Treatment of autonomic disorders

Abstract

To provide improved treatment of autonomic disorders, that circumvent the clinical issues currently observed in patients, such as production of neutralizing antibodies against the toxin, as well as regional and / or systemic side effects.SOLUTION: The present invention relates to the treatment of autonomic disorders with a clostridial neurotoxin comprising a HCC domain from BoNT / B, BoNT / D, BoNT / D-C, BoNT / F or BoNT / G, wherein the dose of the clostridial neurotoxin to be administered to the patient is equivalent to or lower than the dose of BoNT / A used to treat the same autonomic disorder.SELECTED DRAWING: Figure 3

Description

Technical Field

[0001] The present invention relates to the treatment of autonomic neuropathy caused by neurotoxins.

Background Art

[0002] Bacteria of the genus Clostridium produce very potent and specific protein toxins that can harm the delivered nerve cells and other cells. Examples of such Clostridium toxins include neurotoxins produced by C. tetani (TeNT) and C. botulinum (BoNT) serotypes A to G, as well as those produced by C. baratii and C. butyricum.

[0003] Clostridium neurotoxins can be fatal because they cause muscle paralysis by inhibiting cholinergic transmission at the peripheral nervous system, particularly at the neuromuscular junction. In nature, Clostridium neurotoxins are synthesized as single-chain polypeptides and are post-translationally modified by a proteolytic event to form two polypeptide chains linked to each other by disulfide bonds. The cleavage occurs at a specific cleavage site, often called the activation site, which is located between cysteine residues that provide the inter-chain disulfide bond. This two-chain form is the active form of the toxin. The 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 (H N domain) and a C-terminal targeting component (H C domain). The cleavage site is located between the L chain and the H N domain.

[0004] The mechanism of action of Clostridium neurotoxins depends on the following five individual steps: (1) binding of the H C domain to the cell membrane of its target nerve cell, followed by (2) internalization of the bound toxin into the cell via endosomes, (3) H NTranslocation of the L chain into the cytosol via the endosomal membrane by the domain, (4) protein cleavage of an intracellular transport protein known as a SNARE protein by the L chain that provides a non-cytotoxic protease function, and (5) inhibition of cell secretion from target cells.

[0005] In this series of events, SNARE proteins (Soluble N-ethylmaleimide-Sensitive Factor Attachment protein REceptor) are essential for the fusion of intracellular vesicles and thus the secretion of molecules via vesicular transport from cells. Examples of SNARE proteins present in nerve cells include, in particular, SNAP-25, VAMP, or syntaxin. On the other hand, the non-cytotoxic protease function of the L chain is a zinc-dependent endopeptidase activity that exhibits high substrate specificity for SNARE proteins. Therefore, when delivered to nerve target cells, the non-cytotoxic protease of Clostridium neurotoxin inhibits the release of neurotransmitters by cleaving its substrate SNARE protein, resulting in nerve paralysis.

[0006] Due to its unique properties, Clostridium neurotoxins such as botulinum toxin have been successful in a wide range of therapeutic applications, particularly for movement disorders and autonomic neuropathies, for example in their use to return the activity of overactive nerve endings to normal levels. To date, at least seven antigenically distinct BoNT serotypes, namely BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, and BoNT / G, have been described (Rossetto, O. et al., "Botulinum neurotoxins: genetic, structural and mechanistic insights." Nature Reviews Microbiology 12.8 (2014): 535-549). Despite this diversity, BoNT / A remains the serotype of choice for treatment, with three commonly available commercial formulations (Botox®, Dysport®, and Xeomin®), while there is only one commercially available BoNT / B product (Neurobloc® / Myobloc®). To date, these BoNT / A and BoNT / B products are the only two BoNT serotypes that are toxins purified from Clostridium strains and approved by regulatory authorities for use in humans, and their uses include, in particular, spasticity, bladder dysfunction, hyperhidrosis (for BoNT / A) (see, e.g., https: / / www.medicines.org.uk / emc / medicine / 112, https: / / www.medicines.org.uk / emc / medicine / 870, https: / / www.medicines.org.uk / emc / medicine / 2162, the disclosures of which are hereby incorporated by reference in their entireties), to cervical dystonia (for BoNT / B) (see, e.g., https: / / www.medicines.org.uk / emc / medicine / 20568, herein incorporated by reference in its entirety).

[0007] However, the limitations currently seen in these therapeutic uses include the generation of neutralizing antibodies in patients, which renders future treatments ineffective. There have been few reports of antigenicity problems with Neurobloc® / MyoBloc®; however, such antibody responses have hitherto been reported mainly with BoNT / A products, which are still the generally chosen botulinum toxins. These immunological responses have been confirmed to correlate directly with the dose of the toxin administered (Lange, O., et al., Neutralizing antibodies and secondary therapy failure after treatment with botulinum toxin type A: much ado about nothing? Clin Neuropharmacol, 2009, 32, 213-218). There is also a risk of side effects due to the diffusion of the toxin to other areas of the body, and the potential for toxin diffusion is directly related to the injection volume.

[0008] Since both the generation of neutralizing antibodies and the diffusion of the toxin are directly related to the injection volume, there is a need in the art to treat patients with products such as alternative BoNT serotypes that can achieve the same level of therapeutic effect with a lower dose.

[0009] A related problem is the difference in the mode of operation of the various BoNT serotypes, which is thought to result in differences in the therapeutic effect against the same disorder.

[0010] In fact, to exert clinical activity, botulinum toxin needs to enter the nerve terminals of its target cells. To do so, BoNT has a receptor-binding domain (BoNT-H) that is clearly defined in the literature CIt enters neurons by binding to its specific receptor via (Schiavo, G., Matteoli, M. & Montecucco, C. Neurotoxins affecting neuroexocytosis, Physiol Rev, 2000, 80, 717-766). Receptor binding generally determines the efficacy and specificity of BoNT for recognizing neurons. BoNT / B, D-C, and G share the two homologous synaptic vesicle proteins synaptotagmin I and II (Syt I / II) as receptors, while BoNT / A, E, D, and F use another synaptic vesicle protein, SV2. In this regard, it should be noted that the binding affinity of BoNT serotypes for protein receptors may vary by species. For example, BoNT / D-C and / G show a much higher affinity for SytII than for SytI at rodent motor nerve terminals, but in humans, they have a higher affinity for SytI than for SytII, and this difference is due to unique amino acid changes between the rodent and human SytII sequences. As a result of this amino acid change, human SytII is much less efficient at mediating the entry of BoNT / B, / D-C, and / G compared to mouse SytII. This means that in humans, high-affinity receptors for BoNT / B, / D-C, and / G appear to be limited to the minor receptor SytI, at least in motor neurons.All of these findings could explain the clinical observation that much higher doses of BoNT / B (which binds to different receptors) than BoNT / A are required to achieve the same level of therapeutic effect in patients with cervical dystonia (Brashear A. et al., Safety and efficacy of NeuroBloc (botulinum toxin type B) in type A-resistant cervical dystonia, 1999, Neurology 53, 1431-1438, Pappert, E.J. & Germanson, T. Botulinum toxin type B vs. type A in toxin-naive patients with cervical dystonia: Randomized, double-blind, noninferiority trial, 2008 Mov Disord 23, 510-517). In particular, for use at the neuromuscular junction, a conversion ratio of 1:40 has been suggested between BOTOX® and MyoBloc. TM (Dressler et al., Botulinum toxin type B for treatment of axillar hyperhidrosis., 2002, J. Neurol. 249,1729-1732; Comella et al., 2005).

[0011] In addition to protein receptors, all BoNT serotypes require lipid co-receptor gangliosides, which are abundant on the surface of nerve cells and can vary between serotypes, and cleave different SNARE protein substrates, which can further affect the therapeutic effect depending on the type of target cell. Indeed, the L-chain proteases of BoNT / B, BoNT / D, BoNT / F, and BoNT / G cleave VAMP, while the L-chain proteases of BoNT / A and BoNT / E cleave SNAP25, and the L-chain protease of BoNT / C cleaves both SNAP25 and syntaxin.

[0012] The initial therapeutic use of botulinum neurotoxins relied on the inhibition of acetylcholine release at the neuromuscular junctions of skeletal muscles to treat neuromuscular conditions such as dystonia and spasm. However, these neurotoxins have been found to be effective on glands and smooth muscles by inhibiting acetylcholine release at autonomic nerve endings, and thus, can be used for the treatment of various autonomic disorders such as hyperhidrosis, sialorrhea, overactive bladder, etc.

[0013] The recommended dosage of Botox® (also known as Onabotulinumtoxin A) for the treatment of hyperhidrosis is 50 units per axilla (https: / / www.medicines.org.uk / emc / medicine / 112), which is a dosage of approximately 0.365 ng as the 150 kD BoNT / A toxin. Neurobloc® / Myobloc® (also known as Rimabotulinumtoxin B) is not approved for the treatment of hyperhidrosis, but dosages in the range of 2000 to 4000 units, i.e., in the range of approximately 4 to 40 ng as the 150 kD BoNT / A toxin, have been disclosed in the literature. However, such high dosages are not without side effects at both local and systemic levels, such as dry mouth, dysphagia, and heartburn (Dressler D. and Eleopra R., Clinical use of non-A botulinum toxins: botulinum toxin type B., Neurotoxicity research 9.2-3 (2006): 121-125, Tintner R. et al., Autonomic function after botulinum toxin type A or B: a double-blind, randomized trial., Neurology 65.5 (2005): 765-767, Birklein F. et al. Botulinum toxin type B blocks sudomotor function effectively: a 6 month follow up., Journal of investigative dermatology 121.6 (2003): 1312-1316). Nevertheless, it has not been studied whether a therapeutic effect on autonomic neuropathy can be achieved with low dosages of the toxin. Summary of the Invention Problems to be Solved by the Invention

[0014] An object of the present invention is to provide an improved treatment for autonomic neuropathy that produces neutralizing antibodies against toxins and avoids clinical problems currently seen in patients, such as local and / or systemic side effects.

Means for Solving the Problems

[0015] [Aspect 1] A Clostridium neurotoxin for use in the treatment of autonomic neuropathy in a human patient, comprising an H domain derived from BoNT / B, BoNT / F, BoNT / D, BoNT / D-C, or BoNT / G, wherein the dose of the Clostridium neurotoxin to be administered to the patient is equal to or less than the dose of BoNT / A for treating the autonomic neuropathy. CC Clostridium neurotoxin. [Aspect 2] Use of a Clostridium neurotoxin in the manufacture of a drug for treating autonomic neuropathy in a human patient, wherein the Clostridium neurotoxin comprises an H domain derived from BoNT / B, BoNT / F, BoNT / D, BoNT / D-C, or BoNT / G, and the dose of the Clostridium neurotoxin to be administered to the patient is equal to or less than the dose of BoNT / A for treating the autonomic neuropathy. CC Use. [Aspect 3] A method for treating autonomic neuropathy in a human patient in need thereof, comprising administering to the patient a Clostridium neurotoxin comprising an H domain derived from BoNT / B, BoNT / F, BoNT / D, BoNT / D-C, or BoNT / G in a dose equal to or less than the dose of BoNT / A for treating the autonomic neuropathy. CC Method. [Aspect 4] The dose of the Clostridium neurotoxin is from about 1 / 1.1 to about 1 / 100 of the dose of BoNT / A for treating the autonomic neuropathy, and the dose is preferably quantified in nanogram units, the Clostridium neurotoxin for the use according to Aspect 1, the use according to Aspect 2, or the method according to Aspect 3. [Aspect 5] The dosage of the Clostridium neurotoxin is in the range of about 0.00025 ng to about 3 ng, the Clostridium neurotoxin, use, or method of use according to any of the preceding aspects. [Aspect 6] The autonomic neuropathy is selected from smooth muscle disorders, hypersecretion disorders, respiratory disorders, inflammatory disorders involving autonomic elements, neuroendocrine disorders, and other autonomic neuropathies directly related to central nervous disorders, the Clostridium neurotoxin, use, or method of use according to any of the preceding aspects. [Aspect 7] The said H CC domain is the H CC domain of BoNT / B or the H CC domain of BoNT / F, the Clostridium neurotoxin, use, or method of use according to any of the preceding aspects. [Aspect 8] The said H CC domain of BoNT / B is the H CC domain of natural BoNT / B, and contains a mutation of at least one amino acid residue that increases its binding affinity for the human Syt II receptor by at least 50% compared to the natural BoNT / B H [Aspect 9] The mutation of the at least one amino acid residue is selected from the group consisting of 1118M, 1183M, 1191M, 1191I, 1191Q, 1191T, 1199Y, 1199F, 1199L, 1201V, 1191C, 1191V, 1191L, 1191Y, 1199W, 1199E, 1199H, 1178Y, 1178Q, 1178A, 1178S, 1183C, 1183P, and any combination thereof, the Clostridium neurotoxin, use, or method of use according to Aspect 8. [Aspect 10] The mutation of the at least one amino acid residue consists of the mutation of two amino acid residues, 1191M and 1199Y, the Clostridium neurotoxin, use, or method of use according to Aspect 8 or 9. [Aspect 11] The said H CC domain of BoNT / F is the H CCA Clostridium neurotoxin, use, or method of use according to any of aspects 1 to 7, comprising a mutation of at least one amino acid residue that increases its binding affinity for one or more gangliosides as compared to the domain. [Aspect 12] The Clostridium neurotoxin, use, or method of use according to aspect 11, wherein the ganglioside is selected from GD1a and / or GM1a, and / or the mutation of the amino acid residue is 1241K. [Aspect 13] The Clostridium neurotoxin, use, or method of use according to any of aspects 1 to 10, wherein the Clostridium neurotoxin is a BoNT / B neurotoxin. [Aspect 14] The Clostridium neurotoxin, use, or method of use according to any of aspects 1 to 7, 11 or 12, wherein the Clostridium neurotoxin is a BoNT / F neurotoxin. [Aspect 15] The Clostridium neurotoxin, use, or method of use according to any of the preceding aspects, wherein the Clostridium neurotoxin is a chimeric neurotoxin. [Aspect 16] The chimeric neurotoxin comprises the H C domain of BoNT / B and the LH N domain of BoNT / A, for the use of the Clostridium neurotoxin according to aspect 15, use, or method. [Aspect 17] The chimeric neurotoxin comprises the LH N domain derived from a first BoNT / F subtype and an activation loop derived from a second BoNT / F subtype, wherein the second BoNT / F subtype is different from the first BoNT / F subtype, for the use of the Clostridium neurotoxin according to aspect 15, use, or method. [Aspect 18] The LH N domain is the LH N domain of BoNT / F7, and / or the activation loop is the BoNT / F1 activation loop, for the use of the Clostridium neurotoxin according to aspect 17, use, or method. [Aspect 19] The Clostridium neurotoxin is the Clostridium neurotoxin, use, or method for the use according to any of the preceding aspects, comprising an amino acid sequence selected from any of SEQ ID NOs: 2 to 13, or an amino acid sequence having at least 70% sequence identity thereto, preferably at least 90% or 95% sequence identity thereto. [Aspect 20] The Clostridium neurotoxin is the Clostridium neurotoxin, use, or method for the use according to any of the preceding aspects, comprising: a. the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence having at least 70% sequence identity thereto, preferably at least 90% or 95% sequence identity thereto, or b. the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence having at least 70% sequence identity thereto, preferably at least 90% or 95% sequence identity thereto, or c. the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 70% sequence identity thereto, preferably at least 90% or 95% sequence identity thereto, or d. the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having at least 70% sequence identity thereto, preferably at least 90% or 95% sequence identity thereto, or e. the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having at least 70% sequence identity thereto, preferably at least 90% or 95% sequence identity thereto. In one aspect, the present invention is a Clostridium neurotoxin for use in the treatment of autonomic neuropathy in a human patient, comprising an H CC domain derived from BoNT / B, BoNT / D, BoNT / D-C, BoNT / F, or BoNT / G, and the dosage of the Clostridium neurotoxin to be administered to the patient is equal to or less than the dosage of BoNT / A for treating the autonomic neuropathy. In other words, the present invention is a method for treating the autonomic neuropathy of a human patient in need thereof, comprising an H CCA method is provided that includes administering to the patient a Clostridium neurotoxin containing an H domain in an amount equal to or less than the dosage of BoNT / A that treats the autonomic neuropathy. More precisely, the present invention relates to an H derived from BoNT / B, BoNT / D, BoNT / D-C, BoNT / F, or BoNT / G for manufacturing a drug for treating autonomic neuropathy in a human patient CC Use of a Clostridium neurotoxin containing an H domain, wherein the dosage of the Clostridium neurotoxin to be administered to the patient is equal to or less than the dosage of BoNT / A that treats the autonomic neuropathy.

[0016] In a particular aspect, the present invention is a Clostridium neurotoxin for use in treating autonomic neuropathy in a human patient, the Clostridium neurotoxin containing an H domain derived from BoNT / B, BoNT / D, BoNT / D-C, BoNT / F, or BoNT / G CC and the dosage of the Clostridium neurotoxin to be administered to the patient is from about 1 / 1.1 to about 1 / 100, preferably from about 1 / 1.3 to about 1 / 90 of the dosage of BoNT / A that treats the autonomic neuropathy. In other words, the present invention is a method for treating autonomic neuropathy in a human patient in need thereof, the method including administering to the patient a Clostridium neurotoxin containing an H domain derived from BoNT / B, BoNT / D, BoNT / D-C, BoNT / F, or BoNT / G CC in an amount from about 1 / 1.1 to about 1 / 100, preferably from about 1 / 1.3 to about 1 / 90 of the dosage of BoNT / A that treats the autonomic neuropathy. More precisely, the present invention relates to an H derived from BoNT / B, BoNT / D, BoNT / D-C, BoNT / F, or BoNT / G for manufacturing a drug for treating autonomic neuropathy in a human patient CC Use of a Clostridium neurotoxin containing an H domain, wherein the dosage of the Clostridium neurotoxin to be administered to the patient is from about 1 / 1.1 to about 1 / 100, preferably from about 1 / 1.3 to about 1 / 90 of the dosage of BoNT / A that treats the autonomic neuropathy. The dosage is preferably quantified in nanogram units.

[0017] Furthermore, in certain embodiments, the invention provides a Clostridium neurotoxin for use in treating autonomic neuropathy in a human patient, the Clostridium neurotoxin comprising an H CC domain from BoNT / B, BoNT / D, BoNT / D-C, BoNT / F, or BoNT / G, wherein the Clostridium neurotoxin should be administered to the patient at a dosage in the range of about 0.00025 ng to about 3 ng. In other words, the invention provides a method of treating autonomic neuropathy in a human patient in need thereof, the method comprising administering to the patient a Clostridium neurotoxin comprising an H CC domain from BoNT / B, BoNT / D, BoNT / D-C, BoNT / F, or BoNT / G at a dosage in the range of about 0.00025 ng to about 3 ng.

[0018] More specifically, the invention relates to the use of a Clostridium neurotoxin comprising an H CC domain from BoNT / B, BoNT / D, BoNT / D-C, BoNT / F, or BoNT / G for the manufacture of a medicament for treating autonomic neuropathy in a human patient, wherein the dosage of the Clostridium neurotoxin to be administered to the patient is in the range of about 0.00025 ng to about 3 ng.

[0019] The invention is first based on the unexpected finding by the inventors that BoNT / B has at least an effect similar to BoNT / A on human smooth muscle tissue.

[0020] This discovery is unexpected because, as described above, much higher doses of BoNT / B (Neurobloc® / Myobloc®) are required for the treatment of skeletal muscle disorders compared to BoNT / A (e.g., Botox®). Only higher doses of BoNT / B (Neurobloc® / Myobloc®) than BoNT / A (e.g., Botox®) have been tested and reported in the literature for the treatment of, for example, hyperhidrosis involving smooth muscle, which seems to be based on this clinical observation.

[0021] The difference in the efficacy of this BoNT / B in these two tissues is hypothesized to be due to the fact that disorders of smooth muscle and skeletal muscle do not actually depend on the same neural pathway. Smooth muscle contraction is indeed driven by the autonomic nervous system, while skeletal muscle contraction is driven by the somatic nervous system. Many other disorders treated with botulinum neurotoxins, such as cervical dystonia and spasticity, are caused by excessive contraction of skeletal muscle, while other disorders such as overactive bladder (OAB) or neurogenic detrusor overactivity (NDO) are caused by excessive contraction of smooth muscle.

[0022] The somatic nervous system (SoNS or voluntary nervous system) is a part of the peripheral nervous system related to the voluntary regulation of skeletal muscles of body movement. The SoNS consists of afferent nerves and efferent nerves. The SoNS afferent nerves are involved in relaying sensations from the body to the central nervous system (CNS), and the SoNS efferent nerves are involved in sending commands from the CNS to the body and stimulating muscle contraction, which includes all non-sensory nerve cells connected to skeletal muscles and the skin.

[0023] The autonomic nervous system (ANS) is another component of the peripheral nervous system and consists of afferent and efferent nerves. The ANS is composed of two branches, the sympathetic nervous system (SNS) and the parasympathetic nervous system (PNS). The SNS controls more active responses such as increased heart rate and blood pressure. In addition to smooth muscle, the ANS also controls secretions such as sweat, saliva, and tears.

[0024] Smooth muscle is found within the urinary system (e.g., bladder, ureter), digestive system (e.g., gastric wall, gastrointestinal tract, intestine, sphincter of Oddi, anal sphincter, trachea, bile duct), genital system (e.g., prostate, uterus), airways, vascular system (e.g., vessel wall, aorta, artery, arteriole, vein), and iris of the eye. Contraction of smooth muscle is involved in numerous body functions such as the release of urine from the bladder, movement of food through the digestive tract, regulation of airflow in the lungs, regulation of blood pressure in arteries and veins, and constriction of the pupil size.

[0025] The synaptic nerve terminals of cholinergic neurons that supply eccrine sweat glands, salivary glands, and lacrimal glands can be targeted by botulinum neurotoxins. Neurological disorders associated with sweating or increased secretory activity include hyperhidrosis, particularly focal hyperhidrosis of the palms, axillae, or feet, gustatory sweating (Frey's syndrome), increased lacrimation during eating (crocodile tears syndrome), and excessive salivation (sialorrhea, salivary flow).

[0026] While not wishing to be bound by theory, the difference in efficacy observed between BoNT / A and BoNT / B in the somatic system with respect to the autonomic nervous system is hypothesized to be related to the fact that BoNT / A and BoNT / B differ with respect to their protein receptors (SV2 and Syt I / II, respectively) and / or their target SNAREs (SNAP25 and VAMP, respectively).

[0027] Based on this assumption, further, other BoNT serotypes that use Syt I / II as a protein receptor and / or cleave VAMP, such as Bont / D, BoNT / D-C, BoNT / F, or BoNT / G, are hypothesized to exhibit at least in humans, if not with better potency, a potency similar to BoNT / B against the smooth muscle / autonomic nervous system.

[0028] Accordingly, one aspect of the present invention is a Clostridium neurotoxin for use in the treatment of autonomic neuropathy in a human patient, the Clostridium neurotoxin comprising an H CC domain derived from BoNT / B, BoNT / D, BoNT / D-C, BoNT / F, or BoNT / G, and the dosage of the Clostridium neurotoxin to be administered to the patient is equal to or less than the dosage of BoNT / A for treating the autonomic neuropathy. In other words, the present invention is a method for treating autonomic neuropathy in a human patient in need thereof, the method comprising administering to the patient a Clostridium neurotoxin comprising an H CC domain derived from BoNT / B, BoNT / D, BoNT / D-C, BoNT / F, or BoNT / G at a dosage equal to or less than the dosage of BoNT / A for treating the autonomic neuropathy. More precisely, the present invention relates to the use of a Clostridium neurotoxin comprising an H CC domain derived from BoNT / B, BoNT / D, BoNT / D-C, BoNT / F, or BoNT / G for the manufacture of a medicament for the treatment of autonomic neuropathy in a human patient, wherein the dosage of the Clostridium neurotoxin to be administered to the patient is equal to or less than the dosage of BoNT / A for treating the autonomic neuropathy.

[0029] In one embodiment, the autonomic neuropathy treated by the Clostridium neurotoxin of the present invention is selected from the group consisting of smooth muscle disorders, hypersecretion disorders, respiratory disorders, inflammatory disorders involving autonomic elements, neuroendocrine disorders, and other autonomic neuropathies directly related to central nervous disorders.

[0030] Examples of such autonomic neuropathies include, without limitation: ● Smooth muscle disorders, especially those caused by excessive, abnormal, and / or prolonged muscle contractions (spastic disorders), for example, including the following ○ Neurogenic detrusor overactivity (NDO), overactive bladder (OAB), especially idiopathic OAB (iOAB), bladder spasm, detrusor-sphincter dyssynergia (DSD), urinary incontinence, urinary retention, nocturia, urge incontinence, frequency of urination, and other urinary disorders, Oddi sphincter dysfunction, esophageal spasm, intestinal spasm, achalasia, gastric paresis, spastic colitis, anal fissure, constipation, occasional diarrhea, dysphagia, oropharyngeal dysphagia, swallowing disorder, and other gastrointestinal disorders ○ Vascular and cardiovascular disorders such as Raynaud's disease, anastomotic thrombosis, atrial fibrillation after cardiac surgery, orthostatic hypotension, and blood pressure disorders ○ Prostate disorders such as benign prostate hyperplasia (BPH), prostatitis, prostatodynia, and prostate enlargement, and ○ Sexual disorders: erectile dysfunction, priapism, ejaculation disorders, vaginismus ● Hypersecretion disorders, for example, hyperhidrosis (axillary, palmar, plantar hyperhidrosis, diffuse sweating, Frey syndrome, etc.), excessive salivation (salivary flow, drooling), gustatory sweating, excessive lacrimation, crocodile tear syndrome, excessive mucus secretion, bromhidrosis, gastric acid secretion, acne ● Respiratory disorders with hypersecretion and / or muscle components, for example, rhinorrhea, chronic rhinitis, asthma, chronic obstructive pulmonary disease (COPD), bronchial hypersensitivity, wheezing, involuntary shortness of breath, apnea episodes, and other respiratory disorders ● Inflammatory disorders with autonomic components, for example, otitis media, pruritus, urticaria, inflammatory bowel syndrome ● Neuroendocrine disorders: diabetes, hyperinsulinemia, hyperglucagonemia, pancreatic disorders, thyroid disorders, hypocalcemia, hyperthyroidism, metabolic disorders, excessive lipolysis, and ● Parkinson's disease and other autonomic disorders directly related to central nervous disorders such as cerebellar / pyramidal function.

[0031] In a preferred embodiment, the autonomic neuropathy treated by the present invention is a smooth muscle disorder selected from the group consisting of urinary disorders, gastrointestinal disorders, vascular and cardiovascular disorders, prostate disorders, and sexual disorders.

[0032] As used herein, the term "Clostridium neurotoxin" means any polypeptide that enters a nerve cell and inhibits the release of neurotransmitters. This process involves the binding of the neurotoxin to low or high affinity receptors, the internalization of the neurotoxin, the translocation of the endopeptidase portion of the neurotoxin into the cytoplasm, and the enzymatic modification of the neurotoxin substrate. More specifically, the term "neurotoxin" includes any polypeptide produced by Clostridium bacteria (Clostridium neurotoxin) that enters a nerve cell and inhibits the release of neurotransmitters, and such polypeptides produced by recombinant or chemical techniques. This two-chain form is the active form of the toxin. The two chains are referred to as 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. Preferably, the Clostridium neurotoxin is botulinum neurotoxin (BoNT).

[0033] BoNT serotypes A through G are distinguishable based on inactivation by specific neutralizing antisera, and such classification by serotype correlates with the percentage sequence identity at the amino acid level. The BoNT protein of a given serotype is further classified into different subtypes based on the percentage amino acid sequence identity.

[0034] Examples of the BoNT / A neurotoxin amino acid sequence are provided as SEQ ID NO: 1 (UniProt accession number A5HZZ9). Examples of the BoNT / B neurotoxin amino acid sequence are provided as SEQ ID NO: 2 (UniProt accession number B1INP5). Examples of the BoNT / C neurotoxin amino acid sequence are provided as SEQ ID NO: 3 (UniProt accession number P18640). Examples of the BoNT / D neurotoxin amino acid sequence are provided as SEQ ID NO: 4 (UniProt accession number P19321). Examples of the BoNT / E neurotoxin amino acid sequence are provided as SEQ ID NO: 5 (accession number WP_003372387). Examples of the BoNT / F neurotoxin amino acid sequence are provided as SEQ ID NO: 6 (UniProt accession number Q57236) or SEQ ID NO: 11 (UniProt / UniParc accession number UPI0001DE3DAC). Examples of the BoNT / G neurotoxin amino acid sequence are provided as SEQ ID NO: 7 (accession number WP_039635782). Examples of the BoNT / D-C neurotoxin amino acid sequence are provided as SEQ ID NO: 8 (accession number BAM65681).

[0035] As used herein, the term "H" C "domain" refers to a functionally distinct region of the heavy chain of the neurotoxin, having a molecular weight of approximately 50 kDa, that enables binding of the neurotoxin to a receptor located on the surface of the target cell. The H C domain consists of two structurally distinct subdomains, the "H" CN "subdomain" (the N-terminal portion of the H C domain) and the "H" CC "subdomain" (the C-terminal portion of the H C domain, also referred to as the H CC domain), each having a molecular weight of approximately 25 kDa. The H CC domain can bind to the Clostridium neurotoxin protein receptor.

[0036] As used herein, the term "LH" N "domain" is different from the H C domain and consists of an endopeptidase domain ("L" or "light chain") and a domain (the H of the heavy chain) involved in translocation of the endopeptidase into the cytoplasm.N It shows a neurotoxin consisting of an endopeptidase domain (denoted as "L" or "light chain") that can cleave SNARE proteins.

[0037] L, H N , H CN , and H CC Examples of the domain are shown in Table 1.

Table 1

[0038] The above-mentioned reference sequences should be regarded as a guide, and slight variations may occur depending on the sub-serotype. As an example, in US2007 / 0166332 (the entire disclosure of which is incorporated herein by reference), slightly different Clostridium sequences are cited.

[0039] The term "activation loop" refers to a polypeptide domain containing a protein cleavage site. The activation loop of the neurotoxin is described in, for example, WO2016156113 (the entire disclosure of which is incorporated herein by reference) in the art.

[0040] In one embodiment of the present invention, the H CC domain of the Clostridium neurotoxin consists of, or includes, an amino acid sequence selected from the group consisting of: - Amino acid residues 1082 to 1291 of SEQ ID NO: 2, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, - Amino acid residues 1083 to 1276 of SEQ ID NO: 4, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, - Amino acid residues 1088 to 1278 of SEQ ID NO: 6, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, - The amino acid residues 1077 to 1268 of SEQ ID NO: 11, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, - The amino acid residues 1090 to 1297 of SEQ ID NO: 7, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, and - The amino acid residues 1092 to 1285 of SEQ ID NO: 8, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

[0041] As used throughout this specification, the term "percent sequence identity" between two or more amino acid sequences is a function of the number of identical amino acids at the same positions shared by the aligned amino acid sequences. Thus, the percent identity used herein can be calculated by multiplying by 100 the number of identical amino acids at each position in the alignment, divided by the total number of amino acids in the aligned sequences. In calculating percent sequence identity, consideration may be given to the number of gaps and the length of each gap that needs to be introduced to optimize the alignment of two or more sequences. The sequence comparison and determination of percent identity between two or more sequences can be performed using specific mathematical algorithms well known to those skilled in the art, particularly global alignment mathematical algorithms (such as those described in Needleman and Wunsch, J. Mol. Biol. 48(3), 443-453, 1972, etc.).

[0042] In a preferred embodiment, the H CC domain consists of, or comprises, an amino acid sequence selected from the group consisting of: - The amino acid residues 1082 to 1291 of SEQ ID NO: 2, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, - The amino acid residues 1088 to 1278 of SEQ ID NO: 6, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, and - The amino acid residues 1077 to 1268 of SEQ ID NO: 11, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

[0043] In a preferred embodiment, the H CC domain consists of, or comprises, an amino acid sequence corresponding to the amino acid residues 1082 to 1291 of SEQ ID NO: 2, or an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

[0044] In a preferred embodiment, the H CC domain consists of, or comprises, an amino acid sequence corresponding to the amino acid residues 1088 to 1278 of SEQ ID NO: 6, or an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

[0045] In a preferred embodiment, the H CC domain consists of, or comprises, an amino acid sequence corresponding to the amino acid residues 1077 to 1268 of SEQ ID NO: 11, or an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

[0046] In one embodiment, the Clostridium neurotoxin consists of, or comprises, an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 11.

[0047] In a preferred embodiment, the Clostridium neurotoxin consists of or comprises an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any of SEQ ID NO: 2, SEQ ID NO: 6, or SEQ ID NO: 11.

[0048] In a preferred embodiment, the Clostridium neurotoxin consists of or comprises an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 2.

[0049] In a preferred embodiment, the Clostridium neurotoxin consists of or comprises an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 6.

[0050] In a preferred embodiment, the Clostridium neurotoxin consists of or comprises an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 11.

[0051] In a more preferred embodiment, the Clostridium neurotoxin is a BoNT / B neurotoxin. Preferably, the BoNT / B Clostridium neurotoxin consists of or comprises an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 2.

[0052] In a more preferred embodiment, the Clostridium neurotoxin is a BoNT / F neurotoxin. Preferably, the BoNT / F Clostridium neurotoxin consists of or comprises an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 6 or SEQ ID NO: 11.

[0053] In one embodiment, the Clostridium neurotoxin is a chimeric neurotoxin.

[0054] As used herein, the term "chimeric neurotoxin" means a neurotoxin comprising one or more domains derived from a first neurotoxin and one or more domains derived from a second neurotoxin. For example, a chimeric neurotoxin may comprise an LH N domain derived from a first neurotoxin serotype or subtype and an H C domain derived from a second neurotoxin serotype or subtype. Another example of a chimeric neurotoxin is an LH N H CN domain derived from a first neurotoxin serotype or subtype and an H CC domain derived from a second neurotoxin serotype or subtype. Other examples of chimeric neurotoxins are neurotoxins comprising an LH N domain derived from a first neurotoxin serotype or subtype and an activation loop derived from a second neurotoxin serotype or subtype. Examples of chimeric neurotoxins are described in WO2017191315 and WO2016156113, the disclosures of which are incorporated herein by reference in their entirety.

[0055] In one embodiment, the Clostridium neurotoxin is a chimeric neurotoxin comprising an H C domain derived from BoNT / B and an LH N domain derived from BoNT / A, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, or BoNT / D-C.

[0056] In one embodiment of the chimeric neurotoxin according to the present invention, the H C domain consists of, or comprises, an amino acid sequence selected from the group consisting of: - amino acid residues 860 to 1291 of SEQ ID NO: 2, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, - The amino acid residues 864 to 1276 of SEQ ID NO: 4, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, - The amino acid residues 866 to 1278 of SEQ ID NO: 6, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, - The amino acid residues 863 to 1268 of SEQ ID NO: 11, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, - The amino acid residues 865 to 1297 of SEQ ID NO: 7, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, and - The amino acid residues 864 to 1285 of SEQ ID NO: 8, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, and the LH N domain consists of, or includes, an amino acid sequence selected from the group consisting of: - The amino acid residues 1 to 872 of SEQ ID NO: 1, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, - The amino acid residues 1 to 859 of SEQ ID NO: 2, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, - The amino acid residues 1 to 867 of SEQ ID NO: 3, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, - The amino acid residues 1 to 863 of SEQ ID NO: 4, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, - The amino acid residues 1 to 846 of SEQ ID NO: 5, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, - The amino acid residues 1 to 865 of SEQ ID NO: 6, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, - The amino acid residues 1 to 862 of SEQ ID NO: 11, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, - The amino acid residues 1 to 864 of SEQ ID NO: 7, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, and - The amino acid residues 1 to 863 of SEQ ID NO: 8, or a polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

[0057] In a preferred embodiment, the H C domain consists of, or comprises, an amino acid sequence corresponding to the amino acid residues 860 to 1291 of SEQ ID NO: 2, or an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, and the LH N domain consists of, or comprises, an amino acid sequence selected from the group consisting of: - The amino acid residues 1 to 872 of SEQ ID NO: 1, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, - The amino acid residues 1 to 867 of SEQ ID NO: 3, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, - Amino acid residues 1 to 863 of SEQ ID NO: 4, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, - Amino acid residues 1 to 846 of SEQ ID NO: 5, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, - Amino acid residues 1 to 865 of SEQ ID NO: 6, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, - Amino acid residues 1 to 862 of SEQ ID NO: 11, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, - Amino acid residues 1 to 864 of SEQ ID NO: 7, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, and - Amino acid residues 1 to 863 of SEQ ID NO: 8, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

[0058] In a more preferred embodiment, the Clostridium neurotoxin is a chimeric neurotoxin comprising an H C domain derived from BoNT / B and an LH N domain derived from BoNT / A.

[0059] In a more preferred embodiment, the H C domain consists of, or comprises, an amino acid sequence corresponding to amino acid residues 860 to 1291 of SEQ ID NO: 2, or an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, and the LH NThe domain includes an amino acid sequence corresponding to amino acid residues 1 to 872 of SEQ ID NO: 1, or an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

[0060] In one embodiment, the Clostridium neurotoxin is an H derived from BoNT / B CC domain and an LH derived from BoNT / A, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, or BoNT / D-C N H CN domain and is a chimeric neurotoxin.

[0061] In one embodiment of the chimeric neurotoxin according to the present invention, H CC the domain consists of, or includes, an amino acid sequence selected from the group consisting of: - amino acid residues 1082 to 1291 of SEQ ID NO: 2, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, - amino acid residues 1083 to 1276 of SEQ ID NO: 4, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, - amino acid residues 1088 to 1278 of SEQ ID NO: 6, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, - amino acid residues 1077 to 1268 of SEQ ID NO: 11, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, - amino acid residues 1090 to 1297 of SEQ ID NO: 7, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, and - The amino acid residues 1092 to 1285 of SEQ ID NO: 8, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, and, LH N H CN The domain consists of, or includes, an amino acid sequence selected from the group consisting of: - The amino acid residues 1 to 1094 of SEQ ID NO: 1, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, - The amino acid residues 1 to 1081 of SEQ ID NO: 2, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, - The amino acid residues 1 to 1095 of SEQ ID NO: 3, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, - The amino acid residues 1 to 1082 of SEQ ID NO: 4, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, - The amino acid residues 1 to 1069 of SEQ ID NO: 5, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, - The amino acid residues 1 to 1087 of SEQ ID NO: 6, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, - The amino acid residues 1 to 1076 of SEQ ID NO: 11, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, - The amino acid residues 1 to 1089 of SEQ ID NO: 7, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, and - The amino acid residues 1 to 1091 of SEQ ID NO: 8, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

[0062] In a preferred embodiment, H CC The domain consists of, or comprises, an amino acid sequence corresponding to the amino acid residues 1082 to 1291 of SEQ ID NO: 2, or an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, LH N H CN The domain consists of, or comprises, an amino acid sequence selected from the group consisting of: - The amino acid residues 1 to 1094 of SEQ ID NO: 1, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, - The amino acid residues 1 to 1095 of SEQ ID NO: 3, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, - The amino acid residues 1 to 1082 of SEQ ID NO: 4, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, - The amino acid residues 1 to 1069 of SEQ ID NO: 5, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, - The amino acid residues 1 to 1087 of SEQ ID NO: 6, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, - The amino acid residues 1 to 1076 of SEQ ID NO: 11, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, - The amino acid residues 1 to 1089 of SEQ ID NO: 7, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, and - The amino acid residues 1 to 1091 of SEQ ID NO: 8, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

[0063] In one embodiment, the Clostridium neurotoxin is a chimeric neurotoxin comprising an LH N domain derived from a first BoNT / F subtype and an activation loop derived from a second BoNT / F subtype, wherein the second BoNT / F subtype is different from the first BoNT / F subtype. Such BoNT / F subtypes and BoNT / F chimeric neurotoxins are described in the art, such as in WO2016156113 (the entire disclosure of which is incorporated herein by reference).

[0064] In a preferred embodiment, the LH N domain is the BoNT / F7 subtype LH N domain, and / or the activation loop is the BoNT / F1 subtype activation loop.

[0065] In a preferred embodiment, the activation loop comprises (or consists of) the amino acid sequence of SEQ ID NO: 14.

[0066] In a preferred embodiment, the chimeric neurotoxin comprises (or consists of) the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

[0067] The Clostridium neurotoxin can be a modified neurotoxin and its derivatives, including but not limited to those described below. The modified neurotoxin or derivative can contain one or more modified amino acids compared to the native neurotoxin. Such modifications, also known as mutations, include, but are not limited to, substitutions, additions, or deletions of amino acid residues. In the context of the present invention, the modified Clostridium neurotoxin can have a modified amino acid sequence in one or more domains, for example, compared to the amino acid sequence of the native Clostridium neurotoxin. The terms native, unmodified, natural, naturally occurring, or wild-type can be used interchangeably herein.

[0068] Such modifications can affect functional aspects of the neurotoxin, such as biological activity or persistence. However, in the context of the present invention, the modified neurotoxin is considered to have a function. In other words, the modified neurotoxin of the present invention retains the neurotoxin function(s) selected from the ability to bind to low or high affinity neurotoxin receptors on target cells, the ability to translocate the endopeptidase portion (light chain) of the neurotoxin into the cytoplasm, and the ability to cleave SNARE proteins.

[0069] The modified neurotoxin according to the present invention may have one or more modifications in the amino acid sequence of the heavy chain (H C domain, etc.), and the modified heavy chain binds to target nerve cells with higher or lower affinity than the native neurotoxin. H C Such modifications in the H CC domain can include modifications of amino acid residues at the ganglioside binding site of the H CC domain, which can change the binding to gangliosides of target nerve cells, and / or modifications of amino acid residues at the protein receptor binding site of the H

[0070] In one embodiment of the modified Clostridium neurotoxin according to the present invention, the H derived from BoNT / B, BoNT / D, BoNT / D-C, BoNT / F, or BoNT / G CC domain is modified compared to the native H of said BoNT serotype CC domain.

[0071] In a preferred embodiment, the H derived from the BoNT / B, BoNT / D, BoNT / D-C, BoNT / F, or BoNT / G neurotoxin CC domain contains at least one amino acid residue mutation that increases the binding affinity of said H CC domain for human Syt II compared to the native H of BoNT / B, BoNT / D, BoNT / F, BoNT / G, or BoNT / D-C. The inventors have actually discovered that such amino acid residue mutations can increase the potency of the neurotoxin against human smooth muscle. CC In a more preferred embodiment, the H derived from the BoNT / B neurotoxin

[0072] domain contains at least one amino acid residue mutation that increases the binding affinity of said H CC domain for human Syt II compared to the native H of BoNT / B. Further preferably, said at least one amino acid residue mutation increases the binding affinity of said H CC domain for human Syt II by at least 50% compared to the native H of BoNT / B. CC Such appropriate amino acid residue mutations in the H domain of BoNT / B are described in WO2013180799 and WO2016154534, which are incorporated herein by reference in their entirety in the art. CC domain for human Syt II compared to the native H of BoNT / B. CC domain for human Syt II compared to the native H of BoNT / B.

[0073] Specifically, the H of native BoNT / B CC domain for human Syt II compared to the native H of BoNT / B.

[0074] In particular, the H of native BoNT / B CCCompared with the domain, the mutation of at least one amino acid residue suitable for increasing the binding affinity of the H domain of BoNT / B for human Syt II by at least 50% is a substitution, addition, or deletion of an amino acid residue selected from the group consisting of 1118M, 1183M, 1191M, 1191I, 1191Q, 1191T, 1199Y, 1199F, 1199L, 1201V, 1191C, 1191V, 1191L, 1191Y, 1199W, 1199E, 1199H, 1178Y, 1178Q, 1178A, 1178S, 1183C, 1183P, and any combination thereof. Preferably, the mutation of at least one amino acid residue in the H domain of BoNT / B CC is a substitution, addition, or deletion of two amino acid residues selected from the group consisting of 1191M and 1199L, 1191M and 1199Y, 1191M and 1199F, 1191Q and 1199L, 1191Q and 1199Y, 1191Q and 1199F, 1191M and 1199W, 1191M and 1178Q, 1191C and 1199W, 1191C and 1199Y, 1191C and 1178Q, 1191Q and 1199W, 1191V and 1199W, 1191V and 1199Y, or 1191V and 1178Q. More preferably, the mutation of at least one amino acid residue in the H domain of BoNT / B CC is a substitution, addition, or deletion of three amino acid residues: 1191M, 1199W, and 1178Q. Even more preferably, the mutation of at least one amino acid residue in the H domain of BoNT / B CC is a substitution, addition, or deletion of two amino acid residues: 1191M and 1199Y. CC In a more preferred embodiment, compared with the H domain of natural BoNT / B, the H domain of BoNT / B for human Syt II

[0075] CC Compared with the domain, the H domain of BoNT / B for human Syt II CCThe mutation of said at least one amino acid residue suitable for increasing the binding affinity of the domain by at least 50% is an amino acid residue substitution 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 any combination thereof. Preferably, the H of BoNT / B CC The mutation of said at least one amino acid residue in the domain consists of a substitution of two amino acid residues selected from the group consisting of E1191M and S1199L, E1191M and S1199Y, E1191M and S1199F, E1191Q and S1199L, E1191Q and S1199Y, E1191Q and S1199F, E1191M and S1199W, E1191M and W1178Q, E1191C and S1199W, E1191C and S1199Y, E1191C and W1178Q, E1191Q and S1199W, E1191V and S1199W, E1191V and S1199Y, or E1191V and W1178Q. More preferably, the H of BoNT / B CC The mutation of said at least one amino acid residue in the domain consists of a three-amino acid residue substitution: E1191M, S1199W, and W1178Q. More preferably, the H of BoNT / B CC The mutation of said at least one amino acid residue in the domain consists of a substitution of two amino acid residues: E1191M and S1199Y.

[0076] In a preferred embodiment, the H of BoNT / B to be modified CC domain corresponds to amino acid residues 1082 to 1291 of SEQ ID NO: 2 (the H domain of natural BoNT / B CC domain), or an amino acid sequence having at least 70%, preferably at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

[0077] In a more preferred embodiment, the modified Clostridium neurotoxin of the present invention comprises (or consists of) the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

[0078] In a preferred embodiment, the H CC domain derived from BoNT / B, BoNT / D, BoNT / D-C, BoNT / F, or BoNT / G neurotoxin is the H CC domain of natural BoNT / B, BoNT / D, BoNT / D-C, BoNT / F, or BoNT / G, and CC comprises at least one amino acid residue mutation that increases the binding affinity of the H

[0079] domain to one or more gangliosides. CC In a more preferred embodiment, the H CC domain derived from BoNT / F is the H CC domain of natural BoNT / F, and

[0080] comprises at least one amino acid residue mutation that increases the binding affinity of the H

[0081] domain to one or more gangliosides. CC Compared with the H CC domain of natural BoNT / F, amino acid residue mutations suitable for increasing the binding affinity of the H CC domain of BoNT / F to gangliosides such as GD1a and / or GM1a include, but are not limited to, amino acid mutations such as amino acid substitution, addition, or deletion, including 1241K. More preferably, compared with the H CC domain of natural BoNT / F, the at least one amino acid residue mutation that increases the binding affinity of the H

[0082] In a preferred embodiment, the H domain of BoNT / F to be modified CC corresponds to amino acid residues 1088 to 1278 of SEQ ID NO: 6 or amino acid residues 1077 to 1268 of SEQ ID NO: 11 (the H domain of native BoNT / F), or an amino acid sequence having at least 70%, preferably at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto. CC

[0083] In a preferred embodiment, the chimeric Clostridium neurotoxin of the present invention corresponds to the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

[0084] In one embodiment, the Clostridium neurotoxin of the present invention is chimeric and may be modified as described above. For example, in a preferred embodiment, the Clostridium neurotoxin comprises (or consists of) the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

[0085] The Clostridium neurotoxin of the present invention can be produced using recombinant techniques. Thus, in one embodiment, the Clostridium neurotoxin of the present invention is a recombinant Clostridium neurotoxin.

[0086] In one embodiment, the Clostridium neurotoxin is associated with a BoNT complex protein, also known as neurotoxin-associated proteins (NAP). In other words, the Clostridium neurotoxin is administered to a human patient in association with or in combination with the BoNT complex protein. Thus, in one embodiment, the Clostridium neurotoxin is complexed with one or more BoNT complex proteins.

[0087] In another embodiment, the Clostridium neurotoxin does not contain (or is not associated with, or combined with) the BoNT complex protein. In other words, the Clostridium neurotoxin is administered to a human patient without being associated with or combined with the BoNT complex protein.

[0088] Preferably, the Clostridium neurotoxin (e.g., for the uses described herein) is part of a pharmaceutical composition together with at least one pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" means any component that is compatible with the other components of the pharmaceutical composition, particularly the Clostridium neurotoxin, and is not harmful to the human patient. Pharmaceutically acceptable carriers can be selected according to standard pharmaceutical practice, based on the desired route of administration, and include, without limitation, excipients, diluents, adjuvants, propellants, and salts.

[0089] Accordingly, the present invention further relates to a pharmaceutical composition for use in the treatment of autonomic neuropathy in a human patient, said composition comprising the Clostridium neurotoxin of the present invention and at least one pharmaceutically acceptable carrier, and the dosage of the Clostridium neurotoxin to be administered to the patient is as described above. Further included are corresponding uses and methods of treating autonomic neuropathy, including administering the pharmaceutical composition of the present invention to a human patient.

[0090] The Clostridium neurotoxin of the present invention can be formulated for oral, parenteral (i.e., injection), continuous infusion, inhalation, or topical administration. Compositions suitable for injection can be in the form of solutions, suspensions, or emulsions, or as dry powders to be dissolved or suspended in a suitable solvent prior to use.

[0091] In the case of a neurotoxin to be delivered locally, the neurotoxin can be formulated as a cream (e.g., for topical application) or for subcutaneous injection.

[0092] Local delivery means can include an aerosol or other nebulizer (e.g., a nebulizer). In this regard, an aerosol formulation of the neurotoxin enables delivery to the lungs and / or other nasal and / or bronchial or airway passages.

[0093] The Clostridium neurotoxin of the present invention can be administered to a patient by intrathecal or epidural injection in the spine at the level of the spinal segment involved in the innervation of the affected organ.

[0094] In one embodiment, the Clostridium neurotoxin or pharmaceutical composition is for intramuscular, intradermal, or subcutaneous administration.

[0095] In one embodiment, the Clostridium neurotoxin or pharmaceutical composition is for topical administration, for example, by infusion.

[0096] A preferred route of administration is by intramuscular injection.

[0097] However, it is also possible to administer the Clostridium neurotoxin to muscle without using an injection. For example, for treating urinary disorders, injecting a liquid or semi-solid preparation of the Clostridium neurotoxin into the patient's bladder, placing a gel preparation containing the Clostridium neurotoxin at an appropriate location in the patient's bladder, spraying a spray preparation containing the Clostridium neurotoxin at an appropriate location in the patient's bladder, or, as described in WO2005053733, the disclosure of which is incorporated herein by reference in its entirety, topically administering a solid (e.g., lyophilized), semi-solid, or liquid botulinum toxin preparation applied or coated on the outer wall of a balloon that is later inflated in the bladder to contact the wall of the bladder, the Clostridium neurotoxin can be administered to the bladder.

[0098] As described above, the dosage of the Clostridium neurotoxin suitable for achieving a desired therapeutic effect in a human patient suffering from autonomic neuropathy, or, in other words, the therapeutic amount of said Clostridium neurotoxin, is equal to or less than the dosage of BoNT / A for treating the same autonomic neuropathy.

[0099] Preferably, the BoNT / A for treating the same autonomic neuropathy is purified BoNT / A. As used herein, the term "purified BoNT / A" means botulinum neurotoxin type A purified from a Clostridium strain that naturally produces botulinum neurotoxin type A (naturally occurring Clostridium strain). Purified BoNT / A may or may not be associated with a complex protein. Examples of commercially available purified BoNT / A include Botox®, Dysport®, and Xeomin®.

[0100] In one embodiment, the dosage (therapeutic dose) of the Clostridium neurotoxin administered to treat autonomic neuropathy in a human patient is from about 1 / 1.1 to about 1 / 100, preferably from about 1 / 1.3 to about 1 / 90 of the dosage of BoNT / A for treating the autonomic neuropathy.

[0101] In a preferred embodiment, the Clostridium neurotoxin is H derived from BoNT / B CCWhen including a domain, the therapeutic dose of the Clostridium neurotoxin is about 1 / 1.1 to about 1 / 20, preferably about 1 / 1.3 to about 1 / 19.8 of the dose of BoNT / A that treats the same autonomic neuropathy. For example, when the Clostridium neurotoxin of the present invention is a natural BoNT / B neurotoxin such as the neurotoxin of the amino acid sequence of SEQ ID NO: 2, the therapeutic dose of the Clostridium neurotoxin is about 1 / 1.1 to about 1 / 1.5, preferably about 1 / 1.3 of the dose of BoNT / A that treats the same autonomic neuropathy. As another example, when the Clostridium neurotoxin of the present invention is a modified BoNT / B neurotoxin with increased binding affinity for the human SytII receptor (compared to natural BoNT / B) such as the neurotoxin of the amino acid sequence of SEQ ID NO: 9, the therapeutic dose of the Clostridium neurotoxin is about 1 / 15 to about 1 / 20, preferably about 1 / 19.8 of the dose of BoNT / A that treats the same autonomic neuropathy. Further, as another example, when the Clostridium neurotoxin of the present invention is a modified chimeric BoNT / B neurotoxin with increased binding affinity for the human SytII receptor such as the neurotoxin of the amino acid sequence of SEQ ID NO: 10, the therapeutic dose of the Clostridium neurotoxin is about 1 / 2 to about 1 / 5, preferably about 1 / 3.5 of the dose of BoNT / A that treats the same autonomic neuropathy.

[0102] In a preferred embodiment, the Clostridium neurotoxin is H derived from BoNT / F CCWhen including a domain, the therapeutic dose of the Clostridium neurotoxin is about 1 / 60 to about 1 / 100, preferably about 1 / 61.2 to about 1 / 90 of the dose of BoNT / A that treats the same autonomic neuropathy. For example, when the Clostridium neurotoxin of the present invention is a natural BoNT / F neurotoxin such as the neurotoxin of the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 11, the therapeutic dose of the Clostridium neurotoxin is about 1 / 60 to about 1 / 65, preferably about 1 / 61.2 of the dose of BoNT / A that treats the same autonomic neuropathy. As another example, when the Clostridium neurotoxin of the present invention is a modified BoNT / F neurotoxin with increased binding affinity for one or more gangliosides (compared to natural BoNT / F) such as the neurotoxin of the amino acid sequence of SEQ ID NO: 12, the therapeutic dose of the Clostridium neurotoxin is about 1 / 60 to about 1 / 65, preferably about 1 / 61.2 of the dose of BoNT / A that treats the same autonomic neuropathy. Further, as another example, when the Clostridium neurotoxin of the present invention is a chimeric BoNT / F neurotoxin such as the neurotoxin of the amino acid sequence of SEQ ID NO: 13, the therapeutic dose of the Clostridium neurotoxin is about 1 / 85 to about 1 / 100, preferably about 1 / 90 of the dose of BoNT / A that treats the same autonomic neuropathy.

[0103] As described above, the dose of BoNT / A for treating autonomic neuropathy is well known in the art (https: / / www.medicines.org.uk / emc / medicine / 112).

[0104] The dose of the Clostridium neurotoxin is preferably measured in nanogram units herein.

[0105] The dosage of the Clostridium neurotoxin according to the present invention should be understood as the dosage of the active two-chain Clostridium neurotoxin, that is, not including the amount of the complex protein that can be associated with the neurotoxin. In other words, whether the administration to a patient is carried out in association with the complex protein or without the complex protein, it indicates the dosage of the active two-chain Clostridium neurotoxin. As is well known to those skilled in the art, the active two-chain Clostridium neurotoxin can bind to neuronal receptors, translocate the light chain into the cytoplasm, and cleave SNARE proteins, while the complex protein does not exhibit such biological activity (i.e., is not "active"). In the case of botulinum neurotoxin, the size of the entire active two-chain is generally about 150 kD.

[0106] In fact, as is well known to those skilled in the art, the potency of the Clostridium neurotoxin is related to the amount of the neurotoxin (e.g., the number of nanograms) required to achieve the LD50 (50% lethal dose) unit, and 1 LD50 unit is defined as the median of the lethal intraperitoneal dose (measured in mice). However, in the currently marketed BoNT pharmaceuticals, the 150 kD neurotoxin is contained in various amounts, and the LD50 units also vary. Moreover, in these preparations, the neurotoxin may or may not be associated with (or combined with) a non-toxic neurotoxin-related protein (NAP), also known as a complex protein. To facilitate conversion: - Botox® (also known as Onabotulinumtoxin A) contains approximately 0.73 ng of 150 kD BoNT / A and complex protein in 100 units. - Dysport® (also known as Abobotulinumtoxin A) contains approximately 0.65 ng of 150 kD BoNT / A and complex protein in 100 units. - Xeomin® (also known as Incobotulinumtoxin A) contains approximately 0.44 ng of 150 kD BoNT / A in 100 units and does not contain complex protein. - Each 100 units of Neurobloc / Myobloc (registered trademark), also known as Rimabotulinumtoxin B, contains from about 0.2 ng to about 1 ng of 150 kD BoNT / B and a complex protein.

[0107] The amount of Clostridium neurotoxin can be measured by methods conventionally used in the art for quantifying proteins, preferably at the nanogram level, and these include, in particular, mass spectrometry such as isotope dilution mass spectrometry (Munoz et al., Quantification of protein calibrants by amino acid analysis using isotope dilution mass spectrometry, Anal. Biochem. 2011, 408, 124 - 131), or fluorescence assays (Poras et al., Detection and Quantification of Botulinum Neurotoxin Type A by a Novel Rapid In Vitro Fluorimetric Assay, Appl Environ Microbiol. 2009 Jul; 75(13): 4382 - 4390).

[0108] When a range of values is presented herein, each intervening value, to the tenth of a unit, between the upper and lower limits of that range is also specifically disclosed, unless the context clearly indicates otherwise. Each smaller range between any of the stated values or intervening values within the stated range is included in the present disclosure. Further, any range of numerical values indicated herein by the expression "a to b" is to be understood as meaning a range of numerical values extending from a to b (i.e., including the exact endpoints a and b).

[0109] Furthermore, the term "about" is to be understood herein as meaning plus or minus (±) 5% of the numerical value of the number for which it is used, preferably ±4%, ±3%, ±2%, ±1%, ±0.5%, ±0.1%.

[0110] In a preferred embodiment, the dosage (i.e., the therapeutic amount) of the Clostridium neurotoxin of the present invention to be administered for treating autonomic neuropathy in a human patient ranges from about 0.00025 ng to about 3 ng.

[0111] In a preferred embodiment, the therapeutic amount of the Clostridium neurotoxin ranges from about 0.0003 ng to about 2 ng, preferably from about 0.0004 ng to about 1.5 ng, from about 0.0005 ng to about 1 ng, and more preferably from about 0.0006 ng to about 0.5 ng of the Clostridium neurotoxin.

[0112] In a preferred embodiment, the Clostridium neurotoxin comprises the H CC domain of BoNT / B or the H CC domain of BoNT / F, is administered to a human patient at any of the dosages of the Clostridium neurotoxin described herein, and is used for the treatment of autonomic neuropathy as described above.

[0113] In a more preferred embodiment, the Clostridium neurotoxin comprises the H CC domain of BoNT / B or the H CC domain of BoNT / F, is administered to a human patient at any of the dosages of the Clostridium neurotoxin described herein, and is used for the treatment of smooth muscle disorders as described above. More preferably, the smooth muscle disorder is selected from the group consisting of urinary disorders, gastrointestinal disorders, vascular and cardiovascular disorders, prostate disorders, and sexual disorders.

[0114] For example, the therapeutic amount of the Clostridium neurotoxin comprising the H CC domain of BoNT / B preferably ranges from about 0.001 ng to about 2 ng.

[0115] Furthermore, for example, the therapeutic amount of the Clostridium neurotoxin comprising the H CC domain of BoNT / B preferably ranges from about 0.0003 ng to about 0.05 ng.

[0116] However, it will be understood that the necessary dosage range will be determined according to the exact nature of the Clostridium neurotoxin, autonomic neuropathy, route of administration, nature of the formulation, age of the patient, nature, degree, or severity of the patient's condition, contraindications if any, and the judgment of the attending physician. Such variations in dosage levels can be adjusted using standard empirical routines for optimization.

[0117] For reference, examples of appropriate dosages of natural BoNT / A (such as the neurotoxin having the amino acid sequence of SEQ ID NO: 1) for treating some specific autonomic neuropathies are shown below. - NDO (neurogenic detrusor overactivity): 1.46 ng of natural BoNT / A into the detrusor muscle, - OAB (overactive bladder): 0.73 ng of natural BoNT / A into the detrusor muscle, - Axillary hyperhidrosis: 0.365 ng of natural BoNT / A into each axilla, - Sialorrhea in adult patients, especially Parkinson's disease patients: 0.146 ng of natural BoNT / A per submandibular gland and / or parotid gland, - Sialorrhea in pediatric cerebral palsy patients: 0.073 ng of natural BoNT / A per submandibular gland and / or parotid gland.

[0118] Examples of appropriate dosage ranges of natural BoNT / B (such as the neurotoxin having the amino acid sequence of SEQ ID NO: 2) for treating the above-mentioned disorders according to the present invention are as follows. - NDO (neurogenic detrusor overactivity): 0.5 ng to 2 ng of natural BoNT / B into the detrusor muscle, - OAB (overactive bladder): 0.25 ng to 1 ng of natural BoNT / B into the detrusor muscle, - Axillary hyperhidrosis: 0.125 ng to 0.5 ng of natural BoNT / B into each axilla, - Sialorrhea in adult patients, especially Parkinson's disease patients: 0.05 ng to 0.2 ng of natural BoNT / B per submandibular gland and / or parotid gland, - Sialorrhea in pediatric cerebral palsy patients: 0.025 ng to 0.1 ng of natural BoNT / B per submandibular gland and / or parotid gland.

[0119] Examples of appropriate dosage ranges of modified BoNT / B (such as a neurotoxin having the amino acid sequence of SEQ ID NO: 9) with increased binding affinity for the human SytII receptor for treating the above-described disorders according to the present invention are as follows. - NDO (neurogenic detrusor overactivity): 0.025 ng to 0.2 ng of modified BoNT / B into the detrusor muscle, - OAB (overactive bladder): 0.0125 ng to 0.1 ng of modified BoNT / B into the detrusor muscle, - Axillary hyperhidrosis: 0.0075 ng to 0.05 ng of modified BoNT / B into each axilla, - Salivation in adult patients, particularly Parkinson's disease patients: 0.0025 ng to 0.02 ng of modified BoNT / B per submandibular gland and / or parotid gland, - Salivation in pediatric cerebral palsy patients: 0.001 ng to 0.01 ng of modified BoNT / B per submandibular gland and / or parotid gland.

[0120] Examples of appropriate dosage ranges of chimeric modified BoNT / B (such as a neurotoxin having the amino acid sequence of SEQ ID NO: 10) with increased binding affinity for the human SytII receptor for treating the above-described disorders according to the present invention are as follows. - NDO (neurogenic detrusor overactivity): 0.2 ng to 1 ng of chimeric modified BoNT / B into the detrusor muscle, - OAB (overactive bladder): 0.1 ng to 0.5 ng of chimeric modified BoNT / B into the detrusor muscle, - Axillary hyperhidrosis: 0.05 ng to 0.2 ng of chimeric modified BoNT / B into each axilla, - Salivation in adult patients, particularly Parkinson's disease patients: 0.02 ng to 0.1 ng of chimeric modified BoNT / B per submandibular gland and / or parotid gland, - Salivation in pediatric cerebral palsy patients: 0.01 ng to 0.05 ng of chimeric modified BoNT / B per submandibular gland and / or parotid gland.

[0121] Examples of appropriate dosage ranges of native BoNT / F (such as a neurotoxin having the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 11) for treating the above-described disorders according to the present invention are as follows. - NDO (neurogenic detrusor overactivity): 0.01 ng to 0.05 ng of native BoNT / F into the detrusor muscle, - OAB (overactive bladder): 0.005 ng to 0.025 ng of native BoNT / F into the detrusor muscle, - Axillary hyperhidrosis: 0.0025 ng to 0.01 ng of native BoNT / F into each axilla; - Salivation in adult patients, particularly Parkinson's disease patients: 0.001 ng to 0.005 ng of native BoNT / F around the submandibular gland and / or parotid gland, - Salivation in pediatric cerebral palsy patients: 0.0005 ng to 0.0025 ng of native BoNT / F around the submandibular gland and / or parotid gland.

[0122] Examples of appropriate dosage ranges of modified BoNT / B (neurotoxin etc. having the amino acid sequence of SEQ ID NO: 12) with increased binding affinity to one or more gangliosides for treating the above-mentioned disorders according to the present invention are as follows. - NDO (neurogenic detrusor overactivity): 0.01 ng to 0.05 ng of modified BoNT / F into the detrusor muscle, - OAB (overactive bladder): 0.005 ng to 0.025 ng of modified BoNT / F into the detrusor muscle, - Axillary hyperhidrosis: 0.0025 ng to 0.01 ng of modified BoNT / F into each axilla, - Salivation in adult patients, particularly Parkinson's disease patients: 0.001 ng to 0.005 ng of modified BoNT / F around the submandibular gland and / or parotid gland, - Salivation in pediatric cerebral palsy patients: 0.0005 ng to 0.0025 ng of modified BoNT / F around the submandibular gland and / or parotid gland.

[0123] Examples of appropriate dosage ranges of chimeric BoNT / F (neurotoxin etc. having the amino acid sequence of SEQ ID NO: 13) for treating the above-mentioned disorders according to the present invention are as follows. - NDO (neurogenic detrusor overactivity): 0.007 ng to 0.03 ng of chimeric BoNT / F into the detrusor muscle, - Overactive bladder (OAB): 0.003 ng to 0.0015 ng of chimeric BoNT / F into the detrusor muscle, - Axillary hyperhidrosis: 0.001 ng to 0.007 ng of chimeric BoNT / F into each axilla, - Sialorrhea in adult patients, particularly Parkinson's disease patients: 0.0007 ng to 0.003 ng of chimeric BoNT / F around the submandibular and / or parotid glands, - Sialorrhea in pediatric cerebral palsy patients: 0.0003 ng to 0.0015 ng of chimeric BoNT / F around the submandibular and / or parotid glands.

[0124] The present disclosure is not limited to the examples of methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the embodiments of the present disclosure. Unless otherwise indicated, any amino acid sequence is written from left to right in the direction from amino to carboxy.

[0125] Furthermore, it should be noted that in the use in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a clostridial neurotoxin" includes a plurality of such candidate agents, and reference to "the clostridial neurotoxin" includes reference to one or more clostridial neurotoxins known to those skilled in the art and their equivalents, and so on.

[0126] Next, the present invention will be described by way of example only, with reference to the following figures and examples.

Brief Description of the Drawings

[0127]

Figure 1

Figure 2A

Figure 2B

Figure 3

[0128] Amino acid sequence

[0129]

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

[0130] The following examples serve to illustrate specific embodiments of the present invention and in no way limit the scope of the present invention defined in the claims.

[0131] Example 1: Study of Human Bladder Tissue Samples

[0132] Briefly, human bladder tissue samples were obtained from patients who underwent radical cystectomy for bladder cancer without bladder dysfunction as determined by the medical record, and the collection and use of the tissues and other samples were carried out in accordance with all relevant laws, regulations, and codes of conduct, including obtaining written (with data privacy obligations described) informed consent from the patients, and in accordance with the patients' medical histories.

[0133] After the surgical procedure, the samples were immediately transferred from the operating room to the pathology facility, and normal fragments of the bladder dome, i.e., those without visible tumor tissue, were selected for the experiment. Immediately after removal, the tissue samples were stored at 4 °C in Krebs-HEPES buffer (having the following mM composition: NaCl 118.0, KCl 4.7, MgSO4 1.2, KH2PO4 1.2, CaCl2 2.5, NaHCO3 4.2, glucose 11.1, HEPES 20.8, pH 7.4) containing penicillin (100 IU / ml) and streptomycin (0.1 mg / ml) to optimally preserve them until use (within a maximum of 24 hours) and transported to the research facility.

[0134] To prepare the strips, the urothelium was carefully removed and eight sections (4×2×2 mm) of detrusor muscle were excised from the bladder of each donor for each experiment. The remaining bladder tissue was weighed, divided into two cryotubes, immediately frozen in liquid nitrogen, and stored at -80 °C for later analysis.

[0135] Ex vivo experiments were performed using a setup placed under a laboratory hood (Captair Chem Filtair XL1346A) composed of an organ chamber filled with Krebs-HEPES buffer maintained at 37 °C and continuously bubbled with 95% O2 and 5% CO2 to maintain a pH of 7.4. The bladder strips were suspended in 5 ml organ chambers and connected to force transducers (Pioden controls Ltd, UK) for isometric tension recording. An initial tension of 1 g was applied. Following amplification, changes in tension were digitized by a Mac Lab TM / 8 using Chart TM5 software (AD Instruments Ltd). After thorough washing, the strips were contracted by electrical field stimulation (EFS) via two platinum electrodes positioned on both sides of the strip and connected to a stimulator (Bionic System Nozay, France).

[0136] Tension was adjusted to reach a resting tension of 1 g during a 60-minute equilibration period, during which the buffer was replaced every 15 minutes.

[0137] Following the equilibration period, KCl (100 mM, 10 minutes) and then carbachol (3.10 -6 M, 10 minutes) were added sequentially to prime the detrusor smooth muscle strips, with wash steps interspersed between each compound addition. Then, 0.5% gelatin was injected into the organ chamber before applying an EFS train.

[0138] The EFS trains (20 Hz, pulse duration 1 ms, train duration 5 s, 300 mA) were given at 1-min intervals, followed by a 3-min rest period, and were carried out continuously with three trains of stimuli (stimulus conditions selected to give strong and stable contractions in our human bladder tissue in human bladder strips). The stimulation was continued until a stable response was obtained (the reaction was considered stable if the percentage of variation in the amplitude of the EFS contraction, calculated for the last three groups of EFS contractions during the stabilization period, was 90% or more or 110% or less).

[0139] Individual strips were incubated with the solvent (Krebs containing 0.5% gelatin), 0.1, 1, 3, 5, or 10 nM of botulinum neurotoxin, and EFS stimulation was continued for 3 h. At the end of the experiment, carbachol (3.10 -6 M, 10 min) was added to the organ bath to contract the bladder strips by directly activating the muscarinic receptors. Comparison of carbachol-induced strip contractions before and after incubation with botulinum neurotoxin serves as a viability test for the strips during the experiment.

[0140]

Table 2

[0141] Eight non-compound botulinum neurotoxins were tested: - nBoNT / A, native botulinum neurotoxin type A (SEQ ID NO: 1) obtained from List Boological Laboratories, Inc., - nBoNT / B, native botulinum neurotoxin type B (SEQ ID NO: 2) obtained from List Biological Laboratories, Inc., - rBoNT / B MY 、H C domain containing two mutations, E1191M and S1199Y, recombinant botulinum neurotoxin type B (SEQ ID NO: 9), - rBoNT / AB MY 、H CA recombinant chimeric botulinum neurotoxin (LH domain from BoNT / A and H domain from BoNT / B) containing two mutations, E1191M and S1199Y, in the domain (SEQ ID NO: 10), N - nBoNT / F, native botulinum neurotoxin subtype F1 obtained from Metabiologics Inc. (SEQ ID NO: 6), C - mrBoNT / F, a recombinant botulinum neurotoxin subtype F1 (SEQ ID NO: 12) containing a single point mutation, H1241K, in the H domain, - nBoNT / F, native botulinum neurotoxin subtype F1 obtained from Metabiologics Inc. (SEQ ID NO: 6), - mrBoNT / F, a recombinant botulinum neurotoxin subtype F1 (SEQ ID NO: 12) containing a single point mutation, H1241K, in the H domain, CC - mrBoNT / F7-1, a recombinant botulinum neurotoxin subtype F7 (SEQ ID NO: 13) in which the native BoNT / F7 activation loop is replaced with the BoNT / F1 activation loop. - mrBoNT / F7-1, a recombinant botulinum neurotoxin subtype F7 (SEQ ID NO: 13) in which the native BoNT / F7 activation loop is replaced with the BoNT / F1 activation loop.

[0142] Incubation of botulinum neurotoxin in the organ bath was performed by adding 0.75 μl, 7.5 μl, 22.5 μl, 37.5 μl, or 75 μl of the botulinum toxin stock solution (666 nM) to 5 ml of Krebs-HEPES buffer containing 0.5% gelatin, such that the final concentration in the organ bath was 0.1, 1, 3, 5, or 10 nM, respectively.

[0143] The contractile response to the drug was quantified for the mean developed tension. The mean developed tension (mg, corresponding to the mean of the data points in the selected tracing) was measured in a 1-minute section before and at the end of the stable response of each strip following the addition of KCl or carbachol.

[0144] The contractile response to EFS-induced contraction was quantified as follows. Briefly, as shown in Figure 1, after applying three stimuli at 1-minute intervals, a 3-minute rest follows.

[0145] Each EFS-induced response was analyzed over a time frame between two consecutive stimuli, i.e., 60 seconds. This analysis was performed over 3 hours for the last two responses for each stimulus group.

[0146] For each stimulus train, the values obtained in the last two responses (peaks 2 and 3) were averaged.

[0147] To quantify the EFS-evoked response, the amplitude (mg) of the EFS-evoked contraction was calculated as the difference between the basal tension before stimulation and the maximal evoked tension during the response to EFS, i.e., the difference between the selected maximum (Max) and minimum (Min) data points.

[0148] These amplitude values were expressed as a percentage of the value corresponding to the maximal EFS contraction measured during the last train of stimuli during the stabilization period before addition of botulinum neurotoxin.

[0149] The inhibitory effect of botulinum neurotoxin was evaluated by determining the value of the paralysis time 50 (T50) (min), i.e., the time required to inhibit 50% of the maximal response. This value was calculated using the variable slope model of GraphPad Prism 6.05 (Y = Bottom+(Top - Bottom) / (1 + 10^((LogIC50 - X)*HillSlope)). This is also called the four-parameter dose-response logistic curve.

[0150] The results are shown in Table 3 and Figure 2.

[0151]

Table 3

[0152] nBoNT / A, nBoNT / B, rBoNT / B MY , rBoNT / AB MY , nBoNT / F, mrBoNT / F, and mrBoNT / F7-1 showed concentration-dependent inhibition of the EFS-evoked contraction response with respect to amplitude. nBoNT / A and nBoNT / B induced equivalent inhibitory effects on the EFS-evoked contraction of human bladder strips at the same concentration. Furthermore, at all doses, rBoNT / B MY , rBoNT / AB MY, the decrease in EFS-induced contractions of bladder strips caused by nBoNT / F, mrBoNT / F, and mrBoNT / F7-1 was higher compared to either nBoNT / A and / or nBoNT / B. All BoNT / Fs tested, as well as rBoNT / B MY and rBoNT / AB MY appeared to require lower concentrations to achieve the same reduction in contractions as nBoNT / A or nBoNT / B. Among all BoNTs tested, mrBoNT / F7-1 resulted in the greatest reduction in contractions.

[0153] Based on the observations described above, the potency of the botulinum toxins tested against human bladder tissue was quantified by plotting T50 against the botulinum toxin protein concentration, as particularly shown in Table 4 and Figure 3 below, according to the method described by Weisemann et al. (Generation and Characterization of Six Recombinant Botulinum Neurotoxins as Reference Material to Serve in an International Proficiency Test, Toxins, 2015, 7(12): 5035-5054; doi:10.3390 / toxins7124861), and a logarithmic function was fitted to obtain an excellent R 2 value.

[0154]

Table 4

[0155] The results show that the potency of BoNT / F is the highest against human bladder tissue, followed by BoNT / B and then BoNT / A. In particular, nBoNT / B has a potency approximately 1.5 times that of nBoNT / A, mrBoNT / B has a potency approximately 20 times that of nBoNT / A and approximately 15 times that of nBoNT / B, mrBoNT / AB has a potency approximately 4 times that of nBoNT / A and approximately 3 times that of nBoNT / B, nBoNT / F has a potency approximately 60 times that of nBoNT / A and approximately 50 times that of nBoNT / B, and mrBoNT / F7-1 has a potency approximately 90 times that of nBoNT / A and approximately 70 times that of nBoNT / B. Furthermore, mrBoNT / F shows the same T50 value as nBoNT / F at 1 nM and 10 nM, and it can be reasonably inferred that mrBoNT / F has a potency similar to that of nBoNT / F.

[0156] Therefore, based on the surprising data disclosed herein, BoNT / B and BoNT / F neurotoxins, and Clostridium neurotoxins exhibiting similar binding, uptake, translocation, and / or SNARE cleavage properties such as BoNT / D, BoNT / D-C, or BoNT / G, can be used at dosages equal to or lower than that of a type of BoNT / A used to treat the same disorder for treating disorders of the human autonomic nervous system, particularly smooth muscle disorders such as urinary disorders (NOD, OAB, etc.). In particular, depending on the relative potency values confirmed in Table 4, the therapeutic dose of the BoNT / B, BoNT / F, BoNT / D, BoNT / D-C, or BoNT / G Clostridium neurotoxin administered to the subject can be determined based on the known dosage of BoNT / A capable of treating the same autonomic disorder.

[0157] All publications mentioned in the above specification are hereby incorporated by reference as part of the present specification. Various modifications and variations of the described methods and systems of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention claimed in the claims should not be unduly limited to such specific embodiments. Indeed, various modifications of the described embodiments of the present invention that are obvious to those skilled in the art of biochemistry and biotechnology or related fields are intended to be included within the scope of the following claims.

Explanation of Reference Numerals

[0158] [Figure 1] Developped tension (mg): Developed tension (mg) Peak 1: Peak 1 Peak 2: Peak 2 Peak 3: Peak 3 1 min window of analysis: 1-minute analysis window 3 min: 3 minutes 1st EFS stimulation: First EFS stimulation 2nd EFS stimulation: Second EFS stimulation 3rd EFS stimulation: Third EFS stimulation 3 min rest: 3-minute rest Zoom (figure 4): Zoom (Figure 4)

Claims

1. A clostridial neurotoxin for use in treating autonomic neuropathy in a human patient, comprising H from BoNT / B, BoNT / F, BoNT / D, BoNT / DC, or BoNT / G. CC A Clostridial neurotoxin comprising a domain, wherein the dosage of the Clostridial neurotoxin to be administered to the patient is equal to or less than the dosage of BoNT / A to treat the autonomic neuropathy.

2. 2. Use of a Clostridial neurotoxin in the manufacture of a medicament for treating autonomic neuropathy in a human patient, wherein the Clostridial neurotoxin is a H from BoNT / B, BoNT / F, BoNT / D, BoNT / DC, or BoNT / G. CC The use of the present invention, wherein the dosage of the Clostridial neurotoxin to be administered to the patient is equal to or less than the dosage of BoNT / A to treat the autonomic neuropathy.

3. A method for treating autonomic neuropathy in a human patient in need thereof, comprising administering to a subject a therapeutically effective amount of H from BoNT / B, BoNT / F, BoNT / D, BoNT / DC, or BoNT / G. CC A method comprising administering to the patient a Clostridial neurotoxin comprising the domain at a dosage equal to or less than the dosage of BoNT / A used to treat the autonomic neuropathy.

4. The Clostridial neurotoxin for use according to claim 1, the use according to claim 2, or the method according to claim 3, wherein the dosage of the Clostridial neurotoxin is about 1.1 to about 100 times lower than the dosage of BoNT / A for treating the autonomic neuropathy, and the dosage is preferably quantified in nanograms.

5. 13. A Clostridial neurotoxin for use, use or method according to any preceding claim, wherein the dosage of said Clostridial neurotoxin ranges from about 0.00025 ng to about 3 ng.

6. 2. A Clostridial neurotoxin for use, use or method according to any of the preceding claims, wherein the autonomic neuropathies are selected from smooth muscle disorders, hypersecretory disorders, respiratory disorders, inflammatory disorders with an autonomic component, neuroendocrine disorders and other autonomic neuropathies directly related to central nervous disorders.

7. The above H CC The domain is H of BoNT / B. CC Domain or H of BoNT / F CC 20. A Clostridial neurotoxin of use, use or method according to any preceding claim which is a domain.

8. The BoNT / B H CC The domain is the H domain of natural BoNT / B. CC 2. A Clostridial neurotoxin for use, use or method according to any of the preceding claims, comprising a mutation in at least one amino acid residue which increases its binding affinity to the human Syt II receptor by at least 50% compared to the domain.

9. 9. The Clostridial neurotoxin of claim 8, wherein the mutation of at least one amino acid residue is selected from the group consisting of 1118M, 1183M, 1191M, 1191I, 1191Q, 1191T, 1199Y, 1199F, 1199L, 1201V, 1191C, 1191V, 1191L, 1191Y, 1199W, 1199E, 1199H, 1178Y, 1178Q, 1178A, 1178S, 1183C, 1183P, and any combination thereof.

10. 10. A Clostridial neurotoxin for use, use or method according to claim 8 or 9, wherein the mutation of at least one amino acid residue consists of the mutations of two amino acid residues 1191M and 1199Y.

11. The BoNT / F H CC The domain is the H domain of natural BoNT / F. CC 8. A Clostridial neurotoxin for use, use or method according to any of claims 1 to 7, comprising a mutation in at least one amino acid residue which increases its binding affinity for one or more gangliosides compared to the domain.

12. The Clostridial neurotoxin, use or method of use according to claim 11, wherein the ganglioside is selected from GD1a and / or GM1a and / or the mutation in the amino acid residue is 1241K.

13. 11. The Clostridial neurotoxin of any one of claims 1 to 10, wherein the Clostridial neurotoxin is a BoNT / B neurotoxin.

14. 13. The Clostridial neurotoxin of any one of claims 1 to 7, 11 or 12, wherein the Clostridial neurotoxin is a BoNT / F neurotoxin.

15. 13. A Clostridial neurotoxin of any of the preceding claims, wherein the Clostridial neurotoxin is a chimeric neurotoxin.

16. The chimeric neurotoxin is the H C Domain and LH of BoNT / A N 16. The Clostridial neurotoxin of claim 15, comprising a domain.

17. The chimeric neurotoxin is an LH neurotoxin derived from the first BoNT / F subtype. N The clostridial neurotoxin, use or method of use according to claim 15, comprising a domain and an activation loop from a second BoNT / F subtype, said second BoNT / F subtype being different from said first BoNT / F subtype.

18. The above LH N The domain is the LH of BoNT / F7. N 18. The Clostridial neurotoxin, use or method of use according to claim 17, wherein said activation loop is a BoNT / F1 activation loop.

19. A Clostridial neurotoxin for use, use or method according to any of the preceding claims, wherein the Clostridial neurotoxin comprises an amino acid sequence selected from any of SEQ ID NOs: 2 to 13, or an amino acid sequence having at least 70% sequence identity thereto, preferably at least 90% or 95% sequence identity thereto.

20. The Clostridial neurotoxin of any of the preceding claims, comprising: a. the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence having at least 70% sequence identity thereto, preferably at least 90% or 95% sequence identity thereto, or b. the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence having at least 70% sequence identity thereto, preferably at least 90% or 95% sequence identity thereto; or c. the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 70% sequence identity thereto, preferably at least 90% or 95% sequence identity thereto; or d. the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having at least 70% sequence identity thereto, preferably at least 90% or 95% sequence identity thereto; or e. The amino acid sequence of SEQ ID NO:6, or an amino acid sequence having at least 70% sequence identity thereto, preferably at least 90% or 95% sequence identity thereto.

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