino acid sequence, thus reducing the total number of amino acid residues in the sequence.

[0121] Preferably, the modification is a substitution that advantageously maintains the same number of amino acid residues in the modified BoNT / A or its fragments. In the amino acid substitution, the amino acid residues forming a portion of the BoNT / A polypeptide sequence or its fragments are replaced with different amino acid residues. The substituted amino acid residues may be one of the 20 standard amino acids as described above. Alternatively, in the amino acid substitution, the substituted amino acid may be a non-standard amino acid (an amino acid not part of the 20 standard groups described above). For example, the substituted amino acid may be a basic non-standard amino acid, such as L-ornithine, L-2-amino-3-guanidinopropionic acid, or a D-isomer 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 an auxotrophic host of *E. coli*.

[0122] In one specific embodiment, the substitution is selected from: replacing an acidic amino acid residue with a basic amino acid residue, replacing an acidic amino acid residue with a non-charged amino acid residue, and replacing a non-charged amino acid residue with a basic amino acid residue. In one specific embodiment, the substitution is wherein an acidic amino acid residue is replaced with a non-charged amino acid residue, wherein the acidic amino acid residue is replaced by its corresponding non-charged acetaminophen amino acid residue (i.e., aspartic acid is replaced by aspartic acid, and glutamic acid is replaced by glutamic acid).

[0123] Preferably, the basic amino acid residue is a lysine residue or an arginine residue. In other words, the substitution is made with lysine or arginine. Most preferably, the modification is made with lysine.

[0124] Preferably, the modified BoNT / A or fragment thereof used in the present invention comprises 4 to 40 amino acid modifications located in the HCN domain of Clostridium toxin. The modified BoNT / A or fragment thereof preferably also has a pI of at least 6.6. The modified BoNT / A preferably comprises modifications of at least 4 amino acids selected from the following: ASN 886, ASN 930, ASN 954, SER 955, GLN 991, ASN 1025, ASN 1026, and ASN 1052; wherein the modification comprises replacing an amino acid with a lysine residue or an arginine residue. For example, the modified BoNT / A or its fragment may contain modifications of at least five amino acids selected from the following: ASN 886, ASN 930, ASN 954, SER 955, GLN 991, ASN 1025, ASN 1026, ASN 1052, and GLN 1229; wherein the modification includes replacing an amino acid with a lysine residue or an arginine residue.

[0125] Methods for modifying proteins by substitution, insertion, or deletion of amino acid residues are known in the art. For example, amino acid modification can be introduced by modifying the DNA sequence encoding a polypeptide (e.g., encoding unmodified BoNT / A or a fragment thereof). This can be achieved using standard molecular selection techniques, such as site-directed mutagenesis, in which a polymerase enzyme is used to replace the original coding sequence with a short strand of DNA (oligonucleotide) encoding the desired amino acid; or by inserting / deleting portions of the gene using various enzymes (e.g., ligases and restriction endonucleases). Alternatively, the modified gene sequence can be chemically synthesized.

[0126] In one specific embodiment, the polypeptide used according to the present invention comprises: a polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 42 and / or a polypeptide sequence encoded by a nucleotide sequence having at least 70% sequence identity with SEQ ID NO: 41. In one specific embodiment, the polypeptide used according to the present invention comprises a polypeptide sequence having at least 80%, 90%, 95%, or 98% sequence identity with SEQ ID NO: 42. Preferably, the polypeptide used according to the present invention comprises the polypeptide sequence shown in SEQ ID NO: 42. In one specific embodiment, the polypeptide used according to the present invention comprises a polypeptide sequence encoded by a nucleotide sequence having at least 80%, 90%, 95%, or 98% sequence identity with SEQ ID NO: 41. Preferably, the polypeptide used according to the present invention comprises a polypeptide sequence encoded by a nucleotide sequence shown in SEQ ID NO: 41.

[0127] In one specific embodiment, the polypeptide used according to the present invention comprises a polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 62. In one specific embodiment, the polypeptide used according to the present invention comprises a polypeptide sequence having at least 80%, 90%, 95%, or 98% sequence identity with SEQ ID NO: 62. Preferably, the polypeptide used according to the present invention comprises (more preferably consists of) the polypeptide sequence shown in SEQ ID NO: 62.

[0128] SEQ ID NO: 42 is an example of a modified BoNT / A fragment and SEQ ID NO: 62 is an example of a non-catalytically active modified BoNT / A polypeptide. Such modified BoNT / A polypeptides and fragments are particularly preferred for use in this invention. The polypeptides shown in SEQ ID NO: 42 and 62, when compared with wild-type BoNT / A, have some amino acid modifications (e.g., substitutions) that increase the isoelectric point of the polypeptide. It is not intended to be limited to theory, but it is believed that the increased net positive charge promotes electrostatic interaction between the polypeptide and anionic extracellular components, thereby promoting the binding of the polypeptide to the cell surface, thus increasing retention at the administration site and / or duration of action. Thus, it is envisioned that treatment using SEQ ID NO: 42 and 62 would be improved compared to an equivalent polypeptide lacking the modification.

[0129] In one specific embodiment, a polypeptide comprising a polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 42 or 62 and / or comprising a polypeptide sequence encoded by a nucleotide sequence having at least 70% sequence identity with SEQ ID NO: 41, is comprising substitutions at one or more of the following positions (preferably two or more, three or more, four or more, five or more, or six or more, more preferably all): positions 930, 955, 991, 1026, 1052, 1229, and 886.

[0130] Preferably, the polypeptide comprising a polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 42 or 62 and / or comprising a polypeptide sequence encoded by a nucleotide sequence having at least 70% sequence identity with SEQ ID NO: 41, comprises lysine or arginine (more preferably lysine) located at one or more of positions 930, 955, 991, 1026, 1052, 1229, and 886. In a specific embodiment, the polypeptide comprises lysine or arginine (more preferably lysine) located 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 lysine or arginine (most preferably lysine) located at all of positions 930, 955, 991, 1026, 1052, 1229, and 886.

[0131] In one specific embodiment, the Clostridium neurotoxin HC domain used in this invention is a modified BoNT / A HC domain, which includes one or more amino acid residues selected from Y1117, F1252, H1253, and L1278. For example, the modified BoNT / A HC domain may include one or more (preferably two or more) of the following modifications: Y1117V, F1252Y, H1253K, and L1278F or L1278H.

[0132] In one specific embodiment, the modified BoNT / A HC domain includes the following modifications: Y1117V and H1253K; or Y1117V, F1252Y, H1253K, and L1278F; or Y1117V, F1252Y, H1253K, and L1278H.

[0133] Preferably, the modified BoNT / A HC field includes the following modifications: Y1117V and H1253K; or Y1117V, F1252Y, H1253K, and L1278H.

[0134] When compared with the non-catalytically active BoNT / A as shown in SEQ ID NO: 2, this modification may be a modification in which the amino acid residue number is determined by comparison with SEQ ID NO: 2. Since the presence of the methionine residue at position 1 of SEQ ID NO: 2 is not essential, when determining the amino acid residue number, those skilled in the art will consider the presence / absence of the methionine residue. For example, in the case where SEQ ID NO: 2 includes methionine, the position number will be as defined above (e.g., Y1117 will be compared with Y1117 of SEQ ID NO: 2). Alternatively, if the methionine is not present in SEQ ID NO: 2, the amino acid residue number should be modified by -1 (e.g., Y1117 would be compared with Y1116 of SEQ ID NO: 2). Similar considerations apply when the methionine at position 1 of other polypeptide sequences described herein is present / absent, and those skilled in the art will readily determine the correct amino acid residue number using conventional techniques in this art.

[0135] The modified BoNT / A HC domain may contain a polypeptide sequence having at least 70% sequence identity with any of the following SEQ ID NOs: 46, 48, or 50; provided that the modified BoNT / A HC domain contains the modifications described above. In one embodiment, the modified BoNT / A HC domain contains a polypeptide sequence having at least 80%, 90%, 95%, or 98% sequence identity with any of the following SEQ ID NOs: 46, 48, or 50; provided that the modified BoNT / A HC domain contains the modifications described above. In one embodiment, the modified BoNT / A HC domain contains a polypeptide sequence having at least 99% or 99.9% sequence identity with any of the following SEQ ID NOs: 46, 48, or 50; provided that the modified BoNT / A HC domain contains the modifications described above. Preferably, the modified BoNT / A HC domain comprises (more preferably consists of) a polypeptide sequence containing any of the following SEQ ID NOs: 46, 48, or 50.

[0136] The modified BoNT / A HC domain may comprise a polypeptide sequence having at least 70% sequence identity with any of the following SEQ ID NOs: 46 or 50; provided that the modified BoNT / A HC domain comprises the modifications described above. In one embodiment, the modified BoNT / A HC domain comprises a polypeptide sequence having at least 80%, 90%, 95%, or 98% sequence identity with any of the following SEQ ID NOs: 46 or 50; provided that the modified BoNT / A HC domain comprises the modifications described above. In one embodiment, the modified BoNT / A HC domain comprises a polypeptide sequence having at least 99% or 99.9% sequence identity with any of the following SEQ ID NOs: 46 or 50; provided that the modified BoNT / A HC domain comprises the modifications described above. Preferably, the modified BoNT / A HC domain comprises (more preferably constitutes) a polypeptide sequence comprising any of the following SEQ ID NOs: 46 or 50.

[0137] The modified BoNT / A HC domain may be encoded by a nucleotide sequence having at least 70% sequence identity with any of the following SEQ ID NOs: 45, 47, or 49; provided that the modified BoNT / A HC domain includes the modifications described above. In one embodiment, the modified BoNT / A HC domain is encoded by a nucleotide sequence having at least 80%, 90%, 95%, or 98% sequence identity with any of the following SEQ ID NOs: 45, 47, or 49; provided that the modified BoNT / A HC domain includes the modifications described above. In one embodiment, the modified BoNT / A HC domain is encoded by a nucleotide sequence having at least 99% or 99.9% sequence identity with any of the following SEQ ID NOs: 45, 47, or 49; provided that the modified BoNT / A HC domain includes the modifications described above. Preferably, the modified BoNT / A HC field is encoded by any of the following SEQ ID NOs: 45, 47, or 49.

[0138] The modified BoNT / A HC domain may be encoded by a nucleotide sequence having at least 70% sequence identity with any of the following SEQ ID NOs: 45 or 49; provided that the modified BoNT / A HC domain includes the modifications described above. In one embodiment, the modified BoNT / A HC domain is encoded by a nucleotide sequence having at least 80%, 90%, 95%, or 98% sequence identity with any of the following SEQ ID NOs: 45 or 49; provided that the modified BoNT / A HC domain includes the modifications described above. In one embodiment, the modified BoNT / A HC domain is encoded by a nucleotide sequence having at least 99% or 99.9% sequence identity with any of the following SEQ ID NOs: 45 or 49; provided that the modified BoNT / A HC domain includes the modifications described above. Preferably, the modified BoNT / A HC field is encoded by any of the following SEQ ID NOs: 45 or 49.

[0139] The polypeptide of the present invention may comprise (or consist of) a heterozygous or chimeric Clostridium neurotoxin (or a fragment of a heterozygous or chimeric Clostridium neurotoxin), wherein the L-chain is non-catalytically active (when present). A heterozygous Clostridium neurotoxin comprises at least a portion of a light chain from a Clostridium neurotoxin or its subtype, and at least a portion of a heavy chain from another Clostridium neurotoxin or its subtype. In one embodiment, a heterozygous Clostridium neurotoxin may contain an intact light chain from a Clostridium neurotoxin subtype and a heavy chain from another Clostridium neurotoxin subtype, wherein the L-chain is non-catalytically active (when present). In another embodiment, a chimeric Clostridium neurotoxin may contain a portion (e.g., a binding domain) of a heavy chain from a Clostridium neurotoxin subtype and another portion of a heavy chain from another Clostridium neurotoxin subtype. Similarly or alternatively, the therapeutic element may comprise a light chain portion from a different Clostridium neurotoxin, wherein the proviso is that the L-chain is non-catalytically active (when present). Such hybrid or chimeric Clostridium neurotoxins can be used as a means of delivering the therapeutic benefits of such Clostridium neurotoxins to, for example, subjects who are immune to a specified Clostridium neurotoxin subtype, subjects who may have a receptor at a below-average concentration for a specified Clostridium neurotoxin heavy chain binding domain, or subjects who may have a protease-resistant variant of a membrane or vesicular toxin receptor (e.g., SNAP-25, VAMP, and synaptic fusion protein). Hybrid and chimeric Clostridium neurotoxins are described in US 8,071,110, the disclosure of which is incorporated herein by reference in its entirety. Thus, in one specific embodiment, the polypeptide of the present invention is or comprises a hybrid or chimeric Clostridium neurotoxin, wherein the proviso is that the L-chain is non-catalytically active.

[0140] In a preferred embodiment, the polypeptide of the present invention may be a chimeric Clostridium neurotoxin comprising (preferably composed of) a non-catalytically active BoNT / A light chain and a transfer domain (LHN domain), and a BoNT / B receptor-binding domain (HC domain) or a portion thereof. Suitable chimeric and / or hybrid Clostridium neurotoxins may be those taught in WO 2017 / 191315 A1, which is incorporated herein by reference, provided that the L chain is non-catalytically active (e.g., modified and not activated). Such preferred sequences include SEQ ID NO: 44 and 61.

[0141] The non-catalytically active BoNT / A LHN domain can be covalently linked to the BoNT / B HC domain. In this paper, this chimeric BoNT / A is also referred to as "BoNT / AB" or "BoNT / AB chimera".

[0142] The C-terminal amino acid residue of the LHN domain can correspond to the first amino acid residue of the 310 helix of the LHN and HC domains of BoNT / A, and the N-terminal amino acid residue of the HC domain can correspond to the second amino acid residue of the 310 helix of the LHN and HC domains of BoNT / B.

[0143] The phrase “the first amino acid residue of the 310 helix separating the LHN and HC domains of BoNT / A” in this article refers to the N-terminal residue of the 310 helix separating the LHN and HC domains.

[0144] The phrase “the second amino acid residue of the 310 helix separating the LHN and HC domains of BoNT / B” in this article refers to the amino acid residue after the N-terminal residue of the 310 helix separating the LHN and HC domains.

[0145] The "310 helix," along with α-helices, β-lamellae, and inversions, is a type of secondary structure found in proteins and polypeptides. In a 310 helix, amino acids are arranged in a right-handed helical configuration, where each complete turn is accomplished by three residues separated by ten atoms through intramolecular hydrogen bonds. Each amino acid in the helix corresponds to a 120° turn (i.e., the helix has three residues at each turn), accompanied by a 2.0 Å (=0.2 nm) translation along the helical axis, and has 10 atoms in the ring formed by hydrogen bonds. Most importantly, the NH group of the amino acid forms a hydrogen bond with the C=O group of the preceding three amino acids; this repeating i+3→i hydrogen bond defines the 310 helix. The 310 helix is ​​a standard concept in structural biology well known to those skilled in the art.

[0146] This 310 helix corresponds to the four residues that form the actual helix, and two cap (or transition) residues at the ends of these four residues. The term "310 helix separating the LHN and HC domains" as used herein consists of these six residues.

[0147] Through structural analysis and sequence alignment, a 310 helix separating the LHN and HC domains was identified. This 310 helix is ​​surrounded by an α-helix at its N-terminus (i.e., the C-terminal portion of the LHN domain) and by a β-lamellae at its C-terminus (i.e., the N-terminal portion of the HC domain). The first (N-terminal) residue (cap or transition residue) of the 310 helix also corresponds to the C-terminal residue of this α-helix.

[0148] The 310 helix separating the LHN and HC domains can be determined, for example, by the crystal structure of a publicly available botulinum neurotoxin, such as 3BTA of botulinum neurotoxin A1 (http: / / www.rcsb.org / pdb / explore / explore.do?structureId=3BTA) and 1EPW of botulinum neurotoxin B1 (http: / / www.rcsb.org / pdb / explore / explore.do?structureId=1EPW).

[0149] Publicly available computer simulation modeling and alignment tools can also be used to determine the position of the 310 helix separating the LHN and HC domains in other neurotoxins, such as the homology modeling server 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 many other tools / servers listed in Internet Resources for Molecular and Cell Biologists (http: / / molbiol-tools.ca / ). In particular, the structure of the region surrounding the "HN / HCN" junction is highly preserved, making it an ideal region for superimposing different serotypes.

[0150] For example, the following methodologies can be used to determine the sequence of this 310 helix in other neurotoxins: 1. Use the structural homology modeling tool LOOP (http: / / loopp.org) to obtain predicted structures of other BoNT serotypes based on the BoNT / A1 crystal structure (3BTA.pdb); 2. Edit the obtained structure (pdb) file to include only the N-terminus of the HCN domain and approximately 80 residues preceding it (which are part of the HN domain), thus preserving the structurally highly preserved "HN / HCN" region; 3. Use the protein stacking server SuperPose (http: / / wishart.biology.ualberta.ca / superpose / ) to stack each serotype onto the 3BTA.pdb structure; 4. Examine the stacked pdb file to locate the 310 helix starting at the HC domain of BoNT / A1, and then identify the corresponding residues in other serotypes; 5. Align the other BoNT serotype sequences with Clustal Omega to check if the corresponding residues are correct.

[0151] Examples of LHN, HC and 310 helical domains determined by this method are presented below: Neurotoxins Login ID (Add serial version after the decimal point) LH N H C 3 10 spiral Non-catalytically active BoNT / A1 (SEQ ID NO: 2) N / A 1-872 873-1296 872 NIINTS 877 BoNT / A2 X73423.3 1-872 873-1296 872 NIVNTS 877 BoNT / A3 DQ185900.1 (Also known as Q3LRX9.1) 1-872 873-1292 872 NIVNTS877 BoNT / A4 EU341307.1 (Also known as Q3LRX8.1) 1-872 873-1296 872 NITNAS 877 BoNT / A5 EU679004.1 (Also known as C1IPK2.1) 1-872 873-1296 872 NIINTS 877 BoNT / A6 FJ981696.1 1-872 873-1296 872 NIINTS 877 BoNT / A7 JQ954969.1 (Also known as K4LN57.1) 1-872 873-1296 872 NIINTS 877 BoNT / A8 KM233166.1 1-872 873-1297 872 NITNTS 877 BoNT / B1 (Also known as SEQ ID NO: 52) B1INP5.1 1-859 860-1291 859 EILNNI 864 BoNT / B2 AB084152.1 (Also known as Q8GR96.1) 1-859 860-1291 859 EILNNI 864 BoNT / B3 EF028400.1 (Also known as A2I2S2.1) 1-859 860-1291 859 EILNNI 864 BoNT / B4 EF051570.1 (Also known as A2I2W0.1) 1-859 860-1291 859 EILNNI 864 BoNT / B5 EF033130.1 (Also known as A2I2U6.1) 1-859 860-1291 859 DILNNI 864 BoNT / B6 AB302852. 1 (also known as A8R089.1) 1-859 860-1291 859 EILNNI 864 BoNT / B7 JQ354985.1 (Also known as H9CNK9.1) 1-859 860-1291 859 EILNNI 864 BoNT / B8 JQ964806.1 (Also known as I6Z8G9.1) 1-859 860-1292 859 EILNNI 864

[0152] Using structural analysis and sequence alignment, it was found that the β-lamellae following the 310 helix separating the LHN and HC domains are structures that are preserved in all botulinum toxins and tetanus neurotoxins, and that they begin at residue 8 from the first residue of the 310 helix separating the LHN and HC domains (e.g., residue 879 of BoNT / A1).

[0153] The BoNT / AB chimera may contain an LHN domain (a non-catalytically active L chain) derived from BoNT / A covalently linked to an HC domain derived from BoNT / B, wherein the C-terminal amino acid residue of the LHN domain corresponds to the eighth amino acid residue at the N-terminus of the β-plate at the beginning (N-terminus) of the HC domain of BoNT / A, and wherein the N-terminal amino acid residue of the HC domain corresponds to the seventh amino acid residue at the N-terminus of the β-plate at the beginning (N-terminus) of the HC domain of BoNT / B.

[0154] The BoNT / AB chimera may contain an LHN domain (with a non-catalytically active L chain) derived from BoNT / A covalently linked to an HC domain derived from BoNT / B, wherein the C-terminal amino acid residue of the LHN domain corresponds to the C-terminal amino acid residue of the α-helix located at the end (C-terminus) of the LHN domain of BoNT / A, and wherein the N-terminal amino acid residue of the HC domain corresponds to the amino acid residue immediately adjacent to the C-terminus for the C-terminal amino acid residue of the α-helix located at the end (C-terminus) of the LHN domain of BoNT / B.

[0155] The design principle of the BoNT / AB chimera is to ensure that the secondary structure is not compromised, thereby minimizing any changes to the tertiary structure. Not wanting to be bound by theory, it is assumed that the optimal conformation of the chimeric neurotoxin is ensured by not disrupting the four central amino acid residues of the 310 helix in the BoNT / AB chimera.

[0156] The non-catalytically inactive LHN domain derived from BoNT / A 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 with it. The non-catalytically inactive LHN domain derived from BoNT / A 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 with it. Preferably, the non-catalytically inactive LHN domain derived from BoNT / A corresponds to amino acid residues 1 to 872 of SEQ ID NO: 2 or 61.

[0157] The HC domain derived from BoNT / B may correspond to amino acid residues 860 to 1291 of SEQ ID NO: 52 or a polypeptide sequence having at least 70% sequence identity with it. The HC domain derived from BoNT / B 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 with it. Preferably, the HC domain derived from BoNT / B corresponds to amino acid residues 860 to 1291 of SEQ ID NO: 52.

[0158] Preferably, the non-catalytically active LHN domain corresponds to amino acid residues 1 to 872 of BoNT / A (SEQ ID NO: 2 or 61) and the HC domain corresponds to amino acid residues 860 to 1291 of BoNT / B (SEQ ID NO: 52).

[0159] Preferably, the BoNT / B HC domain further includes at least one amino acid residue substitution, addition, or deletion in the HCC subdomain, which, when compared with the native BoNT / B sequence, has the effect of increasing the binding affinity of the BoNT / B neurotoxin to human Syt II. Suitable amino acid residue substitutions, additions, or deletions in the BoNT / B HCC subdomain have been disclosed in WO 2013 / 180799 and WO 2016 / 154534 (both of which are incorporated herein by reference).

[0160] Suitable amino acid residue substitutions, additions, or deletions in the BoNT / B HCC subdomain 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.

[0161] at BoNT / B Suitable amino acid residue substitutions, additions, or deletions in the HCC subdomain further include combinations of two substitution mutations selected from the following groups: 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.

[0162] Suitable amino acid residue substitutions, additions or deletions in the BoNT / B HCC subdomain also include combinations of three substitution mutations, namely E1191M, S1199W and W1178Q.

[0163] Preferably, suitable amino acid residue substitutions, additions or deletions in the BoNT / B HCC subdomain include a combination of two substitution mutations, namely E1191M and S1199Y.

[0164] When compared with the unmodified BoNT / B shown in SEQ ID NO: 52, the modification may be a modification in which the amino acid residue number is determined by comparison with SEQ ID NO: 52. Since the presence of the methionine residue at position 1 in SEQ ID NO: 52 is not essential, when determining the amino acid residue number, those skilled in the art will consider the presence / absence of the methionine residue. For example, if SEQ ID NO: 52 includes methionine, the position number will be as defined above (e.g., E1191 will be E1191 of SEQ ID NO: 52). Alternatively, if the methionine is not present in SEQ ID NO: 52, the amino acid residue number should be modified by -1 (e.g., E1191 will be E1190 of SEQ ID NO: 52). When the methionine at position 1 of other polypeptide sequences described herein is present or absent, similar considerations apply, and those skilled in the art will readily determine the correct amino acid residue number using conventional techniques in this art.

[0165] In one specific embodiment, the polypeptide used according to the present invention comprises a polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 61. In one specific embodiment, the polypeptide used according to the present invention comprises a polypeptide sequence having at least 80%, 90%, 95%, or 98% sequence identity with SEQ ID NO: 61. Preferably, the polypeptide used according to the present invention comprises (more preferably consists of) the polypeptide sequence shown in SEQ ID NO: 61.

[0166] The chimeric and / or hybrid Clostridium neurotoxins used in this invention may comprise a portion of the BoNT / A polypeptide and a portion of the BoNT / B polypeptide, examples of which include the polypeptides described herein, such as SEQ ID NO: 44.

[0167] In one specific embodiment, the polypeptide used according to the present invention comprises: a polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 44 and / or a polypeptide sequence encoded by a nucleotide sequence having at least 70% sequence identity with SEQ ID NO: 43. In one specific embodiment, the polypeptide used according to the present invention comprises a polypeptide sequence having at least 80%, 90%, 95%, or 98% sequence identity with SEQ ID NO: 44. Preferably, the polypeptide used according to the present invention comprises the polypeptide sequence shown in SEQ ID NO: 44. In one specific embodiment, the polypeptide used according to the present invention comprises a polypeptide sequence encoded by a nucleotide sequence having at least 80%, 90%, 95%, or 98% sequence identity with SEQ ID NO: 43. Preferably, the polypeptide used according to the present invention comprises a polypeptide sequence encoded by a nucleotide sequence shown in SEQ ID NO: 43.

[0168] Suitable chimeric Clostridium neurotoxins may include BoNT / FA, provided that any present L-chain is non-catalytically active. Thus, the polypeptides of the present invention may comprise BoNT / FA or fragments thereof, provided that any present L-chain is non-catalytically active. Non-catalytically active forms of BoNT / FA are described herein as SEQ ID NO: 26 and 34. Suitable fragments of BoNT / FA are also described herein as SEQ ID NO: 28, 30, and 32.

[0169] In another preferred embodiment, the polypeptide of the present invention may be a chimeric Clostridium neurotoxin comprising a non-catalytically active BoNT / X light chain and a transfer domain (LHN domain), and a receptor-binding domain (HC domain) or a portion thereof derived from a different Clostridium neurotoxin (i.e., non-BoNT / X). Suitable chimeric and / or hybrid Clostridium neurotoxins may be those taught in WO 2020 / 065336 A1, which is incorporated herein by reference, provided that the L chain is non-catalytically active (e.g., modified and not activated). Such preferred sequences include SEQ ID NO: 63-70 as described herein.

[0170] Chimeric Clostridium neurotoxins may comprise a non-catalytically active BoNT / X light chain and a transfer domain (LHN domain), and: (i) a BoNT / A receptor-binding domain (HC domain) or a portion thereof; or (ii) a BoNT / B receptor-binding domain (HC domain) or a portion thereof; or (iii) a BoNT / C receptor-binding domain (HC domain) or a portion thereof; or (iv) a BoNT / D receptor-binding domain (HC domain) or a portion thereof; or (v) a BoNT / E receptor-binding domain (HC domain) or a portion thereof; or (vi) a BoNT / F receptor-binding domain (HC domain) or a portion thereof; or (vii) a BoNT / G receptor-binding domain (HC domain) or a portion thereof; or (viii) a TeNT receptor-binding domain (HC domain) or a portion thereof.

[0171] In one specific embodiment, the receptor-binding domain (HC domain) or a portion thereof derived from different Clostridium neurotoxin (i.e., non-BoNT / X) domains may be binding to synaptic binding proteins I and / or II (Syt I / II).

[0172] Preferably, the chimeric Clostridium neurotoxin may comprise a non-catalytically active BoNT / X light chain and transfer domain (LHN domain), and a BoNT / B receptor binding domain (HC domain) or a portion thereof.

[0173] A polypeptide comprising a non-catalytically active BoNT / X light chain and a transfer domain (LHN domain) and a receptor-binding domain (HC domain) or a portion thereof derived from different Clostridium neurotoxins (i.e., non-BoNT / X), which may contain a polypeptide sequence having at least 70% sequence identity with any of SEQ ID NO: 63-70. In one specific embodiment, a polypeptide comprising a non-catalytically active BoNT / X light chain and a transfer domain (LHN domain) and a receptor-binding domain (HC domain) or a portion thereof derived from different Clostridium neurotoxins (i.e., non-BoNT / X), which contains a polypeptide sequence having at least 80%, 90%, 95%, or 98% sequence identity with any of SEQ ID NO: 63-70. Preferably, the polypeptide comprises a non-catalytically active BoNT / X light chain and a transfer domain (LHN domain) and a receptor-binding domain (HC domain) or a portion thereof derived from various Clostridium neurotoxins (i.e., non-BoNT / X), and comprises (more preferably consists of) any one of SEQ ID NO: 63-70. Among these polypeptides, SEQ ID NO: 63-66 is preferred.

[0174] A polypeptide comprising a non-catalytically active BoNT / X light chain and a transfer domain (LHN domain) and a receptor-binding domain (HC domain) or a portion thereof derived from various Clostridium neurotoxins (i.e., non-BoNT / X), which may contain the following N-terminal amino acid sequence MGS. In the case where SEQ ID NO: 63-66 contains this N-terminal amino acid sequence, the sequence is not essential. In one embodiment, SEQ ID NO: 63-66 lacks the N-terminal amino acid sequence as shown by MGS. In one embodiment, SEQ ID NO: 63-66 contains the N-terminal amino acid sequence as shown by MGS.

[0175] The non-catalytically active LHN domain derived from BoNT / X may correspond to amino acid residues 1 to 899 of SEQ ID NO: 63 or a polypeptide sequence having at least 70% sequence identity with it. Preferably, the non-catalytically active LHN domain derived from BoNT / X 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 with it.

[0176] The non-catalytically active LHN domain derived from BoNT / X may correspond to amino acid residues 4 to 899 of SEQ ID NO: 63 or a polypeptide sequence having at least 70% sequence identity with it. Preferably, the non-catalytically active LHN domain derived from BoNT / X 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 with it.

[0177] The non-catalytically active LHN domain derived from BoNT / X may correspond to amino acid residues 1 to 866 of SEQ ID NO: 65 or a polypeptide sequence having at least 70% sequence identity with it. Preferably, the non-catalytically active LHN domain derived from BoNT / X 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 with it.

[0178] The non-catalytically active LHN domain derived from BoNT / X may correspond to amino acid residues 4 to 866 of SEQ ID NO: 65 or a polypeptide sequence having at least 70% sequence identity with it. Preferably, the non-catalytically active LHN domain derived from BoNT / X 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 with it.

[0179] The HC domain derived from BoNT / B may correspond to amino acid residues 860 to 1291 of SEQ ID NO: 52 or a polypeptide sequence having at least 70% sequence identity with it. The HC domain derived from BoNT / B 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 with it. Preferably, the HC domain derived from BoNT / B corresponds to amino acid residues 860 to 1291 of SEQ ID NO: 52.

[0180] Preferably, the BoNT / B HC domain further includes at least one amino acid residue substitution, addition, or deletion in the HCC subdomain, which, when compared with the native BoNT / B sequence, has the effect of increasing the binding affinity of the BoNT / B neurotoxin to human Syt II. Suitable amino acid residue substitutions, additions, or deletions in the BoNT / B HCC subdomain have been disclosed in WO 2013 / 180799 and WO 2016 / 154534 (both are incorporated herein by reference).

[0181] Suitable amino acid residue substitutions, additions, or deletions in the BoNT / B HCC subdomain 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.

[0182] at BoNT / B Suitable amino acid residue substitutions, additions, or deletions in the HCC subdomain further include combinations of two substitution mutations selected from the following groups: 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.

[0183] Suitable amino acid residue substitutions, additions or deletions in the BoNT / B HCC subdomain also include combinations of three substitution mutations, namely E1191M, S1199W and W1178Q.

[0184] Preferably, suitable amino acid residue substitutions, additions or deletions in the BoNT / B HCC subdomain include a combination of two substitution mutations, namely E1191M and S1199Y.

[0185] When compared with the unmodified BoNT / B shown in SEQ ID NO: 52, the modification may be a modification in which the amino acid residue number is determined by comparison with SEQ ID NO: 52. Since the presence of the methionine residue at position 1 in SEQ ID NO: 52 is not essential, those skilled in the art will consider the presence / absence of the methionine residue when determining the amino acid residue number. For example, in the case where SEQ ID NO: 52 includes methionine, the position number will be as defined above (e.g., E1191 would be E1191 of SEQ ID NO: 52). Alternatively, if the methionine is not present in SEQ ID NO: 52, the amino acid residue number should be modified by -1 (e.g., E1191 would be E1190 of SEQ ID NO: 52). Similar considerations apply when the methionine at position 1 of other polypeptide sequences described herein is present / absent, and those skilled in the art will readily determine the correct amino acid residue number using conventional techniques in this art.

[0186] The HC domain derived from BoNT / A may correspond to amino acid residues 873 to 1296 of SEQ ID NO: 60 or a polypeptide sequence having at least 70% sequence identity with it. The HC domain derived from BoNT / A 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 with it. Preferably, the HC domain derived from BoNT / B corresponds to amino acid residues 873 to 1296 of SEQ ID NO: 60.

[0187] In one specific embodiment, in the case of a peptide system used to treat inflammatory disorders, the peptide used does not contain a non-catalytically active BoNT / XL chain, a BoNT / X transfer domain (HN domain), and a BoNT / A receptor-binding domain (HC domain). Thus, in one specific embodiment, in the case of a peptide system used to treat inflammatory disorders, the peptide may contain a non-catalytically active BoNT / XL chain, a BoNT / X transfer domain (HN domain), and: (i) a BoNT / B receptor-binding domain (HC domain); (ii) a BoNT / D receptor-binding domain (HC domain); or (iii) a BoNT / F receptor-binding domain (HC domain).

[0188] Similarly, in one specific embodiment, in the case of a peptide system used for treating pain, the peptide used does not contain a non-catalytically active BoNT / XL chain, a BoNT / X transfer domain (HN domain), and a BoNT / A receptor binding domain (HC domain). Thus, in one specific embodiment, in the case of a peptide system used for treating pain, the peptide may contain a non-catalytically active BoNT / XL chain, a BoNT / X transfer domain (HN domain), and: (i) a BoNT / B receptor binding domain (HC domain); (ii) a BoNT / D receptor binding domain (HC domain); or (iii) a BoNT / F receptor binding domain (HC domain).

[0189] The HC domain derived from BoNT / D may correspond to amino acid residues 865 to 1276 of SEQ ID NO: 54 or a polypeptide sequence having at least 70% sequence identity with it. The HC domain derived from BoNT / D 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 with it. Preferably, the HC domain derived from BoNT / D corresponds to amino acid residues 865 to 1276 of SEQ ID NO: 54.

[0190] The HC domain derived from BoNT / F may correspond to amino acid residues 866 to 1278 of SEQ ID NO: 56 or a polypeptide sequence having at least 70% sequence identity with it. The HC domain derived from BoNT / F may 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 with it. Preferably, the HC domain derived from BoNT / F corresponds to amino acid residues 866 to 1278 of SEQ ID NO: 56, having a substitution (H1241K) at position 1241 for histidine to lysine.

[0191] Preferably, the chimeric Clostridium neurotoxin may comprise (preferably constitute) a non-catalytically active BoNT / X light chain and a transfer domain (LHN domain), and a receptor-binding domain (HC domain) or a portion thereof derived from different Clostridium neurotoxins (i.e., non-BoNT / X), and Cys-(Xaa)a-Ile-Asp / Glu-Gly-Arg-(Yaa)b-Cys (SEQ ID NO: 71), wherein a=1-10 and b=4-15. SEQ ID NO: 71 is a consensus sequence based on the BoNT / C1 activation zone.

[0192] However, any polypeptide of the present invention may comprise Cys-(Xaa)a-Ile-Asp / Glu-Gly-Arg-(Yaa)b-Cys (SEQ ID NO: 71), wherein a=1-10 and b=4-15. This activation zone may suitably replace any activation zone sequence present in the Clostridium neurotoxin L chain and / or transfer domain (HN domain) of the polypeptide described herein.

[0193] Xaa or Yaa used in the context of SEQ ID NO: 71 may be any amino acid. The number of amino acids at position Xaa and Yaa is represented by the letters "a" and "b", respectively. In one embodiment, "a" and "b" may be any integer that allows proteolytic cleavage of the activation zone and the generation of active double-stranded Clostridium difficile 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 specific embodiment, "b" is ≤20, ≤19, ≤18, ≤17, ≤16, ≤15, ≤14, ≤13, ≤12, ≤11, ≤10 or ≤9.

[0194] In one specific embodiment, "a" is 1-12, such as 1-10. Preferably, "a" is 1-7, such as 2-4. More preferably, "a" is 3. In one specific embodiment, "b" is 1-20, such as 4-15. Preferably, "b" is 6-10. More preferably, "b" is 8.

[0195] There is no intention to limit Xaa or Yaa to only one type of amino acid. Thus, one or more residues present at position Xaa may be independently selected from standard amino acids: aspartic acid, glutamic acid, arginine, lysine, histidine, aspartic acid, glutamic acid, serine, threonine, tyrosine, methionine, tryptophan, cysteine, alanine, glycine, valine, leucine, isoleucine, proline, and phenylalanine. One or more residues present at position Yaa may be independently selected from standard amino acids: aspartic acid, glutamic acid, arginine, lysine, histidine, aspartic acid, glutamic acid, serine, threonine, tyrosine, methionine, tryptophan, cysteine, alanine, glycine, valine, leucine, isoleucine, proline, and phenylalanine. Preferably, the amino acid at position Yaa (more preferably the one immediately adjacent to the C-terminus of the Arg residue in SEQ ID NO: 71) is not proline.

[0196] Alternatively / additionally, one or more residues present at position Xaa or Yaa may be independently selected from non-standard amino acids (amino acids not part of the above 20 standard groups). For example, non-standard amino acids may include 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline, α-methylserine, trans-3-methylproline, 2,4-methano-proline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allethamine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, and 2-pipecolic acid. The amino acids include tertiary leucine, n-valine, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, L-ornithine, L-2-amino-3-guanidinylpropionic acid, or lysine, arginine, and / or the D-isomers of ornithine, and 4-fluorophenylalanine. Methods for introducing non-standard amino acids into proteins are known in the art and include the synthesis of recombinant proteins using auxotrophic host organisms of *Escherichia coli*.

[0197] The sequence Ile-Asp / Glu-Gly-Arg contained in SEQ ID NO: 71 refers to the site recognized by enterokinase (and factor Xa) that was surprisingly found in WO 2020 / 065336 A1. This document describes a suitable method for cleaving Ile-Asp / Glu-Gly-Arg to generate a double-chain polypeptide. Preferably, the sequence is Ile-Asp-Gly-Arg, for example, Cys-(Xaa)a-Ile-Asp-Gly-Arg-(Yaa)b-Cys. Enterokinase and factor Xa hydrolyze the peptide bond immediately adjacent to the C-terminus of Arg in SEQ ID NO: 71 (i.e., the peptide bond between Arg and Yaa).

[0198] In one specific embodiment, the amino acid residue of Xaa located immediately adjacent to the N-terminus of Ile of SEQ ID NO: 71 is an uncharged hydrophobic amino acid, preferably alanine. In some specific embodiments, "a" is at least 2, and Xaa comprises at least one C-terminus of an uncharged polar amino acid and an immediately adjacent N-terminus of a charged basic amino acid. The charged basic amino acid is preferably lysine. Thus, in the case where "a" is at least 2 in the specific embodiment, Xaa may comprise at least Lys-Ala, wherein Ala is immediately adjacent to the N-terminus of Ile of SEQ ID NO: 71.

[0199] In one specific embodiment, Xaa includes or is composed of the sequence HKA.

[0200] In one specific embodiment, the amino acid residue of Yaa at the C-terminus of Arg immediately adjacent to SEQ ID NO: 71 is an uncharged polar amino acid, preferably serine. In some specific embodiments, "b" is at least 2, and Yaa comprises at least one N-terminus of uncharged polar amino acid and an uncharged hydrophobic amino acid immediately adjacent to its C-terminus. The uncharged hydrophobic amino acid is preferably leucine. Thus, in the case of the specific embodiment where "b" is at least 2, Yaa may comprise at least Ser-Leu, wherein Ser is immediately adjacent to the C-terminus of Arg in SEQ ID NO: 71.

[0201] In one specific embodiment, Yaa comprises or is composed of the sequence SLYNKTLDC.

[0202] In some specific embodiments, the polypeptide herein includes an activation zone having at least 70% sequence identity with SEQ ID NO: 72. In one specific embodiment, the polypeptide herein includes an activation zone having at least 80%, 85%, or 90% sequence identity with SEQ ID NO: 72. Preferably, the polypeptide herein includes an activation zone having at least 95% sequence identity with SEQ ID NO: 72. More preferably, the polypeptide herein includes an activation zone having at least 99% sequence identity with SEQ ID NO: 72.

[0203] In a preferred embodiment, the polypeptide herein includes an activation zone comprising SEQ ID NO: 72, more preferably composed of SEQ ID NO: 72.

[0204] The activation zone may also be a variant of SEQ ID NO: 72, such as SEQ ID NO: 73 or a sequence having at least 70% sequence identity with it. SEQ ID NO: 73 is a variant of SEQ ID NO: 72 in which the enterokinase recognition site IDGR has been mutated to IEGR. In one specific embodiment, the polypeptide herein comprises an activation zone having at least 70% sequence identity with SEQ ID NO: 73. In one specific embodiment, the polypeptide herein comprises an activation zone having at least 80%, 85%, or 90% sequence identity with SEQ ID NO: 73. Preferably, the polypeptide herein comprises an activation zone having at least 95% sequence identity with SEQ ID NO: 73. More preferably, the polypeptide herein comprises an activation zone having at least 99% sequence identity with SEQ ID NO: 73.

[0205] In a preferred embodiment, the polypeptide herein includes an activation zone comprising SEQ ID NO: 73, more preferably composed of SEQ ID NO: 73.

[0206] In one specific embodiment, the activation zone described herein (e.g., SEQ ID NO: 71, 72, or 73) may be modified to include additional or alternative protease sites. For example, a protease site as shown in SEQ ID NO: 77. An example of such a modified activation zone is shown in SEQ ID NO: 78. Thus, in one specific embodiment, the polypeptide herein comprises an activation zone having at least 70% sequence identity with SEQ ID NO: 78. In one specific embodiment, the polypeptide herein comprises an activation zone having at least 80%, 85%, or 90% sequence identity with SEQ ID NO: 78. Preferably, the polypeptide herein comprises an activation zone having at least 95% sequence identity with SEQ ID NO: 78. More preferably, the polypeptide herein comprises an activation zone having at least 99% sequence identity with SEQ ID NO: 78. In a particularly preferred embodiment, the polypeptide herein comprises an activation zone comprising SEQ ID NO: 78, more preferably composed of SEQ ID NO: 78.

[0207] In one specific embodiment, the polypeptide of the present invention may contain (or consist of) a retargeted Clostridium neurotoxin, wherein any present L-chain is non-catalytically active. In a retargeting clostridium neurotoxin, the clostridium neurotoxin is modified to include an exogenous ligand known as the Targeting Moiety (TM). The TM is selected to provide binding specificity to the desired target cells, and as part of the retargeting process, the natural binding moiety of the clostridium neurotoxin (e.g., the HC domain or HCC domain) can be removed. Retargeting techniques 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; 2000 / 62814; WO 2000 / 04926; WO 1993 / 15766; WO 2000 / 61192; and WO 1999 / 58571; all of its contents are incorporated herein by reference in their entirety. Thus, in one specific embodiment, the polypeptide of the present invention is a retargeted Clostridium neurotoxin, wherein the proviso states that any present L-chain is non-catalytically active. The polypeptide of the present invention may lack the functional HC domain of the Clostridium neurotoxin and may also lack any functionally equivalent TM.

[0208] In a specific embodiment, the polypeptide described herein has a purification tag (e.g., a His tag) and / or a linker, which is not essential.

[0209] The polypeptide of the present invention may not contain the complexing protein present in naturally occurring Clostridium neurotoxin complexes.

[0210] The polypeptide of the present invention can be produced using recombinant nucleic acid technology. Thus, in a specific embodiment, the polypeptide (as described above) is a recombinant polypeptide.

[0211] 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 may be selected from any nucleic acid sequence described herein.

[0212] In a preferred embodiment, the carrier has a promoter selected from the following: promoter Inducing agent Typical induction conditions Tac (hybrid) IPTG 0.2 mM (0.05-2.0 mM) AraBAD L-arabinose 0.2% (0.002-0.4%) T7-lac manipulator IPTG 0.2 mM (0.05-2.0 mM)

[0213] In another preferred embodiment, the carrier has promoters selected from the following: promoter Inducing agent Typical induction conditions Tac (hybrid) IPTG 0.2 mM (0.05-2.0 mM) AraBAD L-arabinose 0.2% (0.002-0.4%) T7-lac manipulator IPTG 0.2 mM (0.05-2.0 mM) T5-lac operator IPTG 0.2 mM (0.05-2.0 mM)

[0214] Nucleic acid molecules can be prepared using any suitable method known in the art. Thus, nucleic acid molecules can be prepared using chemical synthesis techniques. Alternatively, the nucleic acid molecules of this invention can be prepared using molecular biotechnology.

[0215] The DNA construct of the present invention is preferably designed by computer simulation and then synthesized by conventional DNA synthesis technology.

[0216] Depending on the final host cell (e.g., Escherichia coli) expression system used, the above nucleic acid sequence information is selectively modified for codon preference.

[0217] The terms "nucleotide sequence" and "nucleic acid" are used synonymously in this document. Preferably, the nucleotide sequence is a DNA sequence.

[0218] The polypeptides of the present invention (and particularly any Clostridium neurotoxin portion thereof) may exist in single-chain or double-chain form. However, it is preferred that the polypeptides exist in double-chain form, wherein the non-catalytically active L chain is linked to the H chain (or a component thereof, such as the HN domain) via a disulfide bond.

[0219] This invention provides a method for producing a single-chain polypeptide having non-catalytically active light and heavy chains, the method comprising: expressing the nucleic acid described herein in a host cell; dissolving host cells to provide a host cell homogenate containing the single-chain polypeptide; and isolating the single-chain polypeptide. Alternatively, this invention provides a method for proteolytically processing the polypeptide described herein, the method comprising contacting the polypeptide with a protease, the protease hydrolyzing the peptide bonds of the polypeptide's activation loop, thereby converting the (single-chain) polypeptide into a corresponding double-chain polypeptide (e.g., wherein the non-catalytically active light and heavy chains are linked together by disulfide bonds).

[0220] Therefore, the present invention provides a double-chain polypeptide that can be obtained by the method of the present invention.

[0221] As used herein, “object” may refer to mammals, such as humans or other mammals. Preferably, “object” means human object.

[0222] The term "disorder" as used herein also includes "disease". In one specific embodiment, the disorder is a disease.

[0223] As used herein, the terms “treatment” or “management” encompass preventive treatment (e.g., prevention of the onset of a disorder [e.g., pain]) and corrective treatment (treatment of an individual who already suffers from a disorder [e.g., pain]. Preferably, as used herein, “treatment” or “management” means corrective treatment.

[0224] As used herein, the terms “treatment” or “management” refer to disorders (e.g., pain) and / or their symptoms.

[0225] Therefore, the polypeptide of the present invention can be administered to a subject in a therapeutically effective amount or a preventatively effective amount. Preferably, the polypeptide of the present invention is administered to a subject in a therapeutically effective amount.

[0226] "Therapeutic effective amount" refers to any amount of polypeptide that is sufficient to achieve such treatment of the disorder (e.g., pain) (or its symptoms) when administered alone or in combination with another agent to a subject for the treatment of the disorder (e.g., pain) (or its symptoms).

[0227] "Prophylactic effective dose" refers to any amount of polypeptide that, when administered to a subject alone or in combination with another agent, inhibits or delays the onset or recurrence of an disorder (e.g., pain) (or its symptoms). In some specific embodiments, the preventive effective dose completely prevents the onset or recurrence of a disorder (e.g., pain). "Inhibit" an onset means reducing the likelihood of a disorder onset (e.g., a situation where the disorder is pain) (or its symptoms) or completely preventing an onset.

[0228] The polypeptide of the present invention can be formulated in any suitable manner for delivery to a subject, for example, as part of a pharmaceutical composition. Thus, in one embodiment, the present invention provides a pharmaceutical composition comprising the polypeptide of the present invention and pharmaceutically acceptable carriers, excipients, adjuvants, propellants, and / or salts.

[0229] The polypeptides of the present invention can be formulated for oral, non-oral, continuous infusion, inhalation, or topical administration. Suitable injectable components may be in the form of solutions, suspensions, emulsions, or dry powders (which are dissolved or suspended in a suitable medium before use).

[0230] In the case of local delivery of the polypeptide, the polypeptide may be formulated as a cream (e.g., for local administration) or for subcutaneous injection.

[0231] Local delivery methods may include aerosols or other sprays (e.g., nebulizers). In this respect, aerosol formulations of peptides can be delivered to the lungs and / or other nasal and / or bronchial or airway pathways.

[0232] The polypeptide of the present invention can be delivered to the target by intrathecal or epidural injection into the spinal cord at the level of the spinal segment involving the nerve innervation of the affected organ.

[0233] The administration route may be via laparoscopy and / or local injection. In one specific embodiment, the polypeptide of the present invention is administered at or near the treatment site, preferably at the treatment site. For example, the polypeptide may be administered intrathecally or intraspinally. In one specific embodiment, the administration route of the polypeptide of the present invention may be perineural, intraneural, intraspinal, and / or intrathecal.

[0234] In one specific embodiment, the polypeptide of the present invention may be administered peripherally to a nerve. In one specific embodiment, the polypeptide may be administered intradermally, subcutaneously, or intramuscularly. Preferably, the polypeptide of the present invention is administered intradermally.

[0235] The dosage range of the polypeptides of the present invention administered is for producing the desired therapeutic and / or preventive effects. It should be understood that the required dosage range depends on the exact nature of the Clostridium neurotoxin or composition, the route of administration, the nature of the formulation, the age of the subject, the nature, extent or severity of the subject's condition, contraindications (if any), and the judgment of the attending physician. These dosage variations can be adjusted using standard empirical practices for optimization.

[0236] In one specific embodiment, the dose of the peptide is a flat dose. The flat dose can range from 50 pg to 250 µg, preferably from 100 pg to 100 µg. In one specific embodiment, the flat dose can be at least 50 pg, 100 pg, 500 pg, 1 ng, 50 ng, 100 ng, 500 ng, 1 µg, or 50 µg. The dose can be a single flat dose.

[0237] In a preferred embodiment, the polypeptide can be administered in a dose greater than 250 µg. In a specific embodiment, the polypeptide of the present invention can be administered in a dose greater than 500 µg, 1 mg, 10 mg, 100 mg, 500 mg, 1 g, or 5 g. In a specific embodiment, the polypeptide of the present invention can be administered in a dose 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 a dose 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, for example, 251 µg to 500 µg. In one specific embodiment, the polypeptide of the present invention is administered in amounts from 500 µg to 5 g, for example, from 1 mg to 1 g or from 1 g to 3 g. This is made possible by the non-toxic nature (e.g., substantially non-toxic) of the polypeptide of the present invention.

[0238] Liquid dosage forms are generally prepared using peptides and pyrogen-free sterile media. Clostridium neurotoxins, depending on the media and concentration used, can be dissolved or suspended in the media. In preparing the solution, the peptide can be dissolved in the media, and if necessary, the solution can be made isotonic by adding sodium chloride, and sterilized by filtration through a sterile filter using aseptic techniques before being filled into suitable sterile vials or ampoules and sealed. Alternatively, if the solution stability is appropriate, the solution in its sealed container can be sterilized by autoclaving. Advantageously, additives such as buffers, solubilizers, stabilizers, preservatives or bactericides, suspending agents or emulsifiers, and / or local anesthetics can be dissolved in the media.

[0239] Dry powder dissolved or suspended in a suitable solvent before use can be prepared by filling a sterile container with pre-sterilized ingredients in a sterile area using aseptic techniques. Alternatively, the ingredients can be dissolved in a suitable container in a sterile area using aseptic techniques. The product is then freeze-dried and the container is aseptically sealed.

[0240] Non-oral suspensions suitable for the administration routes described herein are prepared in substantially the same manner, except that sterile components are suspended in a sterile medium instead of dissolved, and sterilization cannot be achieved by filtration. Components may be isolated under sterile conditions, or they may be sterilized after isolation by, for example, irradiation with gamma rays.

[0241] Advantageously, the composition includes a suspending agent, such as polyvinylpyrrolidone, to promote uniform distribution of the components.

[0242] The delivery according to the present invention can utilize a variety of delivery technologies, including microparticle encapsulation or high-pressure aerosol impingement.

[0243] The polypeptide of the present invention is preferably administered repeatedly (e.g., up to 5, 10, 15, or 20 times) as part of a treatment regimen. Repeated administration means administration at least twice, for example, at least 5, 10, 15, or 20 times. Thus, in one embodiment, the polypeptide of the present invention can be administered two or more times to treat a subject (e.g., pain in the subject). This is particularly suitable for the treatment of chronic conditions (such as chronic pain) that typically require continuous treatment. In one embodiment, the polypeptide of the present invention can be administered weekly, twice a month, monthly, every two months, every six months, or annually, preferably at least twice a year or once a year. In one embodiment, the polypeptide of the present invention is administered more than twice over a period of 10 years, 5 years, 2 years, or 1 year. Preferably, the polypeptide of the present invention is administered more than twice over a period of 1 year. Treatment can last for at least 6 months, 1 year, 2 years, 3 years, 5 years, 10 years, 15 years, 20 years, 25 years, or 30 years.

[0244] Preferably, the polypeptide is not administered with any other therapeutic or diagnostic agent (e.g., nucleic acid, protein, peptide, or small molecule therapeutic or diagnostic agent) other than the L chain, HN domain, and / or HC domain of a non-catalytically active Clostridium neurotoxin. For example, in one embodiment, the polypeptide is not administered with other analgesics and / or anti-inflammatory agents. In one embodiment, the polypeptide of the present invention is not administered with covalently linked therapeutic agents. In one embodiment, the polypeptide of the present invention is not administered with non-covalently linked therapeutic agents.

[0245] The polypeptides described herein may be used to treat subjects suffering from one or more types of pain. The pain may be chronic or acute. The pain may be selected from one or more of the following four categories: nociceptive pain; neuropathic pain; mixed pain; and pain of unknown origin. Nociceptive pain may be caused by known noxious stimulation of pain receptors and may be somatic or visceral. Neuropathic pain may be caused by or triggered by a primary lesion or dysfunction of the nervous system. Mixed pain may be a combination of nociceptive pain and neuropathic pain.

[0246] Examples of pain treated by the present invention (e.g., chronic pain) include neuropathic pain, inflammatory pain, headache, somatic pain, visceral pain, referred pain, allodynia, mixed pain, and postoperative pain.

[0247] The term "pain" as used herein refers to any unpleasant sensory experience, usually associated with bodily dysfunction. Bodily dysfunction may or may not be obvious to the clinician. There are two types of pain: chronic and acute. "Acute pain" is pain that occurs suddenly and for a short period. One type of acute pain is skin pain felt due to injury to the skin or other superficial tissues, such as a cut or burn. Skin pain receptors terminate just beneath the skin, and due to the high concentration of nerve endings, a distinct, localized pain of short duration is produced. "Chronic pain" is pain that is not acute.

[0248] The polypeptide of the present invention can be used to treat pain caused by or associated with any of the following neuropathic pain conditions. "Neuropathy" refers to abnormal sensory input from the peripheral nervous system, central nervous system, or both, resulting in discomfort. Symptoms of neuropathic pain may involve persistent, spontaneous pain, as well as tenderness to touch (a painful response to stimuli that would normally not cause pain), hyperalgesia (an aggravated response to painful stimuli that would normally cause only mild discomfort, such as a needle prick), or hyperpathia (transient discomfort turning into prolonged, severe pain). Neuropathic pain can be caused by any of the following: 1. Traumatic injury, such as nerve compression injury (e.g., nerve crush, nerve stretching, nerve entrapment, or incomplete nerve transection); spinal cord injury (e.g., hemisection); limb amputation; contusion; inflammation (e.g., myelitis); or surgical procedures. 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., solvents, fumes from glue), or nitrous oxide. 5. Diseases, including, for example, inflammatory disorders, neoplastic tumors, acquired immunodeficiency syndrome (AIDS), Lyme disease, leprosy, metabolic diseases, peripheral neuropathy, neuroma, mononeuropathy, or polyneuropathy.

[0249] Types of neuropathic pain include the following: 1. Neuropathic pain. Neuropathic pain is a type of pain that radiates along one or more specific nerves, usually without any obvious pathological changes in the nerve structure. The causes of neuropathic pain are varied. Chemical irritation, inflammation, trauma (including surgery), compression from nearby structures (such as tumors), and infection can all cause neuropathic pain. However, in many cases, the cause is unknown or undetermined. Neuropathic pain is most common in the elderly, but it can occur at any age. Neuropathic pain includes, but is not limited to, trigeminal neuralgia, postherpetic neuralgia, postherpetic neuralgia, glossopharyngeal neuralgia, sciatica, and atypical facial pain.

[0250] Neuralgia is pain distributed in one or more nerves. Examples include trigeminal neuralgia, atypical facial pain, and postherpetic neuralgia (caused by shingles or herpes zoster). The affected nerve is responsible for sensing touch, temperature, and pressure in the facial area from the jaw to the forehead. This disorder usually causes brief episodes of intense pain, typically lasting less than two minutes and occurring only on one side of the face. The pain can be described in many ways, such as "piercing," "sharp," "like lightning," "burning," or even "itching." In atypical forms of TN, the pain may also be severe or just aching and last for a longer period. Pain associated with TN is considered one of the most intense pains one can experience.

[0251] Epilepsy can be triggered by simple stimuli such as eating, talking, washing one's face, or any light touch or sensation (even the sensation of a gentle breeze). Seizures can occur in clusters or singly.

[0252] Symptoms include: sharp, stabbing, or persistent burning pain in any location, usually on or near the body surface, in the same location with each episode; pain along a specific nerve pathway; impaired function of the affected body part due to pain, or muscle weakness due to accompanying motor nerve damage; increased skin sensitivity or numbness in the affected skin area (similar to the sensation of local anesthesia, such as a novocaine injection); and any touch or pressure being interpreted as pain. Movement may also be painful.

[0253] Trigeminal neuralgia is the most common form of neuralgia. It affects the main sensory nerve in the face: the trigeminal nerve (the name "trigeminal nerve" literally means "three sources," referring to the nerve's division into three branches). This condition involves sudden, brief episodes of intense pain on one side of the face, along the area served by the trigeminal nerve on that side. These pain attacks can be severe enough to cause facial contortions, traditionally known as tic douloureux. Sometimes, 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, but the cause is not always definitively identified. In such cases, certain actions (such as chewing, speaking, swallowing, or touching the facial area) may trigger intense spasms of pain.

[0254] Related, but rather uncommon, neuralgia affects the glossopharyngeal nerve, which produces sensation in the throat. Symptoms of this type of neuralgia include brief, shock-like episodes of pain in the throat.

[0255] Postherpetic neuralgia can occur after infection with shingles, caused by the varicella-zoster virus (a type of herpesvirus). This neuralgia causes a persistent burning pain after the shingles rash has healed. Moving or touching the affected area worsens the pain. Not everyone diagnosed with shingles will continue to experience postherpetic neuralgia, which can be more painful than the shingles rash itself. This pain and sensitivity can last for months or even years. The pain is often manifested as unbearable sensitivity to any touch, especially light touch. Postherpetic neuralgia is not limited to the face; it can occur anywhere on the body, but usually at the site of the shingles rash. Depression is not uncommon due to the pain and social isolation during the illness.

[0256] Postherpetic neuralgia can be debilitating for a long time after the initial signs of herpes infection have disappeared. Other infectious diseases that can cause neuralgia include syphilis and Lyme disease.

[0257] Diabetes is another common cause of neuralgia. This very common medical problem affects nearly one in 20 Americans during adulthood. Diabetes damages the tiny arteries that supply nerve circulation, leading to nerve fiber dysfunction and sometimes even nerve loss. Diabetes can cause almost any type of neuralgia, including trigeminal neuralgia, carpal tunnel syndrome (pain and numbness in the hand and wrist), and paresthesia of the femoral thigh (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 those who do suffer from neuralgia.

[0258] Other medical conditions that may be associated with neuralgia include chronic renal insufficiency and porphyria—a hereditary disorder in which the body is unable to remove certain substances produced by the breakdown of normal blood. Some medications may also cause this problem.

[0259] 2. Deafferentation: Deafferentation indicates the loss of sensory input from a part of the body and can be caused by interruption of peripheral sensory fibers or nerves from the central nervous system. Deafferentation pain syndromes include, but are not limited to, brain or spinal cord injury, post-stroke pain, phantom pain, paraplegia, brachial plexus avulsion, and lumbar nerve root lesions.

[0260] 3. Complex Regional Pain Syndromes (CRPSs) CRPS is a chronic pain syndrome caused by sympathetically-maintained pain, presenting in two forms. CRPS 1 is now the alternative term "reflex sympathetic dystrophy syndrome." It is a chronic neurological disorder, most commonly seen in the arms or legs after mild or severe injury. CRPS 1 is associated with: severe pain; changes in nails, bones, and skin; and increased sensitivity of the affected limb to touch. CRPS 2 is the alternative term "causalgia," caused by definite nerve damage. CRPS includes, but is not limited to, type I CRPS (reflex sympathetic dystrophy) and type II CRPS (causalgia).

[0261] 4. Neuropathy Neuropathy refers to functional or pathological changes in nerves, clinically characterized by abnormalities in sensory or motor neurons.

[0262] Central nervous system lesions are functional or pathological changes in the central nervous system.

[0263] Peripheral neuropathy is a functional or pathological change in one or more peripheral nerves. Peripheral nerves transmit 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. When these nerves are unable to transmit information between the brain and spinal cord, it leads to peripheral neuropathy, causing pain, loss of sensation, or inability to control muscles. In some cases, nerve failure controlling blood vessels, the small intestine, and other organs can lead to abnormal blood pressure, digestive problems, and loss of other basic bodily processes. Risk factors for neuropathy include diabetes, excessive alcohol consumption, 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. Prolonged immobility (such as during prolonged surgery or chronic illness) or pressure on nerves from casts, splints, braces, canes, or other devices can cause pressure injuries. Polyneuropathy means a widespread process that typically affects both sides of the body equally. Symptoms depend on the type of nerve affected. The three main types of nerves are sensory nerves, motor nerves, and autonomic nerves. Neuropathy can affect any one, a combination of, or all of the three types of nerves. Symptoms also depend on whether the condition affects the whole body or only one nerve (due to injury). Chronic inflammatory polyneuropathy is caused by an abnormal immune response. Specific antigens, immune processes, and triggering factors are variable and, in many cases, unknown. Its occurrence may be associated with HIV, inflammatory bowel disease, lupus, chronic active hepatitis, and other conditions with abnormal blood cell counts.

[0264] Peripheral neuropathy may involve functional or pathological changes in a single nerve or nerve group (mononeuropathy) or affect the function or pathological changes in multiple nerves (polyneuropathy).

[0265] Peripheral neuropathy can include the following: Hereditary disorders Charcot-Marie-Tooth disease, Friedreich's ataxia, systemic or metabolic diseases such as diabetes (diabetic neuropathy), nutritional deficiencies (especially vitamin B-12), excessive alcohol consumption (alcoholic neuropathy), uremia (due to kidney failure), cancer, infectious or inflammatory conditions, AIDS, hepatitis, Colorado tick fever, Guillain-Barre syndrome, HIV infection without AIDS, leprosy, Lyme disease, polyarteritis nodosa, rheumatoid arthritis, sarcomatoid disease, Sjogren syndrome, syphilis, systemic lupus erythematosus, amyloidosis, exposure to toxic compounds, blister packs or other toxic compounds, nitrous oxide, industrial reagents—especially solvents, heavy metals (lead, arsenic, mercury, etc.). Drug-induced neuropathy, such as analgesic nephropathy, other causes of ischemia (reduced oxygen / blood flow), prolonged exposure to cold temperatures. a. Polyneuropathy: Polyneuropathy is a peripheral neuropathy involving loss of motor or sensory function in an area due to damage or destruction of multiple peripheral nerves. Polyneuropathy-related pain includes, but is not limited to, post-polio syndrome, postmastectomy syndrome, diabetic neuropathy, alcoholic neuropathy, amyloidosis, toxins, AIDS, hypothyroidism, uremia, vitamin deficiency, chemotherapy-induced pain, 2',3'-didecytidine (ddC) treatment, Gba II syndrome, or Fabry's disease. b. Mononeuropathy: Mononeuropathy is a peripheral neuropathy involving loss of motor or sensory function in an area due to damage or destruction of a single peripheral nerve or nerve group. Mononeuropathy is most commonly caused by localized damage from injury or trauma, although occasional systemic disorders can also cause independent nerve damage (such as mononeuritis multiplex). Common causes include direct trauma, prolonged pressure on the nerve, and nerve compression due to swelling or injury to nearby body structures. Damage involves destroying the myelin sheath (covering) of the nerve or part of the nerve cells (axons). This damage slows or blocks impulse conduction through the nerve. Mononeuropathy can involve any part of the body.Mononeuropathy-related pain includes, but is not limited to, sciatic nerve dysfunction, common peroneal nerve dysfunction, radial nerve dysfunction, ulnar nerve dysfunction, cranial mononeuropathy VI, cranial mononeuropathy VII, cranial mononeuropathy III (compression type), cranial 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 sixth (abducens) nerve palsy. c. Systemic peripheral neuropathy: Systemic peripheral neuropathy is symmetrical and usually caused by various systemic diseases and disease processes that comprehensively affect the peripheral nervous system. These can be further subdivided into several categories: i. Distal axonopathy: This results from certain metabolic or toxic disturbances in neurons. These may be caused by conditions such as diabetes, kidney failure, deficiency syndromes such as malnutrition, alcoholism, or the effects of toxins or drugs. Distal axonopathy (also known as retrograde neuropathy) is a type of peripheral neuropathy caused by certain metabolic or toxic disorders of neurons in the peripheral nervous system (PNS). It is the most common response of neurons to metabolic or toxic disorders and can therefore be caused by metabolic diseases such as diabetes, kidney failure, deficiency syndromes such as malnutrition, alcoholism, or the effects of toxins or drugs. The most common cause of distal axonopathy is diabetes, and the most common type of distal axonopathy is diabetic neuropathy. ii. Myelinopathy is an acute failure of impulse conduction due to a primary attack on myelin. The most common cause is acute inflammatory demyelinating polyneuropathy (AIDP; also known as Gba's syndrome), although other causes include chronic inflammatory demyelinating syndrome (CIDP), inherited metabolic disorders (e.g., leukodystrophy), or toxins. Myelin leukoencephalopathy results from the primary destruction of myelin or myelinated Schwann cells, leaving the axons intact but causing acute failure of impulse conduction. This demyelination slows or completely blocks the conduction of electrical impulses through the nerve. The most common cause is acute inflammatory demyelinating polyneuropathy (AIDP, more commonly known as Gba's syndrome), although other causes include chronic inflammatory demyelinating polyneuropathy (CIDP), inherited metabolic disorders (e.g., leukodystrophy or Shamadulosan disease), or toxins. iii. Neuropathies result from the destruction of neurons in the peripheral nervous system (PNS). These can be caused by motor neuron disease, sensory neuropathy (e.g., herpes zoster), toxins, or autonomic dysfunction. Neurotoxins, such as the chemotherapy drug vincristine, can cause neuropathy.Neuropathy is a functional disorder caused by damage to neurons in the peripheral nervous system (PNS). It can be caused by motor neuron disease, sensory neuropathy (e.g., shingles), toxic substances, or autonomic dysfunction. People with neuropathy may present with different symptoms depending on the cause, how it affects nerve cells, and the type of nerve cells most severely affected. iv. Focal entrapment neuropathy (e.g., carpal tunnel syndrome).

[0266] In cases where the pain is neuropathic pain, in one specific embodiment, the polypeptide used does not contain a non-catalytically active BoNT / XL chain, a BoNT / X transfer domain (HN domain), and / or a BoNT / X receptor-binding domain (HC domain). In one specific embodiment, in cases where the pain is neuropathic pain, the polypeptide used does not contain a non-catalytically active BoNT / XL chain and a transfer domain (HN domain), and may optionally combine HC domains derived from different Clostridium neurotoxins (i.e., non-BoNT / X) (e.g., HC domains derived from BoNT / B). Preferably, in cases where the pain is neuropathic pain, the polypeptide used does not contain a non-catalytically active BoNT / XL chain and a transfer domain (HN domain), and a BoNT / B HC domain.

[0267] The polypeptides of the present invention can be used to treat pain caused by or related to any of the following inflammatory conditions. Similarly, the polypeptides of the present invention can be used to treat one or more of the following inflammatory conditions.

[0268] A. Arthritic Dysfunctions Arthritic dysfunctions include, for example: rheumatoid arthritis; juvenile rheumatoid arthritis; systemic lupus erythematosus (SLE); gouty arthritis; scleroderma; osteoarthritis; psoriatic arthritis; adhesive spondylitis; Reiter's syndrome (reactive arthritis); adult Still's disease; arthritis caused by viral infections; arthritis caused by bacterial infections, such as gonococcal arthritis and non-gonococcal bacterial arthritis (septic arthritis); grade 3 Lyme disease; tuberculous arthritis; and arthritis caused by fungal infections, such as blastomycosis.

[0269] B. Autoimmune diseases Autoimmune diseases include, for example: Graham-Barth syndrome, Hashimoto's thyroiditis, pernicious anemia, Addison's disease, type I diabetes, systemic lupus erythematosus, dermatomyositis, Hugh Grant's syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis, Wright's syndrome, and Grave's disease.

[0270] C. Connective tissue disorders Connective tissue disorders include, for example, spondyloarthritis, dermatomyositis, and fibromyalgia.

[0271] D. Injury Inflammation caused by injury (including, for example, compression, puncture, or stretching of tissues or joints) can cause chronic inflammatory pain.

[0272] E. Infection Inflammation caused by infection (including, for example, tuberculosis or interstitial keratitis) can cause chronic inflammatory pain.

[0273] F. Neuropathy Neuropathy is an inflammatory process affecting a nerve or group of nerves. Symptoms depend on the nerve involved but may include pain, paresthesia, mild paralysis, or numbness. Examples include: a. Brachial neuritis b. Retrobulbar neuropathy, an inflammatory process affecting the portion of the optic nerve located just behind the eyeball. c. Optic neuropathy, an inflammatory process affecting the optic nerve that causes a sudden decrease in vision in the affected eye. The cause of optic neuritis is unclear. Sudden inflammation of the optic nerve (the nerve connecting the eye and brain) leads to swelling and destruction of the myelin sheath. Inflammation may sometimes be the result of a viral infection or may be caused by autoimmune diseases such as multiple sclerosis. Risk factors are associated with possible causes. d. Vestibular neuritis, a viral infection that causes an inflammatory process affecting the vestibular nerve.

[0274] G. Joint inflammation Joint inflammation, such as that caused by bursitis or tendinitis, can cause chronic inflammatory pain.

[0275] H. Sunburn and / or damage caused by ultraviolet radiation

[0276] The polypeptide of the present invention can be used to treat pain caused by or related to any of the following headache conditions. Headache (medically known as cephalgia) is a condition of mild to severe pain in the head; sometimes neck or upper back pain can also be interpreted as headache. It may indicate an underlying local or systemic disease, or it may be a disease itself.

[0277] A. Muscle / Myogenic Headache Muscle / myogenic headaches appear to be associated with tightness or tension in the muscles of the face and neck;these may spread to the forehead. Tension headache is the most common form of myogenic headache. Tension headaches are conditions involving pain or discomfort in the head, scalp, or neck and are often associated with muscle tightness in these areas. Contractions of the neck and scalp muscles can lead to tension headaches. One of the causes of this muscle contraction is a response to stress, depression, or anxiety. Any activity that causes the head to remain immobile for long periods of time can cause headaches. Such activities include typing or using a computer, performing fine operations by hand and using a microscope. Sleeping in a cold room or sleeping in an abnormal neck position may also trigger this type of headache. Tension-type headaches include, but are not limited to, paroxysmal tension headaches and chronic tension headaches.

[0278] B. Vascular headache The most common type of vascular headache is migraine. Other types of vascular headaches include cluster headaches (recurrent attacks that cause intense pain) and headaches caused by hypertension. 1. Migraine Migraine is a heterogeneous arrhythmia that usually involves recurrent headaches. Migraines differ from other headaches because they are accompanied by other symptoms such as nausea, vomiting, or sensitivity to light. In most people, throbbing pain is felt on only one side of the head. In subgroups of subjects with different underlying pathophysiological and genetic mechanisms, clinical features, such as the type of aura symptom, the presence of prodrome or associated symptoms, such as vertigo, were seen. Migraines include, but are not limited to, migraines without antecedents (ordinary migraines), migraines with antecedents (typical migraines), menstrual migraines, headache isocratic attacks (migraine equivalent) (migraine without headache (acephalic headache)), complex migraines, abdominal migraines and mixed tension migraines. 2. Cluster headache Cluster headache affects one side of the head (unilateral) and may be associated with eye tearing and nasal congestion. It occurs in clusters, repeating at the same time every day for weeks, and then remissions.

[0279] D. Hypertensive headache

[0280] E. Traction and inflammatory headache Traction and inflammatory headache are usually symptoms of other disorders that range from stroke to sinus infection.

[0281] F. Hormonal headache

[0282] G. Rebound headache Rebound headache occurs when medications are taken excessively frequently to relieve headaches, also known as medication overuse headache. Rebound headaches often occur daily and can be very painful.

[0283] H. Chronic Sinusitis and Headache. Sinusitis is an inflammation of the nasal and paranasal sinuses, whether 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; malaise; thick green or yellow discharge; and a feeling of "fullness" in the face that worsens when bending over. In rare cases, chronic maxillary sinusitis can also be caused by the spread of bacteria from dental infections. Chronic proliferative eosinophilic sinusitis is a non-infectious form of chronic sinusitis.

[0284] I. Organic headache

[0285] J. Epilepsy headache Epilepsy headache is a headache associated with epileptic seizures.

[0286] The polypeptides of the invention may be used to treat pain arising from or associated with any of the following somatosensory pain conditions. Somatosensory pain originates from ligaments, tendons, bones, blood vessels, and even the nerves themselves. which is detected by somatosensory nociceptors. The lack of pain receptors in these areas produces blunt pain lacking locality that lasts longer than skin pain;e.g., including sprains and fractures. Other examples include the following. A. Excessive muscle tension Excessive muscle tension can result from a sprain or strain. B. Recurrent ataxia Recurrent ataxia can be caused by overuse of the hands, wrists, elbows, shoulders, neck, back, hips, knees, legs, legs, or ankles. C. Muscle Arrhythmia Muscle arrhythmias that cause somatosensory pain include, for example, polymyositis, dermatomyositis, lupus, fibromyalgia, rheumatic polymyalgia, and rhabdomyolysis. D. Myalgia Myalgia is muscle pain and is a symptom of many diseases and disorders. The most common cause of myalgia is overuse or overstretching of a muscle or muscle group. Myalgia without a history of trauma is usually caused by a viral infection. Prolonged myalgia may indicate metabolic myopathy, certain nutritional deficiencies, or chronic fatigue syndrome. E. Infection Infection can cause somatosensory pain. Examples of such infections include, for example, muscle abscess, trichinellosis, influenza, Lyme disease, malaria, Rocky Mountain spotted fever, avian influenza, common cold, community-acquired pneumonia, meningitis, monkeypox, severe acute respiratory syndrome, toxic shock syndrome, trichinellosis, typhoid fever, and upper respiratory tract infections. F. Drugs Drugs can cause somatosensory pain. Such drugs include, for example: cocaine, statins (such as atorvastatin, simvastatin and ovastatin) for lowering cholesterol, and ACE inhibitors (such as enalapril and captopril) for lowering blood pressure.

[0287] The polypeptide of the present invention can be used to treat pain caused by or associated with any of the following visceral pain conditions. Visceral pain originates from the viscera or organs of the body. Visceral pain receptors are located in the organs and cavities of the body. Compared with somatic pain, a greater lack of pain receptors in these areas produces pain that is generally more painful and lasts longer than somatic pain. Visceral pain is difficult to localize, and some injuries to visceral tissues can present as "migratory" pain, in which the sensation is localized to an area completely unrelated to the site of injury. Examples of visceral pain include the following: 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. 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; ulcers, kidney stones, urinary tract infections, pancreatitis, and hernias. C. Autoimmune pain: Autoimmune pain includes, for example, sarcoidosis and vasculitis. D. Organic visceral pain: Organic visceral pain includes, for example, pain caused by traumatic, inflammatory, or degenerative changes in the intestine, or pain caused by tumors affecting sensory nerve innervation. E. Treatment-induced visceral pain: Treatment-induced visceral pain includes, for example, pain caused by chemotherapy or radiation therapy.

[0288] The polypeptide of the present invention can be used to treat pain caused by or associated with any of the following metastatic pain conditions.

[0289] Migratory pain originates in areas different from the site of the painful stimulus. Migratory pain typically occurs when a nerve is compressed or damaged at or near its origin. In such cases, even if the injury originates elsewhere, pain is usually felt in the area served by the nerve. A common example occurs in a herniated disc, where a nerve root originating from the spinal cord is compressed by the material of an adjacent intervertebral disc. Although the pain may originate from the damaged disc itself, it 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 alleviate this migratory pain, provided there is no permanent nerve damage. Myocardial ischemia (reduced blood flow to a portion of the heart muscle) is perhaps the most well-known example of migratory pain; this sensation may appear as a feeling of tightness in the upper chest or as pain in the left shoulder, arm, or even hand.

[0290] The polypeptide of the present invention can be used to treat postoperative pain.

[0291] Postoperative (e.g., post-operative) pain is an unpleasant sensation caused by the surgical procedure. Postoperative pain can be caused by damage to tissues from the incision, the surgery itself, wound closure, and any force applied during the procedure. Postoperative pain (e.g., postoperative pain) may also stem from factors associated with the surgery. For example, a patient may experience back pain due to their position on the operating table, or chest pain due to an incision in the chest area. Sore throat may also occur after general anesthesia because the insertion of a breathing tube can cause irritation. However, the most common cause of postoperative pain is the surgical incision that cuts into the skin and muscle.

[0292] For example, a surgical procedure (or more specifically, a surgical incision) may represent a "noxious stimulus" that causes pain. A noxious stimulus, which can cause tissue damage, can activate the release of neurotransmitters from sensory receptive terminals and neuropeptides such as substance P and calcitonin gene-related peptide (CGRP) from sensory terminals. The noxious information is then transmitted from the peripheral nervous system to the central nervous system, where pain is perceived by the individual.

[0293] The combination of inflammation and nerve tissue damage can cause postoperative pain. For example, the degranulation of mast cells activated in response to tissue damage can lead to the release of various substances, including proteases, cytokines, serotonin, and extracellular space. These substances can sensitize (activate) primary afferent neurons at a lower threshold, resulting in pain hypersensitivity. Because tissues are extensively innervated, any part of the body is susceptible to sensory stimulation from surgical nerve damage.

[0294] When referring to surgery, it means a medical procedure involving the treatment of an injury or disease of a subject, including making an incision in a part of the body (optionally removing or repairing a damaged part of the body). Although the level of invasiveness (e.g., the level of the surgical incision required) may vary between types of surgery, it is intended to cover surgeries that have a level of invasiveness that causes pain in the subject once the surgery is completed.

[0295] The procedure may include incisions in the skin and / or fascia and / or muscles. Preferably, the procedure includes an incision in the skin.

[0296] Surgery is not limited to procedures that can be performed by a physician, but also includes procedures such as dental surgery. Non-limiting examples of surgery include appendectomy, breast examination, breast augmentation or reduction, cosmetic surgery, cholecystectomy, coronary artery bypass surgery, debridement (e.g., wounds, burns, or infections), skin grafts, organ transplants, and tonsillectomy.

[0297] Preferably, "postoperative" may refer to a period of up to one day following the surgery (e.g., after the operation). In other words, the term "postoperative" may refer to a period of no more than one day following the surgery. For example, the term "postoperative" may refer to a time point of 1-20 hours following the surgery; alternatively, 2-15 hours; alternatively, 5-10 hours. This time may represent a period beginning at a temporal interface during which the analgesic effect of the surgical anesthetic administered to the subject diminishes (e.g., gradually weakens), and the subject begins to perceive pain.

[0298] Furthermore, the term “postoperative” can be used interchangeably with the term “postoperative”, since “surgery” is used in this text in the sense of “surgical procedure”.

[0299] Similarly, the term "postoperative pain" can refer to pain perceived (or more specifically, pain that begins to be perceived) within a period of no more than one day after surgery (e.g., postoperative). In other words, the term "postoperative" can refer to pain perceived by the subject within a period of no more than one day after surgery. For example, the term "postoperative" can refer to pain perceived within a period of 1-20 hours after surgery; alternatively, 2-15 hours after surgery; alternatively, 5-10 hours after surgery.

[0300] This period can be 1-50 weeks after surgery; for example, 5-45 weeks, 10-40 weeks or 10-35 weeks.

[0301] This is in contrast to the term “peri-operative”, which can refer to, for example, the time before or after the subject undergoes surgery (e.g., the time when the subject is in the operating room), and is suitable as a period that begins at least 1 hour before surgery and / or ends less than 1 hour after surgery.

[0302] This invention addresses a wide range of pain conditions, such as chronic pain. In some specific embodiments, the polypeptides of this invention are used to treat both cancerous and non-cancer pain.

[0303] Preferably, the polypeptide of the present invention is used to treat neuropathic pain. Neuropathic pain can be acute or chronic. In one specific embodiment, the neuropathic pain is injury-induced neuropathic pain (injury-related neuropathic pain). In another specific embodiment, the neuropathic pain is chemotherapy-induced neuropathic pain (chemotherapy-related neuropathic pain).

[0304] Preferably, the polypeptide of the present invention is used to treat inflammatory pain. The inflammatory pain can be acute or chronic. In one specific embodiment, the inflammatory pain can be a burn. For example, the inflammatory pain can be caused by UV damage (e.g., UV-B damage).

[0305] Preferably, the polypeptide of the present invention is used to treat bladder pain syndrome, phantom limb pain, or migraine. Bladder pain syndrome may be caused by or related to interstitial cystitis.

[0306] Better treatment for pain means relief of pain. In other words, in a specific embodiment, administration of the polypeptide of the present invention to a subject relieves pain.

[0307] More specifically, the reference to "reducing" or "lowering" (in terms of pain) preferably means that the subject experiences a lower level of pain after administration of the polypeptide of the present invention compared to the level of pain perceived by the subject before administration. For example, the level of pain perceived after administration may be reduced by at least 15%, 25%, 35%, 45%, 55%, 65%, 75%, 85%, or 95% compared to before administration. For example, the level of pain perceived after administration may be reduced by at least 75%; preferably at least 85%; more preferably at least 95%.

[0308] Various methods for assessing pain perception are known to those of ordinary skill in the art. For example, the assessment of mechanical touch pain (static or dynamic) is commonly used in human pain research, as described by Pogatzki-Zahn et al. (Pain Rep. 2017 Mar; 2(2): e588), which is incorporated herein by reference.

[0309] Suitable (though not limiting) methods for assessing pain perception in a subject include the following: Numerical Rating Scale (NRS) scores; although other methods known to those skilled in the art as being applicable or alternative to these methods may be used, such as sensory thresholds, pain perception thresholds, static mechanical touch pain, dynamic mechanical touch pain, time-additive, pressure pain thresholds, conditioned pain modulation, and temperature thresholds.

[0310] Other non-limiting examples of pain perception measurements include: changes from baseline SF-36 scores at each planned time point; the amount of emergency medication taken during the study period and the time of first administration of emergency medication. These may be considered "exploratory" endpoints or pain perception assessment measurements.

[0311] Thus, in a preferred embodiment, after administration of the polypeptide of the present invention, pain perception can be assessed by one or more of the following: (a) Numerical Rating Scale (NRS); (b) Stimulus-induced NRS; (c) Temperature of the pain area; (d) Size of the pain area; (e) Time of onset of analgesia; (f) Peak analgesia; (g) Time to reach peak analgesia; (h) Duration of analgesia; and (i) SF-36 quality of life.

[0312] Such methods for assessing pain perception are known to those skilled in the art. For convenience, further descriptions of the Numerical Rating Scale and the Short Form-36 Quality of Life Questionnaire are provided below.

[0313] Numerical Rating Scale (NRS): Typically, the Numerical Rating Scale (NRS) is used for pain perception according to the present invention. The NRS is an 11-point rating scale to assess a subject's pain perception. Subjects are asked to give a number between 0 and 10 that best represents their pain intensity. 0 represents "no pain at all," while the upper limit of 10 represents "possibly the most severe pain."

[0314] The NRS can be used to assess many aspects of pain, including spontaneous average pain, spontaneous most severe pain, and spontaneous current pain. Spontaneous average pain is assessed by asking the subject to select a number that best describes the subject's average pain (e.g., perceived pain) over a period of time (e.g., at least 6 hours, 12 hours, 24 hours, or at least 48 hours). Spontaneous most severe pain is assessed by asking the subject to select a number that best describes the subject's most severe pain within a specified period, such as at least the previous 6 hours, 12 hours, 24 hours, or the previous 48 hours. Spontaneous current pain is assessed by asking the subject to select a number that best describes the subject's level of pain at the time of assessment.

[0315] The NRS can also be used to assess a subject's pain perception in response to various stimuli. To assess pain perception in response to stimuli, the subject will receive stimuli of various natures applied to the pain area. The subject's current NRS score will be asked before administration and after stimulation.

[0316] Examples of stimuli used include: (i) light touch (which can be assessed by measuring pain on the surface of the pain area of ​​the radial spoke after the application of the von Frey filament described herein); (ii) pressure (tenderness threshold), which can be assessed by asking the subject to give an NRS score when increased pressure is applied using a pressure sensor; and (iii) temperature (which can be assessed by asking the subject to give an NDS score for warm, cold and hot stimuli when a thermometer is applied to the pain area).

[0317] Preferably, when compared with the NRS score of the object before administration, administration of the polypeptide of the present invention reduces the NRS score of the object after administration (e.g., from a rating of ≥7 to a rating of ≤6).

[0318] Quality of Life Questionnaire Short Form-36 (SF-36): The SF-36 Quality of Life Questionnaire can be used to assess a subject's perceived pain. The SF-36 is a 36-item survey of a subject's reported health. The SF-36 consists of eight rating items (vitality, physical functioning, body pain, general health perception, bodily role functioning, emotional role functioning, social role functioning, and mental health). Assuming equal weight for each item, each scale is directly converted to a 0-100 scale. Higher scores on the SF-36 indicate a lower degree of disability.

[0319] The relevant parameters for pain treatment that are commonly tested in clinical trials are known in the art and can be readily selected by a person of ordinary skill in the art. Examples of such parameters include, but are not limited to, NRS; stimulus-induced NRS; temperature of the pain area; size of the pain area; time of onset of analgesia; peak analgesia; time to peak analgesia; duration of analgesia; and / or SF-36 quality of life as described herein. Methods for assessing these parameters are also known in the art and can be performed by a person of ordinary skill in the art using conventional methods and procedures.

[0320] Preferably, when compared with the SF-36 score of the object before administration, the administration of the polypeptide of the present invention increases the SF-36 score of the object after administration (e.g., from a score of ≤50 to a score of ≥50).

[0321] The inflammatory disorders treated by the polypeptide of the present invention may be the following inflammatory disorders: nervous system, cardiovascular system, respiratory system, digestive system, skin system, musculoskeletal system, urinary system, reproductive system, endocrine system, or lymphatic system.

[0322] Inflammatory disorders of the nervous system may be selected from one or more of the following groups: inflammation of the central nervous system (e.g., encephalitis, myelitis, meningitis or arachnoiditis), inflammation of the peripheral nervous system (e.g., neuritis), inflammation of the eye (e.g., dacryoadenitis, scleritis, episcleritis, keratitis, retinitis, chorioretinitis, blepharitis, conjunctivitis or uveitis), and inflammation of the ear (e.g., otitis externa, otitis media, labyrinthitis and mastoiditis).

[0323] Inflammatory disorders of the cardiovascular system may be selected from one or more of the following groups: carditis (e.g., endocarditis, myocarditis or pericarditis) and vasculitis (e.g., arteritis, phlebitis or microvasculitis).

[0324] Inflammatory disorders of the respiratory system may be selected from one or more of the following groups: inflammatory diseases of the upper respiratory tract (e.g., sinusitis, rhinitis, pharyngitis, or laryngitis), inflammatory disorders of the lower respiratory tract (e.g., tracheitis, bronchitis, bronchiolitis, pneumonia, or pleurisy), and mediastinitis.

[0325] Inflammatory disorders of the digestive system may be selected from one or more of the following groups: oral inflammation (e.g., stomatitis, gingivitis, gingivostomatitis, glossitis, tonsillitis, sialadenitis / mumps, cheilitis, pulpitis or jaw inflammation), gastrointestinal inflammation (e.g., esophagitis, gastritis, gastroenteritis, enteritis, colitis, enterocolitis, duodenitis, ileitis, cecal inflammation, appendicitis or proctitis), and inflammation of accessory digestive organs (e.g., hepatitis, ascending cholangitis, cholecystitis, pancreatitis or peritonitis).

[0326] Inflammatory disorders of the skin system may be one or more of the following groups: dermatitis (e.g., folliculitis), cellulitis, and hidradenitis.

[0327] Inflammatory disorders of the musculoskeletal system may be one or more of the following groups: arthritis, dermatomyositis, soft tissue inflammation (e.g., myositis, synovitis / tenosynovitis, bursitis, enthesitis, fasciitis, capsulitis, epicondylitis, panniculitis), osteochondritis, osteitis / osteomyelitis, spondylitis, periostitis, and chondritis.

[0328] Inflammatory disorders of the urinary system may be one or more of the following groups: nephritis (e.g., glomerulonephritis or pyelonephritis), ureteritis, cystitis, and urethritis.

[0329] Inflammatory disorders of the reproductive system may be selected from one or more of the following groups: 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, posthitis or balanoposthitis), and inflammation related to pregnancy, childbirth and / or newborn (e.g., chorioamnionitis, funisitis or omphalitis)).

[0330] Inflammatory disorders of the endocrine system may be one or more of the following groups: pancreatitis, pituitary inflammation, thyroiditis, parathyroiditis, adrenalitis.

[0331] Inflammatory disorders of the lymphatic system may be one or more of the following groups: lymphangitis and lymphadenitis.

[0332] Preferably, the inflammatory disorder is selected from one or more of the following: complex regional pain syndrome, endometriosis, rheumatoid arthritis, cystitis, and neuritis. Cystitis is preferably interstitial cystitis. Neuritis is preferably peripheral neuritis.

[0333] Specific embodiments relating to various therapeutic uses of the present invention are intended to be used in the same manner as therapeutic methods, and vice versa.

[0334] Sequence homology can be determined using any of a variety of sequence alignment methods, including but not limited to global methods, local methods, and hybrid methods, such as, for example, the segment approach method. The experimental procedure for determining the percentage of homology is a routine procedure within the scope of ordinary knowledge in the art to which this art belongs. The global method aligns sequences from the beginning to the end of the molecule and determines the best alignment by accumulating the scores of each residue pair and by applying a gap penalty. Non-limiting methods include, for example, CLUSTAL W, see, for example, 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 its iterative refinement, see, for example, Osamu Gotoh, Significant Improvement In Accuracy of Multiple Protein. Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments, 264(4) J. MoI. Biol. 823-838 (1996). Local methods align sequences by identifying one or more conserved motifs common to all input sequences.Non-restrictive methods include, for example: Match-box, see, for example, 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, for example, CE Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131) Science 208-214 (1993); Align-M, see, for example, Ivo Van Waille et al., Align-M - A New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics:1428-1435 (2004).

[0335] Thus, the percentage of sequence identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992. Briefly, two amino acid sequences are aligned using a gap opening penalty of 10, a gap extension penalty of 1, and the Henikoff and Henikoff (ibid.) “blosum 62” scoring matrix (amino acids are represented by standard single-letter codes) as shown below to optimize the alignment score; preferably, this method is used to align a sequence with the SEQ ID NO described herein to define the amino acid position number, as described herein.

[0336] The "sequence identity percentage" between two or more nucleic acid or amino acid sequences is a function of the number of common positions in the sequences. Thus, the identity percentage can be calculated as follows: the number of identical nucleotides / amino acids divided by the total number of nucleotides / amino acids multiplied by 100. The calculation of the sequence identity percentage can also take into account the number of gaps required to optimize the alignment of two or more sequences, and the length of each gap. The sequence comparison and determination of the identity percentage between two or more sequences can be performed using specific mathematical algorithms, such as BLAST, which are familiar to those skilled in the art.

[0337] Alignment score used to determine sequence identity

[0338] Then, the percentage of identity is calculated as follows:

[0339] A substantially homologous polypeptide is characterized by having one or more amino acid substitutions, deletions, or additions. These changes are preferably minor, i.e., retained amino acid substitutions (see below) and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically from 1 to about 30 amino acids; and small amino or carboxyl terminal extensions, such as amino-terminal methionine residues, small linker peptides of up to about 20-25 residues, or affinity tags.

[0340] Retained amino acid substitution alkaline: Arginine lysine histidine Acidity: glutamic acid Aspartic acid polarity: glutamic acid aspartic acid Hydrophobicity: Leucine Isoleucine Valine Aromatics: Phenylan tryptophan Tyrosine Small: Glycine alanine serine threonine Methionine

[0341] In addition to the 20 standard amino acids, non-standard amino acids (such as 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovalinic acid, and α-methylserine) may also replace the amino acid residues of the peptides of the present invention. A limited number of non-conservative amino acids, amino acids not encoded by the genetic code, and non-natural amino acids may replace the amino acid residues of the peptides. The peptides of the present invention may also contain non-naturally occurring amino acid residues.

[0342] Non-naturally occurring amino acids include, but are not limited to, trans-3-methylproline, 2,4-methylproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allethamine, methylthreonine, hydroxyethylcysteine, hydroxyethyl cytocysteine, nitroglutamine, cytoglutamine, 2-piperidinic acid, tertiary leucine, n-valine, 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, in vitro systems can be used where chemically aminolated suppressor tRNA is used to suppress nonsense mutations. Methods for synthesizing amino acids and aminolated tRNA are known in the art. Transcription and translation of plastids containing meaningless mutations are performed in a cell-free system comprising E. coli S30 extract and commercially available enzymes and other reagents. Proteins can be purified by chromatography. See, for example: 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 the second method, translation occurs in Xenopus oocytes via microinjection of mutated mRNA and chemically aminolated repressed 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 desired natural amino acid (e.g., phenylalanine) and in the presence of the desired non-natural amino acid (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The non-natural amino acid is incorporated into the polypeptide to replace its natural counterpart. See Koide et al., *Biochem.* 33:7470-6, 1994. Naturally occurring amino acid residues can be converted into non-natural types through 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).

[0343] A limited number of non-reserved amino acids, amino acids not encoded by the genetic code, non-naturally occurring amino acids, and non-natural amino acids may replace the amino acid residues of the polypeptides of the present invention.

[0344] The essential amino acids in the polypeptides of the present 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). The location of biological interactions can also be determined by structural physics analysis, such as by combining the following techniques with the mutation of the presumed contact amino acid: nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, combined with the mutation of the presumed contact amino acid. See, for example: 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 homology analysis of related components (e.g., transfer or protease components) of the polypeptide of the present invention.

[0345] Multiple amino acid substitutions can be performed and tested using known mutagenesis and screening methods, 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). In short, these authors disclose methods for simultaneously randomizing two or more positions in a peptide, selecting functional peptides, and then sequencing the mutagenized peptide to determine the permissible range of 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., US Patent No. 5,223,409; Huse, WIPO Publication WO 92 / 06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

[0346] 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 pertains. 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 a general dictionary of many terms used in this disclosure to one of ordinary skill in the art.

[0347] This disclosure is not limited to the exemplary methods and materials disclosed herein, and any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of specific embodiments of this disclosure. Numerical ranges include numbers defining the range. Unless otherwise stated, any nucleic acid sequences are written from left to right in the 5' to 3' direction; amino acid sequences are written from left to right in the direction from amino to carboxyl groups.

[0348] The headings provided herein are not intended to limit the various forms or specific embodiments disclosed herein.

[0349] References to amino acids herein refer to the names, three-letter abbreviations, or single-letter abbreviations of amino acids. The term "protein" as used herein includes proteins, polypeptides, and peptides. The term "amino acid sequence" as used herein is synonymous with the terms "polypeptide" and / or "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. Common one-letter and three-letter codes for amino acid residues may be used within the scope of this disclosure and the claims. The three-letter codes for amino acids are defined in accordance with the Joint Commission on Biochemical Nomenclature (JCBN) of IUPACIUB. It should also be understood that, due to the degeneracy of the genetic code, a polypeptide can be encoded by more than one nucleotide sequence.

[0350] Other definitions of terms may appear throughout the specification. Before describing the exemplary embodiments in more detail, it should be understood that this disclosure is not limited to the specific embodiments described and is therefore subject to variation. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the invention, as the scope of this disclosure is defined only by the appended claims.

[0351] When providing a numerical range, it should be understood that, unless the context explicitly indicates otherwise, each intervening value between the upper and lower limits of the range, up to one-tenth of the lower limit unit, is also specifically disclosed. This disclosure includes each smaller range between "any value or intervening value within the range" and "any other value or intervening value within the range." The upper and lower limits of these smaller ranges may be independently included or excluded from the range, and each range that includes any one, neither, or both of these limits is also included in this disclosure, subject to any explicit exclusion from the range. When the range includes one or both of these limits, ranges excluding one or both of the included limits are also included in this disclosure.

[0352] It should be noted that when used herein and in the appended claims, the singular forms “a,” “an,” and “the” include the plural referents, unless the context clearly specifies otherwise. Thus, for example, reference to “a Clostridium neurotoxin” includes multiple such candidate agents, and reference to “the Clostridium neurotoxin” includes one or more Clostridium neurotoxins and their equivalents known to those skilled in the art.

[0353] The publications discussed herein are provided only for disclosures made by them prior to the filing date of this application. Nothing in this document should be construed as an admission that such publications constitute prior art within the scope of the appended patent application.

[0354] When the sequence listing shows a starting Met amino acid residue or the corresponding start codon in any of the following SEQ ID NOs, the residue / codon is not required. SEQ ID NO: 1 - Nucleotide sequence of recombinant non-catalytically active BoNT / A (rBoNT / A(0)) SEQ ID NO: 2 - Peptide sequence of rBoNT / A(0) SEQ ID NO: 3 - Nucleotide sequence of rLHN / A (light chain only plus transfer domain) SEQ ID NO: 4 - Peptide sequence of rLHN / A SEQ ID NO: 5 - Nucleotide sequence of rL / A (light chain only) SEQ ID NO: 6 - Peptide sequence of rL / A SEQ ID NO: 7 - Nucleotide sequence of rHC / A SEQ ID NO: 8 - Peptide sequence of rHC / A SEQ ID NO: 9 - Nucleotide sequence of rBoNT / B(0) SEQ ID NO: 10 - Peptide sequence of rBoNT / B(0) SEQ ID NO: 11 - Nucleotide sequence of rBoNT / C(0) SEQ ID NO: 12 - Peptide sequence of rBoNT / C(0) SEQ ID NO: 13 - Nucleotide sequence of rBoNT / E(0) NO: 14-rBoNT / E(0) polypeptide sequence SEQ ID NO: 15-rBoNT / F(0) SEQ ID NO: 16-rBoNT / F(0) polypeptide sequence SEQ ID NO: 17-rBoNT / A(0)(His tag) SEQ ID NO: 18-rBoNT / A(0)(His tag) polypeptide sequence SEQ ID NO: 19-rLHN / A(His tag) SEQ ID NO: 20-rLHN / A(His tag) polypeptide sequence SEQ ID NO: 21-rHC / A(His tag) SEQ ID NO: 22-rHC / A(His tag) polypeptide sequence SEQ ID NO: 23-rLC / A(His tag) SEQ ID NO: 24-rLC / A(His tag) polypeptide sequence SEQ ID NO: 25-rBoNT / FA(0)(His tag) SEQ ID NO: 26-rBoNT / FA(0) (His tag) polypeptide sequence SEQ ID NO: 27-rLHN / FA (His tag) nucleotide sequence SEQ ID NO: 28-rLHN / FA (His tag) polypeptide sequence SEQ ID NO: 29-rHC / FA (His tag) nucleotide sequence SEQ ID NO: 30-rHC / FA (His tag) polypeptide sequenceSEQ ID NO: 31 - Nucleotide sequence of rLC / FA (His tag); SEQ ID NO: 32 - Peptide sequence of rLC / FA (His tag); SEQ ID NO: 33 - Nucleotide sequence of rBoNT / F(0)(His tag); SEQ ID NO: 34 - Peptide sequence of rBoNT / F(0)(His tag); SEQ ID NO: 35 - Nucleotide sequence of rLHN / F (His tag); SEQ ID NO: 36 - Peptide sequence of rLHN / F (His tag); SEQ ID NO: 37 - Nucleotide sequence of rHC / F (His tag); SEQ ID NO: 38 - Peptide sequence of rHC / F (His tag); SEQ ID NO: 39 - Nucleotide sequence of rLC / F (His tag); SEQ ID NO: 40 - Peptide sequence of rLC / F (His tag); SEQ ID NO: 41 - Nucleotide sequence of cationic rHC / A (His tag); SEQ ID NO: 42 - Peptide sequence of cationic rHC / A (His tag). SEQ ID NO: 43-rHC / AB (His tag) nucleotide sequence; SEQ ID NO: 44-rHC / AB (His tag) polypeptide sequence; SEQ ID NO: 45-rHC / A variant Y1117V H1253K (His tag) nucleotide sequence; SEQ ID NO: 46-rHC / A variant Y1117V H1253K (His tag) polypeptide sequence; SEQ ID NO: 47-rHC / A variant Y1117V F1252Y H1253K L1278F (His tag) nucleotide sequence; SEQ ID NO: 48-rHC / A variant Y1117V F1252Y H1253K L1278F (His tag) polypeptide sequence; SEQ ID NO: 49-rHC / A variant Y1117V F1252Y H1253K L1278H (His tag) nucleotide sequence; SEQ ID NO: ... SEQ ID NO: 50-rHC / A variant Y1117V F1252Y H1253K L1278H (His tag) polypeptide sequence; SEQ ID NO: 51-BoNT / A-UniProt P10845 polypeptide sequence; SEQ ID NO: 52-BoNT / B-UniProt P10844 polypeptide sequence; SEQ ID NO: 53-BoNT / C-UniProt P18640 polypeptide sequence; SEQ ID NO: 54-BoNT / D-UniProt P19321 polypeptide sequence; SEQ ID NO: 55-BoNT / E-UniProtThe polypeptide sequence SEQ ID NO of Q00496: 56-BoNT / F-UniProt; the polypeptide sequence SEQ ID NO of A7GBG3: 57-BoNT / G-UniProt; the polypeptide sequence SEQ ID NO of Q60393: 58-TeNT-UniProt; the polypeptide sequence SEQ ID NO of P04958: 59-BoNT / X; the polypeptide sequence SEQ ID NO of unmodified BoNT / A1: 60-; the polypeptide sequence SEQ ID NO of mrBoNT / AB(0): 61-; the polypeptide sequence SEQ ID NO of mrBoNT / A(0): 62-; the polypeptide sequence SEQ ID NO of mrBoNT / A(0): 63-; the polypeptide sequence SEQ ID NO of BoNT / XB(0)(His tag): 64-; the polypeptide sequence SEQ ID NO of BoNT / XB(0): 65-; the polypeptide sequence SEQ ID NO of the BoNT / XB(0) variant (His tag): 66-; the polypeptide sequence SEQ ID of the BoNT / XB(0) variant: 66-. SEQ ID NO: 67-BoNT / XA(0) polypeptide sequence SEQ ID NO: 68-BoNT / XA(0) variant polypeptide sequence SEQ ID NO: 69-BoNT / XD(0) polypeptide sequence SEQ ID NO: 70-BoNT / XF(0) polypeptide sequence SEQ ID NO: 71-C1 activation zone common sequence SEQ ID NO: 72-C1 activation zone SEQ ID NO: 73-C1 activation zone variant SEQ ID NO: 74-rLC / A(0)(His tag) polypeptide sequence SEQ ID NO: 75-rLHN / A(0)(His tag) polypeptide sequence SEQ ID NO: 76-rLC / X(0) polypeptide sequence SEQ ID NO: 77-PreScission protease site SEQ ID NO: 78-C1 activation zone variant 2

[0355] Example

[0356] Example 1: The catalytic activity of BoNT / A(0) (SEQ ID NO: 2) was tested in an in vitro cell line model to determine its non-catalytic activity in vitro and in vivo, measuring the cleavage of SNAP25 (the target SNARE protein of BoNT / A). Figure 1 shows that in human neuron analysis, BoNT / A(0) does not cleave SNAP25, unlike wild-type BoNT / A (SEQ ID NO: 60). Figure 2 confirms this result in rat neuron analysis.

[0357] In vivo DAS analysis was performed using BoNT / A and BoNT / A(0) in a confirmatory manner. DAS analysis was performed by injecting 20 μl of Clostridium perfringens toxin prepared in gelatin phosphate buffer into the gastrocnemius / soleus complex of mice, followed by assessment of the digital abduction score using Aoki's method (Aoki KR, Toxicon 39: 1815-1820; 2001). For DAS analysis, mice were briefly suspended by their tails to elicit a characteristic startle response (Fig. 3A), in which the mice extended their hind limbs and abducted their hind toes. The degree of change in toe abduction after injection of the Clostridium perfringens neurotoxin was scored on a five-point scale (0 = normal to 4 = maximum reduction in toe abduction - Fig. 3B). This provides a functional measure of the paralysis caused by the activity of the neurotoxin at the neuromuscular junction. In addition, changes in mouse body weight were assessed within 7 days of administration. This provides a measure of toxicity and the undesirable effects of toxin diffusion from the administration site. The results are presented in Table 1 below.

[0358] Table 1. DAS score (24 hours later) and weight change after administering BoNT / A(0) or BoNT / A. DAS score Observed weight changes BoNT / A 4 have BoNT / A(0) 0 none

[0359] This result confirms that BoNT / A(0) is non-catalytically active in vivo and does not produce any toxic symptoms. Thus, BoNT / A(0) is a safe and substantially non-toxic therapeutic agent.

[0360] Example 2 Treatment of chronic neuropathic pain using non-catalytically active BoNT (Chronic Constriction Injury (CCI) rat model)

[0361] [Materials & Methods] Chronic compression injury (CCI) was performed as previously described in Bennett and Xie (1988), Pain, 33(1):87-107. Adult male Sprague-Dawley rats (220-250 g) were anesthetized on day -14, and a segment of the left sciatic nerve was exposed and ligated with four loose silk sutures. On day 0 (D0), rats were administered BoNT / A (30 pg / kg), BoNT / A (60 pg / kg), BoNT / A(0) (60 pg / kg), or GPB solution (n=10-11 / group) via paw (i.pl.), while the positive control (gabapentin (100 mg / kg)) was administered orally (po) (n=8 / group). Animals treated with gabapentin were tested at 1, 2, and 4 h post-treatment. Animal lines treated with BoNT / A, BoNT / A(0), or carboxylic acid were tested on days 3, 5, and 9. The mechanical sensitivity of the animals was assessed in the von Frey test.

[0362] [Results] The experiment showed that administration of non-catalytically active BoNT (BoNT / A(0)) reduced mechanosensitivity in the ipsilateral paw (Fig. 4B). Furthermore, BoNT / A(0) was more effective in reducing mechanosensitivity compared to an equivalent dose of BoNT / A. At later time points, BoNT / A(0) was also more effective than gabapentin. To confirm that the reduction in sensitivity was a result of BoNT / A(0) administration, mechanosensitivity in the contralateral paw was also tested. The results showed no significant difference in mechanosensitivity in the contralateral paw under different conditions (Fig. 4C). Furthermore, as confirmation of the substantially non-toxic nature of BoNT / A(0), the change in body weight over time was equivalent to that in rats administered the carton or gabapentin (Fig. 4D). This contrasts with the changes observed in rats administered catalytically active BoNT / A, which showed a statistically significant difference on day 9.

[0363] In summary, the non-catalytically active Clostridium neurotoxin is surprisingly able to alleviate pain (e.g., chronic neuropathic pain), thus suggesting that such neurotoxin is a suitable pain treatment agent.

[0364] Example 3 Treatment of acute neuropathic pain using non-catalytically active BoNT (oxaliplatin rat model)

[0365] [Materials & Methods] An experimental model for inducing oxaliplatin-induced peripheral sensory neuropathy by intraperitoneal injection of oxaliplatin was established (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 sham-treatment (5% glucose) or intraperitoneal injection of oxaliplatin (10 mg / kg). Immediately afterwards, animals treated with sham therapy received intravenous (i.p.) injections of the carrier solution, while animals treated with oxaliplatin received intravenous (i.p.) injections of BoNT / A(0) (1000 pg / kg), BoNT / A (50 pg / kg), BoNT / A (100 pg / kg), BoNT / A (160 pg / kg), or the carrier solution (GPB; n=10 / group). On day 3, a positive control, duloxetine (100 mg / kg), was administered orally. The animals' heat (cold) sensitivity was assessed on days 3 and 5.

[0366] [Results] The experiment showed that the cold sensitivity of the ipsilateral foot was reduced by administering non-catalytically active BoNT (BoNT / A(0)) (Fig. 5B). There was no difference in heat sensitivity of the ipsilateral foot among the groups treated with BoNT / A, BoNT / A(0) or the medium liquid (Fig. 5C).

[0367] In summary, the non-catalytically active Clostridium neurotoxin is surprisingly able to alleviate acute neuropathic pain, thus suggesting that this neurotoxin has a general application in the treatment of pain.

[0368] Example 4 Treatment of chronic neuropathic pain using non-catalytically active BoNT (oxaliplatin rat model)

[0369] [Materials & Methods] Adult male Sprague-Dawley rats (180-210 g) were administered oxaliplatin (10 mg / kg) via i.pl. on day 0, prior to treatment with BoNT / A (100 pg / kg), BoNT / A(0) (100 pg / kg), or mediator (GPB), on day-2 (D-2) (n=11-12 / group). Positive controls were administered pregabalin on day 3 (n=12). Mechanical sensitivity (von Frey test) and heat (cold) sensitivity (cold plate test) of the experimental animals were assessed on days 3, 6, and 9.

[0370] [Results] This experiment showed that by administering non-catalytically active BoNT (BoNT / A(0)), the mechanosensitivity (Fig. 6B) and cold sensitivity (Fig. 6D) of the ipsilateral foot were reduced.

[0371] In summary, non-catalytically active Clostridium neurotoxins were surprisingly able to alleviate chronic neuropathic pain in different chemotherapy-induced pain patterns.

[0372] Example 5 Treatment of inflammatory pain using non-catalytically active BoNT (UV-B sunburn rat model)

[0373] [Materials & Methods] In human and rodent models, ultraviolet (UV)B radiation induces both mechanosensitive and thermal hyperalgesia. Adult male Wistar rats (180–210 g) were administered BoNT / A (100 pg / kg), BoNT / A(0) (100 pg / kg), or a medium (GPB; n=12 / group) via i.pl. injection. 24 hours later, the ipsilateral plantar surface of the paw was exposed to UVB radiation for approximately 5 minutes at a dose of 500 mJ / cm². Mechanosensitive animals were tested in the von Frei test after injection of BoNT / A, BoNT / A(0), or a medium 48 and 72 hours after UVB exposure. Another group of UVB-exposed animals were injected with a positive control (indomethacin) 48 hours later and tested in the von Frei test 1 hour after injection (n=12 / group).

[0374] [Results] Experiments showed that administration of non-catalytically active BoNT (BoNT / A(0)) reduced mechanosensitivity (Fig. 7B). In conclusion, the non-catalytically active Clostridium neurotoxin surprisingly alleviated inflammatory pain (e.g., acute inflammatory pain), thus confirming the general applicability of this neurotoxin for pain treatment.

[0375] The surprising discovery that non-catalytically active Clostridium neurotoxins alleviate inflammatory pain suggests their potential efficacy in treating underlying inflammatory conditions, including at least one symptom of the inflammatory condition, namely, associated pain. Thus, it is plausible that non-catalytically active Clostridium neurotoxins can be used to treat inflammatory conditions.

[0376] Example 6 Treatment of inflammatory pain (CFA-induced inflammatory pain pattern) using non-catalytically active chimeric BoNTs

[0377] [Materials & Methods] Before administering BoNT or mediator, the paw withdrawal threshold (PWT, g) of 70 adult male C57 / BL6 mice (22–26 g) was assessed using the incremental force von Fritz scale for three consecutive days. The mean of the last two days was considered the baseline. On day 0, under gas anesthesia, BoNT / XB (0.3 and 30 ng / kg), BoNT / XB(0) (0.3 and 30 ng / kg), BoNT / A (160 pg / kg), or mediator (840 µl / kg) was injected into the plantar pad of the left hind paw (n=10 / group). On day 2, before CFA injection, PWT was reassessed. Then, under isoflurane anesthesia, a fixed volume of 20 µL of CFA (1.5 mg / mL) was injected into the same hind paw. On day 3 (day 1 after CFA), one hour before the PWT assessment, animals assigned to the indomethacin group were orally administered indomethacin (10 mg / kg, n=9).

[0378] [Results] Experiments showed that the non-catalytically active chimeric BoNT (BoNT / XB(0)) containing the non-catalytically active BoNT / XL chain and transfer domain (BoNT / X LHN) and the BoNT / B receptor-binding domain BoNT / X (HC domain) was effective in treating inflammatory pain. More specifically, Figure 8 shows the decrease in mechanosensitivity following CFA-induced inflammatory pain in mice administered catalytically active BoNT / XB and non-catalytically active BoNT / XB(0) at a dose of 30 ng / kg. The decrease in sensitivity was equivalent to that of BoNT / A or the positive control (indomethacin).

[0379] The surprising finding that BoNT / XB(0) relieves inflammatory pain suggests its potential efficacy in treating underlying inflammatory conditions, such as those involving at least one symptom of the inflammatory condition, i.e., associated pain. Thus, further evidence is considered to support the credibility of non-catalytically active Clostridium neurotoxins for the treatment of inflammatory conditions.

[0380] Example 7 Treatment of Atopic Dermatitis Using Non-Catalytically Active Chimeric BoNTs One day prior to exposure to calcipotriol, mediator or BoNT / XB(0) (40 pg / mouse, 100 pg / mouse, or 400 pg / mouse) was subcutaneously administered to the mid-back of adult C57 / BL6 mice. Mice were then treated with calcipotriol for 5 consecutive days. At the end of the study, the animals were euthanized, and the back skin was collected, fixed, and processed for histological analysis. Epidermal thickness was assessed after hematoxylin and eosin staining. Immunolabeling was performed to confirm CD45+ cells.

[0381] Experiments showed that a non-catalytically active chimeric BoNT (BoNT / XB(0)) containing a non-catalytically active BoNT / XL chain and transfer domain (BoNT / X LHN) and a BoNT / B receptor-binding domain BoNT / X (HC domain) was effective in treating atopic dermatitis (a typical inflammatory condition). Results showed that skin thickness improved after BoNT / XB(0) administration. Skin thickness is an indicator of fibrosis (an inflammatory response to calcipotriol), and showed a statistically significant reduction in BoNT / XB(0)-treated animals. Furthermore, the anti-inflammatory effect of BoNT / XB(0) was confirmed by a decrease in the number of CD45-positive cells (CD45 transduces activation signals in inflammatory cells) in BoNT / XB(0)-treated animals.

[0382] Thus, it can be inferred that BoNT / XB(0) has anti-inflammatory properties and is therefore found to be effective in treating inflammatory disorders.

[0383] All publications mentioned in the foregoing specification are incorporated herein by reference. Various modifications and variations of the methods and systems described herein 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 conjunction with specific preferred embodiments, it should be understood that the claims should not be unduly limited to such specific embodiments. Indeed, it will be apparent to those skilled in the art of biochemistry and biotechnology or related fields that various modifications to the modes of carrying out the invention fall within the scope of the following claims. [Sequence List]

Claims

1. A polypeptide for use in the treatment of pain, wherein the polypeptide comprises a clostridium neurotoxin light chain (L chain), a clostridium neurotoxin transfer domain (HN domain) and / or a clostridium neurotoxin receptor binding domain (HC domain), wherein when the polypeptide comprises the clostridium neurotoxin L chain, the L chain is non-catalytically active.

2. A method for treating pain, the method comprising administering a polypeptide to a subject, wherein the polypeptide comprises a Clostridium neurotoxin light chain (L chain), a Clostridium neurotoxin transfer domain (HN domain) and / or a Clostridium neurotoxin receptor binding domain (HC domain), wherein when the polypeptide comprises the Clostridium neurotoxin L chain, the L chain is non-catalytically active.

3. Use of a polypeptide in the manufacture of a medicine for treating pain, wherein the polypeptide comprises a Clostridium neurotoxin light chain (L chain), a Clostridium neurotoxin transfer domain (HN domain) and / or a Clostridium neurotoxin receptor binding domain (HC domain), wherein when the polypeptide comprises a Clostridium neurotoxin L chain, the L chain is non-catalytically active.

4. The polypeptide used as claimed in claim 1, the method as claimed in claim 2, or the use as claimed in claim 3, wherein the polypeptide does not treat pain by promoting neuronal growth, promoting neuronal repair, or promoting both neuronal growth and repair.

5. A polypeptide for use in treating inflammatory disorders, wherein the polypeptide comprises a Clostridium neurotoxin light chain (L chain), a Clostridium neurotoxin transfer domain (HN domain), and / or a Clostridium neurotoxin receptor binding domain (HC domain), wherein when the polypeptide comprises a Clostridium neurotoxin L chain, the L chain is non-catalytically active.

6. A method for treating an inflammatory dysregulation, the method comprising administering a polypeptide to a subject, wherein the polypeptide comprises a Clostridium neurotoxin light chain (L chain), a Clostridium neurotoxin transfer domain (HN domain), and / or a Clostridium neurotoxin receptor binding domain (HC domain), wherein when the polypeptide comprises a Clostridium neurotoxin L chain, the L chain is non-catalytically active.

7. Use of a polypeptide in the manufacture of a medicine for treating inflammatory disorders, wherein the polypeptide comprises a Clostridium neurotoxin light chain (L chain), a Clostridium neurotoxin transfer domain (HN domain) and / or a Clostridium neurotoxin receptor binding domain (HC domain), wherein when the polypeptide comprises a Clostridium neurotoxin L chain, the L chain is non-catalytically active.

8. The polypeptide used as claimed in claim 5, the method as claimed in claim 6, or the use as claimed in claim 7, wherein the polypeptide does not treat inflammatory conditions by promoting neuronal growth, promoting neuronal repair, or promoting both neuronal growth and repair.

9. The polypeptide, method, or use as described in any of the preceding claims, wherein the polypeptide does not contain an additional catalytically active domain.

10. The polypeptide, method, or use as described in any of the preceding claims, wherein the polypeptide does not contain a therapeutic or diagnostic agent (e.g., a covalently or non-covalently linked therapeutic or diagnostic agent) other than the L chain, HN domain, and / or HC domain of Clostridium neurotoxin.

11. The polypeptide, method, or use as described in any of the preceding claims, wherein the polypeptide is not administered together with other therapeutic or diagnostic agents (e.g., sequentially or subsequently).

12. The polypeptide, method, or use as described in any of the preceding claims, wherein the polypeptide comprises a non-catalytically active Clostridium neurotoxin L chain.

13. The polypeptide, method, or use as described in any of the preceding claims, wherein the polypeptide comprises a Clostridium neurotoxin L chain, an HN domain, and an HC domain, wherein the L chain is non-catalytically active.

14. The polypeptide, method, or use as described in any of claims 1 to 12, wherein the polypeptide is substantially composed of a Clostridium neurotoxin light chain (L chain), a Clostridium neurotoxin transfer domain (HN domain), and / or a Clostridium neurotoxin receptor binding domain (HC domain), wherein when the polypeptide comprises or is substantially composed of a Clostridium neurotoxin L chain, the L chain is non-catalytically active.

15. The polypeptide, method, or use as described in any of claims 1 to 12 or 14, wherein the polypeptide is substantially composed of a Clostridium neurotoxin light chain (L chain) and a Clostridium neurotoxin transfer domain (HN domain), wherein the L chain is non-catalytically active.

16. The polypeptide, method, or use as described in any of the preceding claims, wherein the polypeptide is substantially composed of a Clostridium neurotoxin light chain (L chain), a Clostridium neurotoxin transfer domain (HN domain), and a Clostridium neurotoxin receptor binding domain (HC domain), wherein the L chain is non-catalytically active.

17. The polypeptide, method, or use as claimed in any of claims 1 to 12 or 14, wherein the polypeptide comprises a Clostridium neurotoxin light chain (L chain), a Clostridium neurotoxin transfer domain (HN domain), and / or a Clostridium neurotoxin receptor binding domain (HC domain), wherein when the polypeptide comprises or is composed of a Clostridium neurotoxin L chain, the L chain is non-catalytically active.

18. The polypeptide, method, or use as claimed in any of claims 1 to 12, 15, or 17, wherein the polypeptide comprises a Clostridium neurotoxin light chain (L chain) and a Clostridium neurotoxin transfer domain (HN domain), wherein the L chain is non-catalytically active.

19. The polypeptide, method, or use as described in any of claims 1 to 13, 14, 16, or 17, wherein the polypeptide comprises a Clostridium neurotoxin light chain (L chain), a Clostridium neurotoxin transfer domain (HN domain), and a Clostridium neurotoxin receptor binding domain (HC domain), wherein the L chain is non-catalytically active.

20. The polypeptide, method, or use as described in any of claims 1 to 12, 14, 15, 17, or 18, wherein the polypeptide does not contain both the HN domain and the HC domain of Clostridium neurotoxin.

21. The polypeptide, method, or use as described in any of the preceding claims, wherein the polypeptide does not further comprise a non-Clostridium catalytic domain.

22. The polypeptide, method, or use as described in any of the preceding claims, wherein the pain is chronic pain.

23. The polypeptide, method, or use as described in any of claims 1 to 21, wherein the pain is acute pain.

24. The polypeptide, method, or use as described in any of the preceding claims, wherein the pain is inflammatory pain.

25. The polypeptide, method, or use as described in claim 24, wherein the inflammatory pain is caused by or related to: sunburn, UV-induced damage, arthritis, autoimmune disease, connective tissue disorder, trauma, infection, neuritis, joint inflammation, or headache (preferably muscular / myogenic headache, vascular headache, hypertensive headache, hormonal headache, rebound headache, chronic sinusitis headache, organic headache, or ictal headache).

26. The polypeptide, method, or use as described in any of claims 1 to 23, wherein the pain is neuropathic pain.

27. The polypeptide, method, or use as described in claim 26, wherein the neuropathic pain is (or caused by or related to) neuralgia, deafferentation, complex regional pain syndrome (CRPS), or neuropathy (e.g., central or peripheral neuropathy).

28. The polypeptide, method, or use as described in any of claims 1 to 23, wherein the pain is mixed pain.

29. The polypeptide, method, or use as described in any of claims 1 to 23, wherein the pain is allodynia.

30. The polypeptide, method, or use as described in any of claims 1 to 23, wherein the pain is visceral pain.

31. The polypeptide, method, or use as described in claim 30, wherein the visceral pain is (or caused by or related to) functional visceral pain, chronic gastrointestinal inflammation, autoimmune pain, organic visceral pain, or treatment-induced visceral pain.

32. The polypeptide, method, or use as described in any of claims 1 to 23, wherein the pain is a headache (e.g., migraine).

33. The polypeptide, method, or use as described in claim 32, wherein the pain is a migraine.

34. The polypeptide, method, or use as described in claim 32, wherein the headache is caused by or related to: muscle / myogenic headache, vascular headache, hypertensive headache, hormonal headache, rebound headache, chronic sinusitis headache, organic headache, or epileptic headache.

35. The polypeptide, method, or use as described in any of claims 1 to 23, wherein the pain is postoperative pain.

36. The polypeptide, method, or use as described in any of claims 1 to 23, wherein the pain is referred pain.

37. The polypeptide, method, or use as described in any of claims 1 to 23, wherein the pain is somatic pain.

38. The polypeptide, method, or use as described in claim 37, wherein the pain is somatosensory pain caused by or related to: excessive muscle tension, repetitive motion disorder, muscle incoordination, myalgia, infection, or medication.

39. The polypeptide, method, or use as claimed in any of claims 1 to 23, wherein the pain is bladder pain syndrome, preferably wherein the bladder pain is caused by or related to interstitial cystitis.

40. The polypeptide, method, or use as described in any of claims 1 to 23, wherein the pain is phantom limb pain.

41. The polypeptide, method, or use as described in any of claims 5 to 21, wherein the inflammatory disorder is selected from one or more of the following: cystitis, endometriosis, rheumatoid arthritis, complex regional pain syndrome, and neuritis.

42. The polypeptide, method, or use as described in claim 41, wherein the cystitis is interstitial cystitis.

43. The polypeptide, method, or use as described in claim 41, wherein the neuritis is peripheral neuritis.

44. The polypeptide, method, or use as described in any of the preceding claims, wherein a single dose of the polypeptide administered is greater than 250 µg.

45. The polypeptide, method, or use as described in any of the preceding claims, wherein the single dose of the polypeptide administered is from 251 µg to 10 g.

46. ​​The polypeptide, method, or use as described in any of the preceding claims, wherein the single dose of the polypeptide administered is from 251 µg to 1 g.

47. The polypeptide, method, or use as described in any of the preceding claims, wherein the single dose of the polypeptide administered is 251-1000 µg.

48. The polypeptide, method, or use as described in any of the preceding claims, wherein the polypeptide is repeatedly administered (e.g., as part of a pain treatment regimen).

49. The polypeptide, method, or use as described in any of the preceding claims, wherein the polypeptide is administered intradermally.

50. The polypeptide, method, or use as claimed in any of the preceding claims, wherein the polypeptide comprises a polypeptide sequence having at least 70% sequence identity with any of the following 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 Clostridium neurotoxin L-chain, the L-chain is non-catalytically active.

51. The polypeptide, method, or use as claimed in any of the preceding claims, wherein the polypeptide comprises a polypeptide sequence having at least 80% sequence identity with any of the following 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 Clostridium neurotoxin L-chain, the L-chain is non-catalytically active.

52. The polypeptide, method, or use as claimed in any of the preceding claims, wherein the polypeptide comprises a polypeptide sequence having at least 90% sequence identity with any of the following 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 Clostridium neurotoxin L-chain, the L-chain is non-catalytically active.

53. The polypeptide, method, or use as described in any of the preceding claims, wherein the polypeptide comprises a polypeptide sequence of any of the following 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.

54. The polypeptide, method, or use as described in any of the preceding claims, wherein the polypeptide is a non-catalytically active BoNT / A.

55. The polypeptide, method, or use as claimed in any of the preceding claims, wherein the polypeptide is a modified Clostridium neurotoxin, such as a chimeric Clostridium neurotoxin or a hybrid Clostridium neurotoxin, preferably wherein the polypeptide does not contain the H chain of a natural Clostridium neurotoxin.

56. The polypeptide, method, or use as claimed in any of claims 1 to 53 or 55, wherein the polypeptide lacks the functional HCC or HC domain of Clostridium neurotoxin.

57. The polypeptide, method, or use as claimed in any of claims 1 to 53 or 55 to 56, wherein the polypeptide is a retargeted Clostridium neurotoxin comprising a non-Clostridium targeting moiety (TM) portion.

58. The polypeptide, method, or use as claimed in any of claims 1 to 53 or 55 to 56, wherein the polypeptide lacks the functional HC domain of Clostridium neurotoxin and also lacks any functionally equivalent exogenous ligand targeting moiety (TM).

59. The polypeptide, method, or use as described in any of the preceding claims, wherein the polypeptide is not expressed in the cells of the object, for example, wherein the use or method does not involve expressing the nucleic acid encoding the polypeptide in the cells of the object.

60. The polypeptide, method, or use as described in any of the preceding claims, wherein the polypeptide further comprises one or more non-clostridium neurotoxin sequences.

61. The polypeptide, method, or use as described in claim 60, wherein the one or more non-clostridium neurotoxin sequences do not bind to cell receptors.

62. The polypeptide, method, or use as described in claim 60 or 61, wherein the one or more non-clostridium neurotoxin sequences do not contain a ligand for a cell receptor.

63. The polypeptide, method, or use as claimed in any of claims 1 to 53, 55, or 59 to 62, wherein the polypeptide is a chimeric botulinum neurotoxin (BoNT) comprising a non-catalytically active BoNT / A light chain and a transfer domain, and a BoNT / B receptor-binding domain (HC domain).

64. A polypeptide, method, or use as claimed in any of claims 1 to 55 or 59 to 62, wherein the polypeptide comprises a modified BoNT / A HC domain comprising modifications of one or more amino acid residues selected from the following: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274, and THR 1277; wherein the modification is selected from: i. replacing an acidic surface-exposed amino acid residue with a basic amino acid residue; ii. replacing an acidic surface-exposed amino acid residue with a non-charged amino acid residue; iii. replacing a non-charged surface-exposed amino acid residue with a basic amino acid residue; iv. inserting a basic amino acid residue; and v. deleting an acidic surface-exposed amino acid residue.

65. The polypeptide, method, or use as claimed in any of claims 1 to 53 or 55 to 62, wherein the polypeptide comprises a non-catalytically active botulinum neurotoxin serotype X (BoNT / X) L chain, a BoNT / X HN domain, and / or a BoNT / X HC domain.

66. The polypeptide, method, or use as claimed in any of claims 1 to 53, 59 to 62, or 65, wherein the polypeptide is a chimeric botulinum neurotoxin (BoNT) comprising a non-catalytically active BoNT / X light chain and a transfer domain, and a receptor-binding domain (HC domain) derived from a different (i.e., non-BoNT / X) Clostridium neurotoxin.

67. The polypeptide, method, or use as claimed in any of claims 1 to 53, 59 to 62, or 65 to 66, wherein the polypeptide is a chimeric botulinum neurotoxin (BoNT) comprising a non-catalytically active BoNT / X light chain and a transfer domain, and a BoNT / B receptor-binding domain (HC domain).

68. The polypeptide, method, or use as described in any of claims 65 to 67, wherein the pain is inflammatory pain.

69. The polypeptide, method, or use as described in any of the preceding claims, wherein the polypeptide comprises Cys-(Xaa)a-Ile-Asp / Glu-Gly-Arg-(Yaa)b-Cys (SEQ ID NO: 71), wherein a=1-10 and b=4-15.

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