Treatment of autonomic neuropathy

Abstract

To provide a medicine for use in the treatment of an autonomic disorder.SOLUTION: The present invention provides a medicine for use in the treatment of an autonomic disorder in a human patient, wherein the medicine comprises the clostridial neurotoxin comprising HCC domain from BoNT / B.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] The present invention relates to the treatment of autonomic neuropathy with neurotoxins. [Background technology]

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

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

[0004] The mechanism of action of clostridial neurotoxins depends on five distinct steps: (1) H C (2) binding of the domain to the plasma membrane of its target neuron, followed by internalization of the bound toxin into the cell via endosomes; (3) H N(4) proteolytic cleavage of intracellular transport proteins known as SNARE proteins by the L chain providing a non-cytotoxic protease function; and (5) inhibition of cellular secretion from target cells.

[0005] In this sequence of events, SNARE proteins (Soluble N-ethylmaleimide-Sensitive Factor Attachment protein REceptor) are essential for the fusion of intracellular vesicles and therefore the secretion of molecules via vesicular transport from cells. Examples of SNARE proteins present in neuronal cells include, among others, SNAP-25, VAMP, or syntaxin. Meanwhile, the non-cytotoxic protease function of the L chain is a zinc-dependent endopeptidase activity that shows high substrate specificity for SNARE proteins. Thus, when delivered to neuronal target cells, the non-cytotoxic proteases of Clostridial neurotoxins inhibit the release of neurotransmitters by cleaving their substrate SNARE proteins, resulting in neuroparalysis.

[0006] Due to their unique properties, Clostridial neurotoxins such as botulinum toxins have been successfully used in a wide range of therapeutic applications, particularly for movement and autonomic disorders, for example to restore increased activity of nerve endings to normal levels. At least seven antigenically distinct BoNT serotypes have been described so far, namely BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, and BoNT / G (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 preparations (Botox®, Dysport®, and Xeomin®), while only one BoNT / B product is commercially available (Neurobloc® / Myobloc®). To date, these BoNT / A and BoNT / B products are toxins purified from Clostridium strains and are the only two BoNT serotypes approved by regulatory agencies for use in humans for uses ranging from spasticity, bladder dysfunction, and 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), to cervical dystonia (for BoNT / B) (see, e.g., https: / / www.medicines.org.uk / emc / medicine / 20568, herein incorporated by reference in its entirety), among others.

[0007] However, a current limitation of their therapeutic use is the generation of neutralizing antibodies in patients, which renders future treatments ineffective. Although there have been few reported antigenic problems with Neurobloc® / MyoBloc®, such antibody responses have been reported primarily with BoNT / A products, which remain the botulinum toxin of choice to date. These immunological responses have been found to be directly correlated with the dose of 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). Side effects may also occur due to the spread of the toxin to other areas of the body, and the likelihood of toxin spread is directly related to the dose injected.

[0008] Because both the production of neutralizing antibodies and the spread of toxin are directly related to the injected dose, 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 at lower doses.

[0009] A related challenge is the differences in how the various BoNT serotypes operate, which may result in different therapeutic effects for the same disorder.

[0010] Indeed, to exert its clinical activity, botulinum toxins must enter the nerve endings of their target cells. To do so, BoNT has a receptor-binding domain (BoNT-H) that is well defined in the literature. C) to target and invade neurons by binding to their specific receptors (Schiavo, G., Matteoli, M. & Montecucco, C. Neurotoxins affecting neuroexocytosis, Physiol Rev, 2000, 80, 717-766). Receptor binding generally determines the efficacy and specificity of BoNTs to recognize neurons. BoNT / B, DC, and G share two homologous synaptic vesicle proteins, synaptotagmin I and II (Syt I / II), as receptors, whereas 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 between species; for example, BoNT / DC and / G show a much higher affinity for SytII than for SytI in rodent motor nerve endings, whereas in humans they have a higher affinity for SytI than for SytII, a difference that is due to a unique amino acid change 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, / DC and / G compared to mouse SytII. This means that in humans, the high affinity receptor for BoNT / B, / DC and / G appears to be restricted to the minor receptor, SytI, at least in motor neurons.All these findings may explain the clinical observation that much higher doses of BoNT / B than BoNT / A (which binds to a different receptor) are required to achieve the same level of therapeutic efficacy 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, EJ & 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, BOTOX® and MyoBloc. TM A conversion ratio of 1:40 has been suggested between Botulinum toxin type B and axillar hyperhidrosis (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 coreceptor gangliosides that are abundant on neuronal cell surfaces and may differ between serotypes, cleaving different SNARE protein substrates, which may further impact therapeutic efficacy depending on the cell type targeted. Indeed, the L-chain proteases of BoNT / B, BoNT / D, BoNT / F, and BoNT / G cleave VAMP, whereas 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] Early therapeutic uses of botulinum neurotoxins relied on inhibition of acetylcholine release at the neuromuscular junction of skeletal muscle to treat neuromuscular conditions such as dystonia and spasticity, but it was later discovered that these neurotoxins are also effective against glands and smooth muscle by inhibiting acetylcholine release at autonomic nerve endings, and therefore can be used to treat a variety of autonomic disorders, such as hyperhidrosis, salivation, and overactive bladder.

[0013] The recommended dosage of Botox® (also known as Onabotulinumtoxin A) for treating hyperhidrosis is 50 units per axilla (https: / / www.medicines.org.uk / emc / medicine / 112), which is a dose of approximately 0.365 ng of the 150 kD BoNT / A toxin. Neurobloc® / Myobloc® (also known as Rimabotulinumtoxin B) is not approved for the treatment of hyperhidrosis, but has been disclosed in the literature in doses ranging from 2000 to 4000 units, or approximately 4 to 40 ng of the 150 kD BoNT / A toxin. However, such high doses are not without side effects, both at the local and systemic level, 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, the possibility of reaching therapeutic effects in autonomic neuropathy with lower doses of the toxin has not been studied. Summary of the Invention [Problem to be solved by the invention]

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

[0015] [Aspect 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. [Aspect 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. [Aspect 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. [Aspect 4] The Clostridial neurotoxin for use according to embodiment 1, the use according to embodiment 2, or the method according to embodiment 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. [Aspect 5] 2. The Clostridial neurotoxin of any of the preceding aspects, wherein the dosage of said Clostridial neurotoxin is in the range of about 0.00025 ng to about 3 ng. [Aspect 6] The use, use or method of any of the preceding aspects, 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 disorders directly related to central nervous disorders. [Aspect 7] The above H CC The domain is H of BoNT / B. CC Domain or H of BoNT / F CC A Clostridial neurotoxin of any preceding aspect, which is a domain, use, or method. [Aspect 8] The BoNT / B H CC The domain is the H domain of natural BoNT / B. CC The Clostridial neurotoxin of any of the preceding aspects, comprising a mutation of 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. [Aspect 9] 9. The method, use or use of a Clostridial neurotoxin according to 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. [Aspect 10] 10. The Clostridial neurotoxin of use, use or method according to embodiment 8 or 9, wherein said mutation of at least one amino acid residue consists of the two amino acid residue mutations 1191M and 1199Y. [Aspect 11] The BoNT / F H CC The domain is the H domain of natural BoNT / F. CC8. A Clostridial neurotoxin for use, use or method according to any of embodiments 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. [Aspect 12] 12. The Clostridial neurotoxin, use or method of use according to embodiment 11, wherein said ganglioside is selected from GD1a and / or GM1a and / or said mutation in amino acid residue is 1241K. [Aspect 13] 11. The Clostridial neurotoxin of any of the preceding aspects, wherein said Clostridial neurotoxin is a BoNT / B neurotoxin. [Aspect 14] 13. The Clostridial neurotoxin of any of aspects 1 to 7, 11 or 12, wherein said Clostridial neurotoxin is a BoNT / F neurotoxin. [Aspect 15] The Clostridial neurotoxin of any of the preceding aspects, the use or method, wherein said Clostridial neurotoxin is a chimeric neurotoxin. [Aspect 16] The chimeric neurotoxin is the H C Domain and LH of BoNT / A N 16. The Clostridial neurotoxin of embodiment 15, comprising a domain. [Aspect 17] The chimeric neurotoxin is an LH neurotoxin derived from the first BoNT / F subtype. N 16. The Clostridial neurotoxin of 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. [Aspect 18] The above LH N The domain is the LH of BoNT / F7. N 20. The Clostridial neurotoxin, use or method of use according to embodiment 17, wherein said activation loop is a BoNT / F1 activation loop. [Aspect 19] The Clostridial neurotoxin of any of the preceding aspects, 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. [Aspect 20] The Clostridial neurotoxin of any of the preceding aspects, the use or method of use 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 invention provides a Clostridial neurotoxin for use in treating autonomic neuropathy in a human patient, the neurotoxin comprising a BoNT / B, BoNT / D, BoNT / DC, BoNT / F, or BoNT / G derived H CC The present invention provides a method for treating autonomic neuropathy in a human patient in need thereof, comprising administering to the patient a Clostridial neurotoxin comprising a BoNT / B, BoNT / D, BoNT / DC, BoNT / F, or BoNT / G derived H 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 said autonomic neuropathy. CCThe present invention relates to a method comprising administering to said patient a Clostridial neurotoxin comprising a BoNT / B, BoNT / D, BoNT / DC, BoNT / F or BoNT / G-derived H domain in a dose equivalent or lower than the dose of BoNT / A for treating said autonomic neuropathy. More precisely, the present invention relates to a method for the preparation of a medicament for the treatment of autonomic neuropathy in a human patient. CC The present invention relates to the use of a Clostridial neurotoxin comprising a domain, wherein the dosage of the Clostridial neurotoxin to be administered to a patient is equal to or less than the dosage of BoNT / A to treat said autonomic neuropathy.

[0016] In a particular aspect, the present invention provides a Clostridial neurotoxin for use in treating autonomic neuropathy in a human patient, the neurotoxin being an H from BoNT / B, BoNT / D, BoNT / DC, BoNT / F, or BoNT / G. CC The present invention provides a Clostridial neurotoxin comprising a BoNT / B, BoNT / D, BoNT / DC, BoNT / F, or BoNT / G derived H domain, wherein the dosage of the Clostridial neurotoxin to be administered to a patient is about 1.1 to about 100 times lower, preferably about 1.3 to about 90 times lower, than the dosage of BoNT / A to treat the autonomic neuropathy. In other words, the present invention provides a method of treating an autonomic neuropathy in a human patient in need thereof, comprising administering to the patient a BoNT / B, BoNT / D, BoNT / DC, BoNT / F, or BoNT / G derived H domain. CC The present invention relates to a method for the preparation of a medicament for the treatment of autonomic neuropathy in a human patient, comprising administering to said patient a Clostridial neurotoxin comprising a BoNT / B, BoNT / D, BoNT / DC, BoNT / F or BoNT / G-derived H domain in a dose that is about 1.1 to about 100 times lower, preferably about 1.3 to about 90 times lower, than the dose of BoNT / A for treating said autonomic neuropathy. More precisely, the present invention relates to a method for the preparation of a medicament for the treatment of autonomic neuropathy in a human patient, comprising administering to said patient a Clostridial neurotoxin comprising a BoNT / B, BoNT / D, BoNT / DC, BoNT / F or BoNT / G-derived H domain in a dose that is about 1.1 to about 100 times lower, preferably about 1.3 to about 90 times lower, than the dose of BoNT / A for treating said autonomic neuropathy. CC The present invention relates to the use of a Clostridial neurotoxin comprising a domain, wherein the dosage of said Clostridial neurotoxin to be administered to a patient is about 1.1 to about 100 times lower, preferably about 1.3 to about 90 times lower, than the dosage of BoNT / A to treat said autonomic neuropathy, said dosage being preferably quantified in nanograms.

[0017] Moreover, in a particular embodiment, the present invention provides a Clostridial neurotoxin for use in treating autonomic neuropathy in a human patient, the neurotoxin comprising a clostridial neurotoxin derived from BoNT / B, BoNT / D, BoNT / DC, BoNT / F, or BoNT / G. CC The present invention provides a Clostridial neurotoxin comprising a BoNT / B, BoNT / D, BoNT / DC, BoNT / F, or BoNT / G derived H domain, and a dosage of the Clostridial neurotoxin ranging from about 0.00025 ng to about 3 ng should be administered to the patient. In other words, the present invention provides a method of treating autonomic neuropathy in a human patient in need thereof, comprising administering to the patient a BoNT / B, BoNT / D, BoNT / DC, BoNT / F, or BoNT / G derived H domain. CC administering to said patient a Clostridial neurotoxin comprising the domain at a dosage ranging from about 0.00025 ng to about 3 ng.

[0018] More precisely, the present invention relates to a method for the preparation of a drug for the treatment of autonomic neuropathy in a human patient, comprising administering to the patient a medicament derived from BoNT / B, BoNT / D, BoNT / DC, BoNT / F or BoNT / G. CC The present invention relates to the use of a Clostridial neurotoxin comprising a domain, wherein the dosage of said Clostridial neurotoxin to be administered to a patient ranges from about 0.00025 ng to about 3 ng.

[0019] The present invention is based, first, on the unexpected discovery by the inventors that BoNT / B has at least the same effect as BoNT / A on human smooth muscle tissue.

[0020] This finding is unexpected because, as mentioned above, much higher doses of BoNT / B (Neurobloc® / Myobloc®) are required to treat skeletal muscle disorders compared to BoNT / A (e.g., Botox®). It is likely based on this clinical observation that only higher doses of BoNT / B (Neurobloc® / Myobloc®) than BoNT / A (e.g., Botox®) have been tested and reported in the literature so far for the treatment of, for example, hyperhidrosis involving smooth muscle.

[0021] This difference in efficacy of BoNT / B in these two tissues is hypothesized to be due to the fact that smooth and skeletal muscle disorders do not actually depend on the same neural pathways. Smooth muscle contraction is indeed driven by the autonomic nervous system, whereas 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 muscles, whereas other disorders, such as overactive bladder (OAB) or neurogenic detrusor overactivity (NDO), are caused by excessive contraction of smooth muscles.

[0022] The somatic nervous system (SoNS or voluntary nervous system) is the part of the peripheral nervous system involved in the voluntary control of skeletal muscles and body movements. The SoNS consists of afferent and efferent nerves. SoNS afferent nerves are involved in relaying sensations from the body to the central nervous system (CNS), while SoNS afferent nerves are involved in sending commands from the CNS to the body and stimulating muscle contraction, and these include all the 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 is made up of afferent and efferent nerves. The ANS is made up 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. The ANS also controls smooth muscles, as well as secretions such as sweat, saliva, and tears.

[0024] Smooth muscles are found in the urinary system (e.g., bladder, ureters), digestive system (e.g., stomach wall, gastrointestinal tract, intestines, sphincter of Oddi, anal sphincter, trachea, bile duct), reproductive system (e.g., prostate, uterus), respiratory tract, vascular system (e.g., blood vessel walls, aorta, arteries, arterioles, veins), and iris of the eye. Smooth muscle contraction is involved in many bodily functions, such as releasing urine from the bladder, moving food through the digestive tract, regulating airflow in the lungs, regulating arterial and venous blood pressure, and constricting pupil size.

[0025] The synaptic nerve endings of cholinergic neurons supplying the eccrine sweat glands, salivary glands, and lacrimal glands can be targeted with botulinum neurotoxins. Neurological disorders associated with sweating or hypersecretory activity include hyperhidrosis, especially focal hyperhidrosis of the palms, armpits, or feet, gustatory sweating (Frey's syndrome), increased lacrimation while eating (crocodile tears syndrome), hypersalivation (salivation, saliva flow), etc.

[0026] Without wishing to be bound by theory, it is hypothesized that the differences in potency observed between BoNT / A and BoNT / B in the somatic versus autonomic nervous system are 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, it is further hypothesized that other BoNT serotypes that use Syt I / II as a protein receptor and / or cleave VAMP, such as BoNT / D, BoNT / DC, BoNT / F, or BoNT / G, will exhibit similar, if not better, efficacy than BoNT / B against the smooth muscle / autonomic nervous system, at least in humans.

[0028] Thus, one aspect of the present invention is a Clostridial neurotoxin for use in treating autonomic neuropathy in a human patient, the neurotoxin being an H from BoNT / B, BoNT / D, BoNT / DC, BoNT / F, or BoNT / G. CC The present invention provides a method for treating autonomic neuropathy in a human patient in need thereof, comprising administering to the patient a Clostridial neurotoxin comprising a BoNT / B, BoNT / D, BoNT / DC, BoNT / F, or BoNT / G-derived H 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 said autonomic neuropathy. CC The present invention relates to a method comprising administering to said patient a Clostridial neurotoxin comprising a BoNT / B, BoNT / D, BoNT / DC, BoNT / F or BoNT / G-derived H domain in a dose equivalent or lower than the dose of BoNT / A for treating said autonomic neuropathy. More precisely, the present invention relates to a method for the preparation of a medicament for the treatment of autonomic neuropathy in a human patient. CC The present invention relates to the use of a Clostridial neurotoxin comprising a domain, wherein the dosage of the Clostridial neurotoxin to be administered to a patient is equal to or less than the dosage of BoNT / A to treat said autonomic neuropathy.

[0029] In one embodiment, the autonomic neuropathies treated by the clostridial neurotoxins of the invention are selected from the group consisting of smooth muscle disorders, hypersecretory disorders, respiratory disorders, inflammatory disorders with an autonomic component, neuroendocrine disorders, and other autonomic disorders directly related to central nervous disorders.

[0030] Examples of such autonomic neuropathy include, without limitation: Smooth muscle disorders, especially those caused by excessive, abnormal, and / or prolonged muscle contractions (spastic disorders), including, for example: Neurogenic detrusor overactivity (NDO), overactive bladder (OAB), especially idiopathic OAB (iOAB), bladder spasms, detrusor-sphincter dyssynergia (DSD), urinary incontinence, urinary retention, nocturia, urge incontinence, frequent urination, and other urinary disorders, gastrointestinal disorders such as sphincter of Oddi dysfunction, esophageal spasms, intestinal spasms, achalasia, gastroparesis, spastic colitis, anal fissures, constipation, occasional diarrhea, dysphagia, oropharyngeal dysphagia, and swallowing disorders, Raynaud's disease, anastomotic thrombosis, atrial fibrillation after cardiac surgery, orthostatic hypotension, blood pressure disorders and other vascular and cardiovascular disorders, Prostate disorders such as benign prostate hyperplasia (BPH), prostatitis, prostatodynia, and prostate enlargement, Sexual disorders: erectile dysfunction, priapism, ejaculation disorder, vaginismus, Hypersecretory disorders, such as hyperhidrosis (axillary, palmar, plantar hyperhidrosis, diffuse sweating, Frey's syndrome, etc.), hypersalivation (saliva overflow, drooling), gustatory sweating, excessive lacrimation, crocodile tears syndrome, excessive mucus secretion, bromhidrosis, gastric acid secretion, acne, Respiratory disorders with hypersecretory and / or muscular components, e.g., rhinorrhea, chronic rhinitis, asthma, chronic obstructive pulmonary disease (COPD), bronchial hyperresponsiveness, wheezing, involuntary shortness of breath, apneic episodes, etc. Inflammatory disorders with an autonomic component, e.g., otitis media, itching, pruritus, inflammatory bowel syndrome, Neuroendocrine disorders: diabetes, hyperinsulinism, hyperglucagonemia, pancreatic disorders, thyroid disorders, hypocalcemia, hyperthyroidism, metabolic disorders, excessive lipolysis, and • Other autonomic disorders directly related to central nervous system disorders such as Parkinson's disease and 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] The term "Clostridial neurotoxin" as used herein refers to any polypeptide that enters neuronal cells and inhibits neurotransmitter release. This process includes binding of the neurotoxin to low or high affinity receptors, internalization of the neurotoxin, translocation of the endopeptidase portion of the neurotoxin to the cytoplasm, and enzymatic modification of the neurotoxin substrate. More specifically, the term "neurotoxin" includes any polypeptide produced by Clostridial bacteria (Clostridial neurotoxin) that enters neuronal cells and inhibits neurotransmitter release, as well as 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 Clostridial neurotoxin is a botulinum neurotoxin (BoNT).

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

[0034] An example of a BoNT / A neurotoxin amino acid sequence is provided as SEQ ID NO: 1 (UniProt Accession No. A5HZZ9). An example of a BoNT / B neurotoxin amino acid sequence is provided as SEQ ID NO: 2 (UniProt Accession No. B1INP5). An example of a BoNT / C neurotoxin amino acid sequence is provided as SEQ ID NO: 3 (UniProt Accession No. P18640). An example of a BoNT / D neurotoxin amino acid sequence is provided as SEQ ID NO: 4 (UniProt Accession No. P19321). An example of a BoNT / E neurotoxin amino acid sequence is provided as SEQ ID NO: 5 (Accession No. WP_003372387). An example of a BoNT / F neurotoxin amino acid sequence is provided as SEQ ID NO: 6 (UniProt Accession No. Q57236) or SEQ ID NO: 11 (UniProt / UniParc Accession No. UPI0001DE3DAC). An example of a BoNT / G neurotoxin amino acid sequence is provided as SEQ ID NO: 7 (Accession No. WP_039635782). An example of a BoNT / DC neurotoxin amino acid sequence is provided as SEQ ID NO:8 (Accession No. BAM65681).

[0035] As used herein, "H C The term "domain" refers to a functionally distinct region of the neurotoxin heavy chain, with a molecular weight of approximately 50 kDa, that enables the neurotoxin to bind to a receptor located on the surface of a target cell. C The domain is divided into two structurally distinct subdomains, “H CN Subdomains (H C N-terminal part of the domain) and "H CC Subdomains (H C The C-terminal part of the domain, H CC H consists of two domains, each with a molecular weight of approximately 25 kDa. CC The domain is capable of binding to a Clostridial neurotoxin protein receptor.

[0036] As used herein, "LH N The term "domain" is C The domains are distinct from the endopeptidase domain (the "L" or "light chain") and the domain responsible for translocation of the endopeptidase into the cytoplasm (the H of the heavy chain).N The endopeptidase domain ("L" or "light chain") is capable of cleaving SNARE proteins.

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

[0038] The reference sequences given above should be considered as a guideline and slight variations may occur depending on the subserotype, as for example US2007 / 0166332 (the disclosure of which is incorporated herein by reference in its entirety) cites slightly different Clostridium sequences.

[0039] The term "activation loop" refers to a polypeptide domain that contains a proteolytic cleavage site. Activation loops of neurotoxins have been described in the art, such as in WO2016156113, the disclosure of which is incorporated herein by reference in its entirety.

[0040] In one embodiment of the present invention, the H of the Clostridial neurotoxin CC The domain consists of or comprises 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 - 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 as used herein may be calculated by dividing the number of identical amino acids at each position in the alignment by the total number of amino acids in the aligned sequences, multiplied by 100. The calculation of percent sequence identity may take into account 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 comparison of sequences between two or more sequences and the determination of percent identity may be performed using certain mathematical algorithms well known to those skilled in the art, in particular global alignment mathematical algorithms (such as those described in Needleman and Wunsch, J. Mol. Biol. 48(3), 443-453, 1972).

[0042] In a preferred embodiment, H CC The domain consists of or comprises 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 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 - 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, H CC The domain consists of or comprises an amino acid sequence corresponding to 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, H CC The domain consists of or comprises an amino acid sequence corresponding to 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, H CC The domain consists of or comprises an amino acid sequence corresponding to 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 Clostridial 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, or SEQ ID NO:11.

[0047] In a preferred embodiment, the Clostridial 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 Clostridial 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 Clostridial 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 Clostridial 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 Clostridial neurotoxin is a BoNT / B neurotoxin. Preferably, the BoNT / B Clostridial 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 Clostridial neurotoxin is a BoNT / F neurotoxin. Preferably, the BoNT / F Clostridial 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 Clostridial neurotoxin is a chimeric neurotoxin.

[0054] As used herein, the term "chimeric neurotoxin" refers to a neurotoxin that contains 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 contain an LH domain derived from a first neurotoxin serotype or subtype. N domain and an H from a second neurotoxin serotype or subtype. C Another example of a chimeric neurotoxin may include an LH domain derived from a first neurotoxin serotype or subtype. N H CN domain and an H from a second neurotoxin serotype or subtype. CC Another example of a chimeric neurotoxin is a neurotoxin that contains an LH domain derived from a first neurotoxin serotype or subtype. N A chimeric neurotoxin is a neurotoxin that comprises a chimeric domain and an activation loop derived from a second neurotoxin serotype or subtype. Examples of chimeric neurotoxins are described in WO2017191315 and WO2016156113, both of which are incorporated herein by reference.

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

[0056] In one embodiment of the chimeric neurotoxin according to the invention, H C The 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, - 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, - 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, - 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, - 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 - 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 LH N The domain consists of or comprises an amino acid sequence selected from the group consisting of: - 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, - 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, - 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 polypeptide sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto.

[0057] In a preferred embodiment, H C The 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 N The domain consists of or comprises an amino acid sequence selected from the group consisting of: - 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, - 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 clostridial neurotoxin is H from BoNT / B. C Domain and LH from BoNT / A N It is a chimeric neurotoxin comprising the domain.

[0059] In a more preferred embodiment, H C The 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 NThe domain comprises 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 clostridial neurotoxin is H from BoNT / B. CC domain and LH from BoNT / A, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, or BoNT / DC N H CN It is a chimeric neurotoxin comprising the domain.

[0061] In one embodiment of the chimeric neurotoxin according to the invention, H CC The domain consists of or comprises 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 - 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 comprises an amino acid sequence selected from the group consisting of: - 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, - 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, - 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, - 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, - 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, - 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, - 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, - 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 - 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 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, and N H CN The domain consists of or comprises an amino acid sequence selected from the group consisting of: - 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, - 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, - 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, - 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, - 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, - 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, - 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 - 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 Clostridial neurotoxin is an LH neurotoxin from a first BoNT / F subtype. N and an activation loop from a second BoNT / F subtype, the second BoNT / F subtype being distinct 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 disclosure of which is incorporated herein by reference in its entirety).

[0064] In a preferred embodiment, the LH N The domain is BoNT / F7 subtype LH N domain and / or said activation loop is a 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 Clostridial neurotoxin can be a modified neurotoxin and derivatives thereof, including, but not limited to, those described below. The modified neurotoxin or derivative can include one or more amino acids that are modified compared to the native neurotoxin. Such modifications are also known as mutations, and examples include, but are not limited to, substitutions, additions, or deletions of amino acid residues. In the context of the present invention, a modified Clostridial neurotoxin can have, for example, a modified amino acid sequence in one or more domains compared to the amino acid sequence of a native Clostridial neurotoxin. The terms native, unmodified, natural, naturally occurring, or wild-type can be used interchangeably herein.

[0068] Such modifications may 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 be functional. In other words, the modified neurotoxin of the present invention retains a function(s) of the neurotoxin 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 neurotoxins of the present invention comprise a heavy chain (H C The neurotoxin may have one or more modifications in the amino acid sequence of the heavy chain (e.g., domain), such that the modified heavy chain binds to target neurons with higher or lower affinity than the native neurotoxin. C These modifications in the H domain can alter the binding of target neurons to gangliosides. CC Modification of amino acid residues in the ganglioside-binding site of the domain and / or H that can alter binding to protein receptors on target neurons. CC This may include modification of amino acid residues in the protein receptor binding sites of the domains. Examples of such modified neurotoxins are described in WO2006027207 and WO2006114308, both of which are incorporated herein by reference in their entirety.

[0070] In one embodiment of the modified Clostridial neurotoxin according to the invention, the H from BoNT / B, BoNT / D, BoNT / DC, BoNT / F, or BoNT / G is CC The domain is a natural H CC Modified relative to the domain.

[0071] In a preferred embodiment, the H from BoNT / B, BoNT / D, BoNT / DC, BoNT / F, or BoNT / G neurotoxin CC The domain is the H domain of natural BoNT / B, BoNT / D, BoNT / F, BoNT / G, or BoNT / DC. CC The H domain for human Syt II is CC The present inventors have found that mutation of at least one amino acid residue increases the binding affinity of the domain, and that such mutation can increase the efficacy of the neurotoxin against human smooth muscle.

[0072] In a more preferred embodiment, H from BoNT / B neurotoxin CC The domain is the H domain of natural BoNT / B. CC The H domain for human Syt II is CC The mutation of at least one amino acid residue in the H domain of natural BoNT / B is preferably at least one amino acid residue that increases the binding affinity of the domain. CC The H domain for human Syt II is CC Increase the binding affinity of the domain by at least 50%.

[0073] BoNT / B H CC Such suitable mutations of amino acid residues in domains are described in the art in WO2013180799 and WO2016154534, both of which are incorporated herein by reference.

[0074] In particular, the natural BoNT / B H CCCompared with the H domain of BoNT / B against human Syt II, CC The mutation of at least one amino acid residue suitable for increasing the binding affinity of the domain 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. CC The mutation of at least one amino acid residue in the domain 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. CC The mutation of at least one amino acid residue in the H domain of BoNT / B consists of a substitution, addition, or deletion of three amino acid residues: 1191M, 1199W, and 1178Q. CC The mutation of the at least one amino acid residue in the domain consists of a substitution, addition, or deletion of two amino acid residues: 1191M and 1199Y.

[0075] In a more preferred embodiment, the H of natural BoNT / B CC Compared with the H domain of BoNT / B against human Syt II, CCThe mutation of at least one amino acid residue suitable for increasing the binding affinity of the domain by at least 50% is a substitution of an amino acid residue 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. CC The mutation of at least one amino acid residue in the domain is 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. CC The mutation of at least one amino acid residue in the H domain of BoNT / B comprises three amino acid residue substitutions: E1191M, S1199W, and W1178Q. CC The mutation of the 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 the BoNT / B to be modified CC The domain is amino acid residues 1082 to 1291 of SEQ ID NO:2 (the H 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 Clostridial 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 from BoNT / B, BoNT / D, BoNT / DC, BoNT / F, or BoNT / G neurotoxin CC The domain is the H domain of natural BoNT / B, BoNT / D, BoNT / DC, BoNT / F, or BoNT / G. CC domain, the H for one or more gangliosides. CC It comprises a mutation in at least one amino acid residue that increases the binding affinity of the domain.

[0079] In a more preferred embodiment, the H derived from BoNT / F CC The domain is the H domain of natural BoNT / F. CC domain, the H for one or more gangliosides. CC It comprises a mutation in at least one amino acid residue that increases the binding affinity of the domain.

[0080] In a preferred embodiment, the ganglioside is selected from GD1a and / or GM1a.

[0081] Natural BoNT / F H CC Compared with the H domain, the H of BoNT / F against gangliosides such as GD1a and / or GM1a CC Suitable mutations of amino acid residues for increasing the binding affinity of the domain include, but are not limited to, the amino acid mutation 1241K, such as an amino acid substitution, addition, or deletion. More preferably, the H of natural BoNT / F is CC The H domain of BoNT / F is compared to CC The mutation of at least one amino acid residue that increases the binding affinity of the domain is the amino acid substitution H1241K.

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

[0083] In a preferred embodiment, a chimeric Clostridial neurotoxin of the 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 Clostridial neurotoxin of the present invention may be chimeric and modified as described above. For example, in a preferred embodiment, the Clostridial 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 Clostridial neurotoxins of the present invention can be produced using recombinant techniques. Thus, in one embodiment, the Clostridial neurotoxins of the present invention are recombinant Clostridial neurotoxins.

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

[0087] In another embodiment, the Clostridial neurotoxin does not include (or is not associated with or associated with) a BoNT complex protein, in other words, the Clostridial neurotoxin is administered to a human patient without association or combination with a BoNT complex protein.

[0088] Preferably, the Clostridial neurotoxin (e.g., for use as described herein) is part of a pharmaceutical composition together with at least one pharma- ceutically acceptable carrier. By "pharma-ceutically acceptable carrier" herein is meant any component that is compatible with the other components of the pharmaceutical composition, particularly the Clostridial neurotoxin, and is not harmful to a human patient. Pharmaceutically acceptable carriers can be selected based on the desired route of administration, according to standard pharmaceutical practice, and include, but are not limited to, excipients, diluents, adjuvants, propellants, and salts.

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

[0090] The clostridial neurotoxins of the present invention may be formulated for oral, parenteral (i.e., injection), continuous infusion, inhalation, or topical administration. Compositions suitable for injection may be in the form of a solution, suspension, or emulsion, or as a dry powder that is dissolved or suspended in a suitable solvent before use.

[0091] In the case of locally delivered neurotoxins, the neurotoxins may be formulated as a cream (eg, for topical application) or for subcutaneous injection.

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

[0093] The clostridial neurotoxins of the invention may be administered to a patient by intrathecal or epidural injection in the spinal column at the level of the spinal segment involved in the innervation of the affected organ.

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

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

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

[0097] However, it is also possible to administer the Clostridial neurotoxin to the muscle without using an injection. For example, to treat urinary disorders, the Clostridial neurotoxin may be administered to the bladder by injecting a liquid or semi-solid formulation of the Clostridial neurotoxin into the patient's bladder, placing a gel formulation containing the Clostridial neurotoxin in the appropriate location of the patient's bladder, spraying an aerosol formulation containing the Clostridial neurotoxin in the appropriate location of the patient's bladder, or locally administering a solid (e.g., lyophilized), semi-solid, or liquid botulinum toxin formulation applied or coated on the outer wall of a balloon that is then inflated in the bladder and contacts the wall of the bladder, as described in WO2005053733, the disclosure of which is incorporated herein by reference in its entirety.

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

[0099] Preferably, the BoNT / A used to treat the same autonomic neuropathy is purified BoNT / A. As used herein, the term "purified BoNT / A" refers to botulinum neurotoxin type A purified from a Clostridium strain that naturally produces botulinum neurotoxin type A (naturally occurring Clostridium strain). Purified BoNT / A may be associated with complex proteins or may be free of complex proteins. Examples of commercially available purified BoNT / A include Botox®, Dysport®, and Xeomin®.

[0100] In one embodiment, the dosage (therapeutic amount) of a Clostridial neurotoxin administered to treat an autonomic neuropathy in a human patient is about 1.1 to about 100 times lower, preferably about 1.3 to about 90 times lower, than the dosage of BoNT / A to treat said autonomic neuropathy.

[0101] In a preferred embodiment, the clostridial neurotoxin is H from BoNT / B. CCWhen the clostridial neurotoxin contains a domain, the therapeutic dose of the clostridial neurotoxin is about 1.1 to about 1.20 times, preferably about 1.3 to about 19.8 times, of the dose of BoNT / A to treat the same autonomic neuropathy. For example, when the clostridial neurotoxin of the present invention is a native BoNT / B neurotoxin, such as a neurotoxin having an amino acid sequence of SEQ ID NO: 2, the therapeutic dose of the clostridial neurotoxin is about 1.1 to about 1.5 times, preferably about 1.3 times, of the dose of BoNT / A to treat the same autonomic neuropathy. As another example, when the clostridial neurotoxin of the present invention is a modified BoNT / B neurotoxin having an increased binding affinity to the human SytII receptor (compared to native BoNT / B), such as a neurotoxin having an amino acid sequence of SEQ ID NO: 9, the therapeutic dose of the clostridial neurotoxin is about 15 to about 1.20 times, preferably about 19.8 times, of the dose of BoNT / A to treat the same autonomic neuropathy. Furthermore, as another example, when the Clostridial neurotoxin of the present invention is a modified chimeric BoNT / B neurotoxin with increased binding affinity to the human Syt II receptor, such as a neurotoxin having the amino acid sequence of SEQ ID NO: 10, the therapeutic dose of the Clostridial neurotoxin is about half to about one-fifth, preferably about one-three-and-a-half times lower, than the dose of BoNT / A to treat the same autonomic neuropathy.

[0102] In a preferred embodiment, the clostridial neurotoxin is H from BoNT / F. CCWhen the clostridial neurotoxin contains a domain, the therapeutic dose of the clostridial neurotoxin is about 60 to about 100 times lower, preferably about 61.2 to about 190 times lower, of the dose of BoNT / A to treat the same autonomic neuropathy. For example, when the clostridial neurotoxin of the present invention is a native BoNT / F neurotoxin, such as a neurotoxin having the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 11, the therapeutic dose of the clostridial neurotoxin is about 60 to about 65 times lower, preferably about 61.2 times lower, of the dose of BoNT / A to treat the same autonomic neuropathy. As another example, when the clostridial neurotoxin of the present invention is a modified BoNT / F neurotoxin having increased binding affinity for one or more gangliosides (compared to native BoNT / F), such as a neurotoxin having the amino acid sequence of SEQ ID NO: 12, the therapeutic dose of the clostridial neurotoxin is about 60 to about 65 times lower, preferably about 61.2 times lower, of the dose of BoNT / A to treat the same autonomic neuropathy. Furthermore, as another example, when the Clostridial neurotoxin of the present invention is a chimeric BoNT / F neurotoxin, such as a neurotoxin having the amino acid sequence of SEQ ID NO: 13, the therapeutic dose of the Clostridial neurotoxin is about 85 to about 100 times lower, preferably about 90 times lower, than the dose of BoNT / A to treat the same autonomic neuropathic disorder.

[0103] As mentioned above, dosages of BoNT / A to treat autonomic neuropathy are well known in the art (https: / / www.medicines.org.uk / emc / medicine / 112).

[0104] Dosages of clostridial neurotoxins are preferably measured herein in nanograms.

[0105] The dosage of the Clostridial neurotoxin according to the present invention should be understood as the dosage of the active bichain Clostridial neurotoxin, i.e., without including the amount of the complex protein that may be associated with the neurotoxin. In other words, it refers to the dosage of the active bichain Clostridial neurotoxin, whether administered to a patient in association with the complex protein or without the complex protein. As is well known to those skilled in the art, the active bichain Clostridial neurotoxin is capable of binding to neuronal receptors, translocating the light chain to the cytoplasm, and cleaving SNARE proteins, whereas 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 bichain is generally about 150 kD.

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

[0107] The amount of Clostridial neurotoxin can be measured by methods conventionally used in the art for quantifying proteins, preferably at the nanogram level, including, inter alia, 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] Where a range of values ​​is presented herein, unless the context clearly indicates otherwise, each intervening value, to the tenth of a unit, between the upper and lower limit of that range is also understood to be specifically disclosed. Every smaller range between any stated or intervening value in a stated range and any other stated or intervening value in the stated range is included in the disclosure. Additionally, any range of numerical values ​​expressed herein as "a to b" is understood to mean a range of numerical values ​​running from a to b (i.e., including the precise endpoints a and b).

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

[0110] In a preferred embodiment, the dosage (ie, therapeutic amount) of a Clostridial neurotoxin of the invention to be administered to treat 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 a Clostridial 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 said Clostridial neurotoxin.

[0112] In a preferred embodiment, the clostridial neurotoxin is the H CC Domain or H of BoNT / F CC The clostridial neurotoxin may be administered to a human patient in any of the doses described herein and used to treat autonomic neuropathy, as described above.

[0113] In a more preferred embodiment, the clostridial neurotoxin is the H CC Domain or H of BoNT / F CC The neurotoxin may be administered to a human patient in any of the doses of the Clostridial neurotoxin described herein, as described above, for use in treating a smooth muscle disorder, 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, H of BoNT / B CC The therapeutic amount of a Clostridial neurotoxin containing a domain preferably ranges from about 0.001 ng to about 2 ng.

[0115] Furthermore, for example, H of BoNT / B CC The therapeutic amount of a Clostridial neurotoxin containing a domain preferably ranges from about 0.0003 ng to about 0.05 ng.

[0116] It will be understood, however, that the dosage range required will depend on the precise nature of the Clostridial neurotoxin, the autonomic neuropathy, the route of administration, the nature of the formulation, the age of the patient, the nature, extent, or severity of the patient's condition, any contraindications, and the judgment of the attending physician. Variations in such dosage levels can be adjusted using standard empirical routines for optimization.

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

[0118] Examples of suitable dosage ranges for native 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): 0.5ng to 2ng of natural BoNT / B in the detrusor muscle, - OAB (overactive bladder): 0.25 ng to 1 ng of native BoNT / B into the detrusor muscle; - Axillary hyperhidrosis: 0.125 ng to 0.5 ng of natural BoNT / B in each axilla, - salivation in adult patients, especially those with Parkinson's disease: 0.05 ng to 0.2 ng natural BoNT / B per submandibular and / or parotid gland; - Drooling in children with cerebral palsy: 0.025 ng to 0.1 ng of natural BoNT / B per submandibular and / or parotid gland.

[0119] Examples of suitable dosage ranges for modified BoNT / B (such as a neurotoxin having the amino acid sequence of SEQ ID NO:9) with increased binding affinity to the human Syt II receptor according to the present invention for treating the above-mentioned disorders are as follows: - NDO (neurodetrusor 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 in each axilla, - salivation in adult patients, especially those with Parkinson's disease: 0.0025 ng to 0.02 ng of modified BoNT / B per submandibular and / or parotid gland; - Drooling in children with cerebral palsy: 0.001 ng to 0.01 ng of modified BoNT / B per submandibular and / or parotid gland.

[0120] Examples of suitable dosage ranges for chimeric modified BoNT / B (such as a neurotoxin having the amino acid sequence of SEQ ID NO: 10) with increased binding affinity to the human Syt II receptor according to the present invention for treating the above-mentioned disorders are as follows: - NDO (neurodetrusor overactivity): 0.2ng to 1ng 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, especially those with Parkinson's disease: 0.02 ng to 0.1 ng of chimeric modified BoNT / B per submandibular and / or parotid gland; - Drooling in children with cerebral palsy: 0.01 ng to 0.05 ng of chimeric modified BoNT / B per submandibular and / or parotid gland.

[0121] Examples of suitable dosage ranges for 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-mentioned disorders according to the present invention are as follows: - NDO (neurogenic detrusor overactivity): 0.01 ng to 0.05 ng of natural BoNT / F in the detrusor muscle, - OAB (overactive bladder): 0.005ng to 0.025ng of natural BoNT / F in the detrusor muscle; - Axillary hyperhidrosis: 0.0025 ng to 0.01 ng of natural BoNT / F in each axilla; - salivation in adult patients, especially those with Parkinson's disease: 0.001 ng to 0.005 ng of natural BoNT / F per submandibular and / or parotid gland; - Drooling in children with cerebral palsy: 0.0005 ng to 0.0025 ng of natural BoNT / F per submandibular and / or parotid gland.

[0122] Examples of suitable dosage ranges of modified BoNT / B (such as a neurotoxin having the amino acid sequence of SEQ ID NO: 12) with increased binding affinity to one or more gangliosides according to the present invention for treating the above-mentioned disorders are as follows: - NDO (neurodetrusor 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, especially those with Parkinson's disease: 0.001 ng to 0.005 ng of modified BoNT / F per submandibular and / or parotid gland, - Drooling in children with cerebral palsy: 0.0005 ng to 0.0025 ng of modified BoNT / F per submandibular and / or parotid gland.

[0123] Examples of suitable dosage ranges for a chimeric BoNT / F (such as a neurotoxin having the amino acid sequence of SEQ ID NO: 13) according to the present invention for treating the disorders described above are as follows: - NDO (neurodetrusor overactivity): 0.007ng to 0.03ng of chimeric BoNT / F into the detrusor muscle, - OAB (overactive bladder): 0.003ng to 0.0015ng of chimeric BoNT / F into the detrusor muscle; - axillary hyperhidrosis: 0.001 ng to 0.007 ng of chimeric BoNT / F in each axilla, - salivation in adult patients, especially those with Parkinson's disease: 0.0007 ng to 0.003 ng of chimeric BoNT / F per submandibular and / or parotid gland; - Drooling in children with cerebral palsy: 0.0003 ng to 0.0015 ng of chimeric BoNT / F per submandibular and / or parotid gland.

[0124] The disclosure is not limited by the exemplary 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 embodiments of the present disclosure. Unless otherwise indicated, any amino acid sequence is written left to right in amino to carboxy orientation, respectively.

[0125] Further, as used herein and in the appended claims, it should be noted that the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a clostridial neurotoxin" includes a plurality of such candidate agents, reference to "the clostridial neurotoxin" includes reference to one or more clostridial neurotoxins and equivalents thereof known to those skilled in the art, and so forth.

[0126] The invention will now be described, by way of example only, with reference to the following figures and examples. [Brief description of the drawings]

[0127] [Figure 1] Bladder strip contractile responses to EFS-induced contractions after three stimulations, 1 min apart, followed by a 3 min rest period. [Figure 2A]Time to 50% inhibition (T50) of maximum EFS contraction for amplitudes with 1, 3, 5, or 10 nM nBoNT / A, nBoNT / B, rBoNT / BMY, and rBoNT / ABMY. [Figure 2B] Time to 50% inhibition (T50) of maximal EFS contraction for amplitudes with 0.1, 1, or 10 nM nBoNT / F, mrBoNT / F, and mrBoNT / F7-1. [Diagram 3] Concentration-response curves for nBoNT / A, nBoNT / B, rBoNT / BMY, rBoNT / ABMY, nBoNT / F, and mrBoNT / F7-1. Time to 50% inhibition (T50) was plotted versus botulinum toxin protein concentration and fitted with a logarithmic function to obtain excellent R2 values.

[0128] Amino acid sequence

[0129] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] EXAMPLES

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

[0131] Example 1: Study of human bladder tissue samples

[0132] Briefly, human bladder tissue samples were obtained from patients undergoing radical cystectomy for bladder cancer with no documented bladder dysfunction, ensuring that collection and use of tissue and other samples was carried out in accordance with all relevant laws, regulations, and codes of practice, including obtaining written patient informed consent (including data privacy obligations), and consistent with the patient's medical history.

[0133] After the surgical procedure, the samples were immediately transported from the operating room to the pathology facility, and normal pieces of the bladder dome, i.e., those without visible tumor tissue, were selected for the experiments. Immediately after removal, the tissue samples were stored at 4°C in Krebs-HEPES buffer (with 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) for optimal storage until use (maximum 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 each donor's bladder per 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) consisting of an organ chamber maintained at 37°C and filled with Krebs-HEPES buffer, continuously bubbled with 95% O2 and 5% CO2 to maintain a pH of 7.4. Bladder strips were suspended in the 5 ml organ chamber and connected to force transducers (Pioden controls Ltd, UK) for isometric tension recording. An initial tension of 1 g was applied. Following amplification, tension changes 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 located on either side of the strip and connected to a stimulator (Bionic System Nozay, France).

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

[0137] Following the equilibration period, the organ bath was then filled with KCl (100 mM, 10 min), followed by carbachol (3.10 -6 M, 10 min) were added sequentially to prime the detrusor smooth muscle strips, with a wash step between each compound addition, after which 0.5% gelatin was injected into the organ bath prior to application of the EFS train.

[0138] EFS trains (20 Hz, pulse duration 1 ms, train duration 5 s, 300 mA) were administered 1 min apart, followed by a 3 min rest period, with three successive stimulations (stimulation conditions chosen to give robust and stable contractions in human bladder strips based on our experience with human bladder tissue). Stimulation was continued until a stable response was obtained (a response was considered stable if the percentage of variation in EFS contraction amplitude calculated for the last three EFS contractions during the stabilization period was 90% or more or 110% or less).

[0139] Individual strips were incubated with vehicle (Krebs with 0.5% gelatin), 0.1, 1, 3, 5, or 10 nM botulinum neurotoxin, and EFS stimulation continued for 3 h. At the end of the experiment, the organ baths were filled with carbachol (3.10 -6 Bladder strips were contracted by direct activation of muscarinic receptors with the addition of carbachol (10 M, 10 min). Comparison of carbachol-induced strip contractions before and after incubation with botulinum neurotoxin served as a viability test for the strips during the experiment.

[0140] [Table 2]

[0141] Eight uncomplexed 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 Recombinant botulinum neurotoxin type B (SEQ ID NO: 9) containing two mutations in the domain, E1191M and S1199Y; - rBoNT / AB MY , H CA recombinant chimeric botulinum neurotoxin (BoNT / A-derived LH) containing two mutations, E1191M and S1199Y, in the LH domain N Domain and H from BoNT / B C domain) (SEQ ID NO: 10), - nBoNT / F, native botulinum neurotoxin subtype F1 (SEQ ID NO: 6) obtained from Metabiologics Inc.; - mrBoNT / F, H CC Recombinant botulinum neurotoxin subtype F1 (SEQ ID NO: 12), containing a single point mutation in the domain, H1241K; - mrBoNT / F7-1, a recombinant botulinum neurotoxin subtype F7 (SEQ ID NO: 13) in which the native BoNT / F7 activation loop has been replaced by the BoNT / F1 activation loop.

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

[0143] Contractile responses to drugs were quantified in terms of mean developed tension (mg, corresponding to the average of data points in selected tracings) measured during 1 min sections before and at the end of the stable response of each strip following KCl or carbachol addition.

[0144] Contractile responses to EFS-induced contractions were quantified as follows: Briefly, three stimuli were delivered at 1-min intervals, followed by a 3-min rest, as shown in Fig. 1 .

[0145] Each EFS-evoked response was analyzed during the time window between two successive stimuli, i.e., 60 seconds, and this analysis was performed for the last two responses per stimulation group over a 3-hour period.

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

[0147] To quantify the EFS-induced response, the amplitude (mg) of the EFS-induced contraction was calculated as the difference between the tension developed before stimulation and the maximum tension developed in 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 maximum EFS contraction measured during the last train of stimulation during the stabilization period before addition of botulinum neurotoxin.

[0149] The inhibitory effect of botulinum neurotoxins was evaluated by determining the value of paralysis time 50 (T50) (min), i.e., the time required to inhibit 50% of the maximum response. This value was calculated using a variable slope model in GraphPad Prism 6.05 (Y=Bottom+(Top-Bottom) / (1+10^((LogIC50-X)*HillSlope)), also known as a 4-parameter dose-response logistic curve.

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

[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 EFS-induced contractile responses in amplitude. nBoNT / A and nBoNT / B induced comparable inhibitory effects on EFS-induced contractions of human bladder strips at the same concentrations. Furthermore, at all doses, rBoNT / B MY , rBoNT / AB MYnBoNT / F, mrBoNT / F, and mrBoNT / F7-1 caused a greater reduction in bladder strip EFS-induced contractions compared with either nBoNT / A and / or nBoNT / B. All BoNT / Fs tested, as well as rBoNT / B, MY and rBoNT / AB MY Thus, a lower concentration would be required to achieve the same reduction in contraction as nBoNT / A or nBoNT / B. Of all the BoNTs tested, mrBoNT / F7-1 produced the greatest reduction in contraction.

[0153] Based on the above observations, the potency of the tested botulinum toxins against human bladder tissue was quantified by 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), specifically by plotting T50 against botulinum toxin protein concentration as shown in Table 4 and FIG. 3 below, with a logarithmic fit to obtain good R2 values. 2 Got the value.

[0154] [Table 4]

[0155] The results show that BoNT / F was most potent against human bladder tissue, followed by BoNT / B and then BoNT / A. In particular, nBoNT / B was approximately 1.5 times more potent than nBoNT / A, mrBoNT / B was approximately 20 times more potent than nBoNT / A and approximately 15 times more potent than nBoNT / B, mrBoNT / AB was approximately 4 times more potent than nBoNT / A and approximately 3 times more potent than nBoNT / B, nBoNT / F was approximately 60 times more potent than nBoNT / A and approximately 50 times more potent than nBoNT / B, and mrBoNT / F7-1 was approximately 90 times more potent than nBoNT / A and approximately 70 times more potent than nBoNT / B. Furthermore, mrBoNT / F exhibited the same T50 values ​​as nBoNT / F at 1 nM and 10 nM, and it can be reasonably assumed that mrBoNT / F is similarly potent as nBoNT / F.

[0156] Thus, based on the surprising data disclosed herein, BoNT / B and BoNT / F neurotoxins and clostridial neurotoxins that exhibit similar binding, uptake, translocation, and / or SNARE cleavage properties, such as BoNT / D, BoNT / DC, or BoNT / G, can be used to treat disorders of the human autonomic nervous system, such as smooth muscle disorders, particularly urinary disorders (NOD, OAB, etc.), at dosages equal to or less than a single BoNT / A used to treat the same disorder. In particular, based on the relative potency values ​​identified in Table 4, the therapeutic amount of BoNT / B, BoNT / F, BoNT / D, BoNT / DC, or BoNT / G clostridial 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 incorporated herein by reference. Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art of biochemistry and biotechnology or related fields are intended to be within the scope of the following claims. [Explanation of symbols]

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

Claims

1. A pharmaceutical for use in treating autonomic neuropathy in a human patient, the pharmaceutical comprising H from BoNT / B. CC A clostridial neurotoxin comprising a clostridial neurotoxin comprising a clostridial neurotoxin H domain. CC The domain comprises amino acid residues 1082 to 1291 of SEQ ID NO:2, or a sequence having at least 90% sequence identity thereto.

2. The BoNT / B H CC The domain is the H domain of natural BoNT / B. CC 2. The pharmaceutical composition of claim 1, comprising at least one amino acid residue mutation that increases its binding affinity to the human Syt II receptor by at least 50% compared to the domain, wherein the at least one amino acid residue mutation 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.

3. The pharmaceutical composition of claim 2, wherein the mutation of at least one amino acid residue consists of two amino acid residue mutations, 1191M and 1199Y.

4. A pharmaceutical for use in treating autonomic neuropathy in a human patient, the pharmaceutical comprising H derived from BoNT / F. CC A clostridial neurotoxin comprising a clostridial neurotoxin comprising a clostridial neurotoxin H domain. CC Domains include: a. amino acid residues 1088 to 1278 of SEQ ID NO:6, or a sequence having at least 90% sequence identity thereto; or b. Amino acid residues 1077 to 1268 of SEQ ID NO:11, or a sequence having at least 90% sequence identity thereto.

5. The BoNT / F H CC The domain is the H domain of natural BoNT / F. CC The pharmaceutical of claim 4, comprising a mutation in at least one amino acid residue that increases its binding affinity to one or more gangliosides compared to the domain, the gangliosides being selected from GD1a and / or GM1a, and / or the mutation in the amino acid residue is 1241K.

6. The pharmaceutical according to any one of claims 1 to 5, wherein the Clostridial neurotoxin is a chimeric neurotoxin.

7. The chimeric neurotoxin is the H C Domain and LH of BoNT / A N The pharmaceutical composition of claim 6, comprising a domain.

8. The medicament according to claim 6, wherein Chimeric neurotoxin H C The domain comprises amino acid residues 860 to 1291 of SEQ ID NO:2, or a sequence having at least 90% sequence identity thereto, and is selected from the group consisting of the LH domain of the chimeric neurotoxin. N Domains include: a. amino acid residues 1 to 872 of SEQ ID NO:1, or a sequence having at least 90% sequence identity thereto; or b. Amino acid residues 1 to 859 of SEQ ID NO:2, or a sequence having at least 90% sequence identity thereto.

9. Chimeric neurotoxin H C The domain comprises amino acid residues 860 to 1291 of SEQ ID NO:2, or a sequence having at least 90% sequence identity thereto, and is selected from the group consisting of the LH domain of the chimeric neurotoxin. N The pharmaceutical of claim 6, wherein the domain comprises amino acid residues 1 to 872 of SEQ ID NO:1, or a sequence having at least 90% sequence identity thereto.

10. The chimeric neurotoxin is an LH neurotoxin derived from the first BoNT / F subtype. N 7. The pharmaceutical of claim 6, 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.

11. The above LH N The domain is the LH of BoNT / F7. N The pharmaceutical composition of claim 10, wherein the activation loop is a BoNT / F1 activation loop.

12. The medicament according to claim 6, wherein Chimeric neurotoxin H C Domains include: a. amino acid residues 866 to 1278 of SEQ ID NO:6, or a sequence having at least 90% sequence identity thereto; or b. amino acid residues 863 to 1268 of SEQ ID NO:11, or a sequence having at least 90% sequence identity thereto; And the chimeric neurotoxin LH N Domains include: a. amino acid residues 1 to 865 of SEQ ID NO:6, or a sequence having at least 90% sequence identity thereto; or b. Amino acid residues 1 to 862 of SEQ ID NO:11, or a sequence having at least 90% sequence identity thereto.

13. Chimeric neurotoxin H C The domain comprises amino acid residues 863 to 1268 of SEQ ID NO: 11, or a sequence having at least 90% sequence identity thereto, and is selected from the group consisting of the LH domain of the chimeric neurotoxin. N The pharmaceutical composition of claim 6, wherein the domain comprises amino acid residues 1 to 862 of SEQ ID NO:11, or a sequence having at least 90% sequence identity thereto.

14. A pharmaceutical composition according to any one of claims 1 to 3 or 6 to 9, wherein The clostridial neurotoxins include: a. an amino acid sequence of SEQ ID NO:2, or an amino acid sequence having at least 90% sequence identity thereto; b. the amino acid sequence of SEQ ID NO:9, or an amino acid sequence having at least 90% sequence identity thereto; or c. The amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having at least 90% sequence identity thereto.

15. A medicament according to any one of claims 4 to 6 or 10 to 13, wherein The clostridial neurotoxins include: a. an amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 90% sequence identity thereto; b. the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence having at least 90% sequence identity thereto; or c. The amino acid sequence of SEQ ID NO:6, or an amino acid sequence having at least 90% sequence identity thereto.

16. The pharmaceutical composition of claim 14 or 15, wherein the Clostridial neurotoxin is a dichain Clostridial neurotoxin.

17. The pharmaceutical composition according to any one of claims 1 to 16, wherein the autonomic nervous system disorder is selected from smooth muscle disorders, hypersecretory disorders, respiratory disorders, inflammatory disorders involving autonomic nervous elements, neuroendocrine disorders, or other autonomic nervous system disorders directly related to central nervous system disorders.

18. 18. The pharmaceutical composition of claim 17, wherein the smooth muscle disorder is selected from a urinary disorder, a gastrointestinal disorder, a vascular or cardiovascular disorder, a prostate disorder, or a sexual disorder.

19. a. the urinary disorder is selected from neurogenic detrusor overactivity (NDO), overactive bladder (OAB), idiopathic OAB (iOAB), bladder spasms, detrusor-sphincter dysfunction (DSD), urinary incontinence, urinary retention, nocturia, urge incontinence, or urinary frequency; b. the gastrointestinal disorder is selected from sphincter of Oddi dysfunction, esophageal spasm, intestinal spasm, achalasia, gastroparesis, spastic colitis, anal fissure, constipation, occasional diarrhea, dysphagia, oropharyngeal dysphagia, or swallowing disorder; c. the vascular or cardiovascular disorder is selected from Raynaud's disease, anastomotic thrombosis, atrial fibrillation after cardiac surgery, orthostatic hypotension, or blood pressure disorders; d. the prostate disorder is selected from benign prostatic hyperplasia (BPH), prostatitis, prostatodynia, or prostatic hyperplasia; or e. the sexual disorder is selected from erectile dysfunction, priapism, ejaculation disorder, or vaginismus; The pharmaceutical composition according to claim 18.

20. a. the hypersecretion disorder is selected from hyperhidrosis, hypersalivation, gustatory sweating, excessive lacrimation, crocodile tears syndrome, excessive mucus secretion, bromhidrosis, gastric acid secretion, or acne; b. the respiratory disorder is selected from rhinorrhea, chronic rhinitis, asthma, chronic obstructive pulmonary disease (COPD), bronchial hyperresponsiveness, wheezing, involuntary shortness of breath, or apneic episodes; c. the inflammatory disorder having an autonomic component is selected from otitis media, itch, pruritus, or inflammatory bowel syndrome; d. the neuroendocrine disorder is selected from diabetes, hyperinsulinemia, hyperglucagonemia pancreatic disorder, thyroid disorder, hypocalcemia, hyperthyroidism, metabolic disorder, or excessive lipolysis; or e. The other autonomic neuropathy directly related to the central nervous disorder is selected from Parkinson's disease or cerebellar / pyramidal function; The pharmaceutical composition according to claim 17.

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