CN118284427A - Treatment of pain - Google Patents

Abstract

The invention relates in particular to the treatment of pain. For example, provided are chimeric clostridial neurotoxins for use in treating pain by inhibiting the release of a pain mediator from a neuron comprising an aδ or C nerve fiber, wherein the chimeric clostridial neurotoxin binds to a neuron comprising an aδ or C nerve fiber, respectively, and wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin a (BoNT/a) light chain and translocation domain (H _N domain) and a BoNT/B receptor binding domain (H _C domain). Methods, uses, kits and unit dosage forms are also provided.

Description

Treatment of pain

Technical Field

Pain is an unpleasant sensory and emotional experience associated with or similar to actual or potential tissue damage. Pain is also described as a neurological condition characterized by pathological changes in the nervous system, or more precisely, dysfunction of the endogenous nociceptive system (Raffaeli & Arnaudo (2017), J Pain Res, 10, 2003-2008).

Nociception is the process of transmitting information to the brain about actual tissue damage (or the potential for such damage if noxious stimulation continues). The sensory neurons involved in nociception are divided into three main groups: group A; group B; and group C (Yam et al. (2018), Int J Mol Sci, 19, 8, 2164).

Group A nerve fibers are classified as myelinated fibers, which can be further subdivided into Aα, Aβ, Aγ, and Aδ, each with a different set of characteristics. These fibers usually terminate in layers I, III, IV, and V of the dorsal horn of the spinal cord, with some projections within layer II. Type Ia and Ib sensory fibers from muscle spindle endings and Golgi tendons are both Aα-type. Type Aβ fibers are usually low-threshold, cutaneous, slowly or rapidly adapting mechanical receptors, and include type II afferent fibers from stretch receptors. Aβ-fibers usually belong to layers III and IV. Type Aγ fibers may include type II afferent fibers from stretch receptors. Type Aδ fibers may include thermal and mechanical nociceptors terminating in layers I and V of the rexed layer, as well as type III afferent fibers. Aδ-fibers are also usually the smallest myelinated nerves, and may have a relatively fast conduction velocity of ~30 m/s. The diameter of Aδ-fibers is usually about 2-5 μm, and they are usually sensitive to transient pain and tingling.

Group B nerve fibers are usually moderately myelinated with a conduction velocity of 3-14 m/s. Preganglionic nerve fibers of the autonomic nervous system (ANS) and general visceral afferent fibers belong to this group.

C group nerve fibers are unmyelinated, usually less than 2 μm in diameter, and have relatively slow conduction velocities, usually up to about 2 m/s. Nerve fibers of the dorsal root in the ANS (type IV afferent fibers) and postganglionic fibers can be classified in this group. All of these fibers are primarily nociceptive in function, carrying sensory information and gathering about 70% of incoming nociceptive information, which is then passed into the spinal cord. C-fibers may terminate in layers I and II in the gray matter of the spinal cord. In terms of nociception, C-fiber nociceptors may be multimodal because they are activated by thermal, mechanical and/or chemical stimuli. For example, C-fibers may be activated by adverse local stimuli. In terms of neurochemistry, C-fibers may be classified as peptidergic or non-peptidergic, and about 50% of these fibers express neuropeptides, including calcitonin gene-related peptide (CGRP), neurokinin, and substance P (SP).

Multiple neurotransmitters are involved in pain, including all major types of neurotransmitters, such as inflammatory mediators: prostaglandin E2 (PGE2), prostacyclin (PGI2), leukotriene B4 (LTB4), nerve growth factor (NGF), protons, bradykinin (BK), ATP, adenosine, SP, neurokinin A (NKA), neurokinin B (NKB), serotonin (5-HT), histamine, glutamate, norepinephrine (NE) and nitric oxide (NO); and non-inflammatory mediators: CGRP, γ-aminobutyric acid (GABA), opioid peptides, glycine and cannabinoids (Yam et al. (2018), Int J Mol Sci, 19, 8, 2164).

Of particular therapeutic interest is CGRP, which is widely produced in the central and peripheral nervous systems; however, it is mainly located in primary afferent nerves. As a direct derivative of the dorsal root ganglion (DRG), CGRP can be found in the dorsal horn of the spinal cord and is associated with the conduction of noxious stimuli. CGRP is associated with the excitatory effects of SP, which leads to Ca ²⁺ release. The receptors for CGRP (calcitonin receptor-like receptors (CALCRL)) are usually located in the nucleus accumbens, indicating that the CNS can control CGRP-mediated pain transmission. CGRP is widely distributed in the peripheral and central nervous systems, and its receptors are expressed in pain pathways. CGRP-like immunoreactivity (CGRP-LI) is usually found in 40-50% of DRG neurons. In addition, CGRP is often co-localized with other neuropeptides, including substance P and neurokinins in DRG neurons. Peripheral CGRP-LI fibers can terminate in layers I, III, and V of the spinal cord, and DRG neurons containing CGRP innervate joints. Therefore, CGRP and its receptors can be widely distributed in peripheral and central pain pathways (Schou et al. (2017), The Journal of Headache and Pain, 18, 34, 1-17). In animals, CGRP can be released from peripheral and central nerve endings during noxious pain and/or mechanical stimulation of the skin. In rats, a major portion of circulating CGRP can be released from perivascular nerve endings. Acute and chronic nociception can lead to changes in the release of CGRP from sensory nerve endings and central endings into the dorsal horn of the spinal cord. CGRP is known to be one of the most effective vasodilators. Two subtypes have been characterized: α-CGRP and β-CGRP (Russell et al. (2014), Physiol Rev, 94, 4, 1099-1142). The α subtype is mainly expressed in primary sensory neurons, while the β subtype is mainly found in endogenous enteric neurons. The mature form of this neuropeptide consists of 37 amino acids, and its expression is particularly striking in sensory neurons of DRG and trigeminal ganglia. The mature form is stored in vesicles localized to the terminal regions of central and peripheral nerve endings, from where it can be secreted into the dorsal spinal cord or various peripheral tissues, especially into peripheral blood vessels where vascular tone can be modulated. In addition, a network of CGRP-positive nociceptors has been observed in rodent and human meningeal vessels, and approximately 40-50% of trigeminal ganglion neurons have been found to be positive for CGRP. In addition, CGRP expression has been observed in CNS regions such as the hypothalamus, thalamus, periaqueductal gray, superior and inferior colliculi, amygdala, trigeminal complex, and cerebellum. Given the ability of CGRP to alter synaptic and neuronal activity at the trigeminocervical complex, as well as the transmission of nociceptive signals to the thalamus and cortical regions, these mentioned brain regions may be involved in migraine pathophysiology (Tardiolo et al. (2019), Int J Mol Sci, 20(12), 2932).

Conventional treatments for pain (e.g., CGRP-related pain) include monoclonal antibodies and small molecule antagonists that target pain mediators (e.g., CGRP) once they have been released by presynaptic neurons. As an alternative, some conventional therapeutic agents target receptors for pain mediators. These approaches have many disadvantages, including: systemic effects on chemical mediators (e.g., CGRP); nausea; vomiting; indigestion; diarrhea; bradycardia; hypotension; bronchospasm; dyspnea; fatigue; insomnia; dizziness; dry mouth; flushing; hot or cold sensations; chest pain; constipation; itching; drowsiness; tinnitus; restlessness; muscle cramps; injection site pain; upper respiratory tract infection; fatigue; nasopharyngitis; injection site erythema; injection site induration; anxiety; depression; injection site itching; influenza; urinary tract infection; lethargy; paresthesia; increased heart rate; stroke; and/or heart attack (Woo (2020), Nature, 586, S4-S6 and Tardiolo et al. (2019), Int J Mol Sci, 20(12), 2932). Therefore, there is a need for improved pain therapeutics that have fewer side effects and/or block pain mediators at the point of release.

The present invention overcomes one or more of the above-mentioned problems.

Summary of the invention

The present inventors have discovered that, unlike BoNT/A, chimeric clostridial neurotoxins comprising a botulinum neurotoxin A (BoNT/A) light chain and translocation domain (H _N domain) and a BoNT/B receptor binding domain (H _C domain) can bind to neurons comprising Aδ or C nerve fibers that secrete pain mediators and can effectively inhibit the release of said mediators from said neurons. Thus, by such inhibition, the chimeric clostridial neurotoxins of the present invention can be used as analgesics capable of treating pain. In particular, the present inventors have shown that the claimed chimeric clostridial neurotoxins can effectively inhibit the release of CGRP from said neurons. Thus, by such CGRP release inhibition, the chimeric clostridial neurotoxins of the present invention can be used as analgesics capable of treating CGRP-associated pain.

Without wishing to be bound by theory, it is believed that by blocking chemical mediators at the point of secretion, the chimeric clostridial neurotoxins of the present invention can prevent pain mediators (e.g., CGRP) from reaching adjacent and distal cells. Advantageously, this can provide: selective blocking of abnormal mediator release associated with pain, thereby maintaining mediator release elsewhere; therapeutic agents with longer duration of action (less side effects and/or increased safety window compared to non-chimeric clostridial neurotoxins); and/or fewer side effects compared to conventional therapeutic agents. In particular, once CGRP action and/or CGRP receptor blockade by conventional therapeutic agents after release can lead to nausea, fatigue and increased heart rate, stroke and/or heart attack. The present invention can minimize/avoid such side effects.

The inventors have also discovered that chimeric Clostridial neurotoxins are capable of cleaving SNAP25 in central nervous system structures associated with migraine pathophysiology. Advantageously, chimeric Clostridial neurotoxins are particularly effective in treating migraine (e.g., migraine pain).

Detailed ways

In one aspect, the invention provides a chimeric clostridial neurotoxin for use in treating pain, wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin A (BoNT/A) light chain and translocation domain ( _HN domain) and a BoNT/B receptor binding domain ( _HC domain).

In one aspect, the invention provides a method of treating pain, the method comprising administering to a subject a chimeric clostridial neurotoxin, wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin A (BoNT/A) light chain and translocation domain ( _HN domain) and a BoNT/B receptor binding domain ( _HC domain).

In one aspect, the present invention provides a use of a chimeric clostridial neurotoxin in the preparation of a medicament for treating pain, wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin A (BoNT/A) light chain and a translocation domain ( _HN domain) and a BoNT/B receptor binding domain ( _HC domain).

In one aspect, the invention provides a chimeric clostridial neurotoxin for use in treating migraine, preferably migraine pain, wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin A (BoNT/A) light chain and translocation domain ( _HN domain) and a BoNT/B receptor binding domain ( _HC domain).

In one aspect, the invention provides a method of treating migraine, preferably migraine pain, comprising administering to a subject a chimeric clostridial neurotoxin, wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin A (BoNT/A) light chain and translocation domain ( _HN domain) and a BoNT/B receptor binding domain ( _HC domain).

In one aspect, the present invention provides the use of a chimeric clostridial neurotoxin in the preparation of a medicament for treating migraine, preferably migraine pain, wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin A (BoNT/A) light chain and translocation domain ( _HN domain) and a BoNT/B receptor binding domain ( _HC domain).

Migraine can be episodic migraine or chronic migraine (preferably chronic migraine). If the subject experiences headaches (e.g., migraine) less than 15 days per month (e.g., at least 1 day per month but less than 15 days), preferably if the subject experiences headaches (e.g., migraine) at least 4 days per month but less than 15 days, the subject may suffer from episodic migraine. In other words, episodic migraine can be defined as headaches (e.g., migraine) less than 15 days per month (e.g., at least 1 day per month but less than 15 days), preferably headaches (e.g., migraine) at least 4 days per month but less than 15 days. If the subject experiences headaches (e.g., migraine) at least 15 days per month, the subject may suffer from chronic migraine. If the subject experiences headaches (e.g., migraine) at least 15 days per month and lasts for at least 3 months, and has the characteristics of at least 8 days of migraine per month, the subject may suffer from chronic migraine. In other words, chronic migraine can be defined as headaches (e.g., migraine) at least 15 days per month. In other words, chronic migraine can be defined as headaches (e.g., migraine) at least 15 days per month and lasts for at least 3 months, and has the characteristics of at least 8 days of migraine per month. In one embodiment, chronic migraines may last 4 hours or more a day.In addition to the headache, migraines may be associated with one or more other symptoms, including increased light sensitivity, nausea and/or vomiting.

Preferably, when treating migraine, the chimeric Clostridial neurotoxin is used to treat migraine pain.

In one aspect, the invention provides a chimeric clostridial neurotoxin for treating pain by inhibiting the release of pain mediators from neurons comprising Aδ neurofibers or C neurofibers, wherein the chimeric clostridial neurotoxin binds to neurons comprising Aδ neurofibers or C neurofibers, respectively, and wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin A (BoNT/A) light chain and translocation domain ( _HN domain) and a BoNT/B receptor binding domain ( _HC domain).

In a related aspect, the invention provides a method of treating pain by inhibiting the release of pain mediators from neurons comprising Aδ neurofibers or C neurofibers, the method comprising administering to a subject the chimeric clostridial neurotoxin, wherein the chimeric clostridial neurotoxin binds to neurons comprising Aδ neurofibers or C neurofibers, respectively, and wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin A (BoNT/A) light chain and translocation domain ( _HN domain) and a BoNT/B receptor binding domain ( _HC domain).

In another related aspect, the present invention provides for the use of a chimeric clostridial neurotoxin in the preparation of a medicament for treating pain by inhibiting the release of pain mediators from neurons comprising Aδ nerve fibers or C nerve fibers, wherein the chimeric clostridial neurotoxin binds to neurons comprising Aδ nerve fibers or C nerve fibers, respectively, and wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin A (BoNT/A) light chain and translocation domain (H _N domain) and a BoNT/B receptor binding domain (H _C domain).

In one embodiment, the invention provides a chimeric clostridial neurotoxin for treating CGRP-associated pain by inhibiting the release of CGRP from neurons containing Aδ neurofibers or C neurofibers, wherein the chimeric clostridial neurotoxin binds to neurons containing Aδ neurofibers or C neurofibers, respectively, and wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin A (BoNT/A) light chain and translocation domain ( _HN domain) and a BoNT/B receptor binding domain ( _HC domain). Corresponding methods of treatment and uses are also provided.

The mediator may be any molecule released from neurons that plays a role in a condition (eg, pain). Inhibition of the release of the mediator by a chimeric clostridial neurotoxin according to the invention may treat the condition (eg, may treat pain).

The mediator may be a neurotransmitter.

Inhibition of release of mediators from neurons can be partial or complete inhibition, preferably complete inhibition. For example, a chimeric clostridial neurotoxin can inhibit at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of mediators released from neurons. Preferably, a chimeric clostridial neurotoxin inhibits 100% of mediators released from neurons.

Pain mediators can be neurotransmitters.

Inhibition of the release of pain mediators from the neurons can be partial or complete inhibition, preferably complete inhibition. For example, the chimeric clostridial neurotoxin can inhibit at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the pain mediators released from neurons. Preferably, the chimeric clostridial neurotoxin inhibits 100% of the pain mediators released from neurons.

The inhibition is preferably inhibition of SNARE-associated (eg SNAP25-associated) release.

The chimeric clostridial neurotoxins of the present invention preferably inhibit the release of mediators from neurons to a greater extent than BoNT/A (preferably native BoNT/A as set forth in SEQ ID NO:6 [e.g., a di-chain form of SEQ ID NO:6]) inhibits the release of mediators from neurons. At a given dose (e.g., 1 nM), a chimeric clostridial neurotoxin of the present invention may inhibit at least 10% or 20% (preferably at least 30%) more mediators from neurons than BoNT/A at the same dose (e.g., 1 nM). At a given dose (e.g., 1 nM), a chimeric clostridial neurotoxin of the present invention may inhibit 10-90% or 20-90% (preferably 30-85%) more mediators from neurons than BoNT/A at the same dose (e.g., 1 nM). Thus, a much lower dose of the chimeric clostridial neurotoxin may be required to inhibit the release of the same amount of mediators from neurons compared to BoNT/A. For example, the dose of the chimeric Clostridial neurotoxin can be at least 100-fold, 200-fold, or 500-fold lower than the dose of BoNT/A required to inhibit the same amount of mediator release from neurons, and preferably 1000-fold lower. The dose of the chimeric Clostridial neurotoxin can be at least 500-2000-fold, or 750-1750-fold lower, and preferably 1000-1500-fold lower than the dose of BoNT/A required to inhibit the same amount of mediator release from neurons.

The chimeric clostridial neurotoxins of the present invention preferably inhibit the release of pain mediators from neurons to a greater extent than BoNT/A (preferably native BoNT/A as set forth in SEQ ID NO:6 [e.g., a di-chain form of SEQ ID NO:6]) inhibits the release of mediators from neurons. At a given dose (e.g., 1 nM), the chimeric clostridial neurotoxins of the present invention may inhibit at least 10% or 20% (preferably at least 30%) more pain mediators from neurons than BoNT/A at the same dose (e.g., 1 nM). At a given dose (e.g., 1 nM), the chimeric clostridial neurotoxins of the present invention may inhibit 10-90% or 20-90% (preferably 30-85%) more pain mediators from neurons than BoNT/A at the same dose (e.g., 1 nM). Thus, a much lower dose of the chimeric clostridial neurotoxin may be required to inhibit the release of the same amount of pain mediators from neurons compared to BoNT/A. For example, the dose of the chimeric Clostridial neurotoxin can be at least 100-fold, 200-fold, or 500-fold lower than the dose of BoNT/A required to inhibit the same amount of pain mediator release from neurons, preferably 1000-fold lower. The dose of the chimeric Clostridial neurotoxin can be at least 500-2000-fold, or 750-1750-fold lower, preferably 1000-1500-fold lower than the dose of BoNT/A required to inhibit the same amount of pain mediator release from neurons.

Chimeric Clostridial neurotoxins can inhibit the release of multiple mediators from neurons.

Chimeric Clostridial neurotoxins inhibit the release of multiple pain mediators from neurons.

The chimeric clostridial neurotoxins of the present invention preferably have analgesic properties. In other words, the chimeric clostridial neurotoxins of the present invention are preferably analgesic chimeric clostridial neurotoxins.

Preferably, the chimeric clostridial neurotoxins of the invention neither promote neuronal growth nor promote neuronal repair to treat pain. In other words, preferably, the chimeric clostridial neurotoxins do not treat pain by promoting neuronal growth, by promoting neuronal repair, or by promoting both neuronal growth and repair.

Preferably, the chimeric clostridial neurotoxins of the invention neither promote neuronal growth nor promote neuronal repair to treat the conditions described herein. In other words, preferably, the chimeric clostridial neurotoxins do not treat the conditions described herein by promoting neuronal growth, by promoting neuronal repair, or by promoting both neuronal growth and repair.

The term "promoting neuron growth and/or neuron repair" encompasses increasing the rate of neuron growth and/or neuron repair. The term "neuron growth and/or neuron repair" encompasses rebuilding damaged neuron circuits, thereby restoring activity and/or neuron communication in a neuron network or population. Therefore, the term "neuron repair" used herein encompasses the repair of specific neurons and the repair of neuron circuits. The term also encompasses neuronal plasticity. The term "neuronal plasticity" used herein encompasses axon sprouting, dendrite sprouting, neurogenesis (e.g., the generation of new neurons), maturation, differentiation and/or synaptic plasticity (e.g., including changes in synaptic strength, activity, anatomy and/or connectivity). The term "promoting neuron growth and/or neuron repair" also encompasses (e.g., near the site of injury or site of injury) promoting the establishment of functional synapses. The term "neuron growth" used herein encompasses the growth of any part of a neuron, including the growth of axons and/or dendrites. The term encompasses an increase in neurite length, neurite number (e.g., neurite number per cell), and/or an increase in the length and/or number of projections from the cell body or cell membrane of a neuron, such as axonal growth and/or axonal sprouting of a neuron, such as a neuron in a subject. The axonal growth can promote connections and/or chemical communications between neurons.

Preferably, the chimeric clostridial neurotoxins of the present invention do not promote a neuroimmune response to treat pain. Preferably, the chimeric clostridial neurotoxins of the present invention do not promote a neuroimmune response to treat the conditions described herein. The neuroimmune response herein encompasses a microglial response. Thus, in one embodiment, the chimeric clostridial neurotoxins of the present invention do not promote a microglial response to treat pain. Thus, in one embodiment, the chimeric clostridial neurotoxins of the present invention do not promote a microglial response to treat the conditions described herein.

In a preferred embodiment, pain is not pain associated with or caused by a brain disorder. In a preferred embodiment, the disorder described herein is not a disorder associated with or caused by a brain disorder. The term "brain disorder" used herein is interchangeable with "brain disease". "Brain disorder" used herein encompasses disorders originating from within or outside the brain, and includes disorders associated with physical injuries that cause brain tissue damage. Examples of brain disorders encompassed herein include any one (or more) of the following: traumatic brain injury, cancer (e.g., brain tumors), infectious diseases (e.g., encephalitis, meningitis, brain abscesses, and encephalitis), stroke, neurodegenerative disorders (e.g., Alzheimer's disease, Parkinson's disease, Parkinson's disease-related disorders, motor neuron diseases (e.g., amyotrophic lateral sclerosis), prion diseases, Huntington's disease, spinocerebellar ataxia, ataxia, Hallervorden-Spatz disease, and frontotemporal lobar degeneration), cerebral aneurysm, multiple sclerosis, hypoxic injury, toxic injury, and metabolic injury. Brain disorders can be caused by traumatic brain injury, cancer, infectious diseases (e.g., encephalitis, meningitis, brain abscess and encephalitis), stroke, neurodegenerative disorders (e.g., Alzheimer's disease, Parkinson's disease, Parkinson's disease-related disorders, motor neuron diseases (e.g., amyotrophic lateral sclerosis), prion diseases, Huntington's disease, spinocerebellar ataxia, ataxia, Hallervorden-Spatz disease and frontotemporal lobar degeneration), cerebral aneurysm, multiple sclerosis, anoxic injury, toxic injury and/or metabolic injury.

The chimeric clostridial neurotoxin preferably binds to neurons comprising Aδ fibers or C fibers. The binding can be mediated by the BoNT/BH _C domain (e.g., the _HCC portion thereof) of the chimeric clostridial neurotoxin. After binding to neurons, the chimeric clostridial neurotoxin can be internalized by endosomes, and the BoNT/A light chain can be translocated from the endosomes to the cytosol of the neuron by the BoNT/A translocation domain. Once in the cytosol, the light chain can cleave SNARE proteins (e.g., SNAP25), thereby inhibiting neuronal release/secretion (including the release/secretion of pain mediators from neurons).

Neurons containing Aδ fibers or C fibers are described in Pichon & Chesler (2014), Frontiers in Neuroanatomy (https://doi.org/10.3389/fnana.2014.00021) and Yam et al. (2018), Int J Mol Sci, 19, 8, 2164. The term "fiber" (e.g., in the context of Aδ fibers or C fibers) preferably refers to the axon of a neuron. Typically, multiple fibers (e.g., multiple Aδ fibers or multiple C fibers, respectively) together can define a larger nerve/neuron structure in a subject as, for example, a fiber bundle. For example, a bundle of Aδ fibers or a bundle of C fibers. In some embodiments, the multiple fibers may include fibers other than Aδ fibers or C fibers. For example, a nerve may include multiple neurons, including neurons containing Aδ fibers and/or neurons containing C fibers.

Chimeric clostridial neurotoxins can bind to neurons comprising Aδ fibers. Aδ fibers (or neurons comprising them) can be characterized as peptidergic, fast-conducting, lightly myelinated, involving sharp pain/quick pain, involving nociception and/or involving temperature sensation. Preferably, Aδ fibers (or neurons comprising them) can have a conduction velocity of 5-75m/s (e.g., 5-35m/s) and/or a diameter of about 1-5μm (e.g., 2-5μm). Neurons comprising Aδ fibers bound by the chimeric clostridial neurotoxin of the present invention are neurons capable of releasing pain mediators. In particular, the neurons are capable of releasing CGRP and therefore play an effect in CGRP-related pain. By binding to neurons comprising Aδ fibers, the chimeric clostridial neurotoxin inhibits the release of pain mediators from the neurons by cutting their SNARE proteins (e.g., SNAP25), thereby inhibiting the release/secretion of pain mediators from the neurons.

Chimeric clostridial neurotoxins can bind to neurons containing C fibers. C fibers (or neurons containing them) can be characterized as peptidergic, low (e.g., slow) conduction, unmyelinated, involving dull/slow pain, involving neuropathic pain, involving thermal sensation and/or involving itch sensation. Neurons containing C fibers can be polymodal. Preferably, C fibers (or neurons containing them) may have a conduction velocity of 0.5-2 m/s and/or a diameter of about 0.2-1.5 μm (e.g., 0.2-0.5 μm). Neurons containing C fibers bound by the chimeric clostridial neurotoxin of the present invention are neurons capable of releasing pain mediators. In particular, the neurons are capable of releasing CGRP and therefore play an effect in CGRP-related pain. By binding to neurons containing C fibers, chimeric clostridial neurotoxins can inhibit the release of pain mediators from the neurons by cutting their SNARE proteins (e.g., SNAP25), thereby inhibiting the release/secretion of pain mediators from the neurons.

Preferably, the neurons to which the chimeric Clostridial neurotoxin binds are neurons containing C fibers.

The expression of tropomyosin receptor kinase A (TrkA) can be a marker for distinguishing neurons containing Aδ nerve fibers or C nerve fibers (e.g., from neurons containing Aβ fibers). In other words, neurons containing Aδ nerve fibers or C nerve fibers of the present invention can be neurons expressing TrkA.

In use, the chimeric clostridial neurotoxin can bind to a plurality of neurons, including at least neurons comprising Aδ fibers and neurons comprising C fibers. The plurality of neurons can be part of a larger nerve/neuronal structure in a subject, for example including a fiber bundle.

The neurons comprising Aδ neurofibers or C neurofibers can be neurons of the central nervous system (e.g., the hypothalamus, thalamus, periaqueductal gray, superior colliculus, inferior colliculus, amygdala, trigeminal nucleus complex, and/or cerebellum) or the peripheral nervous system. When treating certain conditions such as headache, preferably migraine, the chimeric clostridial neurotoxin can inhibit the release of mediators from neurons of the central nervous system. When treating certain painful conditions such as headache, preferably migraine, the chimeric clostridial neurotoxin can inhibit the release of pain mediators from neurons of the central nervous system.

The neurons comprising Aδ nerve fibers or C nerve fibers according to the present invention are preferably sensory neurons. The sensory neurons may be primary sensory neurons, such as primary afferent neurons. For example, the neurons to which the chimeric clostridial neurotoxin binds may be sensory neurons of the dorsal root ganglion and/or trigeminal ganglion. Additionally or alternatively, the neurons may be endogenous enteric neurons.

The chimeric clostridial neurotoxins of the invention can bind to neurons comprising Aδ fibers or C fibers with an affinity that is greater than the affinity with which BoNT/A (preferably native BoNT/A as set forth in SEQ ID NO:6 [e.g., a two-chain form of SEQ ID NO:6]) binds to neurons. In particular, the chimeric clostridial neurotoxins of the invention can bind to neurons comprising Aδ fibers or C fibers with an affinity that is at least 2×, 5×, 10×, 50×, 100×, 1,000×, or 10,000× greater than the affinity with which BoNT/A binds to neurons.

The chimeric clostridial neurotoxins of the invention can bind to neurons that contain Aδ fibers or C fibers with an affinity that is greater than the affinity with which the chimeric clostridial neurotoxin binds to neurons (preferably sensory neurons) that do not contain Aδ fibers or C fibers, such as neurons that contain Aβ fibers. For example, the chimeric clostridial neurotoxins of the invention can bind to neurons that contain Aδ fibers or C fibers with an affinity that is at least 2×, 5×, 10×, 50×, 100×, 1,000×, or 10,000× greater than the affinity with which the chimeric clostridial neurotoxin binds to neurons (preferably sensory neurons) that do not contain Aδ fibers or C fibers, such as neurons that contain Aβ fibers.

Aβ fibers (or neurons containing them) can generally be characterized as myelinated, fast-conducting, involved in touch and/or responsive to other non-noxious stimuli. Preferably, Aβ fibers (or neurons containing them) may have a conduction velocity of 80-120 m/s and/or a diameter of about 6-20 μm. Expression of neurofilament 200 (NF200) can be a marker for distinguishing neurons containing Aβ nerve fibers (e.g., from neurons containing Aδ fibers or C fibers). In other words, neurons containing Aβ fibers can be neurons expressing NF200.

In other embodiments, the chimeric clostridial neurotoxin may be able to exert an effect at a site distal to the site of administration (e.g., injection). For example, following administration of the chimeric clostridial neurotoxin, SNARE protein cleavage (e.g., SNAP25 cleavage) may occur at a site distal to the site of administration (e.g., injection). Preferably, this effect occurs by neuronal transport of the chimeric clostridial neurotoxin from its site of administration to a distal site. When treating pain (preferably headache, most preferably migraine pain) or migraine, the chimeric clostridial neurotoxin preferably exerts an effect at a site distal to the site of administration (e.g., injection). In one embodiment, this effect may be in addition to a peripheral effect. Therefore, preferably, when treating pain (preferably headache, most preferably migraine pain) or migraine, the chimeric clostridial neurotoxin may be transported by neuronal transport.

Neuronal transport may be retrograde transport or anterograde transport, preferably retrograde transport. The transport may be axonal transport.

"Retrograde transport" may be a form of axonal transport (aka axoplasmic transport or axoplasmic flow); a cellular process that is generally responsible for the movement of mitochondria, lipids, synaptic vesicles, proteins, and other organelles into and out of the neuronal cell body via the axonal cytoplasm, called the axoplasm. Axons are on the order of several meters long, so neurons cannot rely on diffusion to transport products of the nucleus and organelles to the ends of their axons, and therefore use axonal transport. Axonal transport can also be responsible for moving molecules destined for degradation from the axon back to the cell body, where they are broken down by lysosomes.

"Retrograde transport" may refer to movement toward the cell body of a neuron, while "anterograde transport" may refer to movement toward the synapses of a neuron.

In one embodiment, neuronal (eg, retrograde) transport to neurons of the central nervous system can refer to transport of a chimeric Clostridial neurotoxin to neuronal cell bodies located in the vicinity of the central nervous system (eg, axonal transport).

Neuronal (e.g., retrograde) transport is now described in more detail. In one embodiment, a chimeric clostridial neurotoxin can bind to a first neuron (e.g., a primary sensory afferent) at the site of administration. The chimeric clostridial neurotoxin can be internalized by a first neuron, transported within the first neuron, and then released from the first neuron. Preferably, the clostridial neurotoxin binds to a first neuron at a site of intramuscular or intradermal administration (e.g., intramuscular or intradermal injection). Such a neuron can be a peripheral neuron, preferably a neuron comprising Aδ fibers or C fibers. Once released, the chimeric clostridial neurotoxin can bind to a second neuron, be internalized, and cut a SNARE protein (e.g., SNAP25) in the second neuron. Alternatively, the chimeric clostridial neurotoxin can bind to a second neuron, be internalized by a second neuron, be transported within the second neuron, and then be released from the second neuron. This process can be repeated until the chimeric clostridial neurotoxin binds to a neuron (e.g., a third neuron), is internalized, and cuts a SNARE protein (e.g., SNAP25) in the neuron. The second neuron can be a secondary sensory afferent. Preferably, the second neuron is a neuron of the central nervous system, such as a neuron present in the brain, brainstem or spinal cord. The second neuron may be a neuron present in the trigeminal ganglion (eg, SNARE cleavage may occur in its axon).

In some embodiments, when administered intramuscularly, the chimeric Clostridial neurotoxin can be transported neuronally (eg, retrogradely) through a motor neuron, released from the motor neuron, and enter a second neuron, preferably a neuron of the central nervous system. The neuron can be a sensory neuron.

In one embodiment, when administered intramuscularly, a chimeric Clostridial neurotoxin can diffuse to and bind to sensory neurons present in the periosteum or skin (eg, terminate in the periosteum or skin).

Without wishing to be bound by theory, it is believed that the chimeric clostridial neurotoxins of the invention can inhibit secretion of one or more neurons of the central nervous system through the above-mentioned neuronal (e.g., retrograde) transport mechanism. Thus, the chimeric clostridial neurotoxins can reach neurons of the central nervous system through neuronal (e.g., retrograde) transport and cleave the SNARE proteins (e.g., SNAP25) of the neurons.

In a preferred embodiment, the chimeric clostridial neurotoxin can treat pain or conditions described herein by inhibiting secretion (e.g., inhibiting the release of mediators (e.g., pain mediators)) from one or more neurons of the central nervous system. This may be particularly relevant in the treatment of pain or migraine, preferably in the treatment of migraine pain. The chimeric clostridial neurotoxin can reach the neurons of the central nervous system through neuronal (e.g., retrograde) transport and cleave the SNARE protein (e.g., SNAP25) of the neurons. Thus, the chimeric clostridial neurotoxin can treat pain (e.g., headache or migraine pain) or migraine by inhibiting secretion from central nervous system neurons, preferably by inhibiting the secretion of mediators, more preferably pain mediators, from central nervous system neurons.

The neuron of the central nervous system can be a neuron of the brainstem, spinal cord and/or brain. For example, the neuron of the central nervous system can be a neuron at the following position: trigeminal nucleus (for example spinal trigeminal nucleus (spinaltrigeminal nucleus), such as spinal trigeminal sensory nucleus (spinal trigeminal sensory nucleus)), spinal cord (preferably neurons of the dorsal horn of the spinal cord), hypothalamus, thalamus, midbrain periaqueductal gray matter, superior colliculus, inferior colliculus, amygdala, trigeminal cervical nucleus complex, cortex and/or cerebellum. The neuron of the trigeminal nucleus can be a neuron of the trigeminal spinal tract nucleus caudalis (for example, caudal part).

In a preferred embodiment, the chimeric clostridial neurotoxin cleaves SNARE proteins (e.g., SNAP25) in neurons of the brainstem, more preferably neurons of the trigeminal nucleus (even more preferably the spinal trigeminal (sensory) nucleus). The chimeric clostridial neurotoxin can inhibit the secretion of (e.g., mediators, preferably pain mediators) in the neurons. The cleavage of the SNARE protein can occur by the transport of the chimeric clostridial neurotoxin from neurons at the site of administration (e.g., retrograde). Such neurons can be targeted by administering the chimeric clostridial neurotoxin to muscles, periosteum, and/or skin innervated by sensory trigeminal neurons (e.g., muscles, periosteum, and/or skin located on the face and/or scalp of a subject). Alternatively, the neurons may comprise Aδ nerve fibers or C nerve fibers to which the chimeric clostridial neurotoxin can bind and then cleave its SNARE proteins (e.g., after transport/diffusion through the neuronal cytoplasm). The cleavage can be at the end of a neuron present in the spinal trigeminal sensory nucleus. Most preferably, the SNARE cleavage and secretion inhibition can treat migraine or migraine pain.

In one embodiment, the chimeric clostridial neurotoxin cleaves a SNARE protein (e.g., SNAP25) of a neuron of the trigeminal motor nucleus. The chimeric clostridial neurotoxin can inhibit secretion of the neuron. The cleavage of the SNARE protein can occur by transport of the chimeric clostridial neurotoxin from the neuron at the site of administration (e.g., retrograde).

In another preferred embodiment, the chimeric clostridial neurotoxin cleaves SNARE proteins (e.g., SNAP25) in neurons of the spinal cord, such as the cervical spinal cord. More preferably, the neurons are neurons present in the dorsal horn of the spinal cord (e.g., associated with sensory neurons). The chimeric clostridial neurotoxin can inhibit the secretion of (e.g., mediators, preferably pain mediators) in the neurons. The cleavage of the SNARE protein can occur by transport of the chimeric clostridial neurotoxin from neurons at the site of administration (e.g., retrograde). Such neurons can be targeted by administering the chimeric clostridial neurotoxin to muscles, periosteum, and/or skin innervated by sensory spinal neurons (e.g., muscles, periosteum, and/or skin located at the back of the subject's head and/or neck). Alternatively, the neurons may comprise Aδ nerve fibers or C nerve fibers, to which the chimeric clostridial neurotoxin can bind and then cleave its SNARE proteins (e.g., after transport/diffusion through the neuronal cytoplasm). The cleavage can be at the end of a neuron present in the spinal cord. Most preferably, the SNARE cleavage and secretion inhibition can treat migraine or migraine pain.

In one embodiment, the chimeric clostridial neurotoxin cleaves a SNARE protein (e.g., SNAP25) in a neuron of the ventral horn of the spinal cord (e.g., associated with a motor neuron). The chimeric clostridial neurotoxin can inhibit the secretion of (e.g., a mediator, preferably a pain mediator) in the neuron. The cleavage of the SNARE protein can occur by transport of the chimeric clostridial neurotoxin from the neuron at the site of administration (e.g., retrograde).

In another preferred embodiment, the chimeric clostridial neurotoxin cleaves a SNARE protein (e.g., SNAP25) in the neurons of the trigeminal ganglion, e.g., in the axons thereof. The chimeric clostridial neurotoxin can inhibit the secretion of (e.g., mediators, preferably pain mediators) in the neurons. The cleavage of the SNARE protein can occur by transport of the chimeric clostridial neurotoxin from neurons at the site of administration (e.g., retrograde). Alternatively, the neurons may comprise Aδ nerve fibers or C nerve fibers, to which the chimeric clostridial neurotoxin can bind and then cleave its SNARE protein (e.g., after transport/diffusion through the neuronal cytoplasm). Most preferably, the SNARE cleavage and secretion inhibition can treat migraine or migraine pain.

Neuronal (e.g., retrograde) transport of clostridial neurotoxins has been described (see Bomba-Warczak et al. (2016), Cell Rep., 16(7), 1974-1987), and, without wishing to be bound by theory, it is believed that neuronal transport of clostridial neurotoxins occurs by binding of the clostridial neurotoxin to a non-classical receptor (e.g., in the present case by binding to a receptor other than SYTI or SYTII), incorporation into non-acidified organelles, neuronal (e.g., retrograde) transport (e.g., away from the periphery of the body toward the central nervous system), and release from neurons into the extracellular space. In this context, it has been described that the clostridial neurotoxin can remain intact (i.e., the double chain comprising an L-chain and an H-chain linked together by a disulfide bond remains intact), allowing binding to a second neuron via a canonical intoxication route (e.g., via SYTI or SYTII in the case of a chimeric clostridial neurotoxin of the invention).

A portion of the chimeric clostridial neurotoxin administered to a subject may bind to neurons comprising Aδ nerve fibers or C nerve fibers and inhibit the release of mediators (e.g., pain mediators) from the neurons, and a portion of the chimeric clostridium may exert an effect at a site distal to the site of administration. The portion that exerts its effect at a site distal to the site of administration may inhibit the secretion of neurons of the central nervous system, preferably inhibiting the secretion of mediators (e.g., neurotransmitters), more preferably pain mediators, from neurons of the central nervous system. The chimeric clostridial neurotoxin may be transported through neurons (e.g., retrograde) to reach neurons of the central nervous system and cut the SNARE proteins (e.g., SNAP25) of the neurons.

In some embodiments, neuronal (eg, retrograde) transport of a chimeric Clostridial neurotoxin can include transsynaptic movement (eg, transcytosis) of a chimeric Clostridial neurotoxin from one neuron to another neuron.

Inhibition of neuronal secretion can be partial or complete inhibition, preferably complete inhibition. For example, the chimeric clostridial neurotoxin can inhibit at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of neuronal secretion. Preferably, the chimeric clostridial neurotoxin inhibits 100% of neuronal secretion. The secretion herein is preferably SNARE-related (e.g., SNAP25-related) secretion.

In a preferred embodiment, the chimeric clostridial neurotoxins of the invention can treat migraine or disorders (preferably pain) described herein by inhibiting the release of mediators (e.g., pain mediators) from neurons comprising Aδ neurofibers or C neurofibers and by inhibiting the secretion of (e.g., mediators, preferably pain mediators) from neurons of the central nervous system, wherein the chimeric clostridial neurotoxin binds to neurons comprising Aδ neurofibers or C neurofibers, respectively.

Bacteria of the genus Clostridia produce highly efficient and specific protein toxins that can intoxicate neurons and other cells to which they are delivered. Examples of such clostridial toxins include neurotoxins produced by serotypes A-G and X of Clostridium tetani (C. tetani, TeNT) and Clostridium botulinum (C. botulinum, BoNT) (see WO 2018/009903 A2), as well as neurotoxins produced by Clostridium pasteurianum (C. baratii) and Clostridium butyricum (C. butyricum). Both tetanus toxin and botulinum toxin work by inhibiting the function of affected neurons, particularly the release of neurotransmitters. Although botulinum toxins typically act on neuromuscular junctions and inhibit cholinergic transmission in the peripheral nervous system, tetanus toxin acts on the central nervous system.

In nature, clostridial neurotoxins are synthesized as single-chain polypeptides that are modified by proteolytic cleavage events after translation to form two polypeptide chains linked together by disulfide bonds. Cleavage occurs at a specific cleavage site, commonly referred to as an activation site (e.g., an activation loop), which is located between cysteine residues that provide interchain disulfide bonds. It is this two-chain form that is the active form of the toxin. These two chains are called heavy chains (H-chains), which have a molecular weight of approximately 100 kDa; and light chains (L-chains), which have a molecular weight of approximately 50 kDa. The H-chain contains an N-terminal translocation component ( _HN domain) and a C-terminal targeting component ( _HC domain). The cleavage site is located between the L-chain and the translocation domain component. After the _HC domain binds to its target neuron and the bound toxin is internalized into the cell by endosomes, the _HN domain causes the L-chain to translocate across the endosomal membrane and into the cytosol, and the L-chain provides protease function (also known as non-cytotoxic protease).

Non-cytotoxic proteases act by proteolytic cleavage of intracellular transporters called SNARE proteins (e.g., SNAP25, VAMP or Syntaxin, preferably SNAP25 ). The acronym SNARE is derived from the term Soluble NSF Attachment Receptor, where NSF refers to N-ethylmaleimide-Sensitive F actor . SNARE proteins are essential for intracellular vesicle fusion and are therefore essential for transporting secretory molecules from cells via vesicles. The protease function is a zinc-dependent endopeptidase activity and exhibits high substrate specificity for SNARE proteins. Therefore, once delivered to the desired target cells, non-cytotoxic proteases are able to inhibit cellular secretion from target cells. The L-chain protease of Clostridial neurotoxins is a non-cytotoxic protease that cleaves SNARE proteins.

Given the ubiquitous nature of SNARE proteins, Clostridial neurotoxins such as botulinum toxin have been successfully used in a wide range of therapeutic applications.

For more details on the genetic basis of botulinum and tetani toxin production, see Henderson et al. (1997), The Clostridia: Molecular Biology and Pathogenesis, Academic press.

Clostridial neurotoxin domains are described in more detail below.

Examples of L-chain reference sequences include:

Botulinum neurotoxin type A: amino acid residues 1-448

Botulinum neurotoxin type B: amino acid residues 1-440

The reference sequences determined above should be considered as a guide, as slight variations may occur depending on the subserotype. For example, US 2007/0166332 (incorporated herein by reference in its entirety) cites a slightly different Clostridium sequence:

Botulinum neurotoxin type A: amino acid residues M1-K448

Botulinum neurotoxin type B: amino acid residues M1-K441

The translocation domain is a fragment of the H-chain of a clostridial neurotoxin corresponding approximately to the amino-terminal half of the H-chain, or a domain corresponding to this fragment in an intact H-chain.

Examples of reference translocation domains include:

Botulinum neurotoxin type A - amino acid residues (449-871)

Botulinum neurotoxin type B - amino acid residues (441-858)

The reference sequences determined above should be considered as a guide, as slight variations may occur depending on the subserotype. For example, US2007/0166332 (incorporated herein by reference) cites a slightly different Clostridium sequence:

Botulinum neurotoxin type A - amino acid residues (A449-K871)

Botulinum neurotoxin type B - amino acid residues (A442-S858)

In the context of the present invention, a variety of BoNT/AH _N regions comprising a translocation domain can be used in aspects of the present invention. The H _N region from a BoNT/A heavy chain is approximately 410-430 amino acids in length and comprises a translocation domain. Studies have shown that the full length of the H _N region from a Clostridial neurotoxin heavy chain is not necessary for the translocation activity of the translocation domain. Thus, aspects of this embodiment can include a BoNT/AH _N region comprising a translocation domain that is, for example, at least 350 amino acids, at least 375 amino acids, at least 400 amino acids, or at least 425 amino acids in length. Other aspects of this embodiment can include a BoNT/AH _N region comprising a translocation domain that is, for example, at most 350 amino acids, at most 375 amino acids, at most 400 amino acids, or at most 425 amino acids in length.

The term _HN encompasses naturally occurring BoNT/AH _N moieties, as well as modified BoNT/AH _N moieties having amino acid sequences not found in nature and/or synthetic amino acid residues. Preferably, the modified BoNT/AH _N moieties still exhibit the translocation function described above.

Examples of Clostridial neurotoxin receptor binding domain ( _HC ) reference sequences include:

BoNT/A-N872-L1296

BoNT/B-E859-E1291

The approximately 50 kDa _HC domain of clostridial neurotoxins (e.g., BoNTs) contains two distinct structural features, called the _HC and _HC domains, typically about 25 kDa each. The amino acid residues believed to be involved in receptor binding are primarily located in the _HC domain. The _HC domain of a native clostridial neurotoxin may contain approximately 400-440 amino acid residues. This fact is confirmed by the following publications, each of which is incorporated herein by reference in its entirety: Umland TC (1997) Nat. Struct. Biol. 4:788-792; Herreros J (2000) Biochem. J. 347:199-204; Halpern J (1993) J. Biol. Chem. 268:15, pp. 11188-11192; Rummel A (2007) PNAS 104:359-364; Lacey DB (1998) Nat. Struct. Biol. 5:898-902; Knapp (1998) Am. Cryst. Assoc. Abstract Papers 25:90; Swaminathan and Eswaramoorthy (2000) Nat. Struct. Biol. 7:1751-1759; and Rummel A (2004) Mol. Microbiol. 51(3), 631-643.

Examples of (reference) _HCN domains include:

Botulinum neurotoxin type A - amino acid residues (872-1110)

Botulinum neurotoxin type B - amino acid residues (859-1097)

The above sequence positions may vary slightly depending on the serotype/subtype. Other examples of (reference) _HCN domains include:

Botulinum neurotoxin type A - amino acid residues (874-1110)

Botulinum neurotoxin type B - amino acid residues (861-1097)

Examples of (reference) _HCC domains include:

Botulinum neurotoxin type A - amino acid residues (Y1111-L1296)

Botulinum neurotoxin type B - amino acid residues (Y1098-E1291)

WO 2017/191315 A1 (which is incorporated herein by reference) teaches chimeric clostridial neurotoxins and methods for their preparation and manufacture. Thus, the chimeric clostridial neurotoxin comprising a botulinum neurotoxin A (BoNT/A) light chain and a translocation domain (BoNT/A _NH ) and a BoNT/B receptor binding domain ( _HC domain) for use in the present invention may be one taught in WO 2017/191315 A1.

As used herein, the term "chimeric clostridial neurotoxin" or "chimeric neurotoxin" refers to a neurotoxin that comprises (preferably consists of) a clostridial neurotoxin light chain and translocation domain (H _N domain) from a first clostridial neurotoxin serotype and a receptor binding domain ( _HC domain) from a second, different clostridial neurotoxin serotype. Specifically, the chimeric clostridial neurotoxin for use in the present invention comprises a botulinum neurotoxin A (BoNT/A) light chain and translocation domain (H _N domain) and a BoNT/B receptor binding domain ( _HC domain). The BoNT/A LH _N domain of the chimeric clostridial neurotoxin is covalently linked to the BoNT/B _HC domain. The chimeric clostridial neurotoxin of the present invention may be referred to as a chimeric botulinum neurotoxin. The chimeric clostridial neurotoxin is also referred to herein as a "BoNT/AB,""mrBoNT/AB," or "BoNT/AB chimera."

The L-chain and the H _N domain (optionally including a complete or partial activation loop, e.g., a complete activation loop when the chimeric clostridial neurotoxin is in single-chain form and a cleaved/partial activation loop when it is in two-chain form) may be collectively referred to as the LH _N domain. The LH _N domain therefore also does not include the _HC domain.

The chimeric clostridial neurotoxin can consist essentially of a botulinum neurotoxin A (BoNT/A) light chain and translocation domain ( _HN domain) and a BoNT/B receptor binding domain ( _HC domain).

As used herein, the term "consisting essentially of" means that, for example, when administered to a subject, the chimeric clostridial neurotoxin also does not include one or more amino acid residues that confer additional functions to the polypeptide. In other words, a polypeptide that "consists essentially of" a botulinum neurotoxin A (BoNT/A) light chain and translocation domain ( _HN domain) and a BoNT/B receptor binding domain ( _HC domain) may also include one or more amino acid residues (amino acid residues for a botulinum neurotoxin A (BoNT/A) light chain and translocation domain (HN domain) and a BoNT/B receptor binding domain ( _HC domain)), but the one or more additional amino acid residues do not confer additional functions to the polypeptide, for example, when administered to a subject. Additional functions may include enzymatic activity, binding activity, and/or any physiological activity.

In addition to any clostridial neurotoxin sequence, the chimeric clostridial neurotoxin may include a non-clostridial neurotoxin sequence, as long as the non-clostridial neurotoxin sequence does not destroy the ability of the chimeric clostridial neurotoxin to achieve its therapeutic effect (preferably treating pain). Preferably, the non-clostridial neurotoxin sequence is not a sequence with catalytic activity (eg, enzymatic activity). In one embodiment, the chimeric clostridial neurotoxin of the present invention does not include a non-clostridial catalytic activity domain. In one embodiment, the chimeric clostridial neurotoxin does not include additional catalytic activity domains. In one embodiment, the non-clostridial sequence is not a sequence that binds to a cell receptor. In other words, in one embodiment, the non-clostridial sequence is not a ligand for a cell receptor. A cell receptor may be a protein cell receptor, such as an integral membrane protein. Examples of cell receptors can be found in the IUPHAR Pharmacology Database Guide 2019.4, at https://www.guidetopharmacology.org/download.jsp#db_reports. Non-clostridial neurotoxin sequences may include tags that contribute to purification, such as His-tags. In one embodiment, a chimeric Clostridial neurotoxin of the invention does not comprise a label or a site for adding a label, such as a sortase acceptor or donor site.

Preferably, the chimeric clostridial neurotoxin may be composed of a botulinum neurotoxin A (BoNT/A) light chain and translocation domain ( _HN domain) and a BoNT/B receptor binding domain ( _HC domain).

Chimeric clostridial neurotoxins comprise a light chain that is capable of exhibiting non-cytotoxic protease activity and capable of cleaving SNARE proteins in the cytosol of target neurons. As described above, the two-chain form is the active form of the clostridial neurotoxin. Therefore, the present invention excludes the use of chimeric clostridial neurotoxins comprising a light chain that has been catalytically inactivated (e.g., by one or more mutations) ("catalytically inactive light chain"). Such catalytically inactive light chains (and clostridial neurotoxins comprising it) are known in the art. The catalytically inactive L-chain may have one or more mutations that inactivate the catalytic activity. For example, the catalytically inactive L-chain may comprise a mutation of an active site residue. The mutation may be a substitution or a deletion, in particular a substitution with a chemically similar amino acid. Glutamic acid may be substituted by glutamine, histidine may be substituted by tyrosine, arginine may be substituted by glutamine, and/or tyrosine may be substituted by phenylalanine. Alternatively, any residue may be substituted by alanine. A catalytically inactive BoNT/A L-chain can comprise a mutation at H223, E224, H227, E262, R363, and/or Y366, e.g., a mutation at least E224 and H227. A catalytically inactive BoNT/A L-chain can comprise a substitution at E224 with glutamine (E224Q) and a substitution at H227 with tyrosine (H227Y).

As used herein, the term "catalytically inactive" with respect to a clostridial neurotoxin L-chain means that the L-chain is substantially free of non-cytotoxic protease activity, e.g., free of non-cytotoxic protease activity. A catalytically inactive clostridial neurotoxin L-chain may be a chain that does not cleave a protein of the extracellular fusion apparatus in a target cell. The term "substantially free of non-cytotoxic protease activity" means that the clostridial neurotoxin L-chain has less than 5% of the non-cytotoxic protease activity of a catalytically active clostridial neurotoxin L-chain (preferably the L-chain of a native BoNT/A as set forth in SEQ ID NO:6), e.g., less than 2%, 1%, or less than 0.1% of the non-cytotoxic protease activity of a catalytically active clostridial neurotoxin L-chain. The non-cytotoxic protease activity can be determined in vitro by incubating a test clostridial neurotoxin L-chain with a SNARE protein and comparing the amount of SNARE protein cleaved by the test clostridial neurotoxin L-chain to the amount of SNARE protein cleaved by a catalytically active clostridial neurotoxin L-chain (preferably the L-chain of a native BoNT/A as set forth in SEQ ID NO:6) under the same conditions. Conventional techniques (e.g., SDS-PAGE and Western blotting) can be used to quantify the amount of cleaved SNARE proteins. Suitable in vitro assays are described in WO 2019/145577 A1, which is incorporated herein by reference.

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

When administered to a subject, the chimeric clostridial neurotoxin is preferably in its active two-chain form, wherein the light chain and the heavy chain are linked together by a disulfide bond. When a clostridial neurotoxin (e.g., a chimeric clostridial neurotoxin) is defined herein by a polypeptide sequence (SEQ ID NO), the L-chain portion of the sequence (SEQ ID NO) may constitute the first chain of a two-chain clostridial neurotoxin (e.g., a two-chain chimeric clostridial neurotoxin), and the H _N and _HC domains together may constitute the second chain of a two-chain clostridial neurotoxin (e.g., a two-chain chimeric clostridial neurotoxin), wherein the first chain and the second chain are linked together by a disulfide bond. It will be understood by those skilled in the art that the protease may be cut at one or more positions within the activation loop of a clostridial neurotoxin (e.g., a chimeric clostridial neurotoxin), preferably at two positions within the activation loop. When the cleavage occurs at more than one position (preferably at two positions) in the activation loop, a small fragment of the C-terminal L-chain portion of the sequence may not be present in the two-chain clostridial neurotoxin sequence (e.g., a two-chain chimeric clostridial neurotoxin). In view of this, the sequence of a double-stranded clostridial neurotoxin (e.g., a double-stranded chimeric clostridial neurotoxin) may differ slightly from the sequence of a corresponding single-stranded clostridial neurotoxin (e.g., a single-stranded chimeric clostridial neurotoxin). The small fragment may be 1-15 amino acids. In particular, in one embodiment, when Lys-C is used to convert a single-stranded chimeric clostridial neurotoxin into a double-stranded clostridial neurotoxin, the small fragment of the C-terminal L-chain portion of the absent sequence may be SEQ ID NO: 15 or 16.

The C-terminal amino acid residue of the LH _N domain can correspond to the first amino acid residue of the _3'10 helix separating the LH _N and _HC domains of BoNT/A, and the N-terminal amino acid residue of the _HC domain can correspond to the second amino acid residue of the _3'10 helix separating the LH _N and _HC domains of BoNT/B.

An example of a BoNT/A polypeptide sequence is provided as SEQ ID NO:6.

An example of a BoNT/B polypeptide sequence is provided as SEQ ID NO: 7 (UniProt Accession No. B1 INP5).

Reference herein to "the first amino acid residue of the 3 _{' 10} helix separating the LH _N and _HC domains of BoNT/A" refers to the N-terminal residue of the 3 _{' 10} helix separating the LH _N and _HC domains.

Reference herein to the "second amino acid residue separating the 3 _{' 10} helix of the LH _N and _HC domains of BoNT/B" refers to the amino acid residue following the N-terminal residue separating the 3 _{' 10} helix of the LH _N and _HC domains.

A "3 ₁₀ helix" is a type of secondary structure found in proteins and peptides, along with alpha helices, beta sheets, and reverse turns. The amino acids in a _{3 10} helix are arranged in a right-handed spiral structure, where each complete turn is completed by three residues and ten atoms, separated by intramolecular hydrogen bonds between them. Each amino acid corresponds to a 120° turn in the helix (i.e., there are three residues per turn in the helix), and a translation along the helical axis is 120°. And there are 10 atoms in the ring formed by hydrogen bonding. Most importantly, the NH group of the amino acid first forms a hydrogen bond with the C=O group of the amino acid of the first three residues; this repeated i+3→i hydrogen bond defines a 3 ₁₀ helix. The _{3 10} helix is a standard concept in structural biology familiar to those skilled in the art.

This 3 ₁₀ helix corresponds to the four residues that form the actual helix and two cap (or transition) residues, one at each end of the four residues. The term "3 ₁₀ helix separating the LH _N and _HC domains" as used herein consists of 6 residues.

By performing structural analysis and sequence alignment, a 3 ₁₀ helix was identified that separates the LH _N and _HC domains. This 3 ₁₀ helix is surrounded by an α-helix at its N-terminus (i.e., in the C-terminal part of the LH _N domain) and by a β-strand at its C-terminus (i.e., in the N-terminal part of the _HC domain). The first (N-terminal) residue (cap or transition residue) of the _{3 10} helix also corresponds to the C-terminal residue of the α-helix.

The _3'10 helix separating the LH _N and _HC domains can be determined, for example, from publicly available crystal structures of botulinum neurotoxins, such as 3BTA (http://www.rcsb.org/pdb/explore/explore.do?structureld=3BTA) and 1EPW (http://www.rcsb.org/pdb/explore/explore.do?structureld=1EPW) corresponding to botulinum neurotoxins A1 and B1, respectively.

Publicly available computer modeling and alignment tools can also be used to determine the position of the _3'10 helix separating the LH _N and _HC domains in other neurotoxins, such as the homology modeling servers LOOPP (Learning, Observing and Outputting Protein Patterns, http://loopp.org), PHYRE (Protein Homology/analogY Recognition Engine, http://www.sbg.bio.ic.ac.uk/phyre2/) and Rosetta (https://www.rosettacommons.org/), the protein superposition server SuperPose ( http://wishart.biology.ualberta.ca/superpose/ ), the alignment program Clustal Omega (http://www.clustal.org/omega/), and many other tools/services listed on the Internet Resources for Molecular and Cell Biologists (http://molbiol-tools.ca/). In particular, the region surrounding the " _HN / _HCN " junction may be highly conserved in structure, making it an ideal region for superimposing different serotypes.

For example, the following method can be used to determine the sequence of this 3 ₁₀ helix in other neurotoxins:

1. The structural homology modeling tool LOOP (http://loopp.org) can be used to obtain predicted structures of other BoNT serotypes based on the BoNT/A1 crystal structure (3BTA.pdb);

2. The structure (pdb) file thus obtained can be edited to include only the N-terminus of the _HCN domain and the approximately 80 residues preceding it (which are part of the _HN domain), thereby retaining the highly structurally conserved " _HN / _HCN "region;

3. The protein overlay server SuperPose (http://wishart.biology.ualberta.ca/superpose/) can be used to overlay each serotype onto the 3BTA.pdb structure;

4. Overlay pdb files can be examined to locate the 3 ₁₀ helix at the start of the _HC domain of BoNT/A1 and then the corresponding residues in other serotypes can be identified.

5. Other BoNT serotype sequences can be aligned with Clustal Omega to check whether the corresponding residues are correct.

Examples of LH _N , _HC and 3 ₁₀ helical domains determined by this method are shown below:

Through structural analysis and sequence alignment, it was found that the β-strand behind the 3 ₁₀ helix separating the LH _N and _HC domains is a conserved structure in all botulinum neurotoxins and tetanus neurotoxins, and when starting from the first residue of the 3 ₁₀ helix separating the LH _N and _HC domains, the β-strand starts from the 8th residue (e.g., residue 879 of BoNT/A1).

A BoNT/AB chimera can comprise an LH _N domain from BoNT/A covalently linked to an _HC domain from BoNT/B, wherein the C-terminal amino acid residue of the LH _N domain corresponds to the eighth amino acid residue from the N-terminus of the beta strand located at the beginning (N-terminus) of the _HC domain of BoNT/A, and wherein the N-terminal amino acid residue of the _HC domain corresponds to the seventh amino acid from the N-terminus of the beta strand located at the beginning (N-terminus) of the _HC domain of BoNT/B.

A BoNT/AB chimera can comprise an LH _N domain from BoNT/A covalently linked to an _HC domain from BoNT/B, wherein the C-terminal amino acid residue of the LH _N domain corresponds to the C-terminal amino acid residue of an alpha-helix located at the end (C-terminus) of the LH _N domain of BoNT/A, and wherein the N-terminal amino acid residue of _{the HC} domain corresponds to the C-terminal amino acid residue immediately adjacent to the C-terminal amino acid residue of the alpha-helix located at the end (C-terminus) of the LH _N domain of BoNT/B.

The rationale for the BoNT/AB chimera design process is to ensure that the secondary structure is as unaffected as possible, thereby minimizing any changes to the tertiary structure and the function of each domain. Without wishing to be bound by theory, it is hypothesized that by not disrupting the four central amino acid residues of the _3'10 helix in the BoNT/AB chimera, the optimal conformation of the chimeric neurotoxin is ensured, thereby allowing the chimeric neurotoxins to fully exert their full function. In fact, surprisingly, retaining only the first amino acid residue of the _3'10 helix of BoNT/A and the second amino acid residue before the _3'10 helix of BoNT/B not only allows the generation of soluble and functional BoNT/AB chimeras, but further leads to improved properties relative to other BoNT/AB chimeras, particularly increased potency, increased safety ratio, and/or longer duration of action (as well as increased safety ratio and/or duration of action when compared to native BoNT/A [e.g., SEQ ID NO: 6]).

The adverse effects of neurotoxins (caused by diffusion of neurotoxins from the site of administration) can be experimentally assessed by measuring the percentage of weight loss in relevant animal models (e.g., mice, where weight loss is detected within seven days of administration). Conversely, the desired on-target effects of neurotoxins can be experimentally evaluated by the toe abduction score (DAS) assay (a measure of muscle paralysis). The DAS assay can be performed by injecting 20 μL of neurotoxin formulated in gelatin phosphate buffer into the gastrocnemius/soleus complex of mice, followed by assessment of the toe abduction score using the method of Aoki (Aoki KR, Toxicon 39: 1815-1820; 2001). In the DAS assay, mice are suspended briefly by their tails to elicit a characteristic startle response in which the mice extend their hind limbs and abduct their hind toes. After injection of the neurotoxin, varying degrees of toe abduction are scored on a five-point scale (0 = normal to 4 = maximum reduction in toe abduction and leg extension).

The safety ratio of the neurotoxin can then be expressed as the ratio between the amount of neurotoxin required to cause a 10% decrease in mouse body weight (measured at peak effect within the first seven days after administration to mice) and the amount of neurotoxin required for a DAS score of 2. Thus, a high safety ratio score is desirable and indicates that the neurotoxin is able to effectively paralyze the target muscle with few undesirable off-target effects.

A high safety ratio is particularly advantageous in treatment because it represents an increase in the therapeutic index. In other words, this means that reduced doses can be used and/or increased doses can be used without any additional (e.g., deleterious) effects compared to alternative clostridial neurotoxin therapeutics. Detrimental effects may include systemic toxicity and/or undesirable spread to adjacent muscles. The possibility of using higher doses of neurotoxins without additional effects is particularly advantageous because higher doses generally result in a longer duration of action of the neurotoxin.

The potency of a chimeric clostridial neurotoxin can be expressed as the minimum dose of the neurotoxin that results in a given DAS score when administered to the gastrocnemius/soleus complex of mice, such as a DAS score of 2 ( _ED50 dose) or a DAS score of 4. The potency of a chimeric clostridial neurotoxin can also be expressed as an _EC50 dose in a cellular assay that measures SNARE cleavage by the neurotoxin, such as an _EC50 dose in a cellular assay that measures SNAP25 cleavage by the chimeric clostridial neurotoxin.

The duration of action of a chimeric Clostridial neurotoxin can be expressed as the time required to restore a DAS score of 0 following administration of a given dose of neurotoxin (eg, the minimum dose of neurotoxin that results in a DAS score of 4) to the gastrocnemius/soleus complex of mice.

The chimeric clostridial neurotoxin may have a safety ratio greater than 7, wherein the safety ratio is calculated as: the dose of toxin required for a -10% change in body weight measured in pg/mouse divided by the DAS _EC50 measured in pg/mouse, wherein _EC50 = the dose required to produce a DAS score of 2. For example, a chimeric clostridial neurotoxin may have a safety ratio of at least 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50.

Preferably, the chimeric clostridial neurotoxin has a safety ratio of at least 10 (e.g., a safety ratio of 10), more preferably at least 12 or 13 (e.g., 14-15). The chimeric clostridial neurotoxin may have a safety ratio of greater than 7 up to 50, e.g., 8-45, 10-20, or 12-15.

Chimeric clostridial neurotoxins of the invention preferably have a longer duration of action (e.g., at least 5%, 10%, 25%, or 50% improvement in one or more symptoms) when compared to a BoNT/A (e.g., SEQ ID NO: 6, e.g., a double-chain form of SEQ ID NO: 6). The duration of action may be at least 1.25×, 1.5×, 1.75×, 2.0×, or 2.25× greater. The duration of action of the chimeric clostridial neurotoxin may be 4.5 to 9 months or 6 to 9 months. For example, the duration of action may be at least 4.5 months, 5.0 months, 5.5 months, 6 months, 6.5 months, 7.0 months, 7.5 months, 8.0 months, 8.5 months, or 9.0 months (from onset). In specific embodiments, the duration of action may be greater than 9.0 months.

Thus, in one embodiment, a chimeric clostridial neurotoxin can treat a condition (e.g., pain or sensory disturbance, preferably pain) in a subject for a duration (e.g., from administration) that is longer than a subject treated with a BoNT/A (e.g., SEQ ID NO: 6, e.g., SEQ ID NO: 6 in di-stranded form). The duration can be a duration from administration that is consistent with the duration of action of a chimeric clostridial neurotoxin of the invention. Thus, a chimeric clostridial neurotoxin can treat a condition in a subject for a duration after administration that is at least 1.25×, 1.5×, 1.75×, 2.0×, or 2.25× longer than the duration of treatment following administration of a BoNT/A (e.g., SEQ ID NO: 6, e.g., SEQ ID NO: 6 in di-stranded form). The chimeric clostridial neurotoxin can treat a condition in a subject for a duration of 4.5 to 9 months or 6 to 9 months from administration, e.g., at least 4.5 months, 5.0 months, 5.5 months, 6 months, 6.5 months, 7.0 months, 7.5 months, 8.0 months, 8.5 months, or 9.0 months from administration. In specific embodiments, the chimeric clostridial neurotoxin can treat a condition in a subject for a duration of greater than 9.0 months from administration.

Thus, in one aspect, the invention provides a method for treating a condition (e.g., pain or sensory disturbance, preferably pain) in a subject for a duration (e.g., from the start of administration) that is longer than the duration of treatment of a subject treated with BoNT/A (e.g., SEQ ID NO:6, e.g., SEQ ID NO:6 in di-chain form), the method comprising administering to the subject a chimeric clostridial neurotoxin, wherein the chimeric clostridial neurotoxin inhibits the release of mediators (e.g., pain mediators) from neurons comprising Aδ neurofibers or C neurofibers, wherein the chimeric clostridial neurotoxin binds to neurons comprising Aδ neurofibers or C neurofibers, respectively, and wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin A (BoNT/A) light chain and a translocation domain (H _N domain), and a BoNT/B receptor binding domain (H _C domain).

In a related aspect, the invention provides a chimeric clostridial neurotoxin for use in a method of treating a condition (e.g., pain or sensory disturbance, preferably pain) in a subject, wherein the duration of treatment (e.g., from the start of administration) is longer than the duration of treatment of a subject treated with BoNT/A (e.g., SEQ ID NO:6, e.g., SEQ ID NO:6 in di-chain form), the method comprising administering to the subject the chimeric clostridial neurotoxin, wherein the chimeric clostridial neurotoxin inhibits the release of mediators (e.g., pain mediators) from neurons comprising Aδ neurofibers or C neurofibers, wherein the chimeric clostridial neurotoxin binds to neurons comprising Aδ neurofibers or C neurofibers, respectively, and wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin A (BoNT/A) light chain and a translocation domain (H _N domain), and a BoNT/B receptor binding domain (H _C domain).

In another related aspect, the present invention provides use of a chimeric clostridial neurotoxin in the preparation of a medicament for treating a condition (e.g., pain or sensory disturbance, preferably pain) in a subject, wherein the duration of treatment (e.g., from the start of administration) is longer than the duration of treatment of a subject treated with BoNT/A (e.g., SEQ ID NO:6, e.g., SEQ ID NO:6 in di-chain form), wherein the chimeric clostridial neurotoxin inhibits the release of mediators (e.g., pain mediators) from neurons comprising Aδ neurofibers or C neurofibers, wherein the chimeric clostridial neurotoxin binds to neurons comprising Aδ neurofibers or C neurofibers, respectively, and wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin A (BoNT/A) light chain and a translocation domain (H _N domain), and a BoNT/B receptor binding domain (H _C domain).

Thus, in one aspect, the invention provides a method for treating a condition (e.g., pain or sensory disturbance, preferably pain) in a subject for a duration (e.g., from the start of administration) that is longer than the duration of treatment of a subject treated with a BoNT/A (e.g., SEQ ID NO: 6, e.g., SEQ ID NO: 6 in di-chain form), the method comprising administering to the subject a chimeric clostridial neurotoxin, wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin A (BoNT/A) light chain and a translocation domain ( _HN domain), and a BoNT/B receptor binding domain ( _HC domain).

In a related aspect, the invention provides a chimeric clostridial neurotoxin for use in a method of treating a condition (e.g., pain or sensory disturbance, preferably pain) in a subject, wherein the duration of treatment (e.g., from the start of administration) is greater than the duration of treatment of a subject treated with BoNT/A (e.g., SEQ ID NO:6, e.g., SEQ ID NO:6 in di-chain form), the method comprising administering to the subject the chimeric clostridial neurotoxin, wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin A (BoNT/A) light chain and a translocation domain ( _HN domain), and a BoNT/B receptor binding domain ( _HC domain).

In another related aspect, the invention provides use of a chimeric clostridial neurotoxin in the preparation of a medicament for treating a condition (e.g., pain or sensory disturbance, preferably pain) in a subject, wherein the duration of treatment (e.g., from the start of administration) is longer than the duration of treatment of a subject treated with BoNT/A (e.g., SEQ ID NO: 6, e.g., SEQ ID NO: 6 in a two-chain form), wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin A (BoNT/A) light chain and a translocation domain ( _HN domain), and a BoNT/B receptor binding domain ( _HC domain).

The condition is preferably migraine or migraine pain.

The term "treating a subject for a duration of a disorder (e.g., from administration) that is longer than the duration of treatment in a subject treated with BoNT/A" or "treating a subject for a duration of a disorder (e.g., from administration) that is longer than the duration of treatment in a subject treated with BoNT/A" may mean that one or more symptoms of a disorder in the subject are alleviated for a longer period of time following administration of a chimeric clostridial neurotoxin of the invention compared to administration of BoNT/A. The duration of action may be at least 1.25×, 1.5×, 1.75×, 2.0×, or 2.25× greater. The duration of action of a chimeric clostridial neurotoxin may be between 6 and 9 months. For example, the duration of action may be at least: 4.5 months, 5.0 months, 5.5 months, 6 months, 6.5 months, 7.0 months, 7.5 months, 8.0 months, 8.5 months, or 9.0 months (from onset). In specific embodiments, the duration of action may be greater than 9.0 months. The alleviation may be determined by comparison to an equivalent control subject that has been treated with BoNT/A and exhibits equivalent symptoms. A subject treated with a chimeric Clostridial neurotoxin according to the invention may exhibit at least a 5%, 10%, 25%, or 50% improvement in the severity of one or more symptoms compared to the severity of one or more symptoms prior to treatment with the chimeric Clostridial neurotoxin over a period of time in which the severity of one or more symptoms in the control subject is substantially the same (e.g., the same) as prior to BoNT/A treatment.

In one embodiment, a chimeric Clostridial neurotoxin can treat a condition (e.g., pain or sensory disturbance, preferably pain) in a subject more effectively than a subject treated with a BoNT/A (e.g., SEQ ID NO: 6, e.g., a di-chain form of SEQ ID NO: 6).

Thus, in one aspect, the invention provides a method for treating a condition (e.g., pain or sensory disturbance, preferably pain) in a subject that is more effective than treatment of the subject with BoNT/A (e.g., SEQ ID NO:6, e.g., a di-chain form of SEQ ID NO:6), the method comprising administering to the subject a chimeric clostridial neurotoxin, wherein the chimeric clostridial neurotoxin inhibits release of mediators (e.g., pain mediators) from neurons comprising Aδ neurofibers or C neurofibers, wherein the chimeric clostridial neurotoxin binds to neurons comprising Aδ neurofibers or C neurofibers, respectively, and wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin A (BoNT/A) light chain and translocation domain (H _N domain), and a BoNT/B receptor binding domain (H _C domain).

In a related aspect, the invention provides a chimeric clostridial neurotoxin for use in a method of treating a condition (e.g., pain or sensory disturbance, preferably pain) in a subject, wherein the treatment is more effective than treatment of the subject with BoNT/A (e.g., SEQ ID NO:6, e.g., a di-chain form of SEQ ID NO:6), the method comprising administering to the subject the chimeric clostridial neurotoxin, wherein the chimeric clostridial neurotoxin inhibits release of mediators (e.g., pain mediators) from neurons comprising Aδ neurofibers or C neurofibers, wherein the chimeric clostridial neurotoxin binds to neurons comprising Aδ neurofibers or C neurofibers, respectively, and wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin A (BoNT/A) light chain and translocation domain (H _N domain), and a BoNT/B receptor binding domain (H _C domain).

In another related aspect, the present invention provides a use of a chimeric clostridial neurotoxin in the preparation of a medicament for treating a condition (e.g., pain or sensory disturbance, preferably pain) in a subject, wherein the treatment is more effective than treatment of the subject with BoNT/A (e.g., SEQ ID NO:6, e.g., a di-chain form of SEQ ID NO:6), wherein the chimeric clostridial neurotoxin inhibits the release of mediators (e.g., pain mediators) from neurons comprising Aδ neurofibers or C neurofibers, wherein the chimeric clostridial neurotoxin binds to neurons comprising Aδ neurofibers or C neurofibers, respectively, and wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin A (BoNT/A) light chain and translocation domain (H _N domain), and a BoNT/B receptor binding domain (H _C domain).

Thus, in one aspect, the invention provides a method for treating a condition (e.g., pain or sensory disturbance, preferably pain) in a subject that is more effective than treatment of the subject with BoNT/A (e.g., SEQ ID NO:6, e.g., a di-chain form of SEQ ID NO:6), the method comprising administering to the subject a chimeric clostridial neurotoxin, wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin A (BoNT/A) light chain and a translocation domain ( _HN domain), and a BoNT/B receptor binding domain ( _HC domain).

In a related aspect, the invention provides a chimeric clostridial neurotoxin for use in a method of treating a condition (e.g., pain or sensory disturbance, preferably pain) in a subject, wherein the treatment is more effective than treatment of the subject with BoNT/A (e.g., SEQ ID NO: 6, e.g., SEQ ID NO: 6 in di-chain form), the method comprising administering to the subject the chimeric clostridial neurotoxin, wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin A (BoNT/A) light chain and a translocation domain ( _HN domain), and a BoNT/B receptor binding domain ( _HC domain).

In another related aspect, the present invention provides a use of a chimeric clostridial neurotoxin in the preparation of a medicament for treating a condition (e.g., pain or sensory disturbance, preferably pain) in a subject, wherein the treatment is more effective than treatment of the subject with BoNT/A (e.g., SEQ ID NO: 6, e.g., SEQ ID NO: 6 in di-chain form), wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin A (BoNT/A) light chain and a translocation domain ( _HN domain), and a BoNT/B receptor binding domain ( _HC domain).

The condition is preferably migraine or migraine pain.

The term "treating a condition in a subject better than a subject treated with BoNT/A" or "treating a condition in a subject better than a subject treated with BoNT/A" can mean that one or more symptoms of a condition in the subject are reduced to a greater extent following administration of a chimeric clostridial neurotoxin of the invention compared to administration of BoNT/A. The reduction can be determined by comparison to an equivalent control subject that has been treated with BoNT/A and exhibits equivalent symptoms. At a given time period after administration, a subject treated with a chimeric clostridial neurotoxin according to the invention can exhibit at least a 5%, 10%, 25%, or 50% reduction in severity of one or more symptoms compared to the severity of the equivalent one or more symptoms in a control subject at the same time period after administration of BoNT/A (e.g., SEQ ID NO:6, e.g., SEQ ID NO:6 in di-chain form). In another embodiment, better efficacy can mean that the maximum reduction in severity of one or more symptoms in a subject treated with a chimeric clostridial neurotoxin is greater than the maximum reduction in severity of the equivalent one or more symptoms in a control subject treated with a BoNT/A (e.g., SEQ ID NO: 6, e.g., a di-chain form of SEQ ID NO: 6).

In one embodiment, the chimeric Clostridial neurotoxin can reduce pain (eg, migraine) in a subject to a greater extent than a subject treated with a BoNT/A (eg, SEQ ID NO: 6, eg, a di-chain form of SEQ ID NO: 6).

Thus, in one aspect, the invention provides a method for reducing pain in a subject to a greater extent than in a subject treated with BoNT/A (e.g., SEQ ID NO:6, e.g., a di-chain form of SEQ ID NO:6), the method comprising administering to the subject a chimeric clostridial neurotoxin, wherein the chimeric clostridial neurotoxin inhibits the release of pain mediators from neurons comprising Aδ nerve fibers or C nerve fibers, wherein the chimeric clostridial neurotoxin binds to neurons comprising Aδ nerve fibers or C nerve fibers, respectively, and wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin A (BoNT/A) light chain and translocation domain ( _HN domain), and a BoNT/B receptor binding domain ( _HC domain).

In a related aspect, the invention provides a chimeric clostridial neurotoxin for use in a method of reducing pain in a subject to a greater extent than a subject treated with BoNT/A (e.g., SEQ ID NO:6, e.g., SEQ ID NO:6 in di-chain form), the method comprising administering to the subject the chimeric clostridial neurotoxin, wherein the chimeric clostridial neurotoxin inhibits the release of pain mediators from neurons comprising Aδ nerve fibers or C nerve fibers, wherein the chimeric clostridial neurotoxin binds to neurons comprising Aδ nerve fibers or C nerve fibers, respectively, and wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin A (BoNT/A) light chain and translocation domain (H _N domain), and a BoNT/B receptor binding domain (H _C domain).

In another related aspect, the present invention provides a use of a chimeric clostridial neurotoxin in the preparation of a medicament for reducing pain in a subject, which reduces pain in the subject to a greater extent than a subject treated with BoNT/A (e.g., SEQ ID NO:6, e.g., a di-chain form of SEQ ID NO:6), wherein the chimeric clostridial neurotoxin inhibits the release of pain mediators from neurons comprising Aδ nerve fibers or C nerve fibers, wherein the chimeric clostridial neurotoxin binds to neurons comprising Aδ nerve fibers or C nerve fibers, respectively, and wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin A (BoNT/A) light chain and translocation domain (H _N domain), and a BoNT/B receptor binding domain (H _C domain).

Thus, in one aspect, the invention provides a method for reducing pain in a subject to a greater extent than in a subject treated with BoNT/A (e.g., SEQ ID NO:6, e.g., a two-chain form of SEQ ID NO:6), the method comprising administering to the subject a chimeric clostridial neurotoxin, wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin A (BoNT/A) light chain and a translocation domain ( _HN domain), and a BoNT/B receptor binding domain ( _HC domain).

In a related aspect, the invention provides a chimeric clostridial neurotoxin for use in a method of reducing pain in a subject, which reduces pain in the subject to a greater extent than in a subject treated with BoNT/A (e.g., SEQ ID NO:6, e.g., a di-chain form of SEQ ID NO:6), the method comprising administering to the subject the chimeric clostridial neurotoxin, wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin A (BoNT/A) light chain and a translocation domain ( _HN domain), and a BoNT/B receptor binding domain ( _HC domain).

In another related aspect, the invention provides use of a chimeric clostridial neurotoxin in the preparation of a medicament for reducing pain in a subject, which reduces pain in the subject to a greater extent than a subject treated with BoNT/A (e.g., SEQ ID NO: 6, e.g., SEQ ID NO: 6 in di-chain form), wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin A (BoNT/A) light chain and a translocation domain ( _HN domain), and a BoNT/B receptor binding domain ( _HC domain).

The pain is preferably migraine pain.

The term "reducing pain in a subject to a greater extent than in a subject treated with BoNT/A" or "reducing pain in a subject to a greater extent than in a subject treated with BoNT/A" may refer to a greater reduction in pain in a subject following administration of a chimeric clostridial neurotoxin of the invention compared to administration of BoNT/A. The reduction may be determined by comparison to an equivalent control subject that has been treated with BoNT/A and exhibits equivalent pain. At a given time period after administration, a subject treated with a chimeric clostridial neurotoxin according to the invention may exhibit a reduction in pain of at least 5%, 10%, 25%, or 50% compared to the severity of equivalent pain in a control subject at the same time period following administration of BoNT/A (e.g., SEQ ID NO:6, e.g., SEQ ID NO:6 in double-chain form). In another embodiment, the maximum reduction in pain in a subject treated with the chimeric clostridial neurotoxin is greater than the maximum reduction in equivalent pain in a control subject treated with BoNT/A (e.g., SEQ ID NO:6, e.g., SEQ ID NO:6 in double-chain form).

In one embodiment, a chimeric Clostridial neurotoxin can reduce the amount of a pain mediator (e.g., a migraine pain mediator) in a biological fluid and/or brain of a subject to a greater extent than the amount of the same pain mediator in the same biological fluid and/or brain of a subject treated with a BoNT/A (e.g., SEQ ID NO: 6, e.g., a di-chain form of SEQ ID NO: 6).

Thus, in one aspect, the invention provides a method for reducing the amount of a pain mediator in a biological fluid and/or brain of a subject to a greater extent than the amount of the same pain mediator in the biological fluid and/or brain of the subject treated with BoNT/A (e.g., SEQ ID NO:6, e.g., a di-chain form of SEQ ID NO:6), the method comprising administering to the subject a chimeric clostridial neurotoxin, wherein the chimeric clostridial neurotoxin inhibits release of the pain mediator from neurons comprising Aδ neurofibers or C neurofibers, wherein the chimeric clostridial neurotoxin binds to neurons comprising Aδ neurofibers or C neurofibers, respectively, and wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin A (BoNT/A) light chain and translocation domain ( _HN domain), and a BoNT/B receptor binding domain ( _HC domain).

In a related aspect, the invention provides a chimeric clostridial neurotoxin for use in a method of reducing the amount of a pain mediator in a biological fluid and/or brain of a subject, which reduces the amount of a pain mediator in a biological fluid and/or brain of a subject to a greater extent than the amount of the same pain mediator in the same biological fluid and/or brain of the subject treated with BoNT/A (e.g., SEQ ID NO:6, e.g., a di-chain form of SEQ ID NO:6), the method comprising administering to the subject the chimeric clostridial neurotoxin, wherein the chimeric clostridial neurotoxin inhibits release of the pain mediator from neurons comprising Aδ neurofibers or C neurofibers, wherein the chimeric clostridial neurotoxin binds to neurons comprising Aδ neurofibers or C neurofibers, respectively, and wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin A (BoNT/A) light chain and translocation domain (H _N domain), and a BoNT/B receptor binding domain (H _C domain).

In another related aspect, the present invention provides for use of a chimeric clostridial neurotoxin in the preparation of a medicament for reducing the amount of a pain mediator in a biological fluid and/or brain of a subject, which reduces the amount of a pain mediator in a biological fluid and/or brain of a subject to a greater extent than the amount of the same pain mediator in the same biological fluid and/or brain of a subject treated with BoNT/A (e.g., SEQ ID NO:6, e.g., a di-chain form of SEQ ID NO:6), wherein the chimeric clostridial neurotoxin inhibits release of the pain mediator from neurons comprising Aδ neurofibers or C neurofibers, wherein the chimeric clostridial neurotoxin binds to neurons comprising Aδ neurofibers or C neurofibers, respectively, and wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin A (BoNT/A) light chain and translocation domain (H _N domain), and a BoNT/B receptor binding domain (H _C domain).

Thus, in one aspect, the invention provides a method for reducing the amount of a pain mediator in a biological fluid and/or brain of a subject to a greater extent than the amount of the same pain mediator in the biological fluid and/or brain of a subject treated with BoNT/A (e.g., SEQ ID NO:6, e.g., a di-chain form of SEQ ID NO:6), the method comprising administering to the subject a chimeric clostridial neurotoxin, wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin A (BoNT/A) light chain and translocation domain ( _HN domain), and a BoNT/B receptor binding domain ( _HC domain).

In a related aspect, the invention provides a chimeric clostridial neurotoxin for use in a method of reducing the amount of a pain mediator in a biological fluid and/or brain of a subject, which reduces the amount of a pain mediator in a biological fluid and/or brain of a subject to a greater extent than the amount of the same pain mediator in the same biological fluid and/or brain of the subject treated with BoNT/A (e.g., SEQ ID NO:6, e.g., a di-chain form of SEQ ID NO:6), the method comprising administering to the subject the chimeric clostridial neurotoxin, wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin A (BoNT/A) light chain and translocation domain ( _HN domain), and a BoNT/B receptor binding domain ( _HC domain).

In another related aspect, the invention provides for use of a chimeric clostridial neurotoxin in the preparation of a medicament for reducing the amount of a pain mediator in a biological fluid and/or brain of a subject, which reduces the amount of a pain mediator in a biological fluid and/or brain of a subject to a greater extent than the amount of the same pain mediator in the same biological fluid and/or brain of a subject treated with BoNT/A (e.g., SEQ ID NO:6, e.g., a di-chain form of SEQ ID NO:6), wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin A (BoNT/A) light chain and translocation domain ( _HN domain), and a BoNT/B receptor binding domain ( _HC domain).

The term "reducing the amount of a pain mediator in a biological fluid and/or brain of a subject to a greater extent than the amount of the same pain mediator in the same biological fluid and/or brain of a subject treated with BoNT/A" or "reducing the amount of a pain mediator in a biological fluid and/or brain of a subject to a greater extent than the amount of the same pain mediator in the same biological fluid and/or brain of a subject treated with BoNT/A" may refer to a greater reduction in the amount of a pain mediator in a subject following administration of a chimeric Clostridial neurotoxin of the invention compared to administration of BoNT/A. The reduction may be determined by comparison to the amount of the same pain mediator in the same biological fluid and/or brain of an equivalent control subject that has been treated with BoNT/A. At a given time period after administration, a subject treated with a chimeric clostridial neurotoxin according to the invention can exhibit at least a 5%, 10%, 25%, or 50% reduction in the amount of a pain mediator in their biological fluid and/or brain as compared to the amount of the same pain mediator in the same biological fluid and/or brain of a control subject at the same time period after administration of a BoNT/A (e.g., SEQ ID NO: 6, e.g., SEQ ID NO: 6 in di-chain form). In another embodiment, the maximal reduction in the amount of a pain mediator in the biological fluid and/or brain of a subject treated with a chimeric clostridial neurotoxin is greater than the maximal reduction in the same pain mediator in the same biological fluid and/or brain of a control subject treated with a BoNT/A (e.g., SEQ ID NO: 6, e.g., SEQ ID NO: 6 in di-chain form). The pain mediator can be a migraine pain mediator. The pain mediator is preferably CGRP. Preferably, the biological fluid is blood (including a portion thereof).

CGRP can be used as a relevant marker for evaluating the efficacy of analgesics (e.g., analgesics). CGRP can therefore be used as a biomarker for determining the suitability of a clostridial neurotoxin for treating pain (e.g., migraine pain). Therefore, in one aspect, the present invention provides a method for determining whether a clostridial neurotoxin is suitable for treating pain, the method comprising:

(a) comparing the level of CGRP contained in a first sample to the level of CGRP contained in a second sample, wherein the first sample has been obtained from a subject before administration of a clostridial neurotoxin, and wherein the second sample has been obtained from the same subject after administration of a clostridial neurotoxin; and

(b) determining that the Clostridial neurotoxin is suitable for treating pain when the level of CGRP in the second sample is lower than the level of CGRP in the first sample; or

(c) when the CGRP level in the second sample is not lower than (e.g., higher than or equal to) the CGRP level in the first sample, determining that the clostridial neurotoxin is not suitable for treating pain. As used herein, the term "lower than" preferably means statistically significantly lower than, and "not lower than" preferably means no statistically significant difference (e.g., the same) or statistically significantly higher than.

The clostridial neurotoxin can be any suitable clostridial neurotoxin known in the art, such as a chimeric clostridial neurotoxin described herein. The clostridial neurotoxin can be a BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F, BoNT/G, BoNT/X, or a tetanus neurotoxin (TeNT).

The BoNT/A may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 6. For example, the BoNT/A may comprise a polypeptide sequence having at least 80%, 90%, 95%, or 99.9% sequence identity to SEQ ID NO: 6. Preferably, the BoNT/A may comprise (more preferably consist of) SEQ ID NO: 6.

The BoNT/B may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 7. For example, the BoNT/B may comprise a polypeptide sequence having at least 80%, 90%, 95%, or 99.9% sequence identity to SEQ ID NO: 7. Preferably, the BoNT/B may comprise (more preferably consist of) SEQ ID NO: 7.

The BoNT/C may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 8. For example, the BoNT/C may comprise a polypeptide sequence having at least 80%, 90%, 95%, or 99.9% sequence identity to SEQ ID NO: 8. Preferably, the BoNT/C may comprise (more preferably consist of) SEQ ID NO: 8.

The BoNT/D may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 9. For example, the BoNT/D may comprise a polypeptide sequence having at least 80%, 90%, 95%, or 99.9% sequence identity to SEQ ID NO: 9. Preferably, the BoNT/D may comprise (more preferably consist of) SEQ ID NO: 9.

The BoNT/E may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 10. For example, the BoNT/E may comprise a polypeptide sequence having at least 80%, 90%, 95%, or 99.9% sequence identity to SEQ ID NO: 10. Preferably, the BoNT/E may comprise (more preferably consist of) SEQ ID NO: 10.

The BoNT/F may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 11. For example, the BoNT/F may comprise a polypeptide sequence having at least 80%, 90%, 95%, or 99.9% sequence identity to SEQ ID NO: 11. Preferably, the BoNT/F may comprise (more preferably consist of) SEQ ID NO: 11.

The BoNT/G may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 12. For example, the BoNT/G may comprise a polypeptide sequence having at least 80%, 90%, 95%, or 99.9% sequence identity to SEQ ID NO: 12. Preferably, the BoNT/G may comprise (more preferably consist of) SEQ ID NO: 12.

The BoNT/X may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 13. For example, the BoNT/X may comprise a polypeptide sequence having at least 80%, 90%, 95%, or 99.9% sequence identity to SEQ ID NO: 13. Preferably, the BoNT/X may comprise (more preferably consist of) SEQ ID NO: 13.

TeNT may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 14. For example, TeNT may comprise a polypeptide sequence having at least 80%, 90%, 95% or 99.9% sequence identity to SEQ ID NO: 14. Preferably, TeNT may comprise (more preferably consist of) SEQ ID NO: 14.

In one embodiment, prior to using the Clostridial neurotoxin in a method for determining whether the Clostridial neurotoxin is suitable for treating pain, the Clostridial neurotoxin is converted to its two-chain form, eg, as described herein.

The first sample and the second sample can be blood samples, optionally subjected to one or more processing steps. The first sample and the second sample are preferably identical (e.g., of the same type and optionally have been subjected to the same processing steps). The level of CGRP can be determined using any suitable technique, including quantitative Western blotting and/or mass spectrometry.

The BoNT/A light chain, BoNT/A translocation domain, and/or BoNT/BH _C domain can be a modified BoNT/A light chain, BoNT/A translocation domain, and/or BoNT/BH _C domain, or a derivative thereof, including but not limited to those described below. The modified BoNT/A light chain, BoNT/A translocation domain, and/or BoNT/BH _C domain, or derivative can contain one or more amino acids that have been modified compared to a native (unmodified) form of a BoNT/A light chain, BoNT/A translocation domain, and/or BoNT/BH _C domain, or can contain one or more inserted amino acids that are not present in a native (unmodified) form of a BoNT/A light chain, BoNT/A translocation domain, and/or BoNT/BH _C domain. For example, a modified BoNT/A light chain, BoNT/A translocation domain, and/or BoNT/BH _C domain can have a modified amino acid sequence in one or more domains relative to a native (unmodified) BoNT/A light chain, BoNT/A translocation domain, and/or BoNT/BH _C domain sequence. Such modifications can modify aspects of its function, such as biological activity or persistence. Thus, in one embodiment, the BoNT/A light chain, BoNT/A translocation domain, and/or BoNT/BH _C domain is a modified BoNT/A light chain, BoNT/A translocation domain, and/or BoNT/BH _C domain, or a derivative of a modified BoNT/A light chain, BoNT/A translocation domain, and/or BoNT/BH _C domain.

A modified BoNT/BH _C domain can have one or more modifications that modify binding to a target neural cell, e.g., provide higher or lower affinity binding when compared to a native (unmodified) BoNT/BH _C domain. Such modifications in a BoNT/BH _C domain can include modifying residues in a ganglioside binding site or a protein (e.g., synaptotagmin) binding site of the _HC domain that alters binding to a ganglioside receptor and/or protein receptor of a target neural cell. Examples of such modified neurotoxins are described in WO 2006/027207 and WO 2006/114308, both of which are incorporated herein by reference in their entireties.

The modified light chain may have one or more modifications in its amino acid sequence, such as modifications in the substrate binding or catalytic domains, which may alter or modify the SNARE protein specificity of the modified light chain, provided that the modification does not render the light chain catalytically inactive. Examples of such modified neurotoxins are described in WO 2010/120766 and US2011/0318385, both of which are incorporated herein by reference in their entirety.

The LH _N domain from a BoNT/A can correspond to amino acid residues 1 to 872 of SEQ ID NO: 6, or a polypeptide sequence having at least 70% sequence identity thereto. The LH _N domain from a BoNT/A can correspond to amino acid residues 1 to 872 of SEQ ID NO: 6, or a polypeptide sequence having at least 80%, 90%, or 95% sequence identity thereto. Preferably, the LH _N domain from a BoNT/A corresponds to amino acid residues 1 to 872 of SEQ ID NO: 6.

The _HC domain from a BoNT/B can correspond to amino acid residues 860 to 1291 of SEQ ID NO: 7, or a polypeptide sequence having at least 70% sequence identity thereto. The _HC domain from a BoNT/B can correspond to amino acid residues 860 to 1291 of SEQ ID NO: 7, or a polypeptide sequence having at least 80%, 90%, or 95% sequence identity thereto. Preferably, the _HC domain from a BoNT/B corresponds to amino acid residues 860 to 1291 of SEQ ID NO: 7.

Preferably, the BoNT/AB chimera comprises a BoNT/A1 LH _N domain and a BoNT/B1 _HC domain. More preferably, the LH _N domain corresponds to amino acid residues 1 to 872 of BoNT/A1 (SEQ ID NO: 6) and the _HC domain corresponds to amino acid residues 860 to 1291 of BoNT/B1 (SEQ ID NO: 7).

Most preferably, the BoNT/B _HC domain further comprises at least one amino acid residue substitution, insertion, indel, or deletion in the HC _CC subdomain that has the effect of increasing the binding affinity of the BoNT/B neurotoxin to human SytII compared to the native BoNT/B sequence. Suitable amino acid residue substitutions, insertions, indels, or deletions in the BoNT/B HC _CC subdomain have been disclosed in WO 2013/180799 and WO 2016/154534 (each incorporated herein by reference).

Suitable amino acid residue substitutions, insertions, indels, or deletions in the BoNT/BH _CC subdomain can include a substitution mutation selected from the group consisting of V1118M, Y1183M, E1191M, E1191I, E1191Q, E1191T, S1199Y, S1199F, S1199L, S1201V, E1191C, E1191V, E1191L, E1191Y, S1199W, S1199E, S1199H, W1178Y, W1178Q, W1178A, W1178S, Y1183C, Y1183P, and combinations thereof.

Suitable amino acid residue substitutions, insertions, indels, or deletions in the BoNT/BH _CC subdomain can also include a combination of two substitution mutations selected from the group consisting of E1191M and S1199L, E1191M and S1199Y, E1191M and S1199F, E1191Q and S1199L, E1191Q and S1199Y, E1191Q and S1199F, E1191M and S1199W, E1191M and W1178Q, E1191C and S1199W, E1191C and S1199Y, E1191C and W1178Q, E1191Q and S1199W, E1191V and S1199W, E1191V and S1199Y, or E1191V and W1178Q.

Suitable amino acid residue substitutions, insertions, indels, or deletions in the BoNT/BH _CC subdomain may also include a combination of three substitution mutations, which are E1191M, S1199W, and W1178Q.

Preferably, the amino acid residue substitution, insertion, indel or deletion in the BoNT/BH _CC subdomain comprises a combination of two substitution mutations, which are E1191M and S1199Y. Such modifications are present in the chimeric clostridial neurotoxin SEQ ID NO: 1 and SEQ ID NO: 4. E1191M may correspond to position 1204 of SEQ ID NO: 1, and S1199Y may correspond to position 1212. Thus, SEQ ID NO: 1 may comprise 1204M and 1212Y.

The modification may be a modification when compared to an unmodified BoNT/B as set forth in SEQ ID NO:7, wherein the amino acid residue numbering is determined by alignment with SEQ ID NO:7. Since the presence of a methionine residue at position 1 of SEQ ID NO:7 (and SEQ ID NOs corresponding to the chimeric clostridial neurotoxin polypeptides described herein) is optional, one skilled in the art takes the presence/absence of the methionine residue into account when determining the amino acid residue numbering. For example, when SEQ ID NO:7 includes a methionine, the position numbering will be as defined above (e.g., E1191 will be E1191 of SEQ ID NO:7). Alternatively, when a methionine is absent in SEQ ID NO:7, the amino acid residue numbering should be modified to -1 (e.g., E1191 will be E1190 of SEQ ID NO:7). Thus, the initial methionine amino acid residue of the polypeptide sequence of the chimeric clostridial neurotoxin may be optional or absent. Similar considerations apply when a methionine is present/absent at position 1 of other polypeptide sequences described herein, and one skilled in the art can readily determine the correct amino acid residue numbering using routine techniques in the art. Alignment can be performed using any of the methods described herein for determining sequence homology and/or % sequence identity.

The term "deletion" as used herein refers to the removal of one or more amino acid residues of a polypeptide without replacing one or more amino acid residues at the deletion site. Thus, when one amino acid residue is deleted from a polypeptide sequence having x amino acid residues (for example), the resulting polypeptide has x-1 amino acid residues.

As used herein, the term "indel" refers to the deletion of one or more amino acid residues of a polypeptide and the insertion of a different number of amino acid residues (more or fewer amino acid residues) than the number of amino acid residues deleted at the deletion site. Thus, for example, for an indel in which two amino acid residues are deleted from a polypeptide sequence having x amino acid residues, the resulting polypeptide has x-1 amino acid residues or x+≥1 amino acid residues. Insertions and deletions may be performed in any order, sequentially or simultaneously.

As used herein, the term "substitution" refers to the replacement of one or more amino acid residues with the same number of amino acid residues at the same site. Thus, for example, for a substitution of a polypeptide sequence having x amino acid residues, the resulting polypeptide also has x amino acid residues. Preferably, the substitution is at a single amino acid position.

The term "insertion" as used herein refers to the addition of one or more amino acid residues of a polypeptide at an insertion site without deleting one or more amino acid residues of the polypeptide. Thus, for example, when an amino acid residue is inserted into a polypeptide sequence having x amino acid residues, the resulting polypeptide has x+1 amino acid residues.

Methods for modifying proteins by substitution, insertion, deletion or insertion and deletion (indel) of amino acid residues are known in the art. For example, amino acid modifications can be introduced by modifying a nucleic acid sequence (e.g., a DNA sequence) encoding a polypeptide. This can be achieved using standard molecular cloning techniques, such as by site-directed mutagenesis using a polymerase, in which the original coding sequence is replaced with a short DNA (oligonucleotide) encoding the desired amino acid, or by inserting/deleting part of the gene using various enzymes (e.g., ligases and restriction endonucleases). Alternatively, the modified gene sequence can be chemically synthesized. Typically, the modification can be performed by modifying a nucleic acid encoding a natural clostridial neurotoxin (or a portion thereof) so that the modified chimeric clostridial neurotoxin (or a portion thereof) encoded by the nucleic acid comprises the modification. Alternatively, nucleic acids encoding a modified clostridial neurotoxin (or a portion thereof) comprising the modification can be synthesized.

The chimeric clostridial neurotoxin used in the present invention may comprise a polypeptide sequence having at least 70% sequence identity with a polypeptide sequence selected from SEQ ID NOs: 1-5. For example, the chimeric clostridial neurotoxin may comprise a polypeptide sequence having at least 80%, 90%, 95% or 99.9% sequence identity with a polypeptide sequence selected from SEQ ID NOs: 1-5. Preferably, the chimeric clostridial neurotoxin used in the present invention may comprise (more preferably consist of) a polypeptide sequence selected from SEQ ID NOs: 1-5. Among the chimeric clostridial neurotoxins, SEQ ID NO: 1 is preferred.

Thus, the chimeric clostridial neurotoxin preferably comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 1. More preferably, the chimeric clostridial neurotoxin may comprise a polypeptide sequence having at least 80%, 90%, 95% or 99.9% sequence identity to SEQ ID NO: 1. Most preferably, the chimeric clostridial neurotoxin for use in the present invention may comprise (more preferably consist of) SEQ ID NO: 1.

The two-chain chimeric clostridial neurotoxin of the present invention may comprise an L-chain portion of a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to any one of SEQ ID NOs: 1-5, which constitutes the first chain of the two-chain chimeric clostridial neurotoxin, and may comprise HN and HC domains of a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity _to any one of SEQ ID NOs: _1-5 , which together constitute the second chain of the two-chain chimeric clostridial neurotoxin, wherein the first chain and the second chain are linked together by a disulfide bond.

Wherein cleavage occurs at one or more positions (preferably two positions) within the activation loop of the chimeric clostridial neurotoxin, the chimeric clostridial neurotoxin comprises a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to any one of SEQ ID NOs: 1-5, and small fragments of the C-terminal L-chain portion of the sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to any one of SEQ ID NOs: 1-5 may not be present in the double-chain chimeric clostridial neurotoxin. In view of this, the sequence of a double-chain chimeric clostridial neurotoxin (e.g., comprising a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to any one of SEQ ID NOs: 1-5) may differ slightly from the corresponding single-chain chimeric clostridial neurotoxin, comprising a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to any one of SEQ ID NOs: 1-5. The small fragment may be 1-15 amino acids. In particular, in one embodiment, when Lys-C is used to convert a single-chain chimeric clostridial neurotoxin into a double-chain clostridial neurotoxin, the small fragment of the C-terminal L-chain portion of the sequence that is absent may be SEQ ID NOs: 15 or 16.

Preferably, the two-chain chimeric clostridial neurotoxin of the present invention may comprise an L-chain portion of a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to SEQ ID NO: 1, which constitutes the first chain of the two-chain chimeric clostridial neurotoxin, and may comprise HN and HC domains of a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to _SEQ ID NO: ₁ , which together constitute the second chain of the two-chain chimeric clostridial neurotoxin, wherein the first chain and the second chain are linked together by a disulfide bond.

Wherein cleavage occurs at one or more positions (preferably two positions) within the activation loop of the chimeric clostridial neurotoxin, the chimeric clostridial neurotoxin comprises a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to SEQ ID NO:1, and small fragments of the C-terminal L-chain portion of the sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to SEQ ID NO:1 may not be present in the double-chain chimeric clostridial neurotoxin. In view of this, the sequence of a double-chain chimeric clostridial neurotoxin (e.g., comprising a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to SEQ ID NO: 1) can be slightly different from the corresponding single-chain chimeric clostridial neurotoxin, which comprises a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to SEQ ID NO: 1. The small fragment can be 1-15 amino acids. In particular, in one embodiment, when Lys-C is used to convert a single-chain chimeric clostridial neurotoxin into a double-chain clostridial neurotoxin, the small fragment of the C-terminal L-chain portion of the sequence that is absent can be SEQ ID NO: 15 or 16.

In a particularly preferred embodiment, the two-chain chimeric clostridial neurotoxin comprises (or consists of) a light chain comprising a polypeptide sequence having at least 70%, 80%, 90%, 95% or 99.9% sequence identity to SEQ ID NO: 17 or 18 (preferably SEQ ID NO: 17), and a heavy chain comprising a polypeptide sequence having at least 70%, 80%, 90%, 95% or 99.9% sequence identity to SEQ ID NO: 19, wherein the light chain and the heavy chain are linked together by a disulfide bond. More preferably, the two-chain chimeric clostridial neurotoxin comprises (or consists of) a light chain comprising SEQ ID NO: 17 or 18 (preferably SEQ ID NO: 17), and a heavy chain comprising SEQ ID NO: 19, wherein the light chain and the heavy chain are linked together by a disulfide bond. Even more preferably, the two-chain chimeric clostridial neurotoxin comprises (or consists of) a light chain having SEQ ID NO: 17 and a heavy chain having SEQ ID NO: 19, wherein the light chain and the heavy chain are linked together by a disulfide bond. The disulfide bond is preferably formed by and/or between cysteine residue 429 of SEQ ID NO: 17 or 18 and cysteine residue 6 of SEQ ID NO: 19.

In preferred embodiments, the chimeric clostridial neurotoxins of the invention do not contain therapeutic or diagnostic agents (e.g., nucleic acid, protein, peptide, or small molecule therapeutic or diagnostic agents) other than the light and heavy chains. For example, in one embodiment, the chimeric clostridial neurotoxin may not contain a covalently or non-covalently bound therapeutic or diagnostic agent. Thus, the chimeric clostridial neurotoxins of the invention preferably do not serve as delivery vehicles for other therapeutic or diagnostic agents.

In embodiments, a chimeric Clostridial neurotoxin described herein has a tag (eg, a His tag) and/or a linker for purification, which tag and/or linker is optional.

The chimeric Clostridial neurotoxins of the invention may be free of complexing proteins present in naturally occurring Clostridial neurotoxin complexes.

The chimeric clostridial neurotoxins of the invention can be produced using recombinant nucleic acid technology. Thus, in one embodiment, a chimeric clostridial neurotoxin (as described herein) is a recombinant chimeric clostridial neurotoxin.

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

In a preferred embodiment, the vector has a promoter selected from:

In another preferred embodiment, the vector has a promoter selected from:

Nucleic acid molecules can be prepared using any suitable method known in the art. Therefore, chemical synthesis techniques can be used to prepare nucleic acid molecules. Alternatively, molecular biology techniques can be used to prepare nucleic acid molecules of the present invention.

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

The above nucleic acid sequence information is optionally modified for codon preference depending on the final host cell (eg, E. coli) expression system to be employed.

The terms "nucleotide sequence" and "nucleic acid" are used synonymously herein. Preferably, the nucleotide sequence is a DNA sequence.

The chimeric clostridial neurotoxins of the invention may exist as a single chain or as a double chain. However, it is preferred that the chimeric clostridial neurotoxin exists as a double chain, wherein the L-chain is linked to the H-chain (or a component thereof, such as an _HN domain) by a disulfide bond.

The production of a single-chain chimeric clostridial neurotoxin having a light chain and a heavy chain can be achieved using a method comprising the following steps: expressing a nucleic acid encoding a chimeric clostridial neurotoxin in an expression host, lysing the host cells to provide a host cell homogenate containing the single-chain chimeric clostridial neurotoxin, and isolating the single-chain chimeric clostridial neurotoxin. The single-chain chimeric clostridial neurotoxin described herein can be proteolytically processed using a method comprising contacting the single-chain chimeric clostridial neurotoxin with a protease (e.g., Lys-C) that hydrolyzes the peptide bonds in the activation loop of the chimeric clostridial neurotoxin, thereby converting the single-chain chimeric clostridial neurotoxin into a corresponding double-chain chimeric clostridial neurotoxin (e.g., wherein the light chain and the heavy chain are linked together by a disulfide bond). The double-chain chimeric clostridial neurotoxin is preferably obtainable by this method.

Thus, the chimeric clostridial neurotoxin used in the present invention is preferably a two-chain chimeric clostridial neurotoxin produced from a single-chain BoNT/A, wherein the single-chain BoNT/A comprises or consists of a polypeptide sequence as described herein. For example, preferably, the chimeric clostridial neurotoxin used in the present invention is a two-chain chimeric clostridial neurotoxin produced from a polypeptide comprising a polypeptide sequence having at least 70% (e.g., at least 80%, 90%, 95%, or 99.9%) sequence identity to SEQ ID NO: 1. Most preferably, the chimeric clostridial neurotoxin used in the present invention is a two-chain chimeric clostridial neurotoxin produced from a polypeptide comprising (even more preferably consisting of) SEQ ID NO: 1. Thus, in some embodiments, the chimeric clostridial neurotoxin is a two-chain chimeric clostridial neurotoxin, wherein the light chain (L-chain) is linked to the heavy chain (H-chain) by a disulfide bond obtainable by a method comprising contacting a single-chain chimeric clostridial neurotoxin comprising SEQ ID NO: 1 with a protease that hydrolyzes a peptide bond in its activation loop, thereby converting the single-chain chimeric clostridial neurotoxin into the corresponding two-chain chimeric clostridial neurotoxin. In some embodiments, the chimeric clostridial neurotoxin is a two-chain chimeric clostridial neurotoxin, wherein the L-chain is linked to the H-chain by a disulfide bond obtainable by a method comprising contacting a single-chain chimeric clostridial neurotoxin consisting of SEQ ID NO: 1 with a protease that hydrolyzes a peptide bond in its activation loop, thereby converting the single-chain chimeric clostridial neurotoxin into the corresponding two-chain chimeric clostridial neurotoxin.

As used herein, the term "obtainable" also encompasses the term "obtained." In one embodiment, the term "obtainable" refers to obtained.

The protease used to cleave the activation loop is preferably Lys-C. Suitable proteases and methods for cleaving the activation loop to produce a two-chain clostridial neurotoxin are taught in WO 2014/080206, WO 2014/079495, and EP2677029A2, which are incorporated herein by reference. Lys-C can cleave the C-terminus of the activation loop to one or more lysine residues present therein. When Lys-C cleaves the activation loop more than once, one skilled in the art will understand that the small peptide of the activation loop of the two-chain modified BoNT/A may not be present (preferably SEQ ID NO: 15 or 16 may not be present) when compared to the SEQ ID NOs shown herein.

As used herein, the term "one or more" may refer to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20. In one embodiment, where "one or more" precedes a list, "one or more" may refer to all members of the list. Similarly, as used herein, the term "at least one" may refer to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20. In one embodiment, where "at least one" precedes a list, "at least one" may refer to all members of the list.

As used herein, a "subject" may be a mammal, such as a human or other mammal. Preferably, a "subject" refers to a human subject.

A subject treated in accordance with the present invention may be a subject that is not suitable for treatment with a non-chimeric Clostridial neurotoxin. The subject may be a subject that is resistant to treatment with a non-chimeric Clostridial neurotoxin. Resistance may arise due to the development of an immune response in the subject to the Clostridial neurotoxin, including the production of anti-Clostridial neurotoxin antibodies. In one embodiment, a subject treated in accordance with the present invention may be a subject that is not suitable for treatment with a BoNT/A. The subject may be resistant to treatment with a BoNT/A.

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

The terms "treat" or "treating" as used herein encompass preventive treatment (e.g., preventing the onset of pain) as well as corrective treatment (e.g., treating a subject already suffering from pain). Preferably, "treat" or "treating" as used herein means corrective treatment.

In one embodiment, the chimeric clostridial neurotoxin is administered to a subject who is not experiencing symptoms of pain or a condition at the time of treatment. Such administration may be suitable for achieving a prophylactic treatment of pain or a condition as described herein. In one embodiment, the treatment of a migraine (e.g., migraine pain) may be a prophylactic treatment of a migraine. In one embodiment, a chimeric clostridial neurotoxin is administered to a subject who is not experiencing a migraine or symptoms of a migraine at the time of treatment.

As used herein, the term "treat" or "treating" refers to a condition (preferably pain) and/or its symptoms. Thus, the chimeric clostridial neurotoxins of the invention can be administered to a subject in a therapeutically effective amount or a prophylactically effective amount. Preferably, the chimeric clostridial neurotoxins of the invention are administered to a subject in a therapeutically effective amount.

A "therapeutically effective amount" is any amount of a chimeric Clostridial neurotoxin that, when administered alone or in combination (preferably alone) to a subject for treating a condition (preferably pain) (or a symptom thereof), is sufficient to effect such treatment of the condition (preferably pain) or a symptom thereof.

A "prophylactically effective amount" is any amount of a chimeric clostridial neurotoxin that, when administered to a subject alone or in combination with another agent (preferably alone), inhibits or delays the onset or recurrence of a disorder (preferably pain) (or a symptom thereof). In some embodiments, a prophylactically effective amount completely prevents the onset or recurrence of a disorder (preferably pain). "Inhibiting" an attack refers to reducing the likelihood of an attack (preferably pain) (or a symptom thereof), preventing the magnitude of the peak effect of a disorder (preferably pain), and/or completely preventing an attack.

Chimeric Clostridial neurotoxins can treat pain without treating the underlying condition causing the pain.

In addition to treating pain, the chimeric clostridial neurotoxin can also treat one or more other symptoms of the condition. In one embodiment, the chimeric clostridial neurotoxin can treat one or more other symptoms associated with neuronal secretion, such as the release of mediators described herein (e.g., pain mediators). For example, CGRP may be involved in many symptoms associated with migraine, such as photophobia. Therefore, the treatment of migraine or migraine pain according to the present invention can also treat one or more other symptoms of migraine, such as photophobia.

The chimeric clostridial neurotoxins of the invention can be formulated in any suitable manner for administration to a subject, such as as part of a pharmaceutical composition.Such a pharmaceutical composition can comprise a chimeric clostridial neurotoxin of the invention and a pharmaceutically acceptable carrier, excipient, adjuvant, propellant and/or salt.

The chimeric clostridial neurotoxins of the invention may be formulated for oral, parenteral, continuous infusion, inhalation or topical application. Compositions suitable for injection may be in the form of solutions, suspensions or emulsions or dry powders which are dissolved or suspended in a suitable vehicle before use.

In one aspect, the invention provides a unit dosage form of a chimeric clostridial neurotoxin for use in treating pain, the unit dosage form comprising:

a. 0.2 units to a maximum of 707 units of the chimeric clostridial neurotoxin, wherein 1 unit is the amount of the chimeric clostridial neurotoxin corresponding to the calculated median lethal dose (LD ₅₀ ) in mice; or

b. 5 pg to 17,000 pg of chimeric clostridial neurotoxin; and

c. Optionally, a pharmaceutically acceptable carrier, excipient, adjuvant and/or salt.

Preferably, the chimeric clostridial neurotoxin in unit dosage form comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 1. For example, a polypeptide sequence having at least 80%, 90%, 95% or 99.9% sequence identity to SEQ ID NO: 1. Most preferably, the chimeric clostridial neurotoxin may comprise (more preferably consist of) SEQ ID NO: 1.

A unit dosage form for treating pain may contain 0.2 units to a maximum of 707 units of a chimeric clostridial neurotoxin. The upper limit of the range may be 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150 or 100 units of a chimeric clostridial neurotoxin, preferably the upper limit is 666 units. The lower limit of the range may be 40, 45, 50, 60, 65, 70, 75, 80, 85, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650 or 700 units of a chimeric clostridial neurotoxin, preferably the lower limit is 42 units or 31 units. The lower limit of the range may be greater than 125 units. Preferably, the unit dosage form contains 31 units to 707 units of a chimeric clostridial neurotoxin. More preferably, the unit dosage form comprises 42 units to 666 units of the chimeric clostridial neurotoxin, for example 200 units to 400 units of the chimeric clostridial neurotoxin or 41 units to 229 units, such as 83 units to 188 units, 83 units to 125 units (e.g., 104 units) or 145 units to 188 units of the chimeric clostridial neurotoxin. Preferably, the unit dosage form comprises 166 units of the chimeric clostridial neurotoxin. The unit dosage form may comprise 47 units to 707 units of the chimeric clostridial neurotoxin, for example 187 units to 282 units of the chimeric clostridial neurotoxin, or 47 to 258 units, for example 94 units to 211 units, 94 units to 141 units (e.g., 117 units) or 164 to 211 units of the chimeric clostridial neurotoxin. The unit dosage form may comprise 188 units of the chimeric clostridial neurotoxin.

A unit dosage form for treating pain may contain 5 pg to 17,000 pg of chimeric clostridial neurotoxin. The upper limit of the range may be 16,500, 15,500, 14,500, 13,500, 12,500, 11,500, 10,500, 9,500, 8,500, 7,500, 6,500, 5,500, 4,500, 3,500, 2,500, 1,500 or 500 pg of chimeric clostridial neurotoxin, with a preferred upper limit being 16,000 pg. The lower limit of the range may be 750, 850, 950, 1000, 1500, 2000, 2,500, 3,000, 3,500, 4,000, 4,500 or 5,000 pg of chimeric clostridial neurotoxin, preferably the lower limit is 1000 pg or 750 pg. The lower limit of the range may be greater than 3,000 pg. Preferably, the unit dosage form contains 750 pg to 17,000 pg of chimeric clostridial neurotoxin. More preferably, the unit dosage form comprises 1000 pg to 16,000 pg of the chimeric clostridial neurotoxin, e.g., 4,000 pg to 6,000 pg of the chimeric clostridial neurotoxin or 1,000 to 5,500 pg, such as 2,000 pg to 4,500 pg, 2,000 pg to 3,000 pg (e.g., 2,500 pg) or 3,500 to 4,500 pg of the chimeric clostridial neurotoxin. Preferably, the unit dosage form comprises 4,000 pg of the chimeric clostridial neurotoxin.

In some embodiments, a unit dosage form for treating pain can be used to treat headache pain (e.g., migraine pain) or migraine pain, and can include:

a. 42 units to a maximum of 258 units (eg, 42 to 229 units) of a chimeric clostridial neurotoxin, wherein 1 unit is the amount of the chimeric clostridial neurotoxin corresponding to the calculated mouse median lethal dose ( _LD50 ); or

b. 1,000 pg to 5,500 pg of chimeric clostridial neurotoxin; and

c. Optionally, a pharmaceutically acceptable carrier, excipient, adjuvant and/or salt.

The potency of the chimeric clostridial neurotoxin used according to the present invention can be determined by a mouse _LD50 assay according to standard techniques. In said assay, 1 unit is defined as the amount of chimeric clostridial neurotoxin corresponding to the calculated mouse median lethal dose ( _LD50 ). Preferably, the calculated mouse median lethal intraperitoneal dose.

The amount corresponding to 1 unit of the chimeric clostridial neurotoxin in the assay may be 20-24.04 pg, such as 21.3 pg or 24.04 pg. Preferably, the amount corresponding to 1 unit of the chimeric clostridial neurotoxin in the assay may be 24.04 pg.

When referring to units herein, the units are preferably _LD50 units.

In another aspect, the present invention provides a unit dosage form for treating headache pain (e.g., migraine pain) or migraine, the unit dosage form comprising:

a. 42 units to a maximum of 258 units (eg, 42 to 229 units) of a chimeric clostridial neurotoxin, wherein 1 unit is the amount of the chimeric clostridial neurotoxin corresponding to the calculated median lethal dose ( _LD50 ) in mice; or

b. 1,000 pg to 5,500 pg of chimeric clostridial neurotoxin; and

c. Optionally, a pharmaceutically acceptable carrier, excipient, adjuvant and/or salt.

Preferably, the chimeric clostridial neurotoxin in unit dosage form comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 1. For example, a polypeptide sequence having at least 80%, 90%, 95% or 99.9% sequence identity to SEQ ID NO: 1. Most preferably, the chimeric clostridial neurotoxin may comprise (more preferably consist of) SEQ ID NO: 1.

The unit dosage form for treating headache pain (e.g., migraine pain) or migraine can be 42 units to 229 units. The upper limit of the unit dosage form can be 225, 220, 215, 210, 205, 200, 190, 180, 170, 160, 150, 125, 100 or 83 units of chimeric clostridial neurotoxin, preferably the upper limit is 212 units, more preferably 208 units. The lower limit of the unit dosage form can be 46, 50, 55, 60, 65, 70, 75, 80 or 90, 100, 110, 120, 130, 140, 150, 160 or 166 units of chimeric clostridial neurotoxin, preferably the lower limit is 58 units, more preferably 62 units. The lower limit of the range can be greater than 125 units. The unit dosage form may contain 58 units to 212 units (e.g., 62 units to 208 units), 83 units to 212 units, 125 to 212 units, or 125 to 166 units of the chimeric clostridial neurotoxin. The unit dosage form may contain greater than 125 units to a maximum of 229 units of the chimeric clostridial neurotoxin. Preferably, the unit dosage form contains 83 units to 188 units, 83 units to 125 units (e.g., 104 units), or 145 units to 188 units of the chimeric clostridial neurotoxin. Preferably, the unit dosage form contains 166 units of the chimeric clostridial neurotoxin. The unit dosage form may contain 47 units to 258 units of the chimeric clostridial neurotoxin, for example, 94 units to 211 units, 94 units to 141 units (e.g., 117 units), or 164 to 211 units of the chimeric clostridial neurotoxin. The unit dosage form may contain 188 units of the chimeric clostridial neurotoxin.

The unit dosage form for treating headache pain (e.g., migraine pain) or migraine can be 1,000 pg to 5,500 pg. The upper limit of the unit dosage form can be 5,250, 5,200, 5,100, 5,000, 4,500, 4,000, 3,500, 3,000, 2,500, or 2,000 pg of chimeric clostridial neurotoxin, preferably the upper limit is 5,100 pg, more preferably 5,000 pg. The lower limit of the unit dosage form can be 1,100, 1,200, 1,250, 1,300, 1,350, 1,400, or 1,450, 1,500, 2,000, 2,500, 3,000, 3,500, or 4,000 pg of chimeric clostridial neurotoxin, preferably the lower limit is 1,400 pg, more preferably 1,500 pg. The lower limit of the range may be greater than 3,000 pg. The unit dosage form may contain 1,400 pg to 5,100 pg, 2,000 pg to 5,100 pg, 3,000 pg to 5,100 pg, or 3,000 pg to 4,000 pg of chimeric clostridial neurotoxin. The unit dosage form may contain greater than 3,000 pg to a maximum of 5,500 pg of chimeric clostridial neurotoxin. Preferably, the unit dosage form contains 2,000 pg to 4,500 pg, 2,000 pg to 3,000 pg (e.g., 2,500 pg), or 3,500 pg to 4,500 pg of chimeric clostridial neurotoxin. Preferably, the unit dosage form contains 4,000 pg of chimeric clostridial neurotoxin.

Where the chimeric Clostridial neurotoxin is to be delivered locally, the chimeric Clostridial neurotoxin can be formulated as a cream (eg, for topical administration) or for subcutaneous injection.

Local delivery devices may include aerosols or other sprays (eg, nebulizers). In this regard, aerosol formulations of the chimeric Clostridial neurotoxin can be delivered to the lungs and/or other nasal and/or bronchial tubes or airways.

The chimeric clostridial neurotoxin can be administered to the face, neck, and/or skull of a subject. For example, the chimeric clostridial neurotoxin can be administered to the muscle and/or dermal components thereof. The chimeric clostridial neurotoxin can be administered to two or more of the face, neck, and skull, preferably to the face, neck, and skull. In particular, the chimeric clostridial neurotoxin can be administered to the face, neck, and/or skull regions of a subject.

The chimeric Clostridial neurotoxins of the invention may be administered to a subject by intrathecal or epidural injection at the level of a spinal segment involved in the innervation of the affected organ.

The route of administration may be by laparoscopy and/or local injection. In one embodiment, the chimeric clostridial neurotoxin of the present invention is administered at or near the site to be treated, preferably at the site to be treated. For example, the chimeric clostridial neurotoxin may be administered intrathecally or intraspinally. In one embodiment, the route of administration of the chimeric clostridial neurotoxin of the present invention may be intraspinal and/or intrathecal.

In one embodiment, a chimeric clostridial neurotoxin of the invention may be administered peripherally. In one embodiment, a chimeric clostridial neurotoxin may be administered subcutaneously.

The chimeric clostridial neurotoxins of the present invention can be administered by injection. The chimeric clostridial neurotoxin can be administered at least 5, 10, 15, 20, 25, or 30 injection sites during each treatment. The chimeric clostridial neurotoxin can be administered by injection at a maximum of 50, 45, 40, 35, 30, 25, or 20 injection sites during each treatment. The chimeric clostridial neurotoxin can be administered by injection at a maximum of 20 or 15 injection sites during each treatment. Preferably, the chimeric clostridial neurotoxin can be administered by injection at a maximum of 10 (e.g., a maximum of 9, 8, 7, 6, 5, 4, 3, or 2) injection sites during each treatment. In one embodiment, the chimeric clostridial neurotoxin can be administered at 1-40, 5-40, 8-38, 30-40 (e.g., 35), or 15-25 (e.g., 20) injection sites during each treatment. In one embodiment, the chimeric clostridial neurotoxin can be administered at 1-10, 3-10, 5-10, or 7-10 injection sites per treatment. In one embodiment, the chimeric clostridial neurotoxin may be administered at 25-35 (eg, 31) injection sites per treatment period. Preferably, the chimeric clostridial neurotoxin may be administered at 25-30 (eg, 28) injection sites per treatment period.

The chimeric clostridial neurotoxins of the present invention can be administered intradermally, such as by intradermal injection. The chimeric clostridial neurotoxin can be administered by intradermal injection at least 5, 10, 15, 20, 25 or 30 injection sites during each treatment. The chimeric clostridial neurotoxin can be administered by intradermal injection at a maximum of 50, 45, 40, 35, 30, 25 or 20 injection sites during each treatment. The chimeric clostridial neurotoxin can be administered by intradermal injection at a maximum of 20 or 15 injection sites during each treatment. Preferably, the chimeric clostridial neurotoxin can be administered by intradermal injection at a maximum of 10 (e.g., a maximum of 9, 8, 7, 6, 5, 4, 3 or 2) injection sites during each treatment. In one embodiment, the chimeric clostridial neurotoxin can be administered at 1-40, 5-40, 8-38, 30-40 (e.g., 35) or 15-25 (e.g., 20) injection sites during each treatment. In one embodiment, the chimeric clostridial neurotoxin can be administered at 1-10, 3-10, 5-10, or 7-10 injection sites per treatment. In one embodiment, the chimeric clostridial neurotoxin can be administered at 25-35 (e.g., 31) injection sites during each treatment. Preferably, the chimeric clostridial neurotoxin can be administered at 25-30 (e.g., 28) injection sites during each treatment. Intradermal injections can be performed in a muscle area, such as a muscle described herein. In one embodiment, intradermal injections can be performed on the skin covering the muscle.

Most preferably, the chimeric clostridial neurotoxin is administered intramuscularly, for example, by intramuscular injection. The specific muscle to which the chimeric clostridial neurotoxin is administered will depend on the nature and location of the condition (preferably pain) to be treated.

The chimeric Clostridial neurotoxin can be administered to one or more muscles of a subject selected from the group consisting of: frontalis, corrugator (e.g., corrugator supercilii), procerus (e.g., procerus nasalis), occipitalis, temporalis, trapezius, masseter, nasalis, orbicularis oculi, paraspinal cervical muscles, temporalis fascia, supraauricularis, anterior auricular, posterior auricular, sternocleidomastoid, platysma, anterior nasal dilator, posterior nasal dilator, depressor septi, mentalis, orbicularis oris, zygomaticus, risorius, buccinator, occipitofrontalis, levator labii superioris, depressor labii inferioris, depressor anguli oris, thyrohyoid, omohyoid, sternohyoid, splenius cervi, splenius capitis, semispinalis cervi, semispinalis capitis, levator scapulae, digastric, or scalene muscles.

For example, the chimeric Clostridial neurotoxin can be administered to one or more muscles of a subject selected from the group consisting of: frontalis, corrugator, procerus (e.g., procerus nasalis), occipitalis, temporalis, trapezius, masseter, nasalis, orbicularis oculi, paraspinal cervical muscles, temporalis fascia, supraauricularis, anterior auricular, posterior auricular, sternocleidomastoid, platysma, anterior nasal dilator, posterior nasal dilator, depressor septi, mentalis, orbicularis oris, zygomaticus, risorius, buccinator, occipitofrontalis, levator labii superioris, depressor labii inferioris, depressor anguli oris, thyrohyoid, omohyoid, sternohyoid, splenius, levator scapulae, digastric, and scalene muscles.

When the condition is headache pain (e.g., migraine pain) or migraine headache, the invention can comprise administering the chimeric Clostridial neurotoxin to: the frontalis, corrugator (e.g., corrugatorsupercilii) and procerus (e.g., procerus nasalis), occipitalis, temporalis, trapezius, masseter, nasalis, orbicularis oculi, paraspinal cervical muscles, temporalis fascia, supraauricularis, anterior auricular, posterior auricular, sternocleidomastoid, platysma, anterior nasal dilator, posterior nasal dilator, depressor septi, mentalis, orbicularis oris, zygomaticus, risorius, buccinator, occipitofrontalis, levator labii superioris, depressor labii inferioris, depressor anguli oris, thyrohyoid, omohyoid, sternohyoid, splenius, levator scapulae, digastric, and scalene muscles. When the condition is headache pain (e.g., migraine pain) or migraine headache, the chimeric Clostridial neurotoxin can be administered to one or more muscles of the subject selected from the group consisting of: frontalis, corrugator (e.g., corrugator supercilii), and procerus (e.g., procerus nasalis), occipitalis, temporalis, trapezius, masseter, nasalis, orbicularis oculi, paraspinal cervical muscles, temporalis fascia, superior auricular, anterior auricular, posterior auricular, sternocleidomastoid, platysma, anterior nasal dilator, posterior nasal dilator, depressor septi, mentalis, orbicularis oris, zygomaticus, risorius, buccinator, occipitofrontalis, levator labii superioris, depressor labii inferioris, depressor anguli oris, thyrohyoid, omohyoid, sternohyoid, splenius, levator scapulae, digastric, and scalene muscles. Preferably, the chimeric clostridial neurotoxin is administered to one or more of the following: procerus, corrugator supercilii, masseter, temporalis, occipitalis, and trapezius muscles. When there are two forms of the same muscle (e.g., two occipitalis muscles), the chimeric clostridial neurotoxin may be administered to one or both of the muscles, depending on the needs of the subject. Preferably, the chimeric clostridial neurotoxin is administered to both of the muscles.

The chimeric clostridial neurotoxin can be administered intramuscularly, for example, by intramuscular injection. The specific muscle to which the chimeric clostridial neurotoxin is administered will depend on the nature and location of the condition (preferably pain) to be treated. When the condition is headache pain (e.g., migraine pain) or migraine headache, the invention can comprise administering the chimeric Clostridial neurotoxin to: the frontalis, corrugator (e.g., corrugator supercilii), procerus (e.g., procerus nasalis), occipitalis, temporalis, trapezius, masseter, nasalis, orbicularis oculi, paraspinal cervical muscles, temporalis fascia, supraauricularis, anterior auricular, posterior auricular, sternocleidomastoid, platysma, anterior nasal dilator, posterior nasal dilator, depressor septi, mentalis, orbicularis oris, zygomaticus, risorius, buccinator, occipitofrontalis, levator labii superioris, depressor labii inferioris, depressor anguli oris, thyrohyoid, omohyoid, sternohyoid, splenius cervi, splenius capitis, semispinalis cervi, semispinalis capitis, levator scapulae, digastric, or scalene muscles. When the condition is headache pain (e.g., migraine pain) or migraine, the chimeric Clostridial neurotoxin can be administered to a subject in one or more muscles selected from the group consisting of: frontalis, corrugator (e.g., corrugator supercilii), procerus (e.g., procerus nasalis), The chimeric clostridial neurotoxin is preferably administered to one or more of the following: procerus, corrugator supercilii, masseter, temporalis, occipitalis, orbicularis oculi, paraspinal muscles of the neck, temporalis fascia, superior auricle, anterior auricle, posterior auricle, sternocleidomastoid, platysma, anterior nasal dilator, posterior nasal dilator, depressor septi, mentalis, orbicularis oris, zygomaticus, risorius, buccinator, occipitofrontalis, levator labii superioris, depressor labii inferioris, depressor anguli oris, thyrohyoid, omohyoid, sternohyoid, splenius cervi, splenius capitis, semispinalis cervi, semispinalis capitis, levator scapulae, digastric, and scalene muscles. Preferably, the chimeric clostridial neurotoxin is administered to one or more of the following: procerus, corrugator supercilii, masseter, temporalis, occipitalis, and trapezius. When two forms of the same muscle are present (e.g., two occipitalis muscles), the chimeric clostridial neurotoxin can be administered to one or both of the muscles, depending on the needs of the subject. Preferably, the chimeric Clostridial neurotoxin is administered to both of said muscles.

The invention may include administering a chimeric Clostridial neurotoxin to at least one of the following: the frontalis muscle, the corrugator supercilii muscle (e.g., supercilii) muscle, procerus muscle (e.g., procerus nasalis), occipital muscle (e.g., superior or inferior occipital muscle), temporalis muscle, trapezius muscle (e.g., superior, middle or inferior trapezius muscle), masseter muscle, nasalis muscle, orbicularis oculi muscle, paraspinal cervical muscle, temporalis fascia muscle, auricularis suprauricus muscle, auricularis anterior muscle, auricularis posterior muscle, sternocleidomastoid muscle, platysma muscle, anterior nasal dilator muscle, posterior nasal dilator muscle, depressor septi nasalis muscle, mentalis muscle, orbicularis oris muscle, zygomaticus muscle, risorius muscle, buccinator muscle, occipitofrontalis muscle, levator labii superioris muscle, depressor labii inferioris muscle, depressor anguli oris muscle, thyrohyoid muscle, omohyoid muscle, sternohyoid muscle, splenius cervicis muscle, splenius capitis muscle, semispinalis cervicis muscle, semispinalis capitis muscle, levator scapulae muscle, digastric muscle, or scalene muscle. Preferably, the present invention may comprise administering a chimeric clostridial neurotoxin to at least one of the following: the frontalis muscle, the corrugator (e.g., corrugator supercilii) muscle, the nasal muscle, the orbicularis oculi muscle, the temporalis muscle, the occipitalis muscle, or the trapezius muscle. More preferably, the present invention may comprise administering a chimeric clostridial neurotoxin to: the frontalis muscle, the corrugator (e.g., corrugator supercilii) muscle, the nasal muscle, the orbicularis oculi muscle, the temporalis muscle, the occipitalis muscle, and the trapezius muscle. When two forms of the same muscle are present (e.g., two occipitalis muscles), the chimeric clostridial neurotoxin may be administered to one or both of the muscles, depending on the needs of the subject. Preferably, the chimeric clostridial neurotoxin is administered to both of the muscles. Such administration may be particularly relevant in the treatment of headache pain (e.g., migraine pain) or migraine pain.

When the pain is arthritis pain, the chimeric clostridial neurotoxin can be administered to one or more muscles of the subject's hand, wrist, knee and/or foot, for example, depending on the location of the arthritis and/or arthritis pain. When administered to the subject's hand, wrist, knee or foot, administration can be unilateral (e.g., when the arthritis or arthritis pain is present in only one hand, wrist, knee and/or foot) or bilateral (e.g., when the arthritis or arthritis pain is present in both hands, both wrists, both knees and/or both feet). The chimeric clostridial neurotoxin can be administered to one or more muscles of the subject's hand, the muscles selected from: flexor pollicis brevis, palmar interosseous muscles, abductor pollicis brevis, flexor pollicis brevis, abductor pollicis, palmaris opponens, dorsal interosseous muscles, abductor digiti minimi, flexor digiti minimi and palmaris opponens (preferably selected from one or more of the following: flexor pollicis brevis, palmar interosseous muscles, abductor pollicis brevis, flexor pollicis brevis and abductor pollicis). The chimeric Clostridial neurotoxin can be administered to one or more muscles of the subject's wrist selected from the group consisting of the extensor pollicis brevis, the abductor pollicis longus, the extensor digiti minimi, the extensor carpi ulnaris, the flexor carpi ulnaris, the extensor digitorum flexor, the extensor carpi radialis, and the brachioradialis. The chimeric clostridial neurotoxin can be administered to one or more muscles of the knee of a subject, the muscles selected from the group consisting of sartorious, vastus medialis, vastus lateralis, gastrocnemius, plantaris, semimembranosus, perineum longus, gastrocnemius, tibialis anterior, rectus femoris, peroneus longus, iliopsoas, pectineus, adductor, adductor magnus, gracilis, biceps femoris, soleus, soleus, extensor digitorum longus, extensor hallucis longus, peroneus brevis, and flexor digitorum longus (preferably one or more selected from the group consisting of sartorious, vastus medialis, vastus lateralis, gastrocnemius, plantaris, semimembranosus, perineum longus, gastrocnemius, tibialis anterior, rectus femoris, and peroneus longus). The chimeric clostridial neurotoxin can be administered to one or more muscles of the foot of a subject, the muscles selected from the group consisting of extensor digitorum brevis and extensor hallucis brevis.

The chimeric clostridial neurotoxin can be administered by intramuscular injection at least 5, 10, 15, 20, 25 or 30 injection sites during each treatment. The chimeric clostridial neurotoxin can be administered by intramuscular injection at a maximum of 50, 45, 40, 35, 30, 25 or 20 injection sites during each treatment. The chimeric clostridial neurotoxin can be administered by intramuscular injection at a maximum of 20 or 15 injection sites during each treatment. Preferably, the chimeric clostridial neurotoxin can be administered by intramuscular injection at a maximum of 10 (e.g., a maximum of 9, 8, 7, 6, 5, 4, 3 or 2) injection sites during each treatment. In one embodiment, the chimeric clostridial neurotoxin can be administered at 1-40, 5-40, 8-38, 30-40 (e.g., 35) or 15-25 (e.g., 20) injection sites during each treatment. In one embodiment, the chimeric clostridial neurotoxin can be administered at 1-10, 3-10, 5-10 or 7-10 injection sites per treatment. In one embodiment, the chimeric clostridial neurotoxin may be administered at 25-35 (eg, 31) injection sites per treatment period. Preferably, the chimeric clostridial neurotoxin may be administered at 25-30 (eg, 28) injection sites per treatment period.

The chimeric Clostridial neurotoxin may be administered as a unit dose per injection (eg, per injection site).

The chimeric Clostridial neurotoxin can be administered intraneurally, perineurally, or by periganglionic administration.

The chimeric clostridial neurotoxin can be administered to the trigeminal nerve, trigeminal ganglion, sphenopalatine ganglion, semilunar ganglion, nervus intermedius, glossopharyngeal nerve, vagus nerve, otic ganglion, and/or via the occipital nerve to the upper cervical root. Preferably, the chimeric clostridial neurotoxin is administered to the trigeminal nerve, trigeminal ganglion, and/or sphenopalatine ganglion.

The chimeric clostridial neurotoxin can be administered intraarticularly.The chimeric clostridial neurotoxin can be administered intramuscularly and/or intradermally near a joint.

Chimeric Clostridial neurotoxins can be administered by perivascular administration.

The chimeric Clostridial neurotoxins of the invention are administered within a dosage range that produces the desired therapeutic and/or prophylactic effect.

Fluid dosage forms are usually prepared using chimeric clostridial neurotoxins and pyrogen-free sterile vehicles. Depending on the vehicle and concentration used, the chimeric clostridial neurotoxin can be dissolved or suspended in the vehicle. When preparing the solution, the chimeric clostridial neurotoxin can be dissolved in the vehicle, and the solution can be isotonic by adding sodium chloride if necessary, and sterilized by filtering through a sterile filter using aseptic technique, and then filled into a suitable sterile bottle or ampoule and sealed. Alternatively, if the solution stability is sufficient, the solution in the sealed container can be sterilized by autoclaving. Advantageously, additives such as buffers, solubilizers, stabilizers, preservatives or bactericides, suspending agents or emulsifiers and/or local anesthetics can be dissolved in the vehicle.

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

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

Advantageously, a suspending agent such as polyvinylpyrrolidone is included in the composition to facilitate uniform distribution of the ingredients.

Administration according to the present invention may utilize a variety of delivery techniques including microparticle encapsulation or high pressure aerosol bombardment.

Unlike conventional clostridial neurotoxins (e.g., native BoNT/A), the chimeric clostridial neurotoxins of the present invention have improved safety and/or improved activity, e.g., as demonstrated by an improved safety ratio when compared to conventional neurotoxins (see WO 2017/191315 A1 for further details). In view of this, the chimeric clostridial neurotoxins can be administered at low doses while still exhibiting therapeutic efficacy, and can be administered at high doses without causing undesirable toxicity-related side effects. Thus, the present invention provides a wide range of suitable dosage ranges for treating conditions, preferably pain.

For the convenience of the physician, the chimeric clostridial neurotoxin can be administered in a unit dose. The unit dose can be administered at a single site, or alternatively, less than a unit dose can be administered at an administration site (e.g., where there are two or more administration sites and the dose is distributed (equally or unequally) between the sites). In one embodiment, when practicing the present invention, a single unit dose can be administered to each muscle and/or neuron treated.

In one embodiment, when implementing the present invention, at least one unit dose can be administered to muscle and/or neuron. For example, when implementing the present invention, 1-20, 1-10, 1-7 or 1-5 unit doses can be administered to muscle and/or neuron.

In one embodiment, at least 0.25, 0.5, 1 or 2 unit doses may be administered per injection (e.g., per injection site). For example, 0.25, 0.5, 1 or 2 unit doses may be administered per injection (e.g., per injection site). Preferably, 1 unit dose is administered per injection (e.g., per injection site).

When a unit dose (or fraction or multiple thereof) is administered, this may refer to administration of substantially all of the unit dose (or fraction or multiple thereof). For example, a residual amount (e.g., up to 1%, 0.1%, or 0.01%) of a unit dose (or fraction or multiple thereof) may remain in the vial from which the chimeric clostridial neurotoxin was removed (e.g., in which the chimeric clostridial neurotoxin has been reconstituted). However, it is preferred (e.g., at one or more injection sites) to administer all of the unit dose (or fraction or multiple thereof).

A suitable unit dose may be 5 pg to 17,000 pg of the chimeric clostridial neurotoxin. The upper limit of the unit dose range may be 16,500, 15,500, 14,500, 13,500, 12,500, 11,500, 10,500, 9,500, 8,500, 7,500, 6,500, 5,500, 4,500, 3,500, 2,500, 1,500 or 500 pg of the chimeric clostridial neurotoxin, with a preferred upper limit being 16,000 pg. The lower limit of the unit dose range can be 10, 20, 30, 50, 100, 200, 250, 350, 450, 550, 650, 750, 850, 950, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500 or 5,000 pg of chimeric clostridial neurotoxin, preferably the lower limit is 1,000 pg or 750 pg. The lower limit of the range can be greater than 3,000 pg. Preferably, the unit dose is 750 pg to 17,000 pg of chimeric clostridial neurotoxin. The unit dose of chimeric clostridial neurotoxin can be 3,640 pg to 17,000 pg. More preferably, the unit dose of the chimeric clostridial neurotoxin is 1,000 pg to 16,000 pg of the chimeric clostridial neurotoxin, e.g., 4,000 pg to 6,000 pg of the chimeric clostridial neurotoxin or 1,000 to 5,500 pg, such as 2,000 pg to 4,500 pg, 2,000 pg to 3,000 pg (e.g., 2,500 pg) or 3,500 to 4,500 pg of the chimeric clostridial neurotoxin. Preferably, the unit dose comprises 4,000 pg of the chimeric clostridial neurotoxin.

A suitable unit dose may be 0.2 units to a maximum of 707 units of the chimeric clostridial neurotoxin. The upper limit of the range may be 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150 or 100 units of the chimeric clostridial neurotoxin, with a preferred upper limit of 666 units. The lower limit of the range may be 40, 45, 50, 60, 65, 70, 75, 80, 85, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650 or 700 units of the chimeric clostridial neurotoxin, with a preferred lower limit of 42 units or 31 units. The lower limit of the range may be greater than 125 units. Preferably, the unit dose is 31 units to 707 units of the chimeric clostridial neurotoxin. The unit dose of the chimeric clostridial neurotoxin can be 166 units to 707 units. More preferably, the unit dose is 42 units to 666 units of the chimeric clostridial neurotoxin, for example 200 units to 400 units of the chimeric clostridial neurotoxin or 41 units to 229 units such as 83 units to 188 units, 83 units to 125 units (e.g., 104 units) or 145 units to 188 units of the chimeric clostridial neurotoxin. Preferably, the unit dose is 166 units of the chimeric clostridial neurotoxin. The unit dose can be 47 units to 707 units of the chimeric clostridial neurotoxin, for example 187 units to 282 units of the chimeric clostridial neurotoxin, or 47 to 258 units, for example 94 units to 211 units, 94 units to 141 units (e.g., 117 units) or 164 to 211 units of the chimeric clostridial neurotoxin. The unit dose can be 188 units of the chimeric clostridial neurotoxin.

Suitable unit doses may be 1,000 pg to 5,500 pg. The upper limit of the unit dose range may be 5,250, 5,200, 5,100, 5,000, 4,500, 4,000, 3,500, 3,000, 2,500 or 2,000 pg of chimeric clostridial neurotoxin, preferably the upper limit is 5,100 pg, more preferably 5,000 pg. The lower limit of the unit dose range may be 1,100, 1,200, 1,250, 1,300, 1,350, 1,400 or 1,450, 1,500, 2,000, 2,500, 3,000, 3,500 or 4,000 pg of chimeric clostridial neurotoxin, preferably the lower limit is 1,400 pg, more preferably 1,500 pg. The lower limit of the range may be greater than 3,000 pg. The unit dose may be 1,400 pg to 5,100 pg (e.g., 1,500 pg to 5,000 pg), 2,000 pg to 5,100 pg, 3,000 to 5,100 pg, or 3,000 to 4,000 pg of chimeric clostridial neurotoxin. The unit dose may contain greater than 3,000 pg to a maximum of 5,500 pg of chimeric clostridial neurotoxin. The unit dose of the chimeric clostridial neurotoxin may be 2,000 pg to 4,500 pg, 2,000 pg to 3,000 pg (e.g., 2,500 pg), or 3,500 to 4,500 pg of chimeric clostridial neurotoxin. Preferably, the unit dose contains 4,000 pg of chimeric clostridial neurotoxin.

Suitable unit doses may be 42 units to 258 units (e.g., up to 229 units). The upper limit of the unit dose range may be 225, 220, 215, 210, 205, 200, 190, 180, 170, 160, 150, 125, 100, or 83 units of chimeric clostridial neurotoxin, preferably the upper limit is 212 units, more preferably 208 units. The lower limit of the unit dose range may be 46, 50, 55, 60, 65, 70, 75, 80, or 90, 100, 110, 120, 130, 140, 150, 160, or 166 units of chimeric clostridial neurotoxin, preferably the lower limit is 58 units, more preferably 62 units. The lower limit of the range may be greater than 125 units. The unit dose may be 58 units to 212 units (e.g., 62 units to 208 units), 83 units to 212 units, 125 to 212 units, or 125 to 166 units of a chimeric clostridial neurotoxin. The unit dose may contain greater than 125 units to a maximum of 229 units of a chimeric clostridial neurotoxin. The unit dose of a chimeric clostridial neurotoxin may be 83 units to 188 units, 83 units to 125 units (e.g., 104 units), or 146 units to 188 units of a chimeric clostridial neurotoxin. Preferably, the unit dose contains 166 units of a chimeric clostridial neurotoxin. The unit dose may contain 47 units to 258 units of a chimeric clostridial neurotoxin, for example, 94 units to 211 units, 94 units to 141 units (e.g., 117 units), or 164 to 211 units of a chimeric clostridial neurotoxin. The unit dose may be 188 units of a chimeric clostridial neurotoxin.

The total dose administered per treatment period may be up to 255,000 pg of the chimeric clostridial neurotoxin. This may correspond to 15× the unit dose. The total dose administered may be up to 255,000 pg of the chimeric clostridial neurotoxin and may correspond to 28×, 31×, or 39× the unit dose. In other words, the total amount of the chimeric clostridial neurotoxin administered during a given treatment period may be up to 255,000 pg. The total dose may be up to 240,000, 220,000, 200,000, 180,000, 160,000, 140,000, 110,000, 100,000, 90,000, 80,000, 70,000, 60,000, 50,000, 40,000, 30,000, 20,000, 10,000 or 5,000 pg. Preferably, the total dose may be up to 240,000 pg chimeric clostridial neurotoxin. The total dose may be at least 900, 1,000, 2,000, 3,000, 4,000, 5,000, 7,500, 10,000, 12,500, 15,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 120,000, 150,000, 175,000, 200,000 or 220,000 pg. Preferably, the total dose may be at least 1,500 pg, more preferably at least 2,000 pg of chimeric clostridial neurotoxin, more preferably greater than 3,000 pg, such as at least 12,000 pg. The total dose may be 3,640 pg to 255,000 pg of chimeric clostridial neurotoxin. The total dose may be 2,000-240,000 pg, preferably 128,000-240,000 pg. More preferably, the total dose administered is 15,000-240,000 pg. The total dose may be 75,000 pg or 115,000 pg. The total dose may be 70,000 pg or 112,000 pg.

The total dose administered during each treatment period may be up to 10,607 units of the chimeric clostridial neurotoxin. This may correspond to 15× the unit dose. The total dose administered may be up to 10,607 units of the chimeric clostridial neurotoxin and may correspond to 28×, 31×, or 39× the unit dose. In other words, the total amount of the chimeric clostridial neurotoxin administered during a given treatment period may be up to 10,607 units. The total dose may be up to 10,500, 10,000, 9,500, 9,000, 8,500, 8,000, 7,500, 7,000, 6,500, 6,000, 5,500, 5,000, 4,500, 4,000, 3,500, 3,000, 2,500, 2,000, 1,500, 1,000, 500, or 207 units. Preferably, the total dose may be up to 11,268 or 9,983 units of the chimeric clostridial neurotoxin. The total dose may be at least 37, 50, 100, 150, 200, 250, 500, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 5,000, 5,500, 6,000, 6,500, 7,000, 7,500, 8,000, 8,500, 9,000, 9,500, 9,151, 10,000 or 10,328 units. Preferably, the total dose may be at least 62 units or 70 units, more preferably at least 83 units or 94 units of the chimeric clostridial neurotoxin, more preferably greater than 125 units or 141 units, for example at least 499 units or 563 units. The total dose may be 165 units to 10,607 units or 171 units to 10,607 units of chimeric clostridial neurotoxin. The total dose may be 83-9,983 units or 94-10,607 units, preferably 5,324-9,983 units or 6,009-10,607 units. More preferably, the total dose administered is 624-9,983 units or 704-10,607 units. The total dose may be 3,120 or 4,784 units. The total dose may be 2,911 or 4,659 units. The total dose may be 3,521 units or 5,399 units. The total dose may be 3,286 units or 5,258 units.

A suitable unit dose may be 2,500 pg, and a total dose may be up to 70,000 pg. For example, a suitable unit dose may be 2,500 pg, and a total dose may be 70,000 pg. A suitable unit dose may be 4,000 pg, and a total dose may be up to 112,000 pg. For example, a suitable unit dose may be 4,000 pg, and a total dose may be 112,000 pg. A suitable unit dose may be 5,000 pg, and a total dose may be up to 155,000 pg. For example, a suitable unit dose may be 5,000 pg, and a total dose may be 155,000 pg.

A suitable unit dose may be 104 units, and a total dose may be at most 2,912 units. For example, a suitable unit dose may be 104 units, and a total dose may be 2,912 units. A suitable unit dose may be 166 units, and a total dose may be at most 4,659 units. For example, a suitable unit dose may be 166 units, and a total dose may be 4,659 units. A suitable unit dose may be 208 units, and a total dose may be at most 6,448 units. For example, a suitable unit dose may be 208 units, and a total dose may be 6,448 units.

Suitable unit doses may be 117 units, and the total dose may be at most 3,286 units. For example, suitable unit doses may be 117 units, and the total dose may be 3,286 units. Suitable unit doses may be 188 units, and the total dose may be at most 5,258 units. For example, suitable unit doses may be 188 units, and the total dose may be 5,258 units. Suitable unit doses may be 235 units, and the total dose may be at most 7,277 units. For example, suitable unit doses may be 235 units, and the total dose may be 7,277 units.

The total number of unit doses administered in a given treatment may be up to 15× unit doses. For example, the total number of unit doses administered may be up to 14×, 13×, 12×, 11×, 10×, 9×, 8×, or 7×. The total number of unit doses administered may be at least 2×, 3×, 4×, 5×, 6×, 7×, preferably at least 2× of the unit doses. The total number of unit doses administered may be from 2× to 15×, from 7× to 15×, or from 10× to 14×. Preferably, the number of unit doses administered is 15×.

The total number of unit doses administered in a given treatment may be up to 39× unit doses (as long as the total dose administered during the treatment does not exceed the upper limit of 255,000 pg or 10,607 units). For example, the total number of unit doses administered may be up to 35×, 31×, 30×, 29×, 28×, 27×, 26×, 25×, or 20× unit doses, preferably the total number of unit doses administered is up to 28× unit doses. The total number of unit doses administered may be at least 2×, 3×, 4×, 5×, 6×, 7×, preferably at least 2× of the unit doses. The total number of unit doses administered may be from 2× to 39×, from 15× to 31×, or from 28× to 31×. The total number of unit doses administered may be 28×, 31×, or 39×.

Thus, the total dose administered per treatment period can be up to 192,500 pg of the chimeric clostridial neurotoxin. For example, the total dose administered can be up to 180,000 pg, or up to 177,000 pg (eg, up to 175,000 pg).

Thus, the total dose administered during each treatment period can be up to 8,007 units of the chimeric clostridial neurotoxin. For example, the total dose administered can be up to 7,488 units, or up to 7,363 units (e.g., up to 7,280 units). The total dose administered during each treatment period can be up to 9,037 units of the chimeric clostridial neurotoxin. For example, the total dose administered can be up to 8,451 units, or up to 8,310 units (e.g., up to 8,216 units).

The total dose administered per treatment period may be up to 110,000 pg of the chimeric clostridial neurotoxin. For example, the total dose administered may be up to 105,000 pg, up to 102,000 pg (eg, up to 100,000 pg).

The total dose administered during each treatment period may be up to 4,576 units of the chimeric clostridial neurotoxin. For example, the total dose administered may be up to 4,368 units, preferably up to 4,243 units (more preferably up to 4,160 units). The total dose administered during each treatment period may be up to 5,165 units of the chimeric clostridial neurotoxin. For example, the total dose administered may be up to 4,929 units, preferably up to 4,789 units (more preferably up to 4,695 units).

The term "up to" when used in reference to a numerical value (e.g., up to 255,000 pg) means up to and including the numerical value. Thus, as an example, reference to administering "up to 255,000 pg" of a chimeric Clostridial neurotoxin encompasses administering 255,000 pg of the chimeric Clostridial neurotoxin as well as administering less than 255,000 pg of the chimeric Clostridial neurotoxin.

When the condition is headache pain (e.g., migraine pain) or migraine, at least a unit dose of the chimeric clostridial neurotoxin can be applied to one or more of the frontalis, corrugator, nasal, orbicularis, temporalis, occipitalis, and trapezius muscles. Preferably, at least a unit dose of the chimeric clostridial neurotoxin can be applied to the frontalis, corrugator, nasal, orbicularis, temporalis, occipitalis, and trapezius muscles. In some embodiments, multiple unit doses are applied to one or more of the following: frontalis, corrugator, nasal, orbicularis, temporalis, occipitalis, and trapezius muscles. In one embodiment, a single unit dose is applied to the corrugator, nasal, and orbicularis muscles, and multiple unit doses are applied to the frontalis, temporalis, occipitalis, and trapezius muscles. Multiple unit doses can be 2-10 unit doses, such as 2-8 unit doses, preferably 2-5 unit doses, such as 2-4 unit doses.

More preferably, the method comprises administering the chimeric clostridial neurotoxin to at least one of the following: the frontalis muscle, the corrugator (e.g., corrugator supercilii) muscle, the nasal muscle, the orbicularis oculi muscle, the temporalis muscle, the occipitalis muscle, or the trapezius muscle. The method may comprise administering the chimeric clostridial neurotoxin to: the frontalis muscle, the corrugator (e.g., corrugator supercilii) muscle, the nasal muscle, the orbicularis oculi muscle, the temporalis muscle, the occipitalis muscle, and the trapezius muscle.

Headache pain (e.g., migraine pain) or migraine treatment may include administration of:

(i) 2 unit doses to the frontalis muscle (preferably 2 unit doses per frontalis muscle);

(ii) 1 unit dose to the corrugator supercilii muscle (preferably 1 unit dose per corrugator supercilii muscle);

(iii) 1 unit dose to the nasalis muscle (preferably 1 unit dose per nasalis muscle);

(iv) 1 unit dose to the orbicularis oculi muscle (preferably 1 unit dose per orbicularis oculi muscle);

(v) 4 unit doses to the temporalis muscle (preferably 4 unit doses per temporalis muscle);

(vi) 3 unit doses to the occipitalis muscle (preferably 3 unit doses per occipitalis muscle); and/or

(vii) 2 unit doses to the trapezius muscle (preferably 2 unit doses per trapezius muscle)

Headache pain (e.g., migraine pain) or migraine treatment may include administration of:

(i) 2 unit doses to the frontalis muscle (preferably 2 unit doses per frontalis muscle);

(ii) 1 unit dose to the corrugator supercilii muscle (preferably 1 unit dose per corrugator supercilii muscle);

(iii) 1 unit dose to the nasalis muscle (preferably 1 unit dose per nasalis muscle);

(iv) 1 unit dose to the orbicularis oculi muscle (preferably 1 unit dose per orbicularis oculi muscle);

(v) 4 unit doses to the temporalis muscle (preferably 4 unit doses per temporalis muscle);

(vi) 3 unit doses to the occipitalis muscle (preferably 3 unit doses per occipitalis muscle); and

(vii) 2 unit doses to the trapezius muscle (preferably 2 unit doses per trapezius muscle).

Treatment of headache pain (e.g., migraine pain) or migraine pain may include bilateral administration of a unit dose of a chimeric Clostridial neurotoxin. Treatment of headache pain (e.g., migraine pain) or migraine pain may include administration of:

(i) 4 unit doses to the frontalis muscle (preferably 2 unit doses to the frontalis muscle on the first side of the face, and 2 unit doses to the frontalis muscle on the second side of the face);

(ii) 2 unit doses to the corrugator supercilii muscle (preferably 1 unit dose to the corrugator supercilii muscle on a first side of the face and 1 unit dose to the corrugator supercilii muscle on a second side of the face);

(iii) 2 unit doses to the nasalis muscle (preferably 1 unit dose to the nasalis muscle on a first side of the face and 1 unit dose to the nasalis muscle on a second side of the face);

(iv) 2 unit doses to the orbicularis oculi muscle (preferably 1 unit dose to the orbicularis oculi muscle on a first side of the face and 1 unit dose to the orbicularis oculi muscle on a second side of the face);

(v) 8 unit doses to the temporalis muscle (preferably 4 unit doses to the temporalis muscle on a first side of the head and 4 unit doses to the temporalis muscle on a second side of the head);

(vi) 6 unit doses to the occipitalis muscle (preferably 3 unit doses to the occipitalis muscle on a first side of the head and 3 unit doses to the occipitalis muscle on a second side of the head); and/or

(vii) 4 unit doses into the trapezius muscle (preferably 2 unit doses into the trapezius muscle on the first side of the neck, and 2 unit doses into the trapezius muscle on the second side of the neck).

Preferably, headache pain (e.g. migraine pain) or migraine treatment comprises administering:

(i) 4 unit doses to the frontalis muscle (preferably 2 unit doses to the frontalis muscle on the first side of the face, and 2 unit doses to the frontalis muscle on the second side of the face);

(ii) 2 unit doses to the corrugator supercilii muscle (preferably 1 unit dose to the corrugator supercilii muscle on a first side of the face and 1 unit dose to the corrugator supercilii muscle on a second side of the face);

(iii) 2 unit doses to the nasalis muscle (preferably 1 unit dose to the nasalis muscle on a first side of the face and 1 unit dose to the nasalis muscle on a second side of the face);

(iv) 2 unit doses to the orbicularis oculi muscle (preferably 1 unit dose to the orbicularis oculi muscle on a first side of the face and 1 unit dose to the orbicularis oculi muscle on a second side of the face);

(v) 8 unit doses to the temporalis muscle (preferably 4 unit doses to the temporalis muscle on a first side of the head and 4 unit doses to the temporalis muscle on a second side of the head);

(vi) 6 unit doses to the occipitalis muscle (preferably 3 unit doses to the occipitalis muscle on a first side of the head and 3 unit doses to the occipitalis muscle on a second side of the head); and

(vii) 4 unit doses into the trapezius muscle (preferably 2 unit doses into the trapezius muscle on the first side of the neck, and 2 unit doses into the trapezius muscle on the second side of the neck).

When treating headache (e.g., migraine pain) or migraine as described in the foregoing embodiments, preferably one unit dose is administered per injection (e.g., injection site). Thus, administration of a chimeric Clostridial neurotoxin may include:

(i) 2 injections into the frontalis muscle (preferably 2 injections into each frontalis muscle);

(ii) 1 injection into the corrugator supercilii muscle (preferably 1 injection into each corrugator supercilii muscle);

(iii) 1 injection into the nasalis muscle (preferably 1 injection into each nasal muscle);

(iv) 1 injection into the orbicularis oculi muscle (preferably 1 injection into each orbicularis oculi muscle);

(v) 4 injections into the temporalis muscle (preferably 4 injections into each temporalis muscle);

(vi) 3 injections into the occipitalis muscle (preferably 3 injections into each occipitalis muscle); and/or

(vii) Inject twice into the trapezius muscle (preferably 2 injections into each trapezius muscle).

Administration of a chimeric Clostridial neurotoxin may include:

(i) 2 injections into the frontalis muscle (preferably 2 injections into each frontalis muscle);

(ii) 1 injection into the corrugator supercilii muscle (preferably 1 injection into each corrugator supercilii muscle);

(iii) 1 injection into the nasalis muscle (preferably 1 injection into each nasal muscle);

(iv) 1 injection into the orbicularis oculi muscle (preferably 1 injection into each orbicularis oculi muscle);

(v) 4 injections into the temporalis muscle (preferably 4 injections into each temporalis muscle);

(vi) 3 injections into the occipitalis muscle (preferably 3 injections into each occipitalis muscle); and

(vii) Inject twice into the trapezius muscle (preferably 2 injections into each trapezius muscle).

Administration of a chimeric Clostridial neurotoxin may include:

(i) 4 injections into the frontalis muscle (preferably 2 injections into the frontalis muscle on a first side of the face and 2 injections into the frontalis muscle on a second side of the face);

(ii) 2 injections into the corrugator supercilii muscle (preferably 1 injection into the corrugator supercilii muscle on a first side of the face and 1 injection into the corrugator supercilii muscle on a second side of the face);

(iii) 2 injections into the nasalis muscle (preferably 1 injection into the nasalis muscle on a first side of the face and 1 injection into the nasalis muscle on a second side of the face);

(iv) 2 injections into the orbicularis oculi muscle (preferably 1 injection into the orbicularis oculi muscle on a first side of the face and 1 injection into the orbicularis oculi muscle on a second side of the face);

(v) 8 injections into the temporalis muscle (preferably 4 injections into the temporalis muscle on the first side of the head and 4 injections into the temporalis muscle on the second side of the head);

(vi) 6 injections into the occipitalis muscle (preferably 3 injections into the occipitalis muscle on a first side of the head and 3 injections into the occipitalis muscle on a second side of the head); and/or

(vii) 4 injections into the trapezius muscle (preferably 2 injections into the trapezius muscle on the first side of the neck and 2 injections into the trapezius muscle on the second side of the neck).

Preferably, administration of the chimeric Clostridial neurotoxin comprises:

(i) 4 injections into the frontalis muscle (preferably 2 injections into the frontalis muscle on a first side of the face and 2 injections into the frontalis muscle on a second side of the face);

(ii) 2 injections into the corrugator supercilii muscle (preferably 1 injection into the corrugator supercilii muscle on a first side of the face and 1 injection into the corrugator supercilii muscle on a second side of the face);

(iii) 2 injections into the nasalis muscle (preferably 1 injection into the nasalis muscle on a first side of the face and 1 injection into the nasalis muscle on a second side of the face);

(iv) 2 injections into the orbicularis oculi muscle (preferably 1 injection into the orbicularis oculi muscle on a first side of the face and 1 injection into the orbicularis oculi muscle on a second side of the face);

(v) 8 injections into the temporalis muscle (preferably 4 injections into the temporalis muscle on the first side of the head and 4 injections into the temporalis muscle on the second side of the head);

(vi) 6 injections into the occipitalis muscle (preferably 3 injections into the occipitalis muscle on a first side of the head and 3 injections into the occipitalis muscle on a second side of the head); and

(vii) 4 injections into the trapezius muscle (preferably 2 injections into the trapezius muscle on the first side of the neck and 2 injections into the trapezius muscle on the second side of the neck).

When the condition is headache pain (e.g., migraine pain) or migraine, at least a unit dose of the chimeric clostridial neurotoxin can be administered intramuscularly or intradermally to one or more of the frontalis, procerus, corrugator, temporalis, occipitalis, trapezius, and paraspinal muscles of the cervical spine. Preferably, at least a unit dose of the chimeric clostridial neurotoxin can be administered intramuscularly or intradermally to the frontalis, procerus, corrugator, temporalis, occipitalis, trapezius, and paraspinal muscles of the cervical spine (e.g., at least a unit dose is administered to each paraspinal muscle of the cervical spine).

When the condition is headache pain (e.g., migraine pain) or migraine, at least a unit dose of the chimeric clostridial neurotoxin can be administered to one or more of the frontalis, procerus, corrugator, temporalis, occipitalis, trapezius, and paraspinal muscles. Preferably, at least a unit dose of the chimeric clostridial neurotoxin can be administered to the frontalis, procerus, corrugator, temporalis, occipitalis, trapezius, and paraspinal muscles (e.g., at least a unit dose is administered to each paraspinal muscle). In one embodiment:

(i) administering a single unit dose to one or more of the following: the procerus muscle; and the corrugator supercilii muscle (preferably administering a single unit dose to the corrugator supercilii muscle on a first side of the face (e.g., the left side) and a second unit dose to the corrugator supercilii muscle on a second side of the face (e.g., the right side); and/or (preferably and)

(iii) administering multiple unit doses to one or more of the following: frontalis muscle; temporalis muscle; occipitalis muscle; trapezius muscle; and paraspinal muscles of the cervical spine (e.g., wherein a single or double unit dose is administered to each muscle of the paraspinal muscles of the cervical spine). The multiple unit doses may be 2-8 unit doses, e.g., 2-5 unit doses.

Treatment of headache pain (e.g., migraine pain) or migraine pain may include bilateral intramuscular or intradermal administration of a unit dose of a chimeric Clostridial neurotoxin. Treatment of headache pain (e.g., migraine pain) or migraine pain may include administration of:

(i) 2 unit doses to the frontalis muscle on the first side of the face, and/or 2 unit doses to the frontalis muscle on the second side of the face;

(ii) 1 unit dose to the procerus muscle;

(iii) 1 unit dose to the corrugator supercilii muscle on the first side of the face, and/or 1 unit dose to the corrugator supercilii muscle on the second side of the face;

(iv) 4 unit doses to the temporalis muscle on the first side of the head, and/or 4 unit doses to the temporalis muscle on the second side of the head;

(v) 3 unit doses to the occipitalis muscle on the first side of the neck/head (preferably the head), and/or 3 unit doses to the occipitalis muscle on the second side of the neck/head (preferably the head);

(vi) 3 unit doses to the temporalis muscle on the first side of the neck, and/or 3 unit doses to the temporalis muscle on the second side of the face; and/or

(vii) 4 unit doses to the cervical paraspinal muscles on a first side of the neck, and/or 4 unit doses to the cervical paraspinal muscles on a second side of the neck (e.g., wherein 1 unit dose is administered to each cervical paraspinal muscle group muscle); or 2 unit doses to the cervical paraspinal muscles on a first side of the neck, and/or 2 unit doses to the cervical paraspinal muscles on a second side of the neck (e.g., wherein 2 unit doses are administered to each cervical paraspinal muscle group muscle).

Headache pain (e.g., migraine pain) or migraine treatment may include administration of:

(i) 2 unit doses to the frontalis muscle on the first side of the face, and 2 unit doses to the frontalis muscle on the second side of the face;

(ii) 1 unit dose to the procerus muscle;

(iii) 1 unit dose to the corrugator supercilii muscle on the first side of the face, and 1 unit dose to the corrugator supercilii muscle on the second side of the face; (iv) 4 unit doses to the temporalis muscle on the first side of the head, and 4 unit doses to the temporalis muscle on the second side of the head;

(v) 3 unit doses to the occipitalis muscle on the first side of the neck/head (preferably the head), and 3 unit doses to the occipitalis muscle on the second side of the neck/head (preferably the head);

(vi) 3 unit doses to the temporalis muscle on the first side of the neck and 3 unit doses to the temporalis muscle on the second side of the face; and/or

(vii) 4 unit doses to the cervical paraspinal muscles on a first side of the neck, and 4 unit doses to the cervical paraspinal muscles on a second side of the neck (e.g., wherein 1 unit dose is administered to each cervical paraspinal muscle group muscle); or 2 unit doses to the cervical paraspinal muscles on a first side of the neck, and 2 unit doses to the cervical paraspinal muscles on a second side of the neck (e.g., wherein 2 unit doses are administered to each cervical paraspinal muscle group muscle).

Headache pain (e.g., migraine pain) or migraine treatment may include administration of:

(i) 2 unit doses to the frontalis muscle on the first side of the face, and 2 unit doses to the frontalis muscle on the second side of the face;

(ii) 1 unit dose to the procerus muscle;

(iii) 1 unit dose to the corrugator supercilii muscle on the first side of the face, and 1 unit dose to the corrugator supercilii muscle on the second side of the face;

(iv) 4 unit doses to the temporalis muscle on the first side of the head, and 4 unit doses to the temporalis muscle on the second side of the head;

(v) 3 unit doses to the occipitalis muscle on the first side of the neck/head (preferably the head), and 3 unit doses to the occipitalis muscle on the second side of the neck/head (preferably the head);

(vi) 3 unit doses to the temporalis muscle on the first side of the neck and 3 unit doses to the temporalis muscle on the second side of the face; and

(vii) 4 unit doses to the cervical paraspinal muscles on a first side of the neck, and 4 unit doses to the cervical paraspinal muscles on a second side of the neck (e.g., wherein 1 unit dose is administered to each cervical paraspinal muscle group muscle); or 2 unit doses to the cervical paraspinal muscles on a first side of the neck, and 2 unit doses to the cervical paraspinal muscles on a second side of the neck (e.g., wherein 2 unit doses are administered to each cervical paraspinal muscle group muscle).

Headache pain (e.g., migraine pain) or migraine treatment may include administration of:

(i) 1 unit dose to the frontalis muscle (preferably 1 unit dose per frontalis muscle);

(ii) 1 unit dose to the procerus muscle (preferably 1 unit dose per procerus muscle);

(iii) 1 unit dose to the corrugator supercilii muscle (preferably 1 unit dose per corrugator supercilii muscle);

(iv) 4 unit doses to the temporalis muscle (preferably 4 unit doses to each temporalis muscle);

(v) 3 unit doses to the occipitalis muscle (preferably 3 unit doses per occipitalis muscle);

(vi) 3 unit doses to the trapezius muscle (preferably 3 unit doses per trapezius muscle); and/or

(vii) 4 unit doses to the cervical paraspinal muscle groups (preferably 4 unit doses to each cervical paraspinal muscle group), or 2 unit doses to the cervical paraspinal muscle groups (preferably 2 unit doses to each cervical paraspinal muscle group).

Headache pain (e.g., migraine pain) or migraine treatment may include administration of:

(i) 1 unit dose to the frontalis muscle (preferably 1 unit dose per frontalis muscle);

(ii) 1 unit dose to the procerus muscle (preferably 1 unit dose per procerus muscle);

(iii) 1 unit dose to the corrugator supercilii muscle (preferably 1 unit dose per corrugator supercilii muscle);

(iv) 4 unit doses to the temporalis muscle (preferably 4 unit doses to each temporalis muscle);

(v) 3 unit doses to the occipitalis muscle (preferably 3 unit doses per occipitalis muscle);

(vi) 3 unit doses to the trapezius muscle (preferably 3 unit doses per trapezius muscle); and

(vii) 4 unit doses to the cervical paraspinal muscle groups (preferably 4 unit doses to each cervical paraspinal muscle group), or 2 unit doses to the cervical paraspinal muscle groups (preferably 2 unit doses to each cervical paraspinal muscle group).

Headache pain (e.g., migraine pain) or migraine treatment may include administration of:

(i) 4 unit doses to the frontalis muscle (preferably 2 unit doses to the frontalis muscle on the first side of the face, and 2 unit doses to the frontalis muscle on the second side of the face);

(ii) 1 unit dose to the procerus muscle;

(iii) 2 unit doses to the corrugator supercilii muscle (preferably 1 unit dose to the corrugator supercilii muscle on a first side of the face and 1 unit dose to the corrugator supercilii muscle on a second side of the face);

(iv) 8 unit doses to the temporalis muscle (preferably 4 unit doses to the temporalis muscle on the first side of the head and 4 unit doses to the temporalis muscle on the second side of the head);

(v) 6 unit doses to the occipitalis muscle (preferably 3 unit doses to the occipitalis muscle on a first side of the head and 3 unit doses to the occipitalis muscle on a second side of the head);

(vi) 6 unit doses to the trapezius muscle (preferably 3 unit doses to the trapezius muscle on the first side of the neck and 3 unit doses to the trapezius muscle on the second side of the neck); and/or

(vii) 8 unit doses to the cervical paraspinal muscles (preferably 4 unit doses to the cervical paraspinal muscles on the first side of the neck, and 4 unit doses to the cervical paraspinal muscles on the second side of the neck (e.g., wherein 1 unit dose is administered to each cervical paraspinal muscle group muscle)); or 4 unit doses to the cervical paraspinal muscles (preferably 2 unit doses to the cervical paraspinal muscles on the first side of the neck, and 2 unit doses to the cervical paraspinal muscles on the second side of the neck (e.g., wherein 2 unit doses are administered to each cervical paraspinal muscle group muscle)).

Headache pain (e.g., migraine pain) or migraine treatment may include administration of:

(i) 4 unit doses to the frontalis muscle (preferably 2 unit doses to the frontalis muscle on the first side of the face, and 2 unit doses to the frontalis muscle on the second side of the face);

(ii) 1 unit dose to the procerus muscle;

(iii) 2 unit doses to the corrugator supercilii muscle (preferably 1 unit dose to the corrugator supercilii muscle on a first side of the face and 1 unit dose to the corrugator supercilii muscle on a second side of the face);

(iv) 8 unit doses to the temporalis muscle (preferably 4 unit doses to the temporalis muscle on the first side of the head and 4 unit doses to the temporalis muscle on the second side of the head);

(v) 6 unit doses to the occipitalis muscle (preferably 3 unit doses to the occipitalis muscle on a first side of the head and 3 unit doses to the occipitalis muscle on a second side of the head);

(vi) 6 unit doses to the trapezius muscle (preferably 3 unit doses to the trapezius muscle on the first side of the neck, and 3 unit doses to the trapezius muscle on the second side of the neck); and

(vii) 8 unit doses to the cervical paraspinal muscles (preferably 4 unit doses to the cervical paraspinal muscles on the first side of the neck, and 4 unit doses to the cervical paraspinal muscles on the second side of the neck (e.g., wherein 1 unit dose is administered to each cervical paraspinal muscle group muscle)); or 4 unit doses to the cervical paraspinal muscles (preferably 2 unit doses to the cervical paraspinal muscles on the first side of the neck, and 2 unit doses to the cervical paraspinal muscles on the second side of the neck (e.g., wherein 2 unit doses are administered to each cervical paraspinal muscle group muscle)).

When treating headache pain (e.g., migraine pain) or migraine as described in the foregoing embodiments, preferably one unit dose is administered per injection site. Thus, the treatment may comprise administering the chimeric clostridial neurotoxin to:

(i) 2 injection sites in the frontalis muscle on the first side of the face and/or 2 injection sites in the frontalis muscle on the second side of the face;

(ii) 1 injection site in the procerus muscle;

(iii) 1 injection site in the corrugator supercilii muscle on the first side of the face and/or 1 injection site in the corrugator supercilii muscle on the second side of the face;

(iv) 4 injection sites in the temporalis muscle on the first side of the head and/or 4 injection sites in the temporalis muscle on the second side of the head;

(v) 3 injection sites in the occipitalis muscle on the first side of the neck/head (preferably the head) and/or 3 injection sites in the occipitalis muscle on the second side of the neck/head (preferably the head);

(vi) 3 injection sites in the trapezius muscle on the first side of the neck and/or 3 injection sites in the trapezius muscle on the second side of the neck; and/or

(vii) 4 injection sites in the cervical paraspinal muscles on a first side of the neck and/or 4 injection sites in the cervical paraspinal muscles on a second side of the neck (e.g., wherein there is 1 injection site for each cervical paraspinal muscle), or 2 injection sites in the cervical paraspinal muscles on a first side of the neck and/or 2 injection sites in the cervical paraspinal muscles on a second side of the neck (e.g., wherein there are 2 injection sites for each cervical paraspinal muscle).

The treatment may comprise administering a chimeric Clostridial neurotoxin to:

(i) 2 injection sites in the frontalis muscle on the first side of the face and 2 injection sites in the frontalis muscle on the second side of the face;

(ii) 1 injection site in the procerus muscle;

(iii) 1 injection site in the corrugator supercilii muscle on the first side of the face and 1 injection site in the corrugator supercilii muscle on the second side of the face;

(iv) 4 injection sites in the temporalis muscle on the first side of the head and 4 injection sites in the temporalis muscle on the second side of the head;

(v) 3 injection sites in the occipitalis muscle on the first side of the neck/head (preferably the head) and 3 injection sites in the occipitalis muscle on the second side of the neck/head (preferably the head);

(vi) 3 injection sites in the trapezius muscle on the first side of the neck and 3 injection sites in the trapezius muscle on the second side of the neck; and/or

(vii) 4 injection sites in the cervical paraspinal muscles on a first side of the neck and 4 injection sites in the cervical paraspinal muscles on a second side of the neck (e.g., wherein there is 1 injection site for each cervical paraspinal muscle), or 2 injection sites in the cervical paraspinal muscles on a first side of the neck and 2 injection sites in the cervical paraspinal muscles on a second side of the neck (e.g., wherein there are 2 injection sites for each cervical paraspinal muscle).

The treatment may comprise administering a chimeric Clostridial neurotoxin to:

(i) 2 injection sites in the frontalis muscle on the first side of the face and 2 injection sites in the frontalis muscle on the second side of the face;

(ii) 1 injection site in the procerus muscle;

(iii) 1 injection site in the corrugator supercilii muscle on the first side of the face and 1 injection site in the corrugator supercilii muscle on the second side of the face;

(iv) 4 injection sites in the temporalis muscle on the first side of the head and 4 injection sites in the temporalis muscle on the second side of the head;

(v) 3 injection sites in the occipitalis muscle on the first side of the neck/head (preferably the head) and 3 injection sites in the occipitalis muscle on the second side of the neck/head (preferably the head);

(vi) 3 injection sites in the trapezius muscle on the first side of the neck and 3 injection sites in the trapezius muscle on the second side of the neck; and

(vii) 4 injection sites in the cervical paraspinal muscles on a first side of the neck and 4 injection sites in the cervical paraspinal muscles on a second side of the neck (e.g., wherein there is 1 injection site for each cervical paraspinal muscle), or 2 injection sites in the cervical paraspinal muscles on a first side of the neck and 2 injection sites in the cervical paraspinal muscles on a second side of the neck (e.g., wherein there are 2 injection sites for each cervical paraspinal muscle).

Administration of a chimeric Clostridial neurotoxin may include:

(i) 2 injections into the frontalis muscle (preferably 2 injections into each frontalis muscle);

(ii) 1 injection into the procerus muscle (preferably 1 injection into each procerus muscle);

(iii) 1 injection into the corrugator supercilii muscle (preferably 1 injection into each corrugator supercilii muscle);

(iv) 4 injections into the temporalis muscle (preferably 4 injections into each temporalis muscle);

(v) 3 injections into the occipitalis muscle (preferably 3 injections into each occipitalis muscle);

(vi) 3 injections into the trapezius muscle (preferably 3 injections into each trapezius muscle); and/or

(vii) Inject 4 times into the cervical paraspinal muscles (preferably 2 injections into each cervical paraspinal muscle group).

Administration of a chimeric Clostridial neurotoxin may include:

(i) 2 injections into the frontalis muscle (preferably 2 injections into each frontalis muscle);

(ii) 1 injection into the procerus muscle (preferably 1 injection into each procerus muscle);

(iii) 1 injection into the corrugator supercilii muscle (preferably 1 injection into each corrugator supercilii muscle);

(iv) 4 injections into the temporalis muscle (preferably 4 injections into each temporalis muscle);

(v) 3 injections into the occipitalis muscle (preferably 3 injections into each occipitalis muscle);

(vi) 3 injections into the trapezius muscle (preferably 3 injections into each trapezius muscle); and

(vii) Inject 4 times into the cervical paraspinal muscles (preferably 2 injections into each cervical paraspinal muscle group).

Administration of a chimeric Clostridial neurotoxin may include:

(i) 4 injections into the frontalis muscle (preferably 2 injections into the frontalis muscle on the first side of the face and 2 injections into the frontalis muscle on the second side of the face);

(ii) 1 injection into the procerus muscle;

(iii) 2 injections into the corrugator supercilii muscle (preferably 1 injection into the corrugator supercilii muscle on a first side of the face and 1 injection into the corrugator supercilii muscle on a second side of the face);

(iv) 8 injections into the temporalis muscle (preferably 4 injections into the temporalis muscle on the first side of the head and 4 injections into the temporalis muscle on the second side of the head);

(v) 6 injections into the occipitalis muscle (preferably 3 injections into the occipitalis muscle on the first side of the head and 3 injections into the occipitalis muscle on the second side of the head);

(vi) 6 injections into the trapezius muscle (preferably 3 injections into the trapezius muscle on the first side of the neck and 3 injections into the trapezius muscle on the second side of the neck); and/or

(vii) 8 injections into the cervical paraspinal muscles (preferably 4 injections into the cervical paraspinal muscles on the first side of the neck, and 4 injections into the cervical paraspinal muscles on the second side of the neck (e.g. 1 intramuscular injection into each cervical paraspinal muscle group)); or 4 injections into the cervical paraspinal muscles (preferably 2 injections into the cervical paraspinal muscles on the first side of the neck, and 2 injections into the cervical paraspinal muscles on the second side of the neck (e.g. 2 intramuscular injections into each cervical paraspinal muscle group)).

Administration of a chimeric Clostridial neurotoxin may include:

(i) 4 injections into the frontalis muscle (preferably 2 injections into the frontalis muscle on the first side of the face and 2 injections into the frontalis muscle on the second side of the face);

(ii) 1 injection into the procerus muscle;

(iii) 2 injections into the corrugator supercilii muscle (preferably 1 injection into the corrugator supercilii muscle on a first side of the face and 1 injection into the corrugator supercilii muscle on a second side of the face);

(iv) 8 injections into the temporalis muscle (preferably 4 injections into the temporalis muscle on the first side of the head and 4 injections into the temporalis muscle on the second side of the head);

(v) 6 injections into the occipitalis muscle (preferably 3 injections into the occipitalis muscle on the first side of the head and 3 injections into the occipitalis muscle on the second side of the head);

(vi) 6 injections into the trapezius muscle (preferably 3 injections into the trapezius muscle on the first side of the neck and 3 injections into the trapezius muscle on the second side of the neck); and

(vii) 8 injections into the cervical paraspinal muscles (preferably 4 injections into the cervical paraspinal muscles on the first side of the neck, and 4 injections into the cervical paraspinal muscles on the second side of the neck (e.g., each cervical paraspinal muscle has 1 injection site)); or 4 injections into the cervical paraspinal muscles (preferably 2 injections into the cervical paraspinal muscles on the first side of the neck, and 2 injections into the cervical paraspinal muscles on the second side of the neck (e.g., 2 intramuscular injections into each cervical paraspinal muscle)).

Headache pain (e.g., migraine pain) or migraine treatment may include administration of:

(i) 2 unit doses to the frontalis muscle (preferably 2 unit doses per frontalis muscle);

(ii) 1 unit dose to the procerus muscle;

(iii) 1 unit dose to the corrugator supercilii muscle (preferably 1 unit dose per corrugator supercilii muscle);

(iv) 5 unit doses to the temporalis muscle (preferably 5 unit doses per temporalis muscle);

(v) 4 unit doses to the occipitalis muscle (preferably 4 unit doses per occipitalis muscle);

(vi) 5 unit doses to the trapezius muscle (preferably 5 unit doses per trapezius muscle); and/or

(vii) 4 unit doses to the cervical paraspinal muscle groups (preferably 4 unit doses to each cervical paraspinal muscle group), or 2 unit doses to the cervical paraspinal muscle groups (preferably 2 unit doses to each cervical paraspinal muscle group).

Headache pain (e.g., migraine pain) or migraine treatment may include administration of:

(i) 2 unit doses to the frontalis muscle (preferably 2 unit doses per frontalis muscle);

(ii) 1 unit dose to the procerus muscle (preferably 1 unit dose per procerus muscle);

(iii) 1 unit dose to the corrugator supercilii muscle (preferably 1 unit dose per corrugator supercilii muscle);

(iv) 5 unit doses to the temporalis muscle (preferably 5 unit doses per temporalis muscle);

(v) 4 unit doses to the occipitalis muscle (preferably 4 unit doses per occipitalis muscle);

(vi) 5 unit doses to the trapezius muscle (preferably 5 unit doses per trapezius muscle); and

(vii) 4 unit doses to the cervical paraspinal muscle groups (preferably 4 unit doses to each cervical paraspinal muscle group), or 2 unit doses to the cervical paraspinal muscle groups (preferably 2 unit doses to each cervical paraspinal muscle group).

Headache pain (e.g., migraine pain) or migraine treatment may include administration of:

(i) 4 unit doses to the frontalis muscle (preferably 2 unit doses to the frontalis muscle on the first side of the face, and 2 unit doses to the frontalis muscle on the second side of the face);

(ii) 1 unit dose to the procerus muscle;

(iii) 2 unit doses to the corrugator supercilii muscle (preferably 1 unit dose to the corrugator supercilii muscle on a first side of the face and 1 unit dose to the corrugator supercilii muscle on a second side of the face);

(iv) 10 unit doses to the temporalis muscle (preferably 5 unit doses to the temporalis muscle on the first side of the head and 5 unit doses to the temporalis muscle on the second side of the head);

(v) 8 unit doses to the occipitalis muscle (preferably 4 unit doses to the occipitalis muscle on a first side of the head and 4 unit doses to the occipitalis muscle on a second side of the head);

(vi) 10 unit doses to the trapezius muscle (preferably 4 unit doses to the trapezius muscle on the first side of the neck and 4 unit doses to the trapezius muscle on the second side of the neck); and/or

(vii) 4 unit doses to the cervical paraspinal muscles (eg, wherein 1 or 2 unit doses are administered to each cervical paraspinal muscle).

Headache pain (e.g., migraine pain) or migraine treatment may include administration of:

(i) 4 unit doses to the frontalis muscle (preferably 2 unit doses to the frontalis muscle on the first side of the face, and 2 unit doses to the frontalis muscle on the second side of the face);

(ii) 1 unit dose to the procerus muscle;

(iii) 2 unit doses to the corrugator supercilii muscle (preferably 1 unit dose to the corrugator supercilii muscle on a first side of the face and 1 unit dose to the corrugator supercilii muscle on a second side of the face);

(iv) 10 unit doses to the temporalis muscle (preferably 5 unit doses to the temporalis muscle on the first side of the head and 5 unit doses to the temporalis muscle on the second side of the head);

(v) 8 unit doses to the occipitalis muscle (preferably 4 unit doses to the occipitalis muscle on a first side of the head and 4 unit doses to the occipitalis muscle on a second side of the head);

(vi) 10 unit doses to the trapezius muscle (preferably 5 unit doses to the trapezius muscle on the first side of the neck and 5 unit doses to the trapezius muscle on the second side of the neck); and

(vii) 4 unit doses to the cervical paraspinal muscles (eg, wherein 1 or 2 unit doses are administered to each cervical paraspinal muscle).

When treating headache pain (e.g., migraine pain) or migraine pain, administration of the chimeric Clostridial neurotoxin may include:

(i) 2 injections into the frontalis muscle (preferably 2 injections into each frontalis muscle);

(ii) 1 injection into the procerus muscle;

(iii) 1 injection into the corrugator supercilii muscle (preferably 1 injection into each corrugator supercilii muscle);

(iv) 5 injections into the temporalis muscle (preferably 5 injections into each temporalis muscle);

(v) 4 injections into the occipitalis muscle (preferably 4 injections into each occipitalis muscle);

(vi) 5 injections into the trapezius muscle (preferably 5 injections into each trapezius muscle); and/or

(vii) 4 injections into the cervical paraspinal muscles (eg, 1 or 2 intramuscular injections into each cervical paraspinal muscle group).

Administration may include:

(i) 2 injections into the frontalis muscle (preferably 2 injections into each frontalis muscle);

(ii) 1 injection into the procerus muscle;

(iii) 1 injection into the corrugator supercilii muscle (preferably 1 injection into each corrugator supercilii muscle);

(iv) 5 injections into the temporalis muscle (preferably 5 injections into each temporalis muscle);

(v) 4 injections into the occipitalis muscle (preferably 4 injections into each occipitalis muscle);

(vi) 5 injections into the trapezius muscle (preferably 5 injections into each trapezius muscle); and

(vii) 4 injections into the cervical paraspinal muscles (eg, 1 or 2 intramuscular injections into each cervical paraspinal muscle group).

Administration may include:

(i) 4 injections into the frontalis muscle (preferably 2 injections into the frontalis muscle on a first side of the face and 2 injections into the frontalis muscle on a second side of the face);

(ii) 1 injection into the procerus muscle;

(iii) 2 injections into the corrugator supercilii muscle (preferably 1 injection into the corrugator supercilii muscle on a first side of the face and 1 injection into the corrugator supercilii muscle on a second side of the face);

(iv) 10 injections into the temporalis muscle (preferably 5 injections into the temporalis muscle on the first side of the head and 5 injections into the temporalis muscle on the second side of the head);

(v) 8 injections into the occipitalis muscles (preferably 4 injections into the occipitalis muscles on a first side of the head and 4 injections into the occipitalis muscles on a second side of the head);

(vi) 10 injections into the trapezius muscle (preferably 4 injections into the trapezius muscle on the first side of the neck and 4 injections into the trapezius muscle on the second side of the neck); and/or

(vii) 4 injections into the cervical paraspinal muscles (eg, 1 or 2 intramuscular injections into each cervical paraspinal muscle group).

Administration may include:

(i) 4 injections into the frontalis muscle (preferably 2 injections into the frontalis muscle on a first side of the face and 2 injections into the frontalis muscle on a second side of the face);

(ii) 1 injection into the procerus muscle;

(iii) 2 injections into the corrugator supercilii muscle (preferably 1 injection into the corrugator supercilii muscle on a first side of the face and 1 injection into the corrugator supercilii muscle on a second side of the face);

(iv) 10 injections into the temporalis muscle (preferably 5 injections into the temporalis muscle on the first side of the head and 5 injections into the temporalis muscle on the second side of the head);

(v) 8 injections into the occipitalis muscles (preferably 4 injections into the occipitalis muscles on a first side of the head and 4 injections into the occipitalis muscles on a second side of the head);

(vi) 10 injections into the trapezius muscle (preferably 4 injections into the trapezius muscle on the first side of the neck and 4 injections into the trapezius muscle on the second side of the neck); and

(vii) 4 injections into the cervical paraspinal muscles (eg, 1 or 2 intramuscular injections into each cervical paraspinal muscle group).

When the condition is headache pain (e.g., migraine pain) or migraine, at least a unit dose of the chimeric clostridial neurotoxin can be administered to one or more of the frontalis, corrugator, nasal, orbicularis, masseter, temporalis, occipitalis, and trapezius muscles. In one embodiment, at least a unit dose of the chimeric clostridial neurotoxin can be administered to the frontalis, corrugator, nasal, orbicularis, masseter, temporalis, occipitalis, and trapezius muscles. In one embodiment:

(i) administering a single unit dose to one or more of the following: the procerus muscle; the corrugator supercilii muscle (preferably a single unit dose is administered to the corrugator supercilii muscle on a first side of the face (e.g., the left side) and a second unit dose is administered to the corrugator supercilii muscle on a second side of the face (e.g., the right side)); the orbicularis oculi muscle; the masseter muscle; and/or (preferably and) the upper trapezius muscle;

(ii) administering half of the unit dose to the nasal muscles; and/or (preferably and)

(iii) administering a plurality of unit doses to one or more of the following: the temporalis muscle; the occipitalis muscle; and/or (preferably and) the lower trapezius muscle. The plurality of unit doses may be 2-6 unit doses, for example 2-5 unit doses.

Headache pain (e.g., migraine pain) or migraine treatment may include administration of:

(i) 1 unit dose to the frontalis muscle (preferably 1 unit dose per frontalis muscle);

(ii) 1 unit dose to the corrugator supercilii muscle (preferably 1 unit dose per corrugator supercilii muscle);

(iii) 0.5 unit dose to the nasalis muscle (preferably 0.5 unit dose to each nasalis muscle);

(iv) 1 unit dose to the orbicularis oculi muscle (preferably 1 unit dose per orbicularis oculi muscle);

(v) 1 unit dose to the masseter muscle (preferably 1 unit dose per masseter muscle);

(vi) 6 unit doses to the temporalis muscle (preferably 6 unit doses per temporalis muscle);

(vii) 6 unit doses to the occipitalis muscle (preferably 6 unit doses per occipitalis muscle);

(viii) 1 unit dose to the upper trapezius muscle (preferably 1 unit dose per upper trapezius muscle); and/or

(ix) 2 unit doses to the lower trapezius muscle (preferably 2 unit doses to each lower trapezius muscle);

Headache pain (e.g., migraine pain) or migraine treatment may include administration of:

(i) 1 unit dose to the frontalis muscle (preferably 1 unit dose per frontalis muscle);

(ii) 1 unit dose to the corrugator supercilii muscle (preferably 1 unit dose per corrugator supercilii muscle);

(iii) 0.5 unit dose to the nasalis muscle (preferably 0.5 unit dose to each nasalis muscle);

(iv) 1 unit dose to the orbicularis oculi muscle (preferably 1 unit dose per orbicularis oculi muscle);

(v) 1 unit dose to the masseter muscle (preferably 1 unit dose per masseter muscle);

(vi) 6 unit doses to the temporalis muscle (preferably 6 unit doses per temporalis muscle);

(vii) 6 unit doses to the occipitalis muscle (preferably 6 unit doses per occipitalis muscle);

(viii) 1 unit dose to the upper trapezius muscle (preferably 1 unit dose per upper trapezius muscle); and

(ix) 2 unit doses to the lower trapezius muscle (preferably 2 unit doses to each lower trapezius muscle);

Headache pain (e.g., migraine pain) or migraine treatment may include administration of:

(i) 2 unit doses to the frontalis muscle (preferably 1 unit dose to the frontalis muscle on the first side of the face, and 1 unit dose to the frontalis muscle on the second side of the face);

(ii) 2 unit doses to the corrugator supercilii muscle (preferably 1 unit dose to the corrugator supercilii muscle on a first side of the face and 1 unit dose to the corrugator supercilii muscle on a second side of the face);

(iii) 1 unit dose to the nasalis muscle (preferably 0.5 unit dose to the nasalis muscle on a first side of the face, and 0.5 unit dose to the nasalis muscle on a second side of the face);

(iv) 2 unit doses to the orbicularis oculi muscle (preferably 1 unit dose to the orbicularis oculi muscle on a first side of the face and 1 unit dose to the orbicularis oculi muscle on a second side of the face);

(v) 2 unit doses to the masseter muscle (preferably 1 unit dose to the masseter muscle on the first side of the face, and 1 unit dose to the frontalis muscle on the second side of the face);

(vi) 12 unit doses to the temporalis muscle (preferably 6 unit doses to the temporalis muscle on the first side of the head and 6 unit doses to the temporalis muscle on the second side of the head);

(vii) 12 unit doses to the occipitalis muscle (preferably 6 unit doses to the occipitalis muscle on a first side of the head and 6 unit doses to the occipitalis muscle on a second side of the head);

(viii) 2 unit doses to the upper trapezius muscle (preferably 1 unit dose to the upper trapezius muscle on a first side of the neck and 1 unit dose to the upper trapezius muscle on a second side of the neck); and/or

(ix) 4 unit doses into the lower trapezius muscle (preferably 2 unit doses into the lower trapezius muscle on the first side of the neck, and/or 2 unit doses into the lower trapezius muscle on the second side of the neck).

Headache pain (e.g., migraine pain) or migraine treatment may include administration of:

(i) 2 unit doses to the frontalis muscle (preferably 1 unit dose to the frontalis muscle on the first side of the face, and 1 unit dose to the frontalis muscle on the second side of the face);

(ii) 2 unit doses to the corrugator supercilii muscle (preferably 1 unit dose to the corrugator supercilii muscle on a first side of the face and 1 unit dose to the corrugator supercilii muscle on a second side of the face);

(iii) 1 unit dose to the nasalis muscle (preferably 0.5 unit dose to the nasalis muscle on a first side of the face, and 0.5 unit dose to the nasalis muscle on a second side of the face);

(iv) 2 unit doses to the orbicularis oculi muscle (preferably 1 unit dose to the orbicularis oculi muscle on a first side of the face and 1 unit dose to the orbicularis oculi muscle on a second side of the face);

(v) 2 unit doses to the masseter muscle (preferably 1 unit dose to the masseter muscle on the first side of the face, and 1 unit dose to the frontalis muscle on the second side of the face);

(vi) 12 unit doses to the temporalis muscle (preferably 6 unit doses to the temporalis muscle on the first side of the head and 6 unit doses to the temporalis muscle on the second side of the head);

(vii) 12 unit doses to the occipitalis muscle (preferably 6 unit doses to the occipitalis muscle on a first side of the head and 6 unit doses to the occipitalis muscle on a second side of the head);

(viii) 2 unit doses to the upper trapezius muscle (preferably 1 unit dose to the upper trapezius muscle on a first side of the neck and 1 unit dose to the upper trapezius muscle on a second side of the neck); and

(ix) 4 unit doses into the lower trapezius muscle (preferably 2 unit doses into the lower trapezius muscle on the first side of the neck, and/or 2 unit doses into the lower trapezius muscle on the second side of the neck).

Administration of a chimeric Clostridial neurotoxin may include:

(i) 1 injection into the frontalis muscle (preferably 1 injection into each frontalis muscle);

(ii) 1 injection into the corrugator supercilii muscle (preferably 1 injection into each corrugator supercilii muscle);

(iii) 1 injection into the nasalis muscle (preferably 1 injection into each nasal muscle);

(iv) 1 injection into the orbicularis oculi muscle (preferably 1 injection into each orbicularis oculi muscle);

(v) 1 injection into the masseter muscle (preferably 1 injection into each masseter muscle);

(vi) 3 injections into the temporalis muscle (preferably 3 injections into each temporalis muscle);

(vii) 3 injections into the occipitalis muscle (preferably 3 injections into each occipitalis muscle);

(viii) 1 injection into the upper trapezius muscle (preferably 1 injection into each upper trapezius muscle); and/or

(ix) 1 injection into the lower trapezius muscle (preferably 1 injection into each lower trapezius muscle);

Administration of a chimeric Clostridial neurotoxin may include:

(i) 1 injection into the frontalis muscle (preferably 1 injection into each frontalis muscle);

(ii) 1 injection into the corrugator supercilii muscle (preferably 1 injection into each corrugator supercilii muscle);

(iii) 1 injection into the nasalis muscle (preferably 1 injection into each nasal muscle);

(iv) 1 injection into the orbicularis oculi muscle (preferably 1 injection into each orbicularis oculi muscle);

(v) 1 injection into the masseter muscle (preferably 1 injection into each masseter muscle);

(vi) 3 injections into the temporalis muscle (preferably 3 injections into each temporalis muscle);

(vii) 3 injections into the occipitalis muscle (preferably 3 injections into each occipitalis muscle);

(viii) 1 injection into the upper trapezius muscle (preferably 1 injection into each upper trapezius muscle); and

(ix) 1 injection into the lower trapezius muscle (preferably 1 injection into each lower trapezius muscle);

Administration of a chimeric Clostridial neurotoxin may include:

(i) 2 injections into the frontalis muscle (preferably 1 injection into the frontalis muscle on a first side of the face and 1 injection into the frontalis muscle on a second side of the face);

(ii) 2 injections into the corrugator supercilii muscle (preferably 1 injection into the corrugator supercilii muscle on a first side of the face and 1 injection into the corrugator supercilii muscle on a second side of the face);

(iii) 2 injections into the nasalis muscle (preferably 1 injection into the nasalis muscle on a first side of the face and 1 injection into the nasalis muscle on a second side of the face);

(iv) 2 injections into the orbicularis oculi muscle (preferably 1 injection into the orbicularis oculi muscle on a first side of the face and 1 injection into the orbicularis oculi muscle on a second side of the face);

(v) 2 injections into the masseter muscle (preferably 1 injection into the masseter muscle on a first side of the face and 1 injection into the masseter muscle on a second side of the face);

(vi) 6 injections into the temporalis muscle (preferably 3 injections into the temporalis muscle on the first side of the head and 3 injections into the temporalis muscle on the second side of the head);

(vii) 6 injections into the occipitalis muscle (preferably 3 injections into the occipitalis muscle on the first side of the head and 3 injections into the occipitalis muscle on the second side of the head);

(viii) 2 injections into the upper trapezius muscle (preferably 1 injection into the upper trapezius muscle on a first side of the neck and 1 injection into the upper trapezius muscle on a second side of the neck); and/or

(ix) 2 injections into the lower trapezius muscle (preferably 1 injection into the lower trapezius muscle on the first side of the neck and 1 injection into the lower trapezius muscle on the second side of the neck).

Administration of a chimeric Clostridial neurotoxin may include:

(i) 2 injections into the frontalis muscle (preferably 1 injection into the frontalis muscle on a first side of the face and 1 injection into the frontalis muscle on a second side of the face);

(ii) 2 injections into the corrugator supercilii muscle (preferably 1 injection into the corrugator supercilii muscle on a first side of the face and 1 injection into the corrugator supercilii muscle on a second side of the face);

(iii) 2 injections into the nasalis muscle (preferably 1 injection into the nasalis muscle on a first side of the face and 1 injection into the nasalis muscle on a second side of the face);

(iv) 2 injections into the orbicularis oculi muscle (preferably 1 injection into the orbicularis oculi muscle on a first side of the face and 1 injection into the orbicularis oculi muscle on a second side of the face);

(v) 2 injections into the masseter muscle (preferably 1 injection into the masseter muscle on a first side of the face and 1 injection into the masseter muscle on a second side of the face);

(vi) 6 injections into the temporalis muscle (preferably 3 injections into the temporalis muscle on the first side of the head and 3 injections into the temporalis muscle on the second side of the head);

(vii) 6 injections into the occipitalis muscle (preferably 3 injections into the occipitalis muscle on the first side of the head and 3 injections into the occipitalis muscle on the second side of the head);

(viii) 2 injections into the upper trapezius muscle (preferably 1 injection into the upper trapezius muscle on a first side of the neck and 1 injection into the upper trapezius muscle on a second side of the neck); and

(ix) 2 injections into the lower trapezius muscle (preferably 1 injection into the lower trapezius muscle on the first side of the neck and 1 injection into the lower trapezius muscle on the second side of the neck).

When the condition is headache pain (e.g., migraine pain) or migraine, at least a unit dose of the chimeric clostridial neurotoxin can be administered to one or more of the frontalis, corrugator, nasal, orbicularis oculi, masseter, temporalis, superior occipital, inferior occipital, and upper and lower trapezius muscles. At least a unit dose of the chimeric clostridial neurotoxin can be administered to the frontalis, corrugator, nasal, orbicularis oculi, masseter, temporalis, superior occipital, inferior occipital, and upper and lower trapezius muscles. In one embodiment:

(i) administering a single unit dose to one or more of the following: the procerus muscle; the corrugator supercilii muscle (preferably administering a single unit dose to the corrugator supercilii muscle on a first side of the face (e.g., the left side) and a second unit dose to the corrugator supercilii muscle on a second side of the face (e.g., the right side)); the nasalis muscle; the orbicularis oculi muscle; the masseter muscle; and the occipitalis muscle; and/or (preferably and)

(iii) administering a plurality of unit doses to one or more of the following: the temporalis muscle; the occipitalis muscle; and the trapezius muscle. The plurality of unit doses may be 2-8 unit doses, such as 2-5 unit doses.

Headache pain (e.g., migraine pain) or migraine treatment may include administration of:

(i) 1 unit dose to the frontalis muscle (preferably 1 unit dose per frontalis muscle);

(ii) 1 unit dose to the corrugator supercilii muscle (preferably 1 unit dose per corrugator supercilii muscle);

(iii) 1 unit dose to the nasalis muscle (preferably 1 unit dose per nasalis muscle);

(iv) 1 unit dose to the orbicularis oculi muscle (preferably 1 unit dose per orbicularis oculi muscle);

(v) 1 unit dose to the masseter muscle (preferably 1 unit dose per masseter muscle);

(vi) 4 unit doses to the temporalis muscle (preferably 4 unit doses per temporalis muscle);

(vii) 2 unit doses to the occipitalis muscle (preferably 2 unit doses to each occipitalis muscle);

(viii) 1 unit dose to the occipitalis muscle (preferably 1 unit dose per occipitalis muscle); and/or

(viii) 2 unit doses to the trapezius muscle (preferably 2 unit doses per trapezius muscle).

Headache pain (e.g., migraine pain) or migraine treatment may include administration of:

(i) 1 unit dose to the frontalis muscle (preferably 1 unit dose per frontalis muscle);

(ii) 1 unit dose to the corrugator supercilii muscle (preferably 1 unit dose per corrugator supercilii muscle);

(iii) 1 unit dose to the nasalis muscle (preferably 1 unit dose per nasalis muscle);

(iv) 1 unit dose to the orbicularis oculi muscle (preferably 1 unit dose per orbicularis oculi muscle);

(v) 1 unit dose to the masseter muscle (preferably 1 unit dose per masseter muscle);

(vi) 4 unit doses to the temporalis muscle (preferably 4 unit doses per temporalis muscle);

(vii) 2 unit doses to the occipitalis muscle (preferably 2 unit doses to each occipitalis muscle);

(viii) 1 unit dose to the suboccipital muscle (preferably 1 unit dose per suboccipital muscle); and

(viii) 2 unit doses to the trapezius muscle (preferably 2 unit doses per trapezius muscle).

Headache pain (e.g., migraine pain) or migraine treatment may include administration of:

(i) 2 unit doses to the frontalis muscle (preferably 1 unit dose to the frontalis muscle on the first side of the face, and 1 unit dose to the frontalis muscle on the second side of the face);

(ii) 2 unit doses to the corrugator supercilii muscle (preferably 1 unit dose to the corrugator supercilii muscle on a first side of the face and 1 unit dose to the corrugator supercilii muscle on a second side of the face);

(iii) 2 unit doses to the nasalis muscle (preferably 1 unit dose to the nasalis muscle on a first side of the face and 1 unit dose to the nasalis muscle on a second side of the face);

(iv) 2 unit doses to the orbicularis oculi muscle (preferably 1 unit dose to the orbicularis oculi muscle on a first side of the face and 1 unit dose to the orbicularis oculi muscle on a second side of the face);

(v) 2 unit doses to the masseter muscle (preferably 1 unit dose to the masseter muscle on the first side of the face, and 1 unit dose to the frontalis muscle on the second side of the face);

(vi) 8 unit doses to the temporalis muscle (preferably 4 unit doses to the temporalis muscle on the first side of the head and 4 unit doses to the temporalis muscle on the second side of the head);

(vii) 4 unit doses to the occipitalis muscle (preferably 2 unit doses to the occipitalis muscle on a first side of the head and 2 unit doses to the occipitalis muscle on a second side of the head);

(viii) 2 unit doses to the suboccipitalis muscle (preferably 1 unit dose to the suboccipitalis muscle on a first side of the head and 1 unit dose to the suboccipitalis muscle on a second side of the head); and/or

(ix) 4 unit doses into the trapezius muscle (preferably 2 unit doses into the trapezius muscle on the first side of the neck, and 2 unit doses into the trapezius muscle on the second side of the neck).

Headache pain (e.g., migraine pain) or migraine treatment may include administration of:

(i) 2 unit doses to the frontalis muscle (preferably 1 unit dose to the frontalis muscle on the first side of the face, and 1 unit dose to the frontalis muscle on the second side of the face);

(ii) 2 unit doses to the corrugator supercilii muscle (preferably 1 unit dose to the corrugator supercilii muscle on a first side of the face and 1 unit dose to the corrugator supercilii muscle on a second side of the face);

(iii) 2 unit doses to the nasalis muscle (preferably 1 unit dose to the nasalis muscle on a first side of the face and 1 unit dose to the nasalis muscle on a second side of the face);

(iv) 2 unit doses to the orbicularis oculi muscle (preferably 1 unit dose to the orbicularis oculi muscle on a first side of the face and 1 unit dose to the orbicularis oculi muscle on a second side of the face);

(v) 2 unit doses to the masseter muscle (preferably 1 unit dose to the masseter muscle on the first side of the face, and 1 unit dose to the frontalis muscle on the second side of the face);

(vi) 8 unit doses to the temporalis muscle (preferably 4 unit doses to the temporalis muscle on the first side of the head and 4 unit doses to the temporalis muscle on the second side of the head);

(vii) 4 unit doses to the occipitalis muscle (preferably 2 unit doses to the occipitalis muscle on a first side of the head and 2 unit doses to the occipitalis muscle on a second side of the head);

(viii) 2 unit doses to the suboccipital muscle (preferably 1 unit dose to the suboccipital muscle on a first side of the head and 1 unit dose to the suboccipital muscle on a second side of the head); and

(ix) 4 unit doses into the trapezius muscle (preferably 2 unit doses into the trapezius muscle on the first side of the neck, and 2 unit doses into the trapezius muscle on the second side of the neck).

Administration of a chimeric Clostridial neurotoxin may include:

(i) 1 injection into the frontalis muscle (preferably 1 injection into each frontalis muscle);

(ii) 1 injection into the corrugator supercilii muscle (preferably 1 injection into each corrugator supercilii muscle);

(iii) 1 injection into the nasalis muscle (preferably 1 injection into each nasal muscle);

(iv) 1 injection into the orbicularis oculi muscle (preferably 1 injection into each orbicularis oculi muscle);

(v) 1 injection into the masseter muscle (preferably 1 injection into each masseter muscle);

(vi) 4 injections into the temporalis muscle (preferably 4 injections into each temporalis muscle);

(vii) 2 injections into the occipitalis muscle (preferably 2 injections into each occipitalis muscle);

(viii) 1 injection into the occipitalis muscle (preferably 1 injection into each occipitalis muscle); and/or

(viii) Inject twice into the trapezius muscle (preferably 2 injections into each trapezius muscle).

Administration of a chimeric Clostridial neurotoxin may include:

(i) 1 injection into the frontalis muscle (preferably 1 injection into each frontalis muscle);

(ii) 1 injection into the corrugator supercilii muscle (preferably 1 injection into each corrugator supercilii muscle);

(iii) 1 injection into the nasalis muscle (preferably 1 injection into each nasal muscle);

(iv) 1 injection into the orbicularis oculi muscle (preferably 1 injection into each orbicularis oculi muscle);

(v) 1 injection into the masseter muscle (preferably 1 injection into each masseter muscle);

(vi) 4 injections into the temporalis muscle (preferably 4 injections into each temporalis muscle);

(vii) 2 injections into the occipitalis muscle (preferably 2 injections into each occipitalis muscle);

(viii) 1 injection into the occipital muscle (preferably 1 injection into each occipital muscle); and

(viii) Inject twice into the trapezius muscle (preferably 2 injections into each trapezius muscle).

Administration of the chimeric Clostridial neurotoxin may include:

(i) 2 injections into the frontalis muscle (preferably 1 injection into the frontalis muscle on a first side of the face and 1 injection into the frontalis muscle on a second side of the face);

(ii) 2 injections into the corrugator supercilii muscle (preferably 1 injection into the corrugator supercilii muscle on a first side of the face and 1 injection into the corrugator supercilii muscle on a second side of the face);

(iii) 2 injections into the nasalis muscle (preferably 1 injection into the nasalis muscle on a first side of the face and 1 injection into the nasalis muscle on a second side of the face);

(iv) 2 injections into the orbicularis oculi muscle (preferably 1 injection into the orbicularis oculi muscle on a first side of the face and 1 injection into the orbicularis oculi muscle on a second side of the face);

(v) 2 injections into the masseter muscle (preferably 1 injection into the masseter muscle on a first side of the face and 1 injection into the masseter muscle on a second side of the face);

(vi) 8 injections into the temporalis muscle (preferably 4 injections into the temporalis muscle on the first side of the head and 4 injections into the temporalis muscle on the second side of the head);

(vii) 4 injections into the occipitalis muscle (preferably 2 injections into the occipitalis muscle on the first side of the head and 2 injections into the occipitalis muscle on the second side of the head);

(viii) 2 injections into the occipitalis muscle (preferably 1 injection into the occipitalis muscle on a first side of the head and 1 injection into the occipitalis muscle on a second side of the head); and/or

(ix) 4 injections into the trapezius muscle (preferably 2 injections into the trapezius muscle on the first side of the neck and 2 injections into the trapezius muscle on the second side of the neck).

Administration of a chimeric Clostridial neurotoxin may include:

(i) 2 injections into the frontalis muscle (preferably 1 injection into the frontalis muscle on a first side of the face and 1 injection into the frontalis muscle on a second side of the face);

(ii) 2 injections into the corrugator supercilii muscle (preferably 1 injection into the corrugator supercilii muscle on a first side of the face and 1 injection into the corrugator supercilii muscle on a second side of the face);

(iii) 2 injections into the nasalis muscle (preferably 1 injection into the nasalis muscle on a first side of the face and 1 injection into the nasalis muscle on a second side of the face);

(iv) 2 injections into the orbicularis oculi muscle (preferably 1 injection into the orbicularis oculi muscle on a first side of the face and 1 injection into the orbicularis oculi muscle on a second side of the face);

(v) 2 injections into the masseter muscle (preferably 1 injection into the masseter muscle on a first side of the face and 1 injection into the masseter muscle on a second side of the face);

(vi) 8 injections into the temporalis muscle (preferably 4 injections into the temporalis muscle on the first side of the head and 4 injections into the temporalis muscle on the second side of the head);

(vii) 4 injections into the occipitalis muscle (preferably 2 injections into the occipitalis muscle on the first side of the head and 2 injections into the occipitalis muscle on the second side of the head);

(viii) 2 injections into the occipitalis muscle (preferably 1 injection into the occipitalis muscle on a first side of the head and 1 injection into the occipitalis muscle on a second side of the head); and

(ix) 4 injections into the trapezius muscle (preferably 2 injections into the trapezius muscle on the first side of the neck and 2 injections into the trapezius muscle on the second side of the neck).

In any aspect or embodiment described herein, administration of the chimeric clostridial neurotoxin can include injecting the chimeric clostridial neurotoxin directly into a muscle or injecting it indirectly into a muscle. For example, when the injection of the chimeric clostridial neurotoxin into a muscle is an indirect injection into a muscle, the chimeric clostridial neurotoxin can be administered to the muscle region. In one embodiment, when the injection of the chimeric clostridial neurotoxin into a muscle is a direct injection into a muscle, the chimeric clostridial neurotoxin can be administered intramuscularly to the muscle. In one embodiment, when the injection of the chimeric clostridial neurotoxin into a muscle is an indirect injection into a muscle, the chimeric clostridial neurotoxin can be administered intradermally.

When the condition is a headache (e.g., migraine pain) or a migraine headache, at least a unit dose of the chimeric clostridial neurotoxin can be administered intradermally to one or more of the following: the trigeminal ocular region; the trigeminal maxillary region; the trigeminal mandibular region; and the back of the head. Preferably, at least a unit dose of the chimeric clostridial neurotoxin can be administered intradermally to: the trigeminal ocular region; the trigeminal maxillary region; the trigeminal mandibular region; and the back of the head.

Intradermal administration in one or more of the described areas can target the chimeric clostridial neurotoxin to a target trigeminal nerve (e.g., a target nerve ending). The target nerve (e.g., a target nerve ending) of the trigeminal nerve in the ocular region can be one or more of the following: the supraorbital nerve; the supratrochlear nerve; and the intratrochlear nerve (e.g., a nerve ending thereof). The target nerve (e.g., a target nerve ending) of the trigeminal nerve in the maxillary region can be one or more of the following: the zygomaticotemporal nerve and the zygomaticofacial nerve (e.g., a nerve ending thereof). The target nerve (e.g., a target nerve ending) of the trigeminal nerve in the mandibular region can be the auriculotemporal nerve (e.g., a nerve ending thereof). The target nerve (e.g., a target nerve ending) of the back of the head can be one or more of the following: the greater occipital nerve and the lesser occipital nerve (e.g., a nerve ending thereof).

Intradermal administration in one or more of the described areas can target the chimeric clostridial neurotoxin to a target trigeminal nerve (e.g., a target nerve ending). The target nerve (e.g., a target nerve ending) of the trigeminal nerve ocular region can be one or more of the following: the supraorbital nerve; the supratrochlear nerve; and the intratrochlear nerve (e.g., a nerve ending thereof). The target nerve (e.g., a target nerve ending) of the trigeminal nerve maxillary region can be one or more of the following: the zygomaticotemporal nerve; and the intraorbital nerve (e.g., a nerve ending thereof). The target nerve (e.g., a target nerve ending) of the trigeminal nerve mandibular region can be one or more of the following: the auriculotemporal nerve; and the mandibular nerve (e.g., a nerve ending thereof). The target nerve (e.g., a target nerve ending) of the back of the head can be one or more of the following: the greater occipital nerve; the lesser occipital nerve; and the suboccipital nerve (e.g., a nerve ending thereof).

In one embodiment:

(i) administering a single unit dose intradermally to one or more of the following areas: the supraorbital nerve (preferably administering a single unit dose to the supraorbital nerve region on a first side of the face (e.g., the left side), and administering a second unit dose to the supraorbital nerve region on a second side of the face (e.g., the right side); the supratrochlear nerve (preferably administering a single unit dose to the supratrochlear nerve region on a first side of the face (e.g., the left side), and administering a second unit dose to the supratrochlear nerve region on a second side of the face (e.g., the right side); the intratrochlear nerve (preferably administering a single unit dose to the intratrochlear nerve region on a first side of the face (e.g., the left side), and administering a second unit dose to the intratrochlear nerve region on a second side of the face (e.g., the right side); zygomaticotemporal nerve (preferably a single unit dose is administered in the zygomaticotemporal nerve region on a first side of the face (e.g., the left side), and a second unit dose is administered in the zygomaticotemporal nerve region on a second side of the face (e.g., the right side); zygomaticofacial nerve (preferably a single unit dose is administered in the zygomaticofacial nerve region on a first side of the face (e.g., the left side), and a second unit dose is administered in the zygomaticofacial nerve region on a second side of the face (e.g., the right side); occipital nerve (preferably a single unit dose is administered in the occipital nerve region on a first side of the neck (e.g., the left side), and a second unit dose is administered in the occipital nerve region on a second side of the neck (e.g., the right side); and/or (preferably and)

(iii) administering multiple unit doses intradermally in one or more of the following areas: supraorbital nerve (preferably a single unit dose is administered in the supraorbital nerve region on a first side of the face (e.g., the left side), and a second unit dose is administered in the supraorbital nerve region on a second side of the face (e.g., the right side); supratrochlear nerve (preferably a single unit dose is administered in the supratrochlear nerve region on a first side of the face (e.g., the left side), and a second unit dose is administered in the supratrochlear nerve region on a second side of the face (e.g., the right side); intratrochlear nerve (preferably a single unit dose is administered in the intratrochlear nerve region on a first side of the face (e.g., the left side), and a second unit dose is administered in the intratrochlear nerve region on a second side of the face (e.g., the right side); The plurality of unit doses may be 2-8 unit doses, for example, 2-5 unit doses.

Preferred injection sites and injection times are shown in Figure 6. In this case, one unit dose of the chimeric clostridial neurotoxin can be administered per injection site.

Preferably, the injection site is in the terminal region of a designated nerve.

Treatment of headache pain (e.g., migraine pain) or migraine pain may include bilateral intradermal administration of a unit dose of a chimeric Clostridial neurotoxin. Treatment of headache pain (e.g., migraine pain) or migraine pain may include administration of:

(i) 1 unit dose to the supraorbital nerve area on a first side of the face and/or 1 unit dose to the supraorbital nerve area on a second side of the face;

(ii) 1 unit dose to the supratrochlear nerve area on a first side of the face and/or 1 unit dose to the supratrochlear nerve area on a second side of the face;

(iii) 1 unit dose to the intratrochlear nerve area on a first side of the face and/or 1 unit dose to the intratrochlear nerve area on a second side of the face;

(iv) 1 unit dose to the zygomaticotemporal nerve area on the first side of the face and/or 1 unit dose to the zygomaticotemporal nerve area on the second side of the face;

(v) 1 unit dose to the zygomaticofacial nerve area on a first side of the face and/or 1 unit dose to the zygomaticofacial nerve area on a second side of the face;

(vi) 2 unit doses to the auriculotemporal nerve area on a first side of the face and/or 2 unit doses to the auriculotemporal nerve area on a second side of the face;

(vii) 2 unit doses to the first lateral occipital nerve area of the neck and/or 2 unit doses to the second lateral occipital nerve area of the neck; and/or

(vii) 1 unit dose to the lesser occipital nerve area on a first side of the neck and/or 1 unit dose to the lesser occipital nerve area on a second side of the neck.

Treatment of headache pain (e.g., migraine pain) or migraine pain may include bilateral administration of a unit dose of a chimeric Clostridial neurotoxin. Treatment of headache pain (e.g., migraine pain) or migraine pain may include administration of:

(i) 1 unit dose to the supraorbital nerve area on a first side of the face and/or 1 unit dose to the supraorbital nerve area on a second side of the face;

(ii) 1 unit dose to the supratrochlear nerve area on a first side of the face and/or 1 unit dose to the supratrochlear nerve area on a second side of the face;

(iii) 1 unit dose to the intratrochlear nerve area on a first side of the face and/or 1 unit dose to the intratrochlear nerve area on a second side of the face;

(iv) 1 unit dose to the zygomaticotemporal nerve area on the first side of the face and/or 1 unit dose to the zygomaticotemporal nerve area on the second side of the face;

(v) 1 unit dose to the zygomaticofacial nerve area on a first side of the face and/or 1 unit dose to the zygomaticofacial nerve area on a second side of the face;

(vi) 2 unit doses to the auriculotemporal nerve area on a first side of the face and/or 2 unit doses to the auriculotemporal nerve area on a second side of the face;

(vii) 2 unit doses to the greater occipital nerve area on the first side of the neck and/or 2 unit doses to the greater occipital nerve area on the second side of the neck; and/or

(vii) 1 unit dose to the lesser occipital nerve area on a first side of the neck and/or 1 unit dose to the lesser occipital nerve area on a second side of the neck.

Preferably, headache pain (e.g. migraine pain) or migraine treatment may comprise administration of:

(i) 1 unit dose to the supraorbital nerve area on the first side of the face and 1 unit dose to the supraorbital nerve area on the second side of the face;

(ii) 1 unit dose to the supratrochlear nerve area on a first side of the face and 1 unit dose to the supratrochlear nerve area on a second side of the face;

(iii) 1 unit dose to the intratrochlear nerve area on a first side of the face and 1 unit dose to the intratrochlear nerve area on a second side of the face;

(iv) 1 unit dose to the zygomaticotemporal nerve area on the first side of the face and 1 unit dose to the zygomaticotemporal nerve area on the second side of the face;

(v) 1 unit dose to the zygomaticofacial nerve area on a first side of the face and 1 unit dose to the zygomaticofacial nerve area on a second side of the face;

(vi) 2 unit doses to the auriculotemporal nerve area on a first side of the face and 2 unit doses to the auriculotemporal nerve area on a second side of the face;

(vii) 2 unit doses to the greater occipital nerve area on the first side of the neck and 2 unit doses to the greater occipital nerve area on the second side of the neck; and/or

(vii) 1 unit dose to the lesser occipital nerve area on the first side of the neck and 1 unit dose to the lesser occipital nerve area on the second side of the neck.

More preferably, headache pain (e.g. migraine pain) or migraine treatment may comprise administering:

(i) 1 unit dose to the supraorbital nerve area on the first side of the face and 1 unit dose to the supraorbital nerve area on the second side of the face;

(ii) 1 unit dose to the supratrochlear nerve area on a first side of the face and 1 unit dose to the supratrochlear nerve area on a second side of the face;

(iii) 1 unit dose to the intratrochlear nerve area on a first side of the face and 1 unit dose to the intratrochlear nerve area on a second side of the face;

(iv) 1 unit dose to the zygomaticotemporal nerve area on the first side of the face and 1 unit dose to the zygomaticotemporal nerve area on the second side of the face;

(v) 1 unit dose to the zygomaticofacial nerve area on a first side of the face and 1 unit dose to the zygomaticofacial nerve area on a second side of the face;

(vi) 2 unit doses to the auriculotemporal nerve area on a first side of the face and 2 unit doses to the auriculotemporal nerve area on a second side of the face;

(vii) 2 unit doses to the greater occipital nerve area on the first side of the neck and/or 2 unit doses to the greater occipital nerve area on the second side of the neck; and

(vii) 1 unit dose to the lesser occipital nerve area on the first side of the neck and 1 unit dose to the lesser occipital nerve area on the second side of the neck.

Treatment of headache pain (e.g., migraine pain) or migraine pain may include bilateral intradermal administration of a unit dose of a chimeric Clostridial neurotoxin. Treatment of headache pain (e.g., migraine pain) or migraine pain may include administration of:

(i) 2 unit doses to the supraorbital nerve area on a first side of the face and/or 2 unit doses to the supraorbital nerve area on a second side of the face;

(ii) 2 unit doses to the supratrochlear nerve area on a first side of the face and/or 2 unit doses to the supratrochlear nerve area on a second side of the face;

(iii) 2 unit doses to the intratrochlear nerve area on a first side of the face and/or 2 unit doses to the intratrochlear nerve area on a second side of the face;

(iv) 2 unit doses to the zygomaticotemporal nerve area on the first side of the face and/or 2 unit doses to the zygomaticotemporal nerve area on the second side of the face;

(v) 2 unit doses to the zygomaticofacial nerve area on a first side of the face and/or 2 unit doses to the zygomaticofacial nerve area on a second side of the face;

(vi) 4 unit doses to the auriculotemporal nerve area on a first side of the face and/or 4 unit doses to the auriculotemporal nerve area on a second side of the face;

(vii) 4 unit doses to the first lateral occipital nerve area of the neck and/or 4 unit doses to the second lateral occipital nerve area of the neck; and/or

(vii) 2 unit doses to the lesser occipital nerve area on a first side of the neck and/or 2 unit doses to the lesser occipital nerve area on a second side of the neck.

Treatment of headache pain (e.g., migraine pain) or migraine pain may include bilateral administration of a unit dose of a chimeric Clostridial neurotoxin. Treatment of headache pain (e.g., migraine pain) or migraine pain may include administration of:

(i) 2 unit doses to the supraorbital nerve area on a first side of the face and/or 2 unit doses to the supraorbital nerve area on a second side of the face;

(ii) 2 unit doses to the supratrochlear nerve area on a first side of the face and/or 2 unit doses to the supratrochlear nerve area on a second side of the face;

(iii) 2 unit doses to the intratrochlear nerve area on a first side of the face and/or 2 unit doses to the intratrochlear nerve area on a second side of the face;

(iv) 2 unit doses to the zygomaticotemporal nerve area on the first side of the face and/or 2 unit doses to the zygomaticotemporal nerve area on the second side of the face;

(v) 2 unit doses to the zygomaticofacial nerve area on a first side of the face and/or 2 unit doses to the zygomaticofacial nerve area on a second side of the face;

(vi) 4 unit doses to the auriculotemporal nerve area on a first side of the face and/or 4 unit doses to the auriculotemporal nerve area on a second side of the face;

(vii) 4 unit doses to the first lateral occipital nerve area of the neck and/or 4 unit doses to the second lateral occipital nerve area of the neck; and/or

(vii) 2 unit doses to the lesser occipital nerve area on a first side of the neck and/or 2 unit doses to the lesser occipital nerve area on a second side of the neck.

Preferably, headache pain (e.g. migraine pain) or migraine treatment may comprise administration of:

(i) 2 unit doses to the supraorbital nerve area on the first side of the face and 2 unit doses to the supraorbital nerve area on the second side of the face;

(ii) 2 unit doses to the supratrochlear nerve area on a first side of the face and 2 unit doses to the supratrochlear nerve area on a second side of the face;

(iii) 2 unit doses to the intratrochlear nerve area on a first side of the face and 2 unit doses to the intratrochlear nerve area on a second side of the face;

(iv) 2 unit doses to the zygomaticotemporal nerve area on the first side of the face and 2 unit doses to the zygomaticotemporal nerve area on the second side of the face;

(v) 2 unit doses to the zygomaticofacial nerve area on a first side of the face and 2 unit doses to the zygomaticofacial nerve area on a second side of the face;

(vi) 4 unit doses to the auriculotemporal nerve area on a first side of the face and 4 unit doses to the auriculotemporal nerve area on a second side of the face;

(vii) 4 unit doses to the greater occipital nerve area on the first side of the neck and 4 unit doses to the greater occipital nerve area on the second side of the neck; and/or

(vii) 2 unit doses to the lesser occipital nerve area on the first side of the neck and 2 unit doses to the lesser occipital nerve area on the second side of the neck.

More preferably, headache pain (e.g. migraine pain) or migraine treatment may comprise administering:

(i) 2 unit doses to the supraorbital nerve area on the first side of the face and 2 unit doses to the supraorbital nerve area on the second side of the face;

(ii) 2 unit doses to the supratrochlear nerve area on a first side of the face and 2 unit doses to the supratrochlear nerve area on a second side of the face;

(iii) 2 unit doses to the intratrochlear nerve area on a first side of the face and 2 unit doses to the intratrochlear nerve area on a second side of the face;

(iv) 2 unit doses to the zygomaticotemporal nerve area on the first side of the face and 2 unit doses to the zygomaticotemporal nerve area on the second side of the face;

(v) 2 unit doses to the zygomaticofacial nerve area on a first side of the face and 2 unit doses to the zygomaticofacial nerve area on a second side of the face;

(vi) 4 unit doses to the auriculotemporal nerve area on a first side of the face and 4 unit doses to the auriculotemporal nerve area on a second side of the face;

(vii) 4 unit doses to the greater occipital nerve area on the first side of the neck and 4 unit doses to the greater occipital nerve area on the second side of the neck; and

(vii) 2 unit doses to the lesser occipital nerve area on the first side of the neck and 2 unit doses to the lesser occipital nerve area on the second side of the neck.

When treating headache pain (e.g., migraine pain) or migraine as described in the foregoing embodiments, preferably one unit dose is administered per injection site. Thus, the treatment may comprise administering the chimeric clostridial neurotoxin to:

(i) 1 injection site in the supraorbital nerve region on the first side of the face and/or 1 injection site in the supraorbital nerve region on the second side of the face;

(ii) 1 injection site in the supratrochlear nerve region on a first side of the face and/or 1 injection site in the supratrochlear nerve region on a second side of the face;

(iii) one injection site in the intratrochlear nerve region on a first side of the face and/or one injection site in the intratrochlear nerve region on a second side of the face;

(iv) 1 injection site in the zygomaticotemporal nerve region on the first side of the face and/or 1 injection site in the zygomaticotemporal nerve region on the second side of the face;

(v) 1 injection site in the zygomaticofacial nerve region on the first side of the face and/or 1 injection site in the zygomaticofacial nerve region on the second side of the face;

(vi) 2 injection sites in the auriculotemporal nerve region on a first side of the face and/or 2 injection sites in the auriculotemporal nerve region on a second side of the face;

(vii) 2 injection sites in the region of the greater occipital nerve on the first side of the neck and/or 2 injection sites in the region of the greater occipital nerve on the second side of the neck; and/or

(vii) one injection site in the region of the lesser occipital nerve on the first side of the neck and/or one injection site in the region of the lesser occipital nerve on the second side of the neck.

Preferably, treatment may comprise administering a chimeric Clostridial neurotoxin to:

(i) 1 injection site in the supraorbital nerve region on the first side of the face and 1 injection site in the supraorbital nerve region on the second side of the face;

(ii) 1 injection site in the supratrochlear nerve region on the first side of the face and 1 injection site in the supratrochlear nerve region on the second side of the face;

(iii) 1 injection site in the intratrochlear nerve region on the first side of the face and 1 injection site in the intratrochlear nerve region on the second side of the face;

(iv) 1 injection site in the zygomaticotemporal nerve area on the first side of the face and 1 injection site in the zygomaticotemporal nerve area on the second side of the face;

(v) 1 injection site in the zygomaticofacial nerve region on the first side of the face and 1 injection site in the zygomaticofacial nerve region on the second side of the face;

(vi) 2 injection sites in the auriculotemporal nerve region on the first side of the face and 2 injection sites in the auriculotemporal nerve region on the second side of the face;

(vii) 2 injection sites in the region of the greater occipital nerve on the first side of the neck and 2 injection sites in the region of the greater occipital nerve on the second side of the neck; and/or

(vii) 1 injection site in the region of the lesser occipital nerve on the first side of the neck and 1 injection site in the region of the lesser occipital nerve on the second side of the neck.

More preferably, treatment may comprise administering a chimeric Clostridial neurotoxin to:

(i) 1 injection site in the supraorbital nerve region on the first side of the face and 1 injection site in the supraorbital nerve region on the second side of the face;

(ii) 1 injection site in the supratrochlear nerve region on the first side of the face and 1 injection site in the supratrochlear nerve region on the second side of the face;

(iii) 1 injection site in the intratrochlear nerve region on the first side of the face and 1 injection site in the intratrochlear nerve region on the second side of the face;

(iv) 1 injection site in the zygomaticotemporal nerve area on the first side of the face and 1 injection site in the zygomaticotemporal nerve area on the second side of the face;

(v) 1 injection site in the zygomaticofacial nerve region on the first side of the face and 1 injection site in the zygomaticofacial nerve region on the second side of the face;

(vi) 2 injection sites in the auriculotemporal nerve region on the first side of the face and 2 injection sites in the auriculotemporal nerve region on the second side of the face;

(vii) 2 injection sites in the region of the greater occipital nerve on the first side of the neck and 2 injection sites in the region of the greater occipital nerve on the second side of the neck; and

(vii) 1 injection site in the region of the lesser occipital nerve on the first side of the neck and 1 injection site in the region of the lesser occipital nerve on the second side of the neck.

When treating headache pain (e.g., migraine pain) or migraine as described in the foregoing embodiments, preferably one unit dose is administered per injection site. Thus, the treatment may comprise administering the chimeric clostridial neurotoxin to:

(i) 2 injection sites in the supraorbital nerve region on the first side of the face and/or 2 injection sites in the supraorbital nerve region on the second side of the face;

(ii) 2 injection sites in the supratrochlear nerve region on a first side of the face and/or 2 injection sites in the supratrochlear nerve region on a second side of the face;

(iii) 2 injection sites in the intratrochlear nerve region on a first side of the face and/or 2 injection sites in the intratrochlear nerve region on a second side of the face;

(iv) 2 injection sites in the zygomaticotemporal nerve region on the first side of the face and/or 2 injection sites in the zygomaticotemporal nerve region on the second side of the face;

(v) 2 injection sites in the zygomaticofacial nerve region on the first side of the face and/or 2 injection sites in the zygomaticofacial nerve region on the second side of the face;

(vi) 4 injection sites in the auriculotemporal nerve region on a first side of the face and/or 4 injection sites in the auriculotemporal nerve region on a second side of the face;

(vii) 4 injection sites in the region of the greater occipital nerve on the first side of the neck and/or 4 injection sites in the region of the greater occipital nerve on the second side of the neck; and/or

(vii) 2 injection sites in the region of the lesser occipital nerve on the first side of the neck and/or 2 injection sites in the region of the lesser occipital nerve on the second side of the neck.

Preferably, treatment may comprise administering a chimeric Clostridial neurotoxin to:

(i) 2 injection sites in the supraorbital nerve region on the first side of the face and 2 injection sites in the supraorbital nerve region on the second side of the face;

(ii) 2 injection sites in the supratrochlear nerve region on the first side of the face and 2 injection sites in the supratrochlear nerve region on the second side of the face;

(iii) 2 injection sites in the intratrochlear nerve region on the first side of the face and 2 injection sites in the intratrochlear nerve region on the second side of the face;

(iv) 2 injection sites in the zygomaticotemporal nerve region on the first side of the face and 2 injection sites in the zygomaticotemporal nerve region on the second side of the face;

(v) 2 injection sites in the zygomaticofacial nerve region on the first side of the face and 2 injection sites in the zygomaticofacial nerve region on the second side of the face;

(vi) 4 injection sites in the auriculotemporal nerve region on the first side of the face and 4 injection sites in the auriculotemporal nerve region on the second side of the face;

(vii) 4 injection sites in the region of the greater occipital nerve on the first side of the neck and 4 injection sites in the region of the greater occipital nerve on the second side of the neck; and/or

(vii) 2 injection sites in the region of the lesser occipital nerve on the first side of the neck and 2 injection sites in the region of the lesser occipital nerve on the second side of the neck.

More preferably, treatment may comprise administering a chimeric Clostridial neurotoxin to:

(i) 2 injection sites in the supraorbital nerve region on the first side of the face and 2 injection sites in the supraorbital nerve region on the second side of the face;

(ii) 2 injection sites in the supratrochlear nerve region on the first side of the face and 2 injection sites in the supratrochlear nerve region on the second side of the face;

(iii) 2 injection sites in the intratrochlear nerve region on the first side of the face and 2 injection sites in the intratrochlear nerve region on the second side of the face;

(iv) 2 injection sites in the zygomaticotemporal nerve region on the first side of the face and 2 injection sites in the zygomaticotemporal nerve region on the second side of the face;

(v) 2 injection sites in the zygomaticofacial nerve region on the first side of the face and 2 injection sites in the zygomaticofacial nerve region on the second side of the face;

(vi) 4 injection sites in the auriculotemporal nerve region on the first side of the face and 4 injection sites in the auriculotemporal nerve region on the second side of the face;

(vii) 4 injection sites in the region of the greater occipital nerve on the first side of the neck and 4 injection sites in the region of the greater occipital nerve on the second side of the neck; and

(vii) 2 injection sites in the region of the lesser occipital nerve on the first side of the neck and 2 injection sites in the region of the lesser occipital nerve on the second side of the neck.

When treating headache pain (e.g., migraine) or migraine pain as described in the foregoing embodiments, it is preferred that more than one unit dose (preferably 2 unit doses) be administered per injection site. Thus, the treatment may comprise administering the chimeric clostridial neurotoxin to:

(i) 1 injection site in the supraorbital nerve region on the first side of the face and/or 1 injection site in the supraorbital nerve region on the second side of the face;

(ii) 1 injection site in the supratrochlear nerve region on a first side of the face and/or 1 injection site in the supratrochlear nerve region on a second side of the face;

(iii) one injection site in the intratrochlear nerve region on a first side of the face and/or one injection site in the intratrochlear nerve region on a second side of the face;

(iv) 1 injection site in the zygomaticotemporal nerve region on the first side of the face and/or 1 injection site in the zygomaticotemporal nerve region on the second side of the face;

(v) 1 injection site in the zygomaticofacial nerve region on the first side of the face and/or 1 injection site in the zygomaticofacial nerve region on the second side of the face;

(vi) 2 injection sites in the auriculotemporal nerve region on a first side of the face and/or 2 injection sites in the auriculotemporal nerve region on a second side of the face;

(vii) 2 injection sites in the region of the greater occipital nerve on the first side of the neck and/or 2 injection sites in the region of the greater occipital nerve on the second side of the neck; and/or

(vii) one injection site in the region of the lesser occipital nerve on the first side of the neck and/or one injection site in the region of the lesser occipital nerve on the second side of the neck.

Preferably, treatment may comprise administering a chimeric Clostridial neurotoxin to:

(i) 1 injection site in the supraorbital nerve region on the first side of the face and 1 injection site in the supraorbital nerve region on the second side of the face;

(ii) 1 injection site in the supratrochlear nerve region on the first side of the face and 1 injection site in the supratrochlear nerve region on the second side of the face;

(iii) 1 injection site in the intratrochlear nerve region on the first side of the face and 1 injection site in the intratrochlear nerve region on the second side of the face;

(iv) 1 injection site in the zygomaticotemporal nerve area on the first side of the face and 1 injection site in the zygomaticotemporal nerve area on the second side of the face;

(v) 1 injection site in the zygomaticofacial nerve region on the first side of the face and 1 injection site in the zygomaticofacial nerve region on the second side of the face;

(vi) 2 injection sites in the auriculotemporal nerve region on the first side of the face and 2 injection sites in the auriculotemporal nerve region on the second side of the face;

(vii) 2 injection sites in the region of the greater occipital nerve on the first side of the neck and 2 injection sites in the region of the greater occipital nerve on the second side of the neck; and/or

(vii) 1 injection site in the region of the lesser occipital nerve on the first side of the neck and 1 injection site in the region of the lesser occipital nerve on the second side of the neck.

More preferably, treatment may comprise administering a chimeric Clostridial neurotoxin to:

(i) 1 injection site in the supraorbital nerve region on the first side of the face and 1 injection site in the supraorbital nerve region on the second side of the face;

(ii) 1 injection site in the supratrochlear nerve region on the first side of the face and 1 injection site in the supratrochlear nerve region on the second side of the face;

(iii) 1 injection site in the intratrochlear nerve region on the first side of the face and 1 injection site in the intratrochlear nerve region on the second side of the face;

(iv) 1 injection site in the zygomaticotemporal nerve area on the first side of the face and 1 injection site in the zygomaticotemporal nerve area on the second side of the face;

(v) 1 injection site in the zygomaticofacial nerve region on the first side of the face and 1 injection site in the zygomaticofacial nerve region on the second side of the face;

(vi) 2 injection sites in the auriculotemporal nerve region on the first side of the face and 2 injection sites in the auriculotemporal nerve region on the second side of the face;

(vii) 2 injection sites in the region of the greater occipital nerve on the first side of the neck and 2 injection sites in the region of the greater occipital nerve on the second side of the neck; and

(vii) 1 injection site in the region of the lesser occipital nerve on the first side of the neck and 1 injection site in the region of the lesser occipital nerve on the second side of the neck.

Thus, when treating headache pain (e.g., migraine pain) or migraine, 1-50, 5-45, or 10-38 unit doses may be administered. Preferably, a maximum of 35 unit doses are administered. Preferably, the total dose administered during each treatment may be a maximum of 192,500 pg of chimeric clostridial neurotoxin. For example, the total dose administered may be a maximum of 180,000 pg, preferably a maximum of 177,000 pg (more preferably a maximum of 175,000 pg). Most preferably, the total dose administered may be a maximum of 115,000 pg or 75,000 pg, for example a maximum of 112,000 pg or 70,000 pg.

Thus, when treating headache pain (e.g., migraine pain) or migraine by intramuscular injection, 1-50, 5-45, or 10-38 unit doses may be administered. Preferably, a maximum of 35 unit doses are administered. Preferably, the total dose administered during each treatment period may be a maximum of 192,500 pg of chimeric clostridial neurotoxin. For example, the total dose administered may be a maximum of 180,000 pg, preferably a maximum of 177,000 pg (more preferably a maximum of 175,000 pg). Most preferably, the total dose administered may be a maximum of 115,000 pg or 75,000 pg, for example a maximum of 112,000 pg or 70,000 pg.

Thus, when treating headache pain (e.g., migraine pain) or migraine by intradermal injection, 1-35, 5-25, or 10-20 unit doses may be administered. Preferably, a maximum of 20 unit doses are administered. Preferably, the total dose administered during each treatment period may be a maximum of 110,000 pg of chimeric clostridial neurotoxin. For example, the total dose administered may be a maximum of 105,000 pg, preferably a maximum of 102,000 pg (more preferably a maximum of 100,000 pg).

When treating headache pain (e.g., migraine pain) or migraine by intradermal injection, 2-70, 10-50, or 20-40 unit doses may be administered. Preferably, a maximum of 40 unit doses are administered. Preferably, the total dose administered during each treatment period may be a maximum of 220,000 pg of chimeric clostridial neurotoxin. For example, the total dose administered may be a maximum of 210,000 pg, preferably a maximum of 204,000 pg (more preferably a maximum of 200,000 pg).

In some embodiments, headache pain (e.g., migraine pain) or migraine treatment can be performed by a mixture of intramuscular and intradermal injections. For example, the chimeric clostridial neurotoxin of the present invention can be administered intradermally to the neck of the subject, and the chimeric clostridial neurotoxin of the present invention can be administered intramuscularly to the face of the subject. Preferably, the chimeric clostridial neurotoxin of the present invention can be administered intradermally to the face of the subject, and the chimeric clostridial neurotoxin of the present invention can be administered intramuscularly to the neck of the subject. For example, in addition to application to the neck and/or face, the chimeric clostridial neurotoxin can also be applied to the head of the subject.

When treating headache pain (e.g., migraine pain) or migraine by intradermal injection or by intramuscular injection, the preferred unit dose may be 1,000 pg to 5,500 pg. The upper limit of the unit dose range may be 5,250, 5,200, 5,100, 5,000, 4,500, 4,000, 3,500, 3,000, 2,500 or 2,000 pg of chimeric clostridial neurotoxin, preferably 5,100 pg, more preferably 5,000 pg. The lower limit of the unit dose range may be 1,100, 1,200, 1,250, 1,300, 1,350, 1,400 or 1,450, 1,500, 2,000, 2,500, 3,000, 3,500 or 4,000 pg of chimeric clostridial neurotoxin, preferably the lower limit is 1,400 pg, more preferably 1,500 pg. The lower limit of the range may be greater than 3,000 pg. The unit dose may be 1,400 pg to 5,100 pg (e.g., 1,500 pg to 5,000 pg), 2,000 pg to 5,100 pg, 3,000 to 5,100 pg, or 3,000 to 4,000 pg of chimeric clostridial neurotoxin. The unit dose may contain greater than 3,000 pg to a maximum of 5,500 pg of the chimeric clostridial neurotoxin. The unit dose of the chimeric clostridial neurotoxin may be 2,000 pg to 4,500 pg, 2,000 pg to 3,000 pg (e.g., 2,500 pg), or 3,500 to 4,500 pg of the chimeric clostridial neurotoxin. Preferably, the unit dose contains 4,000 pg of the chimeric clostridial neurotoxin.

When treating headache pain (e.g., migraine pain) or migraine by intradermal injection or by intramuscular injection, the preferred unit dose may be 42 units to 229 units. The upper limit of the unit dose range may be 225, 220, 215, 210, 205, 200, 190, 180, 170, 160, 150, 125, 100 or 83 units of chimeric clostridial neurotoxin, preferably the upper limit is 212 units, more preferably 208 units. The lower limit of the unit dose range may be 46, 50, 55, 60, 65, 70, 75, 80 or 90, 100, 110, 120, 130, 140, 150, 160 or 166 units of chimeric clostridial neurotoxin, preferably the lower limit is 58 units, more preferably 62 units. The lower limit of the range may be greater than 125 units. The unit dose may be 58 units to 212 units (e.g., 62 units to 208 units), 83 units to 212 units, 125 to 212 units, or 125 to 166 units of a chimeric clostridial neurotoxin. A unit dose may contain greater than 125 units to a maximum of 229 units of a chimeric clostridial neurotoxin. A unit dose of a chimeric clostridial neurotoxin may be 83 units to 188 units, 83 units to 125 units (e.g., 104 units), or 146 units to 188 units of a chimeric clostridial neurotoxin. Preferably, a unit dose contains 166 units of a chimeric clostridial neurotoxin. A unit dose may contain 47 units to 258 units of a chimeric clostridial neurotoxin, e.g., 94 units to 211 units, 94 units to 141 units (e.g., 117 units), or 164 to 211 units of a chimeric clostridial neurotoxin. A unit dosage form may contain 188 units of a chimeric clostridial neurotoxin.

When headache pain (e.g., migraine pain) or migraine is treated by intramuscular injection or intradermal injection (preferably intramuscular injection), the preferred unit dose may be 2,500 pg, and the total dose may be up to 70,000 pg. For example, the preferred unit dose may be 2,500 pg, and the total dose may be 70,000 pg. The preferred unit dose may be 4,000 pg, and the total dose may be up to 112,000 pg. For example, the preferred unit dose may be 4,000 pg, and the total dose may be 112,000 pg. A suitable unit dose may be 5,000 pg, and the total dose may be up to 155,000 pg. For example, a suitable unit dose may be 5,000 pg, and the total dose may be 155,000 pg.

When treating headache pain (e.g., migraine pain) or migraine by intramuscular injection or intradermal injection (preferably intramuscular injection), the preferred unit dose may be 104 units, and the total dose may be up to 2,912 units. For example, the preferred unit dose may be 104 units, and the total dose may be 2,912 units. The preferred unit dose may be 166 units, and the total dose may be up to 4,659 units. For example, the preferred unit dose may be 166 units, and the total dose may be 4,659 units. A suitable unit dose may be 208 units, and the total dose may be up to 6,448 units. For example, a suitable unit dose may be 208 units, and the total dose may be 6,448 units.

When treating headache pain (e.g., migraine pain) or migraine by intramuscular injection or intradermal injection (preferably intramuscular injection), the preferred unit dose may be 117 units, and the total dose may be up to 3,286 units. For example, the preferred unit dose may be 117 units, and the total dose may be 3,286 units. The preferred unit dose may be 188 units, and the total dose may be up to 5,258 units. For example, the preferred unit dose may be 188 units, and the total dose may be 5,258 units. A suitable unit dose may be 235 units, and the total dose may be up to 7,277 units. For example, a suitable unit dose may be 235 units, and the total dose may be 7,277 units.

When headache pain (e.g., migraine pain) or migraine is treated by intramuscular injection, the total dose administered during each treatment period may be up to 8,007 units of the chimeric clostridial neurotoxin. For example, the total dose administered may be up to 7,488 units, or up to 7,363 units (e.g., up to 7,280 units). Most preferably, the total dose administered may be up to 4,784 units or 3,120 units, such as up to 4,659 units or 2,912 units. The total dose administered during each treatment period may be up to 9,037 units of the chimeric clostridial neurotoxin. For example, the total dose administered may be up to 8,451 units, or up to 8,310 units (e.g., up to 8,216 units). Most preferably, the total dose administered may be up to 5,399 units or 3,521 units, such as up to 5,258 units or 3,286 units.

When headache pain (e.g., migraine pain) or migraine is treated by intradermal injection, the total dose administered during each treatment period can be up to 4,576 units of chimeric clostridial neurotoxin. For example, the total dose administered can be up to 4,368 units, or up to 4,243 units (e.g., up to 4,160 units). The total dose administered during each treatment period can be up to 5,165 units of chimeric clostridial neurotoxin. For example, the total dose administered can be up to 4,929 units, or up to 4,789 units (e.g., up to 4,695 units). The total dose administered during each treatment period can be up to 5,165 units of chimeric clostridial neurotoxin. For example, the total dose administered can be up to 4,929 units, or up to 4,789 units (e.g., up to 4,695 units).

In preferred embodiments, when a chimeric clostridial neurotoxin is used to treat pain (e.g., headache or migraine pain) or migraine, the treatment does not induce muscle paralysis. For example, in some embodiments, the unit dose of a chimeric clostridial neurotoxin can be lower than the unit dose of a chimeric clostridial neurotoxin required to induce muscle paralysis. In particular, in some embodiments, the unit dose of a chimeric clostridial neurotoxin administered at a particular site (e.g., an injection site) can be lower than the unit dose of a chimeric clostridial neurotoxin required to induce muscle paralysis (e.g., at that site and/or muscle).

The headache pain is preferably migraine pain. The migraine pain may be paroxysmal migraine pain or chronic migraine pain, such as pain caused by or associated with paroxysmal migraine, or pain caused by or associated with chronic migraine.

The chimeric clostridial neurotoxins of the present invention are preferably administered iteratively (e.g., up to 5, 10, 15, or 20 times) as part of a treatment regimen (preferably on different days, e.g., at least 1 day apart between consecutive treatments). Iterative administration refers to administration at least twice, e.g., at least 5, 10, 15, or 20 times. Therefore, in one embodiment, the chimeric clostridial neurotoxin of the present invention can be administered two or more times to treat a condition (preferably pain) in a subject. This is particularly important for the treatment of chronic conditions, such as chronic pain, which generally requires continuous treatment. In one embodiment, the chimeric clostridial neurotoxin of the present invention can be administered weekly, twice a month, monthly, every two months, every six months, or annually, preferably at least twice a year or annually. In one embodiment, the chimeric clostridial neurotoxin of the present invention is administered twice or more over a period of 10 years, 5 years, 2 years, or 1 year. Preferably, the chimeric clostridial neurotoxin of the present invention is administered twice or more within 1 year. Treatment may continue for at least 6 months, 1 year, 2 years, 3 years, 5 years, 10 years, 15 years, 20 years, 25 years, or 30 years.

In some embodiments, after a first administration of a chimeric clostridial neurotoxin according to the invention (e.g., a first treatment period), a subject may receive a second administration of a chimeric clostridial neurotoxin (e.g., a second treatment period). The time interval between the first administration and the second administration may be at least 5, 6, 7, 8, 9, or 10 months. For example, the time interval between the first administration and the second administration may be 5-10 months, 5-9 months, 5-8 months, 6-10 months, 6-9 months, or 6-8 months.

Preferably, the chimeric clostridial neurotoxin is not administered with other therapeutic agents or diagnostic agents other than the light and heavy chains (e.g., nucleic acid, protein, peptide, or small molecule therapeutic or diagnostic agents). For example, in one embodiment, the chimeric clostridial neurotoxin is not administered with other analgesics. In one embodiment, the chimeric clostridial neurotoxin of the invention is not administered with a covalently bound therapeutic agent. In one embodiment, the chimeric clostridial neurotoxin of the invention is not administered with a non-covalently bound therapeutic agent.

Chimeric Clostridial neurotoxins are preferably used to treat pain, and can be used to treat a subject suffering from one or more types of pain.

The term "pain" as used herein refers to any unpleasant sensory and emotional experience associated with or resembling actual or potential tissue damage. Any associated physical condition may or may not be apparent to the clinician.

Pain may be associated with the release of mediators (e.g., neurotransmitters) from neurons. Neurons are preferably neurons to which the chimeric clostridial neurotoxin of the present invention binds. For example, the mediator may be any mediator associated with pain transmission. The mediator may be a neuropeptide such as substance P, CGRP, or vasoactive intestinal peptide (VIP).

The mediator can be an inflammatory mediator or a non-inflammatory mediator. The mediator can be one or more of the following: CGRP, neurokinin (e.g., tachykinin, substance P, neurokinin A, neurokinin B, hemokinin (hemokinin) and/or endokinin), adrenocorticotropic hormone (ACTH), glucocorticoids, vasopressin, oxytocin, catecholamines, opioids (e.g., opioid peptides and/or brain opioids), angiotensin II, endorphins, enkephalins, vasoactive intestinal peptide (VIP), eicosanoids (e.g., prostaglandins such as prostaglandin E2 (PGE2) and/or leukotrienes), tissue kininogens (e.g., bradykinin)), histamine, serotonin, potassium, Prostacyclin (PGI2), leukotriene B4 (LTB4), nerve growth factor (NGF), protons, ATP, adenosine, serotonin (5-HT), histamine, glutamate, norepinephrine (NE), nitric oxide (NO), gamma-aminobutyric acid (GABA), glycine, acetylcholine, cannabinoids, tissue necrosis factor alpha (TNF-α), cytokines (e.g., interleukin (IL)-6, IL-1 and/or IL-8), platelet activating factor (PAF), neurotrophic growth factor (NGF), glutamate, aspartate, pituitary adenylate cyclase activating peptide (PACAP) and proteolytic enzymes.

The mediator may be calcitonin gene-related peptide (CGRP), amylin, pituitary adenylate cyclase activating peptide (PACAP), oxytocin, neuropeptide Y (NPY), substance P, angiotensin, corticotropin-releasing hormone (CRH), leptin, adiponectin, orexin and/or melanin-concentrating hormone (MCH).

The mediator may be one or more selected from the group consisting of: CGRP, substance P, and glutamate.

The mediator may be one or more of the following: neuropeptides (eg, substance P, CGRP, or VIP), nitric oxide, glutamate, and aspartate.

When the pain is headache pain (preferably migraine pain), the mediator may be one or more of the following: CGRP, VIP, PACAP and proinflammatory cytokines (eg IL-6, IL-8 and/or TNF-α).

Preferably, the mediator may be CGRP, substance P and/or alternative neurokinin.

Most preferably, the mediator is CGRP. CGRP may be α-CGRP or β-CGRP, preferably α-CGRP.

Pain can be associated with the release of pain mediators (e.g., pain neurotransmitters) from neurons containing Aδ nerve fibers or C nerve fibers. Pain mediators can be any pain mediators released/secreted from neurons containing Aδ nerve fibers or C nerve fibers. Pain mediators can be neuropeptides, such as substance P, CGRP, or vasoactive intestinal peptide (VIP).

Pain mediators can be inflammatory mediators or non-inflammatory mediators. Pain mediators can be one or more of the following: CGRP, neurokinins (e.g., tachykinins, substance P, neurokinin A, neurokinin B, hemokinin (hemokinin) and/or endokinin), adrenocorticotropic hormone (ACTH), glucocorticoids, vasopressin, oxytocin, catecholamines, opioids (e.g., opioid peptides and/or brain opioids), angiotensin II, endorphins, enkephalins, vasoactive intestinal peptide (VIP), eicosanoids (e.g., prostaglandins such as prostaglandin E2 (PGE2) and/or leukotrienes), tissue kininogens (e.g., bradykinin)), histamine, serotonin, potassium , prostacyclin (PGI2), leukotriene B4 (LTB4), nerve growth factor (NGF), protons, ATP, adenosine, serotonin (5-HT), histamine, glutamate, norepinephrine (NE), nitric oxide (NO), γ-aminobutyric acid (GABA), glycine, acetylcholine, cannabinoids, tissue necrosis factor alpha (TNF-α), cytokines (such as interleukin (IL) -6, IL-1 and/or IL-8), platelet activating factor (PAF), neurotrophic growth factor (NGF), glutamate, aspartate, pituitary adenylate cyclase activating peptide (PACAP) and proteolytic enzymes.

Pain mediators may be calcitonin gene-related peptide (CGRP), amylin, pituitary adenylate cyclase-activating peptide (PACAP), oxytocin, neuropeptide Y (NPY), substance P, angiotensin, corticotropin-releasing hormone (CRH), leptin, adiponectin, orexin, and/or melanin-concentrating hormone (MCH).

The pain mediators released from neurons comprising Aδ nerve fibers may be one or more selected from the group consisting of CGRP, substance P, and glutamate.

Pain mediators released from neurons containing C nerve fibers may be one or more of the following: neuropeptides (eg, substance P, CGRP, or VIP), nitric oxide, glutamate, and aspartate.

Glutamate may be involved in the initiation of chronic pain and/or neuropathic pain.

When the pain is headache pain (preferably migraine pain), the pain mediator may be one or more of the following: CGRP, VIP, PACAP and proinflammatory cytokines (eg IL-6, IL-8 and/or TNF-α).

Preferably, when the neurons contain Aδ nerve fibers or C nerve fibers, the pain mediator may be CGRP, preferably, when the neurons contain C fibers, the pain mediator is CGRP. When the neurons contain C fibers, the pain mediator may be substance P and/or alternative neurokinin.

Most preferably, the pain mediator is CGRP. CGRP may be α-CGRP or β-CGRP, preferably α-CGRP.

Thus, the chimeric Clostridial neurotoxins of the invention can inhibit the release of CGRP from sensory neurons comprising Aδ nerve fibers or C nerve fibers.

When the pain mediator is CGRP, the pain may be CGRP-related pain.

The term "CGRP-associated pain" as used herein refers to pain associated with the release of CGRP from neurons and any of its effects. CGRP-induced pain can be CGRP-dependent pain. In one embodiment, CGRP-associated pain is CGRP-induced pain induced by the release of CGRP from neurons and any of its effects.

Examples of CGRP-related pain include migraine and pruritus.

In one embodiment, the therapeutic uses or methods of the present invention do not include the treatment of pain associated with any pain mediator other than CGRP. The therapeutic uses or methods of the present invention may exclude the treatment of pain associated with one or more of the following: neurokinins (e.g., tachykinins, substance P, neurokinin A, neurokinin B, hemokinin and/or endokinin), adrenocorticotropic hormone (ACTH), glucocorticoids, vasopressin, oxytocin, catecholamines, opioids (e.g., opioid peptides and/or brain opioids), angiotensin II, endorphins, enkephalins, vasoactive intestinal peptide (VIP), eicosanoids (e.g., prostaglandins such as prostaglandin E2 (PGE2) and/or leukotrienes), tissue kininogens (e.g., bradykinin)), histamine, Serotonin, potassium, prostacyclin (PGI2), leukotriene B4 (LTB4), nerve growth factor (NGF), protons, ATP, adenosine, 5-hydroxytryptamine (5-HT), histamine, glutamate, norepinephrine (NE), nitric oxide (NO), gamma-aminobutyric acid (GABA), glycine, acetylcholine, cannabinoids, tissue necrosis factor alpha (TNF-α), cytokines (e.g., interleukin (IL)-6, IL-1 and/or IL-8), platelet activating factor (PAF), neurotrophic growth factor (NGF), glutamate, aspartate, pituitary adenylate cyclase activating peptide (PACAP), and proteolytic enzymes.

Pain can be chronic or acute. "Acute pain" is pain that comes on suddenly and lasts for a short time. For example, one type of acute pain is cutaneous pain felt when the skin or other superficial tissue is damaged, such as caused by a cut or burn. Cutaneous nociceptors end just below the skin and, because of the high concentration of nerve endings, produce well-defined, localized pain that lasts for a short time. "Chronic pain" is pain other than acute pain.

Thus, pain can be chronic or acute pain. Pain can be one or more selected from the following four categories of pain: nociceptive pain; neuropathic pain; mixed pain; and pain of unknown origin. Nociceptive pain can be caused by known harmful stimulation of nociceptors (pain receptors) and can be somatic or visceral pain. Neuropathic pain can be pain triggered or caused by primary damage or dysfunction in the nervous system. Mixed pain can be a combination of nociceptive pain and neuropathic pain.

The pain (eg, chronic pain) may be one or more selected from the group consisting of neuropathic pain, inflammatory pain, headache pain, somatic pain, visceral pain, referred pain, allodynia, mixed pain, and postoperative pain. However, preferably, the pain is not postoperative pain.

In one embodiment, the pain is not visceral pain.In one embodiment, the disorder is not a visceral pain disorder.

The somatic pain may be one or more selected from the following: headache pain (e.g., post-traumatic headache, head injury headache, or post-traumatic brain injury headache), arthritis pain (e.g., osteoarthritis pain and/or rheumatoid arthritis pain), movement pain, degenerative disc disease pain, carpal tunnel compression pain, soft tissue injury pain, temporomandibular joint pain, musculoskeletal pain, somatic pain caused by or associated with a vascular disorder (e.g., Raynaud's syndrome, thromboangiitis obliterans, peripheral venous disease, peripheral arterial disease, varicose veins, venous thrombosis, coagulopathy, or lymphedema), facial pain, somatic pain caused by or associated with trigeminal autonomic cephalgia; somatic pain caused by or associated with trigeminal neuralgia; and bone pain (e.g., cancer-induced bone pain, e.g., CGRP-related cancer-induced bone pain).

The pain is preferably headache pain. More preferably, the pain is migraine pain.

The visceral pain may be one or more selected from the group consisting of endometriosis pain, pancreatitis pain, gastrointestinal pain, and visceral pain caused by or associated with a vascular disorder.

The inflammatory pain may be one or more selected from chronic pain, wound healing pain, itching pain and burn pain.

The neuropathic pain may be one or more selected from the group consisting of postherpetic neuralgia, diabetic pain, chronic neuropathic pain, and Morton's neuroma pain.

Pain and conditions that can be treated with the chimeric Clostridial neurotoxins of the invention are described in more detail below.

The chimeric clostridial neurotoxins of the invention can be used to treat pain caused by or associated with any of the following neuropathic pain conditions. "Neuropathic pain" refers to abnormal sensory input from the peripheral nervous system, the central nervous system, or both, resulting in discomfort. Symptoms of neuropathic pain can include persistent, spontaneous pain, as well as allodynia (painful response to stimuli that are not normally painful), hyperalgesia (intense response to painful stimuli such as pinpricks that normally cause only mild discomfort), or hyperalgesia (where brief discomfort becomes prolonged, severe pain). Neuropathic pain can be caused by any of the following:

1. Traumatic injury, such as nerve compression injury (e.g., nerve compression, nerve stretch, nerve entrapment, or incomplete nerve transection); spinal cord injury (e.g., spinal cord hemisection); limb amputation; contusion; inflammation (e.g., spinal cord inflammation); or surgical intervention.

2. Ischemic events, including, for example, stroke and heart attack.

3. Infectious factors.

4. Exposure to toxic substances, including, for example, drugs, alcohol, heavy metals (e.g., lead, arsenic, mercury), industrial agents (e.g., solvents, glue fumes), or nitrous oxide.

5. Diseases, including, for example, inflammatory disorders, neoplastic tumors, acquired immune deficiency syndrome (AIDS), Lyme disease, leprosy, metabolic diseases, peripheral nerve disorders such as neuromas, mononeuropathies or polyneuropathies.

Types of neuropathic pain include the following:

1. Neuralgia.

Neuralgia is a type of pain that radiates along the course of one or more specific nerves, usually without any obvious pathological changes in the neural structure. The causes of neuralgia are varied. Chemical irritation, inflammation, trauma (including surgery), compression of nearby structures (such as tumors), and infection can all lead to neuralgia. However, in many cases, the cause is unknown or unidentifiable. Neuralgia is most common in the elderly, but it can occur at any age. Neuralgia includes, but is not limited to, trigeminal neuralgia, postherpetic neuralgia, postherpetic neuralgia, glossopharyngeal neuralgia, sciatica, and atypical facial pain.

Neuralgia refers to pain in one or more nerve distribution areas. Examples are trigeminal neuralgia, atypical facial pain and postherpetic neuralgia (caused by herpes zoster or herpes). The affected nerves are responsible for sensing touch, temperature and pressure in the facial area from the jaw to the forehead. The disease usually causes a short attack of severe pain, usually less than two minutes and only on one side of the face. Pain can be described in a variety of ways, such as "stinging", "sharp pain", "lightning-like", "burning" and even "itching". In the atypical form of TN, pain can also be manifested as severe or just pain and last for a long time. The pain associated with TN is considered to be one of the most severe pain that can be experienced.

Simple stimulation such as eating, talking, washing the face or any light touch or sensation can trigger an attack (even the feeling of a breeze). Attacks can occur in clusters or in isolation.

Symptoms include sharp, stabbing or constant burning pain anywhere, usually on or near the surface of the body, with each episode occurring in the same location; pain along a specific nerve pathway; impaired function of the affected body part due to the pain, or muscle weakness due to concomitant motor nerve damage; increased skin sensitivity or numbness of the affected skin area (feeling similar to an injection of a local anesthetic such as procaine); and any touch or pressure being interpreted as pain. Movement may also be painful.

Trigeminal neuralgia is the most common form of neuralgia. It affects the main sensory nerve in the face, the trigeminal nerve ("trigeminal" literally means "three origins," referring to the fact that the nerve divides into 3 branches). The condition involves sudden, brief, severe pain on one side of the face, along the area innervated by the trigeminal nerve on that side. The pain episodes may be severe enough to cause a facial grimacing, which is often called a painful tic (tic douloureux). Sometimes, the cause of trigeminal neuralgia is compression of the nerve by a blood vessel or a small tumor. Conditions such as multiple sclerosis (an inflammatory disease that affects the brain and spinal cord), certain forms of arthritis, diabetes (high blood sugar) and others may also cause trigeminal neuralgia, but the cause cannot always be determined. In this case, certain movements such as chewing, talking, swallowing, or touching the facial area may trigger spasms of severe pain.

A related but much less common neuralgia affects the glossopharyngeal nerve, which provides sensation to the throat. Symptoms of this neuralgia are brief, shock-like episodes of pain located in the throat.

Nerve pain may occur after an infection such as shingles, which is caused by the varicella zoster virus (a herpes virus). This type of neuralgia produces a persistent, burning pain after the shingles rash has healed. The pain is worsened by movement or touch of the affected area. Not all patients diagnosed with shingles experience postherpetic neuralgia, which can be more painful than shingles itself. The pain and sensitivity can last for months or even years. The pain often presents as an unbearable sensitivity to any touch, especially light touch. Postherpetic neuralgia is not limited to the face; it can occur anywhere on the body, but usually at the location of the shingles rash. Depression is not uncommon due to the pain and social isolation during the illness.

Postherpetic neuralgia can be debilitating after the initial herpes infection symptoms have worn off. Other infectious diseases that can cause neuralgia are syphilis and Lyme disease.

Diabetes is another common cause of nerve pain. Almost 1 in 20 American adults is affected by this very common medical problem. Diabetes damages the tiny arteries that supply circulation to the nerves, causing nerve fiber dysfunction and sometimes nerve loss. Diabetes can produce almost any type of nerve pain, including trigeminal neuralgia, carpal tunnel syndrome (pain and numbness in the hands and wrists), and meralgia paresthesia (numbness and pain in the thigh due to damage to the lateral femoral cutaneous nerve). Tight control of blood sugar can prevent diabetic nerve damage and can speed recovery in subjects with nerve pain.

Other medical conditions that may be associated with nerve pain are chronic renal insufficiency and porphyria - a genetic disorder in which the body is unable to eliminate certain substances produced by the normal breakdown of the blood in the body. Certain medications may also cause this problem.

2. Afferent nerve block.

Deafferentation refers to the loss of sensory input from a part of the body and may be caused by the interruption of peripheral sensory fibers or nerves from the central nervous system. Deafferentation pain syndromes include, but are not limited to, brain or spinal cord injury, post-stroke pain, phantom pain, paraplegia, brachial plexus avulsion, lumbar radiculopathy.

3. Complex Regional Pain Syndrome (CRPS)

CRPS is a chronic pain syndrome caused by sympathetically maintained pain and presents in two forms. CRPS1 currently replaces the term "Reflex Sympathetic Dystrophy Syndrome". It is a chronic nerve condition that most often occurs in the arms or legs after a minor or severe injury. CRPS1 is associated with severe pain; changes in the nails, bones, and skin; and increased sensitivity to touch in the affected limb. CRPS2 replaces the term causalgia and is due to nerve damage. CRPS includes, but is not limited to, CRPS type I (Reflex Sympathetic Dystrophy) and CRPS type II (causalgia).

4. Neuropathy.

Neuropathies are functional or pathological changes in nerves and are clinically characterized by abnormalities of sensory or motor neurons.

Central neuropathy is a functional or pathological change in the central nervous system.

Peripheral neuropathy is a functional or pathological change in one or more peripheral nerves. Peripheral nerves carry messages from the central nervous system (brain and spinal cord) to muscles and other organs and back to the brain from the skin, joints and other organs. Peripheral neuropathy occurs when these nerves cannot carry messages between the brain and spinal cord, resulting in pain, loss of sensation or loss of muscle control. In some cases, failure of nerves that control blood vessels, intestines and other organs can lead to abnormal blood pressure, digestive problems and loss of other basic body processes. Risk factors for neuropathy include diabetes, alcohol abuse and exposure to certain chemicals and drugs. Some people have a genetic predisposition to neuropathy. Prolonged pressure on nerves is another risk for developing nerve damage. Pressure injuries can result from prolonged immobility (such as prolonged surgery or long-term illness) or from pressure on nerves by casts, splints, braces, crutches or other devices. Polyneuropathy means a widespread process that usually affects both sides of the body equally. Symptoms depend on which type of nerve is affected. The three main types of nerves are sensory, motor and autonomic. Neuropathy can affect any one of all three types of nerves or a combination of them. Symptoms also depend on whether the condition affects the whole body or just one nerve (such as from an injury). The cause of chronic inflammatory polyneuropathy is an abnormal immune response. The specific antigens, immune processes, and triggers are variable and in many cases unknown. It may be associated with other conditions such as HIV, inflammatory bowel disease, lupus erythematosus, chronic active hepatitis, and blood cell abnormalities.

Peripheral neuropathy can involve functional or pathological changes in a single nerve or group of nerves (mononeuropathy) or affect multiple nerves (polyneuropathy).

Peripheral neuropathies may include the following:

Genetic disorders

Charcot-Marie-Tooth disease

Friedreich's ataxia

Systemic or metabolic disorders

Diabetes (diabetic neuropathy)

Dietary deficiencies (especially vitamin B-12)

Excessive drinking (alcoholic neuropathy)

Uremia (kidney failure)

cancer

Infectious or inflammatory conditions

AIDS

hepatitis

Colorado Tick Fever

diphtheria

Guillain-Barré syndrome

HIV infection that has not progressed to AIDS

Leprosy

Lyme disease

Polyarteritis nodosa

Rheumatoid arthritis

Sarcoidosis

Sjögren's syndrome

syphilis

Systemic lupus erythematosus

Amyloid

Exposure to toxic compounds

Sniffing glue or other toxic compounds

Nitrous oxide

Industrial reagents - especially solvents

Heavy metals (lead, arsenic, mercury, etc.)

Secondary neuropathy caused by analgesic nephropathy and other drugs

other reasons

Ischemia (reduced oxygen/blood flow)

Long-term exposure to low temperatures

a. Polyneuropathy

Polyneuropathy is a peripheral neuropathy that involves regional loss of movement or sensation caused by damage or destruction of multiple peripheral nerves. Polyneuropathy pain includes, but is not limited to, post-polio syndrome, post-mastectomy syndrome, diabetic neuropathy, alcoholic neuropathy, amyloid, toxins, AIDS, hypothyroidism, uremia, vitamin deficiency, chemotherapy-induced pain, 2',3'-dideoxycytidine (ddC) treatment, Guillain-Barré syndrome, or Fabry's disease.

b. Mononeuropathy

Mononeuropathy is a peripheral neuropathy, which involves the loss of movement or sensation in the area caused by the damage or destruction of a single peripheral nerve or nerve group. Mononeuropathy is most commonly caused by localized regional damage caused by damage or trauma, although occasionally systemic conditions can cause isolated nerve damage (such as multiple mononeuritis). Common causes are direct trauma, long-term compression of nerves, and compression of nerves due to swelling or damage of nearby body structures. Damage includes the destruction of nerve myelin sheaths (coverings) or part of nerve cells (axons). This damage can slow down or prevent the conduction of nerve impulses. Mononeuropathy can involve any part of the body. Mononeuropathy pain includes but is not limited to sciatic nerve dysfunction, common peroneal nerve dysfunction, radial nerve dysfunction, ulnar nerve dysfunction, cranial mononeuropathy VI, cranial mononeuropathy VII, cranial mononeuropathy III (compression type), cranial mononeuropathy III (diabetic type), axillary nerve dysfunction, carpal tunnel syndrome, femoral nerve dysfunction, tibial nerve dysfunction, Bell's palsy, thoracic outlet syndrome, carpal tunnel syndrome and sixth (abducens) nerve palsy.

c. Systemic peripheral neuropathy

Systemic peripheral neuropathies are symmetrical and usually result from a variety of systemic diseases and disease processes that affect the entire peripheral nervous system. They are further subdivided into several categories:

i. Distal axonopathy is the result of certain metabolic or toxic disorders of neurons. They can be caused by metabolic diseases such as diabetes, deficiency syndromes such as renal failure, malnutrition, and alcoholism or the effects of toxins or drugs. Distal axonopathy (also known as dying back neuropathy) is a peripheral neuropathy caused by certain metabolic or toxic disorders of neurons in the peripheral nervous system (PNS). It is the most common reaction of nerves to metabolic or toxic disorders, and therefore can be caused by metabolic diseases such as diabetes, deficiency syndromes such as renal failure, malnutrition, and alcoholism or the effects of toxins or drugs. The most common cause of distal axonopathy is diabetes, and the most common distal axonopathy is diabetic neuropathy.

ii. Myelinopathy is an acute failure of impulse conduction due to a primary attack on the myelin sheath. The most common cause is acute inflammatory demyelinating polyneuropathy (AIDP; also known as Guillain-Barré syndrome), while other causes include chronic inflammatory demyelinating syndrome (CIDP), inherited metabolic disorders (such as leukodystrophy), or toxins. Myelinating neuropathy is due to primary destruction of the myelin sheath or myelin-forming Schwann cells, which keep the axons intact but cause acute failure of impulse conduction. This demyelination slows down or completely blocks the conduction of electrical impulses through the nerves. The most common cause is acute inflammatory demyelinating polyneuropathy (AIDP, more widely known as Guillain-Barré syndrome), while other causes include chronic inflammatory demyelinating polyneuropathy (CIDP), inherited metabolic disorders (such as leukodystrophy or Charcot-Marie-Tooth disease), or toxins.

Iii. neuronopathy is the result of the destruction of neurons in the peripheral nervous system (PNS). They can be caused by motor neuron disease, sensory neuron disease (e.g. herpes zoster), toxins or autonomic dysfunction. Neurotoxins such as the chemotherapy agent vincristine can cause neuronopathy. Neuronopathy is a dysfunction caused by damage to neurons in the peripheral nervous system (PNS), leading to peripheral neuropathy. It can be caused by motor neuron disease, sensory neuron disease (e.g. herpes zoster), toxic substances or autonomic dysfunction. People with neuronopathy can appear in different ways, depending on the cause, the way in which the nerve cells are affected, and the type of nerve cells most seriously affected.

iv. Focal entrapment neuropathy (eg, carpal tunnel syndrome).

The chimeric Clostridial neurotoxins of the invention may be used to treat pain caused by or associated with any of the following inflammatory conditions.

A. Arthritis

Arthritic conditions include, for example, rheumatoid arthritis; juvenile rheumatoid arthritis; systemic lupus erythematosus (SLE); gouty arthritis; scleroderma; osteoarthritis; psoriatic arthritis; ankylosing spondylitis; Reiter's syndrome (reactive arthritis); adult Still's disease; arthritis caused by viral infections; arthritis caused by bacterial infections, such as gonococcal arthritis and non-gonococcal bacterial arthritis (septic arthritis); tertiary Lyme disease; tuberculous arthritis; and arthritis caused by fungal infections, such as blastomycosis.

B. Autoimmune diseases

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

C. Connective tissue disorders

Connective tissue diseases include, for example, spondyloarthritis, dermatomyositis, and fibromyalgia.

D.Injury

Inflammation caused by injury, including, for example, squeezing, puncture, or stretching of tissue or a joint, may cause chronic inflammatory pain.

E. Infection

Inflammation caused by infection, including, for example, tuberculosis or interstitial keratitis, may cause chronic inflammatory pain.

F.Neuritis

Neuritis is an inflammatory process that affects a nerve or group of nerves. Symptoms depend on the nerves involved but may include pain, paresthesias, paralysis, or hypoesthesia (numbness).

Examples include:

a. Brachial neuritis

b. Retrobulbar neuropathy, an inflammatory process affecting the portion of the optic nerve located just behind the eyeball.

Optic neuropathy, an inflammatory process that affects the optic nerve, causing a sudden decrease in vision in the affected eye. The cause of optic neuritis is unknown. Sudden inflammation of the optic nerve (the nerve that connects the eye to the brain) causes swelling and destruction of the myelin sheath. Inflammation can sometimes be the result of a viral infection, or it can be caused by an autoimmune disease such as multiple sclerosis. Risk factors are associated with possible causes.

d. Vestibular neuritis, a viral infection that causes an inflammatory process affecting the vestibular nerve.

G. Joint inflammation

Inflammation of the joints, such as caused by bursitis or tendinitis, may cause chronic inflammatory pain.

H. Sunburn and/or UV damage

The chimeric Clostridial neurotoxins of the invention can be used to treat pain caused by or associated with any of the following headache conditions. Headache (medically known as cephalgia) is a condition of mild to severe pain in the head; sometimes pain in the neck or upper back may also be interpreted as a headache. It may indicate an underlying local or systemic disease or be a condition in itself.

A. Muscular/myogenic headache

Muscular/myogenic headaches seem to involve tightening or tensing of the muscles in the face and neck; they can radiate to the forehead. Tension headaches are the most common form of myogenic headaches.

A tension headache is a condition involving pain or discomfort in the head, scalp, or neck, usually related to muscle tension in these areas. Tension headaches are caused by contraction of the neck and scalp muscles. One cause of this muscle contraction is a response to stress, depression, or anxiety. Any activity that keeps the head in one position for a long time without moving can cause headaches. Such activities include typing or using a computer, fine manual work, and using a microscope. Sleeping in a cold room or sleeping in an abnormal neck position may also trigger this type of headache. Tension-type headaches include, but are not limited to, episodic tension headaches and chronic tension headaches.

B. Vascular headache

The most common type of vascular headache is a migraine. Other types of vascular headaches include cluster headaches, which cause recurring episodes of intense pain, and headaches caused by high blood pressure.

1. Migraine

Migraine is a heterogeneous disorder that typically involves recurrent headaches. Migraines differ from other headaches in that they are accompanied by other symptoms, such as nausea, vomiting, or sensitivity to light. For most people, throbbing pain is felt on only one side of the head. Clinical features such as the type of aura symptoms, the presence of prodromal symptoms, or associated symptoms such as vertigo may be observed in subgroups of subjects with different underlying pathophysiology and genetic mechanisms. Migraines include, but are not limited to, migraine without aura (common migraine), migraine with aura (classic migraine), menstrual migraine, acephalic headache, complicated migraine, abdominal migraine, and mixed tension migraine.

2. Cluster headaches

Cluster headaches affect one side of the head (unilateral) and may be associated with tearing and nasal congestion. They come in clusters, reoccur at the same time each day, last for several weeks, and then subside.

D. Hypertension headache

E. Traction headache and inflammatory headache

Traction headaches and inflammatory headaches are often symptoms of other medical conditions, including stroke and sinus infection.

F. Hormonal headache

G. Rebound headache

Rebound headaches, also called medication overuse headaches, occur as a result of taking medication too often to relieve headache pain. Rebound headaches can occur frequently, every day, and can be very painful.

H. Chronic sinusitis headache

Sinusitis is a bacterial, fungal, viral, allergic, or autoimmune inflammation of the sinuses. Chronic sinusitis is one of the most common complications of the common cold. Symptoms include: nasal congestion; facial pain; headache; fever; general malaise; thick green or yellow discharge; a "full" feeling in the face that worsens when leaning over. In rare cases, chronic maxillary sinusitis may also be caused by bacteria spread from a dental infection. Chronic hyperplastic eosinophilic sinusitis is a noninfectious form of chronic sinusitis.

I. Organic headache

J. Paroxysmal headache

Paroxysmal headaches are headaches that are associated with seizure activity.

The chimeric clostridial neurotoxins of the invention can be used to treat pain caused by or otherwise associated with any of the following somatic pain conditions. Somatic pain originates from ligaments, tendons, bones, blood vessels, and even the nerves themselves. It is detected using somatic nociceptors. These areas lack pain receptors and produce dull, poorly localized pain that lasts longer than cutaneous pain; examples include sprains and broken bones. Other examples include the following.

A. Excessive muscle tension

Excessive muscle tension can be caused by, for example, a sprain or strain.

B. Repetitive movement disorder

Repetitive motion disorders may result from overuse of the hands, wrists, elbows, shoulders, neck, back, hips, knees, feet, legs, or ankles.

C. Muscle disease

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

D. Myalgia

Myalgia is muscle pain and is a symptom of many diseases and conditions. The most common cause of myalgia is overuse or overstretching of a muscle or muscle group. Myalgia without a history of trauma is often due to a viral infection. Long-term myalgia may indicate the presence of a metabolic myopathy, certain nutritional deficiencies, or chronic fatigue syndrome.

E. Infection

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

F.Medications

Medications can cause physical pain. Examples of such medications include cocaine, statins (such as atorvastatin, simvastatin, and lovastatin) used to lower cholesterol, and ACE inhibitors (such as enalapril and captopril) used to lower blood pressure.

The chimeric Clostridial neurotoxins of the present invention can be used to treat pain caused by or otherwise associated with any of the following visceral pain conditions. Visceral pain originates from the internal organs or organs of the body. Visceral nociceptors are located in organs and internal cavities of the body. Nociceptors in these areas are more scarce, and the pain produced is generally more intense and lasts longer than somatic pain. Visceral pain is extremely difficult to localize, and some injuries to visceral tissues exhibit "referred" pain, in which the sensation is localized to an area completely unrelated to the site of injury. Examples of visceral pain include the following.

A. Functional visceral pain

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

B. Chronic gastrointestinal inflammation

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

C. Autoimmune pain

Autoimmune pain includes, for example, sarcoidosis and vasculitis.

D. Organic visceral pain

Organic visceral pain includes, for example, pain caused by traumatic, inflammatory or degenerative changes in the intestine or pain caused by tumors impinging on sensory nerves.

E. Treatment of visceral pain

Treatment-induced visceral pain includes, for example, chemotherapy-associated pain or radiation therapy-associated pain.

The chimeric Clostridial neurotoxins of the invention may be used to treat pain caused by or otherwise associated with any of the below-mentioned painful conditions.

Referred pain is pain that is localized to an area separate from the site of the painful stimulus. Referred pain typically occurs when a nerve is compressed or damaged at or near its origin. In this case, the sensation of pain is usually felt in the area innervated by the nerve, even if the damage originated elsewhere. A common example occurs in a herniated disc, in which a nerve root originating from the spinal cord is compressed by the adjacent disc material. While the pain may be caused by the damaged disc itself, pain may also be felt in the area innervated by the compressed nerve, such as the thigh, knee, or foot. Relieving pressure on the nerve root may relieve referred pain if no permanent nerve damage has occurred. Myocardial ischemia (loss of blood flow to a portion of the heart muscle tissue) is perhaps the best-known example of referred pain; the sensation may appear in the upper chest as a feeling of restriction, or as pain in the left shoulder, arm, or even hand.

The chimeric clostridial neurotoxins of the invention may be used to treat postoperative pain. However, it is preferred that the pain of the invention is not postoperative pain.

Postoperative (e.g., postoperative) pain is an unpleasant sensation caused by a surgical procedure. Postoperative pain may be caused by damage to tissue caused by the incision, the surgery itself, wound closure, and any force applied during the operation. Postoperative pain (e.g., postoperative pain) may also be caused by factors associated with the surgery. For example, the subject may suffer from back pain due to the way they are positioned on the operating table, or may suffer from chest pain due to an incision in the chest area. Sore throat may also occur after general anesthesia because the insertion of a breathing tube may cause irritation. However, the most common is postoperative pain caused by the surgical incision cutting into the skin and muscles. Postoperative pain may also include pain caused by or associated with postoperative scars (e.g., postoperative scar pain).

For example, a surgical procedure (or more specifically, a surgical incision) represents a "noxious stimulus" that causes pain. Noxious stimuli (stimuli that can cause tissue damage) can activate the release of pain mediators from nociceptive afferent terminals and from sensory terminals (e.g., CGRP is released therefrom). The noxious information is then transduced from the peripheral nervous system to the central nervous system, where the individual perceives pain.

Postoperative pain can be caused by a combination of inflammation and neural tissue damage. For example, mast cell degranulation activated in response to tissue damage can lead to the release of a variety of substances including proteases, cytokines, serotonin, and the extracellular space. These substances can sensitize (activate at a lower threshold) primary afferent neurons to produce pain hypersensitivity. Because tissues are widely innervated, any area of the body is susceptible to nerve damage caused by surgery.

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

The surgery may comprise an incision of the skin and/or fascia and/or muscle. Preferably, the surgery comprises an incision of the skin.

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

Preferably, "postoperative" may refer to a time period starting at most one day after surgery (e.g., postoperatively). In other words, the term "postoperative" may refer to a time period starting no more than one day after surgery. For example, the term "postoperative" may refer to a time point starting 1-20 hours after surgery; optionally 2-15 hours after surgery; optionally 5-10 hours after surgery. Such a time may represent a time period starting from a chronological interface at which the analgesic effect of the surgical anesthetic administered to the subject diminishes (e.g., tapers off), such that the subject begins to feel pain.

Furthermore, the term "post-operative" may be used interchangeably with the term "post-surgical" since "procedure" is used herein in the sense of "surgery."

Similarly, the term "postoperative pain" may refer to pain felt (or more specifically, beginning to be felt) within a time period starting up to one day after surgery (e.g., after surgery). In other words, the term "postoperative pain" may refer to pain felt by a subject within a time period not exceeding one day after surgery. For example, the term "postoperative pain" may refer to pain felt within a time period starting 1-20 hours after surgery; optionally 2-15 hours after surgery; optionally 5-10 hours after surgery.

The time period may be 1-50 weeks; for example 5-45 weeks, 10-40 weeks, or 10-35 weeks after surgery.

This is in contrast to the term "peri-operative", which can refer, for example, to the time period during or surrounding a subject undergoing surgery (e.g., while the subject is in the operating room), suitably beginning at least 1 hour before surgery and/or ending less than 1 hour after surgery.

The present invention is relevant to a wide range of pain conditions, such as chronic pain conditions.In some embodiments, the chimeric Clostridial neurotoxins of the invention are used to treat cancerous and/or non-cancer pain.

Preferably, the chimeric clostridial neurotoxins of the invention are used to treat bladder pain syndrome (eg, bladder pain), phantom limb pain, or migraine pain. Bladder pain syndrome (eg, bladder pain) may be caused by or associated with interstitial cystitis.

In particularly preferred embodiments, the pain is bladder pain, such as caused by or associated with interstitial cystitis.

Treating pain preferably means alleviating pain. In other words, in one embodiment, administration of a chimeric Clostridial neurotoxin of the invention reduces pain in a subject.

In more detail, reference to "reduced" or "reducing" (with respect to pain) preferably refers to a lower level of pain perceived by a subject after administration of a chimeric clostridial neurotoxin of the invention (post-administration) compared to the level of pain perceived by the subject before administration (pre-administration). For example, the level of pain perceived after administration may be reduced by at least 15%, 25%, 35%, 45%, 55%, 65%, 75%, 85% or 95% relative to before administration. For example, the level of pain perceived after administration may be reduced by at least 75%; preferably by at least 85%; more preferably by at least 95%.

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

Suitable (although non-limiting) methods for assessing pain perception in a subject include the following: Numerical Rating Scale (NRS) score; although the skilled artisan will appreciate that other methods may additionally or alternatively be used, such as sensory threshold, pain perception threshold, static mechanical allodynia, dynamic mechanical allodynia, temporal summation, pressure pain threshold, conditioned pain modulation, and temperature threshold.

Other non-limiting examples of pain perception measures include: change from baseline in SF-36 scores at each predetermined time point; amount of rescue medication taken during the study and time to first rescue medication intake. These may be considered "exploratory" endpoints or pain perception assessment measures.

Thus, in a preferred embodiment, following administration of the chimeric clostridial neurotoxin of the present invention, pain perception can be assessed by one or more of the following: (a) numerical rating scale (NRS); (b) stimulation-induced NRS; (c) temperature at the site of pain; (d) size of the painful area; (e) time to onset of analgesia; (f) peak analgesic effect; (g) time to peak analgesic effect; (h) duration of analgesic effect; and (i) SF-36 quality of life assessment.

Such methods for assessing pain perception are known to those skilled in the art.For convenience, further description of the numerical rating score and the Quality of Life Questionnaire Short Form 36 is provided below.

Numerical Rating Scale (NRS): Generally, the pain perception according to the present invention uses a Numerical Rating Scale (NRS). The NRS is an 11-point scale for assessing the subject's pain perception. The subject is asked to give a number between 0-10 that best fits their pain intensity. 0 means "no pain at all", while the upper limit of 10 means "the worst possible pain".

NRS can be used to assess multiple aspects of pain, including spontaneous average pain, spontaneous worst pain, and spontaneous current pain. Spontaneous average pain is assessed by asking the subject to select the number that best describes the average pain (e.g., perceived pain) of the subject over a period of time (e.g., at least 6 hours, 12 hours, 24 hours, or at least 48 hours). Spontaneous worst pain is assessed by asking the subject to select the number that best describes the subject's worst pain over a specified time period (e.g., at least in the previous 6 hours, 12 hours, 24 hours, or the previous 48 hours). Spontaneous current pain is assessed by asking the subject to select the number that best describes the subject's pain level at the time of assessment.

NRS can also be used to assess the pain perception of a subject in response to a variety of different stimuli. In order to assess the pain perception in response to stimulation, the subject will be subjected to stimulation of a variety of properties applied to the painful area. The subject will be asked about the current NRS score before and after application.

Examples of stimuli used include: (i) light touch (which can be assessed by measuring pain on the surface of the painful area on the radial spokes after application of von Frey cilia as described herein); (ii) pressure (pressure pain threshold), which can be assessed by asking the subject to give an NRS score when increasing pressure is applied using a pressure analgesia meter; and (iii) temperature (which can be assessed by asking the subject to give an NRS score for warm, cold, and hot stimuli using a thermode applied to the painful area).

Preferably, administration of a chimeric Clostridial neurotoxin of the invention reduces the NRS score of a subject after administration compared to the subject's NRS score before administration (eg, from a rating of ≥ 7 to a rating of ≤ 6).

Short Form 36 Quality of Life Questionnaire (SF-36): The SF-36 Quality of Life Questionnaire can be used to assess the subject's pain perception. The SF-36 is a 36-item subject-reported survey of the subject's health. The SF-36 consists of eight scale scores (vitality, physical functioning, bodily pain, general health perceptions, physical role functioning, emotional role functioning, social role functioning, and mental health). Assuming that each question has equal weight, each scale is directly converted to a 0-100 scale. The higher the score recorded in the SF-36, the fewer deficits there are.

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

Preferably, administration of a chimeric Clostridial neurotoxin of the invention increases the subject's SF-36 score after administration compared to the subject's SF-36 score before administration (eg, from a score of ≤ 50 to a score of ≥ 50).

The present invention may also (e.g., additionally or alternatively) relate to the treatment of any sensory disorder that can be treated by a chimeric clostridial neurotoxin that binds to neurons containing Aδ nerve fibers or C nerve fibers, respectively, and inhibits the release of mediators therefrom. Without wishing to be bound by theory, it is believed that the condition can be treated similarly to pain, as described herein. Therefore, all of the above embodiments for treating pain can be equally effective in the case of treating sensory disorders. The mediator can be any mediator involved in sensation (e.g., a sensory mediator), and in some cases, the mediator can be a pain mediator as described herein. The mediator can be a neurotransmitter. Inhibition of the release of the mediator from neurons can be partial or complete inhibition, preferably complete inhibition. For example, a chimeric clostridial neurotoxin can inhibit at least 80%, 90%, 95%, or 99% of the mediator released from the neuron. Preferably, a chimeric clostridial neurotoxin inhibits 100% of the mediator released from the neuron. A chimeric clostridial neurotoxin can inhibit the release of multiple mediators from neurons. Sensory disorders can be sensory modulation disorders (e.g., sensory hyperreactivity) and/or abnormal sensory processing disorders (e.g., fibromyalgia).

Thus, in one aspect, the invention provides a chimeric clostridial neurotoxin for use in treating sensory disorders by inhibiting the release of mediators from neurons comprising Aδ neurofibers or C neurofibers, wherein the chimeric clostridial neurotoxin binds to neurons comprising Aδ neurofibers or C neurofibers, respectively, and wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin A (BoNT/A) light chain and translocation domain ( _HN domain) and a BoNT/B receptor binding domain ( _HC domain).

In a related aspect, the invention provides a method of treating sensory disorders by inhibiting the release of mediators from neurons comprising Aδ neurofibers or C neurofibers, the method comprising administering to a subject the chimeric clostridial neurotoxin, wherein the chimeric clostridial neurotoxin binds to neurons comprising Aδ neurofibers or C neurofibers, respectively, and wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin A (BoNT/A) light chain and translocation domain (H _N domain) and a BoNT/B receptor binding domain (H _C domain).

In another related aspect, the present invention provides for the use of a chimeric clostridial neurotoxin in the preparation of a medicament for treating sensory disorders by inhibiting the release of mediators from neurons comprising Aδ neurofibers or C neurofibers, wherein the chimeric clostridial neurotoxin binds to neurons comprising Aδ neurofibers or C neurofibers, respectively, and wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin A (BoNT/A) light chain and translocation domain (H _N domain) and a BoNT/B receptor binding domain (H _C domain).

In another aspect, the present invention provides a kit comprising:

(a) a unit dosage form according to the present invention; and

(b) instructions for its use in the treatment of pain; and

(c) optional diluent.

Terms:

1. A chimeric clostridial neurotoxin for use in treating pain by inhibiting the release of pain mediators from neurons comprising Aδ neurofibers or C neurofibers, wherein the chimeric clostridial neurotoxin binds to neurons comprising Aδ neurofibers or C neurofibers, respectively, and wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin A (BoNT/A) light chain and translocation domain ( _HN domain) and a BoNT/B receptor binding domain ( _HC domain).

2. The chimeric clostridial neurotoxin for use according to clause 1, wherein the pain mediator is one or more selected from the group consisting of: calcitonin gene-related peptide (CGRP); substance P; and neurokinin.

3. The chimeric clostridial neurotoxin for use according to clause 1 or 2, wherein the pain mediator is CGRP and the pain is CGRP-related pain.

4. The chimeric clostridial neurotoxin for use according to clause 3, wherein the CGRP-related pain is CGRP-related headache pain.

5. The chimeric clostridial neurotoxin for use according to clause 3 or 4, wherein the CGRP-related pain is CGRP-related migraine pain.

6. The chimeric clostridial neurotoxin for use according to any one of clauses 3 to 5, wherein CGRP-related pain is:

(a) CGRP-related somatic pain is selected from the group consisting of headache pain (e.g., post-traumatic headache, head injury headache, or post-traumatic brain injury headache), arthritis pain (e.g., osteoarthritis pain and/or rheumatoid arthritis pain), movement pain, degenerative disc disease pain, carpal tunnel compression pain, soft tissue injury pain, temporomandibular joint pain, musculoskeletal pain, CGRP-related somatic pain caused by or associated with a vascular disorder (e.g., Raynaud's syndrome, thromboangiitis obliterans, peripheral venous disease, peripheral arterial disease, varicose veins, venous thrombosis, coagulopathy, or lymphedema), facial pain, CGRP-related somatic pain caused by or associated with trigeminal autonomic cephalgia; CGRP-related somatic pain caused by or associated with trigeminal neuralgia; and CGRP-related cancer-induced pain (e.g., CGRP-related cancer-induced bone pain);

(b) CGRP-related visceral pain selected from the group consisting of endometriosis pain, pancreatitis pain, gastrointestinal pain, and CGRP-related visceral pain caused by or associated with vascular disorders;

(c) CGRP-related inflammatory pain selected from the group consisting of chronic pain, wound healing pain, pruritus pain and burn pain; and/or

(d) CGRP-related neuropathic pain is selected from the group consisting of postherpetic neuralgia, diabetic pain, chronic neuropathic pain and Morton's neuroma pain.

7. The chimeric clostridial neurotoxin for use according to any one of the preceding clauses, wherein the neuron is the trigeminal ganglion.

8. The chimeric clostridial neurotoxin for use according to any one of the preceding clauses, wherein the chimeric clostridial neurotoxin is administered to the face, neck and/or skull.

9. The chimeric clostridial neurotoxin for use according to any one of the preceding clauses, wherein the chimeric clostridial neurotoxin is administered intradermally.

1.10. The chimeric clostridial neurotoxin for use according to any of the preceding clauses, wherein the chimeric clostridial neurotoxin is administered by intradermal injection at up to 10 injection sites per treatment period.

11. The chimeric clostridial neurotoxin for use according to any one of clauses 1 to 8, wherein the chimeric clostridial neurotoxin is administered intramuscularly.

12. The chimeric clostridial neurotoxin for use according to any one of clauses 1 to 8 or 11, wherein the chimeric clostridial neurotoxin is administered to one or more muscles of the subject selected from the group consisting of: frontalis, corrugator (e.g., corrugator supercilii), procerus (e.g., procerus nasalis), the nasalis), occipitalis, temporalis, trapezius, masseter, nasalis, orbicularis oculi, paraspinal muscles of the neck, temporalis fascia, superior auricle, anterior auricle, posterior auricle, sternocleidomastoid, platysma, anterior nasal dilator, posterior nasal dilator, depressor septi, mentalis, orbicularis oris, zygomaticus, risorius, buccinator, occipitofrontalis, levator labii superioris, depressor labii inferioris, depressor anguli oris, thyrohyoid, omohyoid, sternohyoid, splenius cervi, splenius capitis, semispinalis cervi, semispinalis capitis, levator scapulae, digastric, and scalene muscles; preferably, wherein the chimeric Clostridial neurotoxin is administered to a subject at least one muscle selected from the group consisting of: frontalis, corrugator, procerus (e.g., procerus-nasalis) The nasalis), occipitalis, temporalis, trapezius, masseter, nasalis, orbicularis oculi, paraspinal muscles of the neck, temporalis fascia, superior auricle, anterior auricle, posterior auricle, sternocleidomastoid, platysma, anterior nasal dilator, posterior nasal dilator, depressor septi, mentalis, orbicularis oris, zygomaticus, risorius, buccinator, occipitofrontalis, levator labii superioris, depressor labii inferioris, depressor anguli oris, thyrohyoid, omohyoid, sternohyoid, splenius cervi, levator scapulae, digastric, and scalene muscles.

13. The chimeric clostridial neurotoxin for use according to any one of clauses 1-8 or 11-12, wherein the chimeric clostridial neurotoxin is administered by intramuscular injection at up to 10 injection sites per treatment period.

14. The chimeric clostridial neurotoxin for use according to any one of clauses 1 to 8, wherein the chimeric clostridial neurotoxin is administered intraneurally, perineurally or by periganglionic administration.

15. The chimeric clostridial neurotoxin for use according to any one of clauses 1 to 8 or 14, wherein the chimeric clostridial neurotoxin is administered to the trigeminal nerve, the semilunar ganglion, the neurula intermedius, the glossopharyngeal nerve, the vagus nerve and/or via the occipital nerve to the superior cervical root.

16. The chimeric clostridial neurotoxin for use according to any one of clauses 1 to 8, wherein the chimeric clostridial neurotoxin is administered by perivascular administration.

17. The chimeric clostridial neurotoxin for use according to any one of the preceding clauses, wherein the chimeric clostridial neurotoxin is administered in a unit dose of 5 pg to 17,000 pg of the chimeric clostridial neurotoxin.

18. The chimeric clostridial neurotoxin for use according to any one of the preceding clauses, wherein the chimeric clostridial neurotoxin is administered in a unit dose of 500 pg to 17,000 pg.

19. The chimeric clostridial neurotoxin for use according to any one of the preceding clauses, wherein the chimeric clostridial neurotoxin is administered in a unit dose of 1,000 pg to 17,000 pg.

20. The chimeric clostridial neurotoxin for use according to any one of the preceding clauses, wherein the total dose administered per treatment period is up to 255,000 pg of the chimeric clostridial neurotoxin, such as 3,640-255,000 pg.

21. The chimeric clostridial neurotoxin for use according to any one of the preceding clauses, wherein the chimeric clostridial neurotoxin is administered in a unit dose of 3,640 pg to 17,000 pg.

22. A chimeric clostridial neurotoxin for use according to any of the preceding clauses, wherein the chimeric clostridial neurotoxin has a safety ratio greater than 7 (preferably a safety ratio of at least 10), wherein the safety ratio is calculated as: the dose of toxin required for a -10% change in body weight measured in pg/mouse divided by the DAS _EC50 measured in pg/mouse, wherein _EC50 = the dose required to produce a DAS score of 2.

23. The chimeric clostridial neurotoxin for use according to any of the preceding clauses, wherein the C-terminal amino acid residue of the _HN domain corresponds to the first amino acid residue of the _3'0 helix separating the _HN and _HC domains of BoNT/A, and wherein the N-terminal amino acid residue of the _HC domain corresponds to the second amino acid residue of the _3'0 helix separating the _HN and _HC domains of BoNT/B.

24. The chimeric clostridial neurotoxin for use according to any one of the preceding clauses, wherein the chimeric clostridial neurotoxin comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO:1.

25. The chimeric clostridial neurotoxin for use according to any of the preceding clauses, wherein the BoNT/BH _C domain comprises one or more substitution mutations selected from the group consisting of: E1191M; S1199Y; V1118M; Y1183M; E1191I; E1191Q; E1191T; S1199F; S1199L; S1201V and combinations thereof, preferably wherein the BoNT/BH _C domain comprises substitution mutations at E1191M and S1199Y.

Embodiments relating to the various therapeutic uses of the invention are intended to apply equally to the methods, compositions (eg, unit dosage forms), and kits of the invention, and vice versa.

Sequence homology

Any of a variety of sequence alignment methods can be used to determine percent identity, including but not limited to global methods, local methods, and hybrid methods, such as segment methods. Protocols for determining percent identity are routine procedures within the capabilities of those skilled in the art. Global methods align sequences from the beginning to the end of the molecule and determine the best alignment by adding the scores for individual residue pairs and applying gap penalties. Non-limiting methods include, for example, CLUSTAL W, see, for example, Julie D. Thompson et al., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position-Specific Gap Penalties and Weight Matrix Choice, 22 (22) Nucleic Acids Research 4673-4680 (1994); and iterative refinement, see, for example, Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein. Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments, 264 (4) J. MoI. Biol. 823-838 (1996). Local methods align sequences by identifying one or more conserved motifs common to all input sequences. Non-limiting methods include, for example, Match-box, see, for example, Eric Depiereux and Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8(5) CABIOS 501-509 (1992); Gibbs sampling, see, for example, C.E. Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131) Science 208-214 (1993); Align-M, see, for example, Ivo Van WaIIe et al., Align-M-A New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics: 1428-1435 (2004).

Therefore, the sequence identity percentage is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915-19, 1992. In brief, as shown below, two amino acid sequences are aligned using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum 62" scoring matrix of Henikoff and Henikoff (supra) to optimize the alignment score (amino acids are represented by standard single-letter codes); preferably, the method is used to align the sequence with the subject sequence of this article (e.g., SEQ ID NO: 7) to define the amino acid position numbering described herein.

"Percentage of sequence identity" between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by the sequences. Therefore, % identity can be calculated as the number of identical nucleotides/amino acids divided by the total number of nucleotides/amino acids, multiplied by 100. The calculation of % sequence identity can also take into account the number of spaces and the length of each space that need to be introduced to optimize the comparison of two or more sequences. Specific mathematical algorithms such as BLAST that are familiar to those skilled in the art can be used to compare sequences and determine the percentage identity between two or more sequences.

Alignment score used to determine sequence identity

The percent identity is then calculated as follows:

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

Conservative amino acid substitution

Essential amino acids: Arginine

Lysine

Histidine

Acidic amino acids: Glutamic acid

Aspartic acid

Polar amino acid: glutamine

Asparagine

Hydrophobic amino acid: Leucine

Isoleucine

Valine

Aromatic amino acids: Phenylalanine

Tryptophan

Tyrosine

Small amino acid: glycine

Alanine

Serine

Threonine

Methionine

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

Non-naturally occurring amino acids include, but are not limited to, trans-3-methylproline, 2,4-formylproline (2,4-methano-proline), cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allo-threonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine and 4-fluorophenylalanine. Several methods for incorporating non-naturally occurring amino acid residues into proteins are known in the art. For example, an in vitro system can be used in which nonsense mutations are suppressed using chemical aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylated tRNAs are known in the art. Transcription and translation of plasmids containing nonsense mutations are performed in a cell-free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. The protein is purified by chromatography. See, e.g., Robertson et al., J. Am. Chem. Soc. 113:2722, 1991; Ellman et al., Methods Enzymol. 202:301, 1991; Chung et al., Science 259:806-9, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA 90:10145-9, 1993). In a second approach, translation is performed in Xenopus oocytes by microinjection of mutant mRNA and chemically aminoacylated suppressor tRNA (Turcatti et al., J. Biol. Chem. 271:19991-8, 1996). In the third method, E. coli cells are cultured in the absence of the natural amino acid to be replaced (e.g., phenylalanine) and in the presence of the desired non-natural amino acid (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturally occurring amino acid is incorporated into the polypeptide in place of its natural counterpart. See Koide et al., Biochem. 33: 7470-6, 1994. Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the scope of substitution (Wynn and Richards, Protein Sci. 2: 395-403, 1993).

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

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

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

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

The present disclosure is not limited to the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used to practice or test the embodiments of the present disclosure. Numerical ranges include the numbers defining the ranges. Unless otherwise indicated, any nucleic acid sequence is written from left to right in the 5' to 3' direction; amino acid sequences are written from left to right in the direction of amino to carboxyl, respectively.

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

The name, three-letter abbreviation or single-letter abbreviation of amino acid is used herein to refer to amino acid. As used herein, the term "protein" includes protein, polypeptide and peptide. As used herein, the term "amino acid sequence" is synonymous with the term "polypeptide" and/or the term "protein". In some cases, the term "amino acid sequence" is synonymous with the term "peptide". In some cases, the term "amino acid sequence" is synonymous with the term "enzyme". The terms "protein" and "polypeptide" are used interchangeably herein. In the present disclosure and claims, conventional single-letter and three-letter codes of amino acid residues can be used. The 3-letter code of amino acid is defined according to the Joint Committee on Biochemical Nomenclature (JCBN) of IUPAC IUB. It should also be understood that due to the degeneracy of the genetic code, a polypeptide can be encoded by more than one nucleotide sequence.

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

Where a numerical range is provided, it is to be understood that, unless the context clearly indicates otherwise, each intermediate value (to one tenth of the lower limit unit) between the upper and lower limits of the range is also specifically disclosed. Each smaller range between the specified value or intermediate value in any specified range and any other specified value or intermediate value in the specified range is included in the present disclosure. The upper and lower limits of these smaller ranges may be independently included or excluded in the range, and each range (wherein the smaller range includes one of the upper and lower limits, does not include, or includes both) is also included in the present disclosure, subject to any explicitly excluded limit in the range. When the range includes one or two limits, the range excluding one or two of these included limits is also included in the present disclosure.

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

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that such publications constitute prior art to the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present invention will now be described by way of example only with reference to the following drawings and examples. Many of the drawings submitted herein may be better understood in color. The color versions of the drawings are part of the application submitted, and the right to present color images of the drawings in subsequent proceedings is therefore reserved.

Figure 1 shows representative single images (n=3) of untreated aDRG neurons stained with an antibody recognizing cleaved SNAP25 (green) and DAPI for nuclear staining. The bottom image represents a merge of the two channels.

Figure 2 shows representative single images (n=3) of aDRG neurons treated with 1 nM rBoNT/A for 24 hours and stained with antibodies recognizing cleaved SNAP25 (green), antibodies against aDRG markers NF200, CGRP, P2X3, TrkA (red), and DAPI for nuclear staining. Arrows indicate specific areas where co-localization is more pronounced or absent. The bottom image represents a merge of the two channels.

Figure 3 shows representative single images (n=3) of aDRG neurons treated with 1 nM mrBoNT/AB for 24 hours and stained with antibodies recognizing cleaved SNAP25 (green), antibodies against aDRG markers NF200, CGRP, P2X3, TrkA (red), and DAPI for nuclear staining. Arrows indicate specific areas where co-localization is more pronounced or absent. The bottom image represents a merge of the two channels.

Figure 4 shows % CGRP release from aDRG neurons treated with BoNT for 24 hours prior to stimulation with KCl. CGRP release was determined by EIA. Basal CGRP release was subtracted from stimulated release and results were normalized to no-BoNT control cells. Nonlinear curves were fit to each experimental dose response (variable slope - four parameters with the bottom of the curve constrained to 20%) to determine _IC50 . Representative curves fit to KCl-stimulated aDRG treated with mrBoNT/AB (n=3) or rBoNT/A (n=3) are shown, and mean ± SEM are shown. Each experiment was performed in triplicate.

Figure 5 shows the maximum % CGRP release inhibition of aDRG treated with 1 nM BoNT for 24 hours. One-way ANOVA with post hoc Tukey test for multiple comparisons: **-p≤0.01.mrBoNT/AB (n=3) and rBoNT/A (n=3). Each experiment was performed in triplicate (mean±SEM).

The location of preferred intradermal injection sites is shown in Figure 6. The table indicates the number of injections per side of the face for a given target nerve ending and the total number of injections for that target nerve ending.

Figure 7 shows concentration response curves based on CGRP release (pg/ml) for cells treated with: (A) rBoNT/A; or (B) mrBoNT/AB. Each mean (▲) represents data +/- standard deviation of 6 (or 5 for rBoNT/A) samples from three different plates. All individual data points (+) are also included.

Figure 8 shows _pEC50 values for SNAP25 cleavage after 24 h of treatment with mrBoNT/AB (□; 12.14) or rBoNT/A (o; 9.67) in rat primary TG neurons in vitro. Data are mean ± sem of n = 3 experiments. ****p < 0.0001 (Student's unpaired t-test).

FIG. 9 shows concentration-response curve values for SNAP25 cleavage in sensory neurons derived from hiPSCs in vitro after treatment with mrBoNT/AB (pEC ₅₀ =12.85) or rBoNT/A (pEC ₅₀ =10.73) for 24 hours.

Figure 10 shows the proportion (%) of the area of the spinal trigeminal sensory nucleus in the brainstem (left) or trigeminal motor nucleus (right) that stained positive for cleaved SNAP25 in rats administered 300 pg/kg mrBoNT/AB by intramuscular (IM) or intradermal (ID) injection. *p<0.05; Mann-Whitney test.

Figure 11 shows the amount of cleaved SNAP25 in the dorsal horn (sensory) (left) or ventral horn (motor) (right) of the cervical spinal cord in rats administered 300 pg/kg mrBoNT/AB by intramuscular (IM) or intradermal (ID) injection. The amount was expressed by a scoring system ("c-SNAP25 IHC score") as explained in Example 12.

Figure 12 shows the amount of cleaved SNAP25 in trigeminal ganglion axons in rats administered 300 pg/kg mrBoNT/AB by intramuscular (IM) or intradermal (ID) injection or botulinum toxin (Botox) by IM injection. "c-SNAP25 IHC score" was assigned based on the scoring system explained in Example 12. **p<0.01; Kruskal-Wallis test followed by Dunn's multiple comparison test.

Sequence Listing

When an initial Met amino acid residue is indicated in any of the following SEQ ID NOs, said residue is optional.

SEQ ID NO: 1 - Polypeptide sequence of chimeric Clostridial neurotoxin 1 (mrBoNT/AB)

SEQ ID NO:2 - Polypeptide sequence of chimeric clostridial neurotoxin 2

SEQ ID NO:3 - Polypeptide sequence of chimeric clostridial neurotoxin 3

SEQ ID NO:4 - Polypeptide sequence of chimeric clostridial neurotoxin 4

SEQ ID NO:5 - Polypeptide sequence of chimeric clostridial neurotoxin 5

SEQ ID NO:6 - Polypeptide sequence of native BoNT/A (rBoNT/A)

SEQ ID NO:7 - Polypeptide sequence of BoNT/B

SEQ ID NO:8—Polypeptide sequence of BoNT/C

SEQ ID NO:9—Polypeptide sequence of BoNT/D

SEQ ID NO: 10 - Polypeptide sequence of BoNT/E

SEQ ID NO: 11 - Polypeptide sequence of BoNT/F

SEQ ID NO: 12 - Polypeptide sequence of BoNT/G

SEQ ID NO: 13 - Polypeptide sequence of BoNT/X

SEQ ID NO: 14 - polypeptide sequence of TeNT

SEQ ID NO:15—C-terminal L-chain fragment

SEQ ID NO:16—C-terminal L-chain fragment 2

SEQ ID NO: 17 - Double-stranded L-strand 1

SEQ ID NO: 18 - Double-stranded L-strand 2

SEQ ID NO: 19 - Double-stranded H-strand

SEQ ID NO: 1 - Polypeptide sequence of chimeric Clostridial neurotoxin 1 (mrBoNT/AB)

MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSED TSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDF VGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNILNNIILNLRYKDNNLIDLSGYGAKVEVYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIHNEYTIINCMKNNSGWKIS IRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTITNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELSQSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKYNQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFKKEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESGIVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTE

SEQ ID NO:2—Chimeric Clostridial neurotoxin 2 polypeptide sequence

MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTS GKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALI FSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKSEILNNIILNLRYKDNNLIDLSGYGAKVEVYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIHNEYTIINCMKNNSGWKISIRGNRII WTLIDINGKTKSVFFEYNIREDISEYINRWFFVTITNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELSQSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKYNQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFKKEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESGIVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTEHHHHHHHHH

SEQ ID NO:3—Chimeric Clostridial neurotoxin 3 polypeptide sequence

MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKF SVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVI LLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIIELGGGGSELSEILNNIILNLRYKDNNLIDLSGYGAKVEVYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIHNEYTIINCMKNNSGWKISIRGN RIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTITNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELSQSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKYNQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFKKEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESGIVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTEHHHHHHHHH

SEQ ID NO:4—Chimeric Clostridial neurotoxin 4 polypeptide sequence

MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTS GKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALI FSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNILNNIILNLRYKDNNLIDLSGYGAKVEVYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIHNEYTIINCMKNNSGWKISIRGNRII WTLIDINGKTKSVFFEYNIREDISEYINRWFFVTITNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELSQSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKYNQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFKKEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESGIVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTEHHHHHHHHH

SEQ ID NO:5—Chimeric Clostridial neurotoxin 5 polypeptide sequence

MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSED TSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDF VGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNILNNIILNLRYKDNNLIDLSGYGAKVEVYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIHNEYTIINCMKNNSGWKIS IRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTITNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELSQSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKYNQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFKKEEEKLFLAPISDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESGIVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTE

SEQ ID NO:6—Polypeptide sequence of native BoNT/A (rBoNT/A)

MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLS EDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLY KDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNS GWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL

SEQ ID NO:7 - Polypeptide sequence of BoNT/B

MPVTINNFNYNDPIDNNNIIMMEPPFARGTGRYYKAFKITDRIWIIPERYTFGYKPEDFNKSSGIFNRDVCEYYDPDYLNTNDKKNIFLQTMIKLFNRIKSKPLGEKLLEMIINGIPYLGDRRVPLEEFNTNIASVTVNKLISNPGEVERKKGIFANLIIFGPGPVLNENETIDIGIQNHFASREGFGGIMQMKFCPEYVSVFNNVQENKGASIFNRRGYFSDPALILMHELIHVLHGLYGIKVDDLPIVPNEKKFFMQSTDAIQAEELYTFGGQDPSIITPSTDKSIYDKVLQNFRGIVDRLNKVLVCISDPNININIYKN KFKDKYKFVEDSEGKYSIDVESFDKLYKSLMFGFTETNIAENYKIKTRASYFSDSLPPVKIKNLLDNEIYTIEEGFNISDKDMEKEYRGQNKAINKQAYEEISKEHLAVYKIQMCKSVKAPGICIDVDNEDLFFIADKNSFSDDLSKNERIEYNTQSNYIENDFPINELILDTDLISKIELPSENTESLTDFNVDVPVYEKQPAIKKIFTDENTIFQYLYSQTFPLDIRDISLTSSFDDALLFSNKVYSFFSMDYIKTANKVVEAGLFAGWVKQIVNDFVIEANKSNTMDKIADISLIVPYIGLALNVGNETAKGNFENAFEI AGASILLEFIPELLIPVVGAFLLESYIDNKNKIIKTIDNALTKRNEKWSDMYGLIVAQWLSTVNTQFYTIKEGMYKALNYQAQALEEIIKYRYNIYSEKEKSNINIDFNDINSKLNEGINQAIDNINNFINGCSVSYLMKKMIPLAVEKLLDFDNTLKKNLLNYIDENKLYLIGSAEYEKSKVNKYLKTIMPFDLSIYTNDTILIEMFNKYNSEILNNIILNLRYKDNNLIDLSGYGAKVEVYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIHNEYTIINCMKNNSGWKISIRG NRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTITNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELSQSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKYNQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFKKEEEKLFLAPISDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESGIVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTE

SEQ ID NO:8—Polypeptide sequence of BoNT/C

MPITINNFNYSDPVDNKNILYLDTHLNTLANEPEKAFRITGNIWVIPDRFSRNSNPNLNKPPRVTSPKSGYYDPNYLSTDSDKDPFLKEIIKLFKRINSREIGEELIYRLSTDIPFPGNNNTPINTFDFDVDFNSVDVKTRQGNNWVKTGSINPSVIITGPRENIIDPETSTFKLTNNTFAAQEGFGALSIISISPRFMLTYSNATNDVGEGRFSKSEFCMDPILILMHELNHAMHNLYGIAIPNDQTISSVTSNIFYSQYNVKLEYAEIYAFGGPTIDLIPKSARKYFEEKALDYYRSIAKRLNSITTANPSSFNKYIGEY KQKLIRKYRFVVESSGEVTVNRNKFVELYNELTQIFTEFNYAKIYNVQNRKIYLSNVYTPVTANILDDNVYDIQNGFNIPKSNLNVLFMGQNLSRNPALRKVNPENMLYLFTKFCHKAIDGRSLYNKTLDCRELLVKNTDLPFIGDISDVKTDIFLRKDINEETEVIYYPDNVSVDQVILSKNTSEHGQLDLLYPSIDSESEILPGENQVFYDNRTQNVDYLNSYYYLESQKLSDNVEDFTFTRSIEEALDNSAKVYTYFPTLANKVNAGVQGGLFLMWANDVVEDFTTNILRKDTLDKISDVSAIIPYIGPALNISNSVRRG NFTEAFAVTGVTILLEAFPEFTIPALGAFVIYSKVQERNEIIKTIDNCLEQRIKRWKDSYEWMMGTWLSRIITQFNNISYQMYDSLNYQAGAIKAKIDLEYKKYSGSDKENIKSQVENLKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNEFDRNTKAKLINLIDSHNIILVGEVDKLKAKVNNSFQNTIPFNIFSYTNNSLLKDIINEYFNNINDSKILSLQNRKNTLVDTSGYNAEVSEEGDVQLNPIFPFDFKLGSSGEDRGKVIVTQNENIVYNSMYESFSISFWIRINKWVSNLPGYTIIDSVKNNSGW SIGIISNFLVFTLKQNEDSEQSINFSYDISNNAPGYNKWFFVTVTNNMMGNMKIYINGKLIDTIKVKELTGINFSKTITFEINKIPDTGLITSDSDNINMWIRDFYIFAKELDGKDINILFNSLQYTNVVKDYWGNDLRYNKEYYMVNIDYLNRYMYANSRQIVFNTRRNNNDFNEGYKIIIKRIRGNTNDTRVRGGDILYFDMTINNKAYNLFMKNETMYADNHSTEDIYAIGLREQTKDINDNIIFQIQPMNNTYYYASQIFKSNFNGENISGICSIGTYRFRLGGDWYRHNYLVPTVKQGNYASLLESTSTHWGFVPVSE

SEQ ID NO:9—Polypeptide sequence of BoNT/D

MTWPVKDFNYSDPVNDNDILYLRIPQNKLITTPVKAFMITQNIWVIPERFSSDTNPSLSKPPRPTSKYQSYYDPSYLSTDEQKDTFLKGIIKLFKRINERDIGKKLINYLVVGSPFMGDSSTPEDTFDFTRHTTNIAVEKFENGSWKVTNIITPSVLIFGPLPNILDYTASLTLQGQQSNPSFEGFGTLSILKVAPEFLLTFSDVTSNQSSAVLGKSIFCMDPVIALMHELTHSLHQLYGINIPSDKRIRPQVSEGFFSQDGPNVQFEELYTFGGLDVEIIPQIERSQLREKALGHYKDIAKRLNNINKTIPSSWISNI DKYKKIFSEKYNFDKDNTGNFVVNIDKFNSLYSDLTNVMSEVVYSSQYNVKNRTHYFSRHYLPVFANILDDNIYTIRDGFNLTNKGFNIENSGQNIERNPALQKLSSESVVDLFTKVCLRLTKNSRDDSTCIKVKNNRLPYVADKDSISQEIFENKIITDETNVQNYSDKFSLDESILDGQVPINPEIVDPLLPNVNMEPLNLPGEEIVFYDDITKYVDYLNSYYYLESQKLSNNVENITLTTSVEEALGYSNKIYTFLPSLAEKVNKGVQAGLFLNWANEVVEDFTTNIMKKDTLDKISDVSVIIPYIGPALNIGNSA LRGNFNQAFATAGVAFLLEGFPEFTIPALGVFTFYSSIQEREKIIKTIENCLEQRVKRWKDSYQWMVSNWLSRITTQFNHINYQMYDSLSYQADAIKAKIDLEYKKYSGSDKENIKSQVENLKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNKFDLRTKTELINLIDSHNIILVGEVDRLKAKVNESFENTMPFNIFSYTNNSLLKDIINEYFNSINDSKILSLQNKKNALVDTSGYNAEVRVGDNVQLNTIYTNDFKLSSSGDKIIVNLNNNILYSAIYENSSVSFWIKISKDLTNSHNEYTIINSIE QNSGWKLCIRNGNIEWILQDVNRKYKSLIFDYSESLSHTGYTNKWFFVTITNNIMGYMKLYINGELKQSQKIEDLDEVKLDKTIVFGIDENIDENQMLWIRDFNIFSKELSNEDINIVYEGQILRNVIKDYWGNPLKFDTEYYIINDNYIDRYIAPESNVLVLVQYPDRSKLYTGNPITIKSVSDKNPYSRILNGDNIILHMLYNSRKYMIIRDTDTIYATQGGECSQNCVYALKLQSNLGNYGIGIFSIKNIVSKNKYCSQIFSSFRENTMLLADIYKPWRFSFKNAYTPVAVTNYETKLLSTSSFWKFISRDPGWVE

SEQ ID NO: 10—Polypeptide sequence of BoNT/E

MPKINSFNYNDPVNDRTILYIKPGGCQEFYKSFNIMKNIWIIPERNVIGTTPQDFHPPTSLKNGDSSYYDPNYLQSDEEKDRFLKIVTKIFNRINNNLSGGILLEELSKANPYLGNDNTPDNQFHIGDASAVEIKFSNGSQDILLPNVIIMGAEPDLFETNSSNISLRNNYMPSNHRFGSIAIVTFSPEYSFRFNDNCMNEFIQDPALTLMHELIHSLHGLYGAKGITTKYTITQKQNPLITNIRGTNIEEFLTFGGTDLNIITSAQSNDIYTNLLADYKKIASKLSKVQVSNPLLNPYKDVFEAKYGLDKD ASGIYSVNINKFNDIFKKLYSFTEFDLRTKFQVKCRQTYIGQYKYFKLSNLLNDSIYNISEGYNINNLKVNFRGQNANLNPRIITPITGRGLVKKIIRFCKNIVSVKGIRKSICIEINNGELFFVASENSYNDDNINTPKEIDDTVTSNNNYENDLDQVILNFNSESAPGLSDEKLNLTIQNDAYIPKYDSNGTSDIEQHDVNELNVFFYLDAQKVPEGENNVNLTSSIDTALLEQPKIYTFFSSEFINNVNKPVQAALFVSWIQQVLVDFTTEANQKSTVDKIADISIVVPYIGLALNIGNEAQKGNFKDAL ELLGAGILLEFEPELLIPTILVFTIKSFLGSSDNKNKVIKAINNALKERDEKWKEVYSFIVSNWMTKINTQFNKRKEQMYQALQNQVNAIKTIIESKYNSYTLEEKNELTNKYDIKQIENELNQKVSIAMNNIDRFLTESSISYLMKIINEVKINKLREYDENVKTYLLNYIIQHGSILGESQQELNSMVTDTLNNSIPFKLSSYTDDKILISYFNKFFKRIKSSSVLNMRYKNDKYVDTSGYDSNININGDVYKYPTNKNQFGIYNDKLSEVNISQNDYIIYDNKYKNFSISFWVRIPNYDNKIVNVNNEYT IINCMRDNNSGWKVSLNHNEIIWTFEDNRGINQKLAFNYGNANGISDYINKWIFVTITNDRLGDSKLYINGNLIDQKSILNLGNIHVSDNILFKIVNCSYTRYIGIRYFNIFDKELDETEIQTLYSNEPNTNILKDFWGNYLLYDKEYYLLNVLKPNNFIDRRKDSTLSINNIRSTILLANRLYSGIKVKIQRVNNSSTNDNLVRKNDQVYINFVASKTHLFPLYADTATTNKEKTIKISSSGNRFNQVVVMNSVGNCTMNFKNNNGNNIGLLGFKADTVVASTWYYTHMRDHTNSNGCFWNFISEEHGWQEK

SEQ ID NO: 11 - Polypeptide sequence of BoNT/F

MPVVINSFNYNDPVNDDTILYMQIPYEEKSKKYYKAFEIMRNVWIIPERNTIGTDPSDFDPPASLENGSSAYYDPNYLTTDAEKDRYLKTTIKLFKRINSNPAGEVLLQEISYAKPYLGNEHTPINEFHPVTRTTSVNIKSSTNVKSSIILNLLVLGAGPDIFENSSYPVRKLMDSGGVYDPSNDGFGSINIVTFSPEYEYTFNDISGGYNSSTESFIADPAISLAHELIHALHGLYGARGVTYKETIKVKQAPLMIAEKPIRLEEFLTFGGQDLNIITSAMKEKIYNNLLANYEKIATRLSRVNSAPPEYDINEYKDY FQWKYGLDKNADGSYTVNENKFNEIYKKLYSFTEIDLANKFKVKCRNTYFIKYGFLKVPNLLDDDIYTVSEGFNIGNLAVNNRGQNIKLNPKIIDSIPDKGLVEKIVKFCKSVIPRKGTKAPPRLCIRVNNRELFFVASESSYNENDINTPKEIDDTTNLNNNYRNNLDEVILDYNSETIPQISNQTLNTLVQDDSYVPRYDSNGTSEIEEHNVVDLNVFFYLHAQKVPEGETNISLTSSIDTALSEESQVYTFFSSEFINTINKPVHAALFISWINQVIRDFTTEATQKSTFDKIADISLVVPYVGLALNIGNEVQKEN FKEAFELLGAGILLEFVPELLIPTILVFTIKSFIGSSENKNKIIKAINNSLMERETKWKEIYSWIVSNWLTRINTQFNKRKEQMYQALQNQVDAIKTVIEYKYNNYTSDERNRLESEYNINNIREELNKKVSLAMENIERFITESSIFYLMKLINEAKVSKLREYDEGVKEYLLDYISEHRSILGNSVQELNDLVTSTLNNSIPFELSSYTNDKILILYFNKLYKKIKDNSILDMRYENNKFIDISGYGSNISINGDVYIYSTNRNQFGIYSSKPSEVNIAQNNDIIYNGRYQNFSISFWVRIPKYFNKVNLNNEYTII DCIRNNNSGWKISLNYNKIIWTLQDTAGNNQKLVFNYTQMISISDYINKWIFVTITNNRLGNSRIYINGNLIDEKSISNLGDIHVSDNILFKIVGCNDTRYVGIRYFKVFDTELGKTEIETLYSDEPDPSILKDFWGNYLLYNKRYYLLNLLRTDKSITQNSNFLNINQQRGVYQKPNIFSNTRLYTGVEVIIRKNGSTDISNTDNFVRKNDLAYINVVDRDVEYRLYADISIAKPEKIIKLIRTSNSNNSLGQIIVMDSIGNNCTMNFQNNNGGNIGLLGFHSNNLVASSWYYNNIRKNTSSNGCFWSFISKEHGWQEN

SEQ ID NO: 12 - Polypeptide sequence of BoNT/G

MPVNIKNFNYNDPINNDDIIMMEPFNDPGPGTYYKAFRIIDRIWIVPERFTYGFQPDQFNASTGVFSKDVYEYYDPTYLKTDAEKDKFLKTMIKLFNRINSKPSGQRLLDMIVDAIPYLGNASTPPDKFAANVANVSINKKIIQPGAEDQIKGLMTNLIIFGPGPVLSDNFTDSMIMNGHSPISEGFGARMMIRFCPSCLNVFNNVQENKDTSIFSRRAYFADPALTLMHELIHVLHGLYGIKISNLPITPNTKEFFMQHSDPVQAEELYTFGGHDPSVISPSTDMNIYNKALQNFQDIANRLNIVSSAQGSGIDISLYKQIYK NKYDFVEDPNGKYSVDKDKFDKLYKALMFGFTETNLAGEYGIKTRYSYFSEYLPPIKTEKLLDNTIYTQNEGFNIASKNLKTEFNGQNKAVNKEAYEEISLEHLVIYRIAMCKPVMYKNTGKSEQCIIVNNEDLFFIANKDSFSKDLAKAETIAYNTQNNTIENNFSIDQLILDNDLSSGIDLPNENTEPFTNFDDIDIPVYIKQSALKKIFVDGDSLFEYLHAQTFPSNIENLQLTNSLNDALRNNNKVYTFFSTNLVEKANTVVGASLFVNWVKGVIDDFTSESTQKSTIDKVSDVSIIIPYIGPALNVGNETAKENFKNAF EIGGAAILMEFIPELIVPIVGFFTLESYVGNKGHIIMTISNALKKRDQKWTDMYGLIVSQWLSTVNTQFYTIKERMYNALNNQSQAIEKIIEDQYNRYSEEDKMNINIDFNDIDFKLNQSINLAINNIDDFINQCSISYLMNRMIPLAVKKLKDFDDNLKRDLLEYIDTNELYLLDEVNILKSKVNRHLKDSIPFDLSLYTKDTILIQVFNNYISNISSNAILSLSYRGGRLIDSSGYGATMNVGSDVIFNDIGNGQFKLNNSENSNITAHQSKFVVYDSMFDNFSINFWVRTPKYNNNDIQTYLQNEYTIISCIKNDSGWKVS IKGNRIIWTLIDVNAKSKSIFFEYSIKDNISDYINKWFSITITNDRLGNANIYINGSLKKSEKILNLDRINSSNDIDFKLINCTDTTKFVWIKDFNIFGRELNATEVSSLYWIQSSTNTLKDFWGNPLRYDTQYYLFNQGMQNIYIKYFSKASMGETAPRTNFNNAAINYQNLYLGLRFIIKKASNSRNINNDNIVREGDYIYLNIDNISDESYRVYVLVNSKEIQTQLFLAPINDDPTFYDVLQIKKYYEKTTYNCQILCEKDTKTFGLFGIGKFVKDYGYVWDTYDNYFCISQWYLRRISENINKLRLGCNWQFIPVDEGWTE

SEQ ID NO: 13 - Polypeptide sequence of BoNT/X

MKLEINKFNYNDPIDGINVITMRPPRHSDKINKGKGPFKAFQVIKNIWIVPERYNFTNNTNDLNIPSEPIMEADAIYNPNYLNTPSEKDEFLQGVIKVLERIKSKPEGEKLLELISSSIPLPLVSNGALTLSDNETIAYQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEGTLSEVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMHELVHVTHNLYGISNRNFYYNFDTGKIETSRQQNSLIFEELLTFGGIDSKAISSLIIKKIIETAKNNYTTLISERLNTVTVENDLLKYIKNKIPVQGRLG NFKLDTAEFEKKLNTILFVLNESNLAQRFSILVRKHYLKERPIDPIYVNILDDNSYSTLEGFNISSQGSNDFQGQLLESSYFEKIESNALRAFIKICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTNVSYPCSLLNGCIEVENKDLFLISNKDSLNDINLSEEKIKPETTVFFKDKLPPQDITLSNYDFTEANSIPSISQQNILERNEELYEPIRNSLFEIKTIYVDKLTTFHFLEAQNIDESIDSSKIRVELTDSVDEALSNPNKVYSPFKNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVIDKSSDTLAIVPY IGPLLNIGNDIRHGDFVGAIELAGITALLEYVPEFTIPILVGLEVIGGELAREQVEAIVNNALDKRDQKWAEVYNITKAQWWGTIHLQINTRLAHTYKALSRQANAIKMNMEFQLANYKGNIDDKAKIKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMIPKVQDNLKNFDLETKKTLDKFIKEKEDILGTNLSSSLRRKVSIRLNKNIAFDINDIPFSEFDDLINQYKNEIEDYEVLNLGAEDGKIKDLSGTTSDINIGSDIELADGRENKAIKIKGSENSTIKIAMNKYLRFSATDNFSISFWIKHPKPTNLLNNGI EYTLVENFNQRGWKISIQDSKLIWYLRDHNNSIKIVTPDYIAFNGWNLITITNNRSKGSIVYVNGSKIEEKDISSIWNTEVDDPIIFRLKNNRDTQAFTLLDQFSIYRKELNQNEVVKLYNYYFNSNYIRDIWGNPLQYNKKYYLQTQDKPGKGLIREYWSSFGYDYVILSDSKTITFPNNIRYGALYNGSKVLIKNSKKLDGLVRNKDFIQLEIDGYNMGISADRFNEDTNYIGTTYGTTHDLTTDFEIIQRQEKYRNYCQLKTPYNIFHKSGLMSTETSKPTFHDYRDWVYSSAWYFQNYENLNLRKHTKTNWYFIPKDEGWDED

SEQ ID NO: 14 - polypeptide sequence of TeNT

MPITINNFRYSDPVNNDTIIMMEPPYCKGLDIYYKAFKITDRIWIVPERYEFGTKPEDFNPPSSLIEGASEYYDPNYLRTDSDKDRFLQTMVKLFNRIKNNVAGEALLDKIINAIPYLGNSYSLLDKFDTNSNSVSFNLLEQDPSGATTKSAMLTNLIIFGPGPVLNKNEVRGIVLRVDNKNYFPCRDGFGSIMQMAFCPEYVPTFDNVIENITSLTIGKSKYFQDPALLLMHELIHVLHGLYGMQVSSHEIIPSKQEIYMQHTYPISAEELFTFGGQDANLISIDIKNDLYEKTLNDYKAIANKLSQVTSCNDPNIDIDSYKQIYQQ KYQFDKDSNGQYIVNEDKFQILYNSIMYGFTEIELGKKFNIKTRLSYFSMNHDPVKIPNLLDDTIYNDTEGFNIESKDLKSEYKGQNMRVNTNAFRNVDGSGLVSKLIGLCKKIIPPTNIRENLYNRTASLTDLGGELCIKIKNEDLTFIAEKNSFSEEPFQDEIVSYNTKNKPLNFNYSLDKIIVDYNLQSKITLPNDRTTPVTKGIPYAPEYKSNAASTIEIHNIDDNTIYQYLYAQKSPTTLQRITMTNSVDDALINSTKIYSYFPSVISKVNQGAQGILFLQWVRDIIDDFTNESSQKTTIDKISDVSTIVPYIGPALNIVKQGY EGNFIGALETTGVVLLLEYIPEITLPVIAALSIAESSTQKEKIIKTIDNFLEKRYEKWIEVYKLVKAKWLGTVNTQFQKRSYQMYRSLEYQVDAIKKIIDYEYKIYSGPDKEQIADEINNLKNKLEEKANKAMININIFMRESSRSFLVNQMINEAKKQLLEFDTQSKNILMQYIKANSKFIGITELKKLESKINKVFSTPIPFSYSKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLS IGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND

SEQ ID NO:15—C-terminal L-chain fragment

TKSLDKGYNK

SEQ ID NO:16—C-terminal L-chain fragment 2

SLDKGYNK

SEQ ID NO: 17 - Double-stranded L-strand 1

PFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAV TLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSK

SEQ ID NO: 18 - Double-stranded L-strand 2

PFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVT LAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTK

SEQ ID NO: 19 - Double-stranded H-strand

ALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAV ILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNILNN IILNLRYKDNNLIDLSGYGAKVEVYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIHNEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTITNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELSQSNIEERYKI QSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKYNQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFKKEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESGIVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTE

Example

Example 1

Chimeric Clostridial neurotoxin BoNT/AB targets neurons of a different type than BoNT/A

A study was designed to identify neuronal subtypes intoxicated by various clostridial neurotoxins. An in vitro model of adult rat dorsal root ganglia (aDRG) was used. During the characterization of this model, different neuronal subtypes were found. These subtypes reflect the characteristics described in Usoskin, D., A. Furlan, S. Islam, H. Abdo, P. Lonnerberg, D. Lou, J. Hjerling-Leffler, J. Haeggstrom, O. Kharchenko, P. V. Kharchenko, S. Linnarsson and P. Ernfors (2015) "Unbiased classification of sensory neuron types by large-scale single-cell RNA sequencing" Nat Neurosci 18(1): 145-153.

Materials and methods

aDRG cultures

aDRG neurons were generated on glass coverslips. In brief, adult rat DRG tissue was dissected from 2-3 month old SD (Sprague Dawley) rats. The dissected tissue was digested with papain, then digested with dispase/collagenase, and spread on poly-D-lysine and laminin coated glass coverslips. Antimitotic agents were used to inhibit the proliferation of glial cell types in the culture. From DIV 7, it was determined that the neuronal culture could be used for determination.

Immunofluorescence

aRDG neurons were treated with 1 nM native recombinant BoNT/A ("rBoNT/A"—SEQ ID NO:6 [converted to two-chain form]) or BoNT/AB chimera ("mrBoNT/AB"—SEQ ID NO:1 [converted to two-chain form]) at DIV7-8 for 24 hours. Control samples were untreated. After treatment, neurons were washed twice in culture medium and fixed in 4% PFA for 30 minutes. Neurons were then permeabilized using 0.1% TritonX-100 in 1× PBS for 15 minutes and then blocked in 10% donkey serum for 30 minutes. Primary and secondary antibody staining was performed as indicated in the table below.

Table 1. Primary and secondary antibody staining.

Ab anti- company Part Number Species Monoclonal/polyclonal antibodies ^1st / ^2nd dilution DEAN10 Ipsen N/A rabbit Polyclonal antibody ^1st 1∶400 C.SNAP25 Origene AM31671SU-N Mouse Monoclonal antibody ^1st 1∶100 NF200(N52) Sigma N0142-.2ML Mouse Monoclonal antibody ^1st 1∶200 NF200 Abcam ab8135 rabbit Polyclonal antibody ^1st 1∶200 CGRP Stratech 311-CGRP-PPS Mouse Monoclonal antibody ^1st 1∶150 CGRP Sigma C8198 rabbit Polyclonal antibody ^1st 1∶150 P2X3 Stratech GP10108-NEU guinea pig Polyclonal antibody ^1st 1∶150 TrkA Fisher 15710644 goat Polyclonal antibody ^1st 1∶100 Mouse Alexa Fluor 594 Fischer 15960296 donkey Polyclonal antibody ^2nd 5×1 ^st Mouse Alexa Fluor 488 Fischer 16051752 donkey Polyclonal antibody ^2nd 5×1 ^st Rabbit Alexa Fluor 594 Fischer 15910767 donkey Polyclonal antibody ^2nd 5X1 ^st Rabbit Alexa Fluor 488 Thermo A32790 donkey Polyclonal antibody ^2nd 5X1 ^st Guinea pig Alexa Fluor 594 Thermo A-11076 goat Polyclonal antibody ^2nd 5X1 ^st Goat Alexa Fluor 594 Fischer 16331974 donkey Polyclonal antibody ^2nd 5×1 ^st

Coverslips were then mounted on slides with Prolong Diamond mounting medium containing 1 μg/ml DAPI for nuclear staining.

Imaging and colocalization analysis

Images were taken using a LSM800 Zeiss confocal microscope at 40× magnification. At least 3 images were taken for each specific sample and antibody combination, and 3 biological replicates were performed. Intoxicated neurons were determined by using two different antibodies that detect the cleaved isoforms of SNAP25. The aDRG subtype markers used were characteristic of aDRG cultures. These were NF200 for the NF class, CGRP for PEP neurons, P2X3 for NP neurons, and TrkA for NP and PEP neurons. Colocalization was performed by merging two colors of interest (usually green (488) for de-cleaved SNAP25 and red (594) for specific neuronal markers).

result

All images presented in Figures 1-3 show a representative example of a single color image of the cleaved SNAP25 signal and the specific marker (at least 3 images were taken for each biological experiment), as well as a color merge of the two channels with the addition of blue nuclear stain. Where an arrow is present, the area of interest of specific marker/cleaved SNAP25 co-localization is indicated. The untreated control of Figure 1 shows a small amount of background given by the antibody used to detect cleaved SNAP25. This background staining was taken into account when analyzing the treated samples.

Natural recombinant BoNT/A targeting Aβ fibrils

Figure 2 shows the results obtained when aDRG neurons were contacted with rBoNT/A (SEQ ID NO:6 [converted into double-chain form]). In particular, there was a clear colocalization between Aβ fibers (NF200) and cleaved SNAP25. In all images analyzed, the expression of cleaved SNAP25 and NF200 was high. No colocalization was observed for Aδ and C fibers (NP, PEP). Neurons expressing specific markers (CGRP, P2X3, TrkA) do not express high levels of cleaved SNAP25. In the images, it can be seen where cleaved SNAP25 is expressed, however, the signal amount is not higher than the background signal shown in Figure 1 and is significantly lower than the signal given by neurons expressing high levels of cleaved SNAP25. In summary, it was found that the intoxication of rBoNT/A in aDRG neurons occurs in Aβ fibers (NF200).

Chimeric BoNT/AB targets Aδ fibers and C fibers

Figure 3 shows the results obtained when aDRG neurons were contacted with mrBoNT/AB (SEQ ID NO: 1 [converted to double-chain form]). In particular, there was no colocalization between Aβ fibers (NF200) and cleaved SNAP25. The neuronal portion highlighted with arrows showed high expression of NF200 without the presence of cleaved SNAP25. Colocalization was observed for Aδ and C fibers (NP, PEP). In particular, strong colocalization was observed in neurons expressing CGRP and TrkA. In summary, intoxication of mrBoNT/AB in aDRG neurons occurred in Aδ (PEP) and C fibers (NP). This is in contrast to what was seen with rBoNT/A (Figure 2).

in conclusion

Immunofluorescence studies of primary aDRG neurons allowed the identification of the subtypes of neurons intoxicated by rBoNT/A and mrBoNT/AB. The studies revealed clear differences between the subtypes of neurons intoxicated by mrBoNT/AB compared to rBoNT/A. rBoNT/A cleaves SNAP25 in Aβ fibers, whereas mrBoNT/AB cleaves SNAP25 in Aδ and C fibers. These last two subpopulations represent particularly important pain targets, given their roles in nociception. The table below summarizes these differences.

Table 2. Schematic summary of aDRG fiber types targeted by different toxins.

In summary, the data show that BoNT/AB chimeras target cells involved in nociception and therefore may exhibit analgesic effects, such as improved analgesia compared to rBoNT/A.

Example 2

Chimeric Clostridial neurotoxin BoNT/AB effectively inhibits calcitonin gene-related peptide (CGRP) from Aδ and C fibers freed

Based on the findings presented in Example 1, experiments were performed to confirm its role as an analgesic by determining whether BoNT/AB chimeras targeting Aδ and C fibers could inhibit calcitonin gene-related peptide (CGRP) release. CGRP is a neuropeptide found primarily in subsets of C and Aδ sensory fibers from the dorsal root and trigeminal ganglia. Recent studies have implicated CGRP in the development of peripheral sensitization and enhanced pain, neuroinflammation, and neuropathic pain. In support of this, blocking CGRP function has been shown to relieve migraine pain.

Materials and methods

aDRG cultures

Following a slightly modified version of the method given in Example 1, aDRG neurons were plated in 96-well half-volume plates.

CGRP release assay

At DIV7-14, aDRG neurons were treated with 10 nM-1 pM Log10 dilutions of Clostridial neurotoxins for 24 hours. Toxins used: rBoNT/A (SEQ ID NO:6 [converted to double-chain form]) and mrBoNT/AB (SEQ ID NO:1 [converted to double-chain form]). Control samples were untreated. After treatment, neurons were washed twice in HBS (110 mM sodium chloride, 3 mM potassium chloride, 2 mM calcium chloride, 1 mM magnesium chloride, 10 mM HEPES, 20 mM glucose, pH 7.2) and returned to the incubator at 37°C for 1 hour. The cell plate was then transferred to a preheated heating block and washed once more with HBS. The HBS was removed and replaced with 50 μL HBS + 0.03% BSA. After 5 minutes, the HBS/BSA superfusate was removed and stored in a separate plate. Immediately after removing the cast solution, 50 μL of stimulation medium (100 nM capsaicin HBS + 0.03% BSA or 65 nM potassium chloride (KCl) HBS + 0.03% BSA) was added to the appropriate wells. After 5 minutes, the cast solution was collected. After collection, the cast solution was immediately used for CGRP EIA assay or stored at -20°C.

CGRP enzyme immunoassay (EIA)

CGRP immunoassay reagents were purchased as part of a commercial kit (Bertin Pharma, France, #A05482) and prepared according to the manufacturer's instructions. The plates were washed 5 times with wash buffer, followed by the addition of 40 μL of standards and samples. 100 μL of CGRP tracer (prepared as follows: a vial of stock EIA buffer #A07000 diluted with 50 ml of distilled water) diluted with 10 ml of EIA buffer) was then added to each well. The plates were then covered with adhesive tape and incubated at 4°C for 16 to 20 hours. After incubation, the plate contents were discarded and the wells were washed 3 times with wash buffer (prepared as follows: 1 ml of wash buffer stock (#A17000) was diluted with 400 ml of distilled water and 200 μl of Tween-20 (#A12000) was added), followed by a 2-minute shaking step in wash buffer and then washed 3 more times. After removing the wash buffer, 200 μL of Ellman's reagent (prepared as follows: Ellman's reagent stock vial #A09000 diluted in 1 ml of stock wash buffer #A17000 and 49 ml of distilled water) was added to each well. The plate was then covered with foil and incubated in the dark at room temperature for 4 hours. Finally, the plate was read at 410 nm using a Clariostar microplate reader. The standards were plotted on an X-Y plot in Prism V8 (Graphpad) and the sample values were interpolated from the standard curve.

result

Figure 4 shows that mrBoNT/AB is much more effective than rBoNT/A in inhibiting CGRP release. The average _pIC50 values for rBoNT/A and mrBoNT/AB are: 6.87±0.44 (7.34 μM for rBoNT/A) and 9.99±0.16 (9.73 nM for mrBoNT/AB), respectively (mean±SEM).

In confirmation of the results shown in Figure 4, comparison of maximum inhibition showed a significant difference between the inhibition of CGRP release caused by 1 nM mrBoNT/AB when compared to 1 nM rBoNT/A. Specifically, mrBoNT/AB was statistically significantly better than rBoNT/A in inhibiting CGRP release (see Figure 5). This is consistent with the surprising finding that mrBoNT/AB specifically targets Aδ-type fibers and C-type fibers (see Example 1).

in conclusion

The rat aDRG CGRP release model allows for functional comparison of different clostridial neurotoxins in an in vitro pain model. Consistent with the fiber subtype binding specificity of the toxins, mrBoNT/AB was significantly more potent than rBoNT/A in inhibiting CGRP release from aDRGs. Thus, chimeric clostridial neurotoxins with the light chain and translocation domain ( _HN domain) of botulinum neurotoxin A (BoNT/A) and the receptor binding domain ( _HC domain) of BoNT/B are improved analgesics.

Example 3

Treatment of patients with chronic migraine pain

Joe, 43 years old, was diagnosed with chronic migraine by his GP and treated with a chimeric clostridial neurotoxin of the invention comprising SEQ ID NO: 1 (converted into a di-stranded form). The chimeric clostridial neurotoxin was administered at a unit dose of 5,000 pg, with a single unit dose administered by intramuscular injection to each procerus, two corrugators, two masseters, two temporalis, two occipitalis, and two trapezius muscles (i.e., a total of 11×unit doses administered). Nine months later, when Joe received his next treatment, his pain was significantly reduced and he no longer had significant pain.

Example 4

Preclinical testing of the chimeric clostridial neurotoxin BoNT/AB (SEQ ID NO: 1)

BoNT/AB chimera SEQ ID NO: 1 (converted to double-stranded form) was tested in a mouse _LD50 assay and yielded a result of 1.202 ng/kg. Thus, in this assay, 1 unit of SEQ ID NO: 1 (converted to double-stranded form) corresponds to 24.04 pg.

Example 5

Calculation of unit dose of chimeric clostridial neurotoxin BoNT/AB (SEQ ID NO: 1) for the treatment of migraine

In view of preclinical pharmacology data, a suitable unit dose range (UD) for administration of the chimeric clostridial neurotoxin BoNT/AB in humans has been calculated.

The DAS ED ₅₀ of SEQ ID NO: 1 (converted into a double-chain form) was calculated to be 13 pg/kg. The ED ₅₀ is considered to be the minimum pharmacologically active dose, which is approximately 300 times lower than the no observed adverse effect level (NOAEL) of 4 ng/kg in the same animal species. The ED ₅₀ of SEQ ID NO: 1 (converted into a double-chain form) in rats is 13 pg/kg, corresponding to a dose of 0.8 ng for a 60 kg body weight human.

Therefore, the lower limit of the unit dose was selected as 1,000 pg. The upper limit of the unit dose was selected as 5,000 pg, which is lower than the NOAEL of 4 ng/kg from non-clinical safety species (rat and monkey) converted to a human dose of 60 kg body weight.

Taking into account the improved safety, the maximum total dose for the treatment of migraine was set at 175,000 pg, which was based on the NOAEL of 4 ng/kg from two nonclinical safety species (rat and monkey) converted to a human dose of 60 kg body weight.

Advantageously, in treating migraine, the chimeric clostridial neurotoxin BoNT/AB (SEQ ID NO: 1 [converted to di-chain form]) can be injected into more muscles before the maximum dose is reached. This is a significant and advantageous discovery that could improve the treatment of migraine while providing clinicians with a wider range of treatment options.

Example 6

Safety and efficacy of chimeric clostridial neurotoxin BoNT/AB (SEQ ID NO: 1) in humans

SEQ ID NO: 1 (converted to di-stranded form) was administered to human subjects via a single unit dose. Different (increasing) amounts of mrBoNT/AB (SEQ ID NO: 1 [converted to di-stranded form]) were administered to 5 cohorts. Cohort 1 was administered 2×1,000 pg unit doses of mrBoNT/AB (i.e., 2,000 pg maximum), while Cohort 5 was administered 2×16,000 pg unit doses of mrBoNT/AB (i.e., 32,000 pg maximum). The results showed that despite the extremely high dose per muscle, all mrBoNT/AB unit doses tested (i.e., up to 16,000 pg unit dose) were effective for muscle paralysis, were safely tolerated, and no adverse effects were observed. This indicates that mrBoNT/AB does not spread away from the injection site and highlights the excellent safety profile of mrBoNT/AB (SEQ ID NO: 1 [converted to di-stranded form]).

Example 7

Safety and efficacy of chimeric clostridial neurotoxin BoNT/AB (SEQ ID NO: 1) in humans

SEQ ID NO: 1 (converted to di-chain form) was administered to human subjects via a single unit dose. Seven cohorts were administered different (increasing) amounts of mrBoNT/AB (SEQ ID NO: 1 [converted to di-chain form]) to facial muscles. Cohort 1 was administered 5×20 pg unit doses of mrBoNT/AB (i.e., 100 pg maximum), while Cohort 7 was administered 5×1,500 pg unit doses of mrBoNT/AB (i.e., 7,500 pg maximum).

Results showed that despite the high per-muscle dose, all mrBoNT/AB unit doses tested (i.e., up to 1,500 pg unit dose) were effective against muscle paralysis, were safely tolerated, and no adverse effects were observed. This suggests that mrBoNT/AB does not spread away from the injection site and highlights the excellent safety profile of mrBoNT/AB (SEQ ID NO: 1 [converted to double-stranded form]).

Example 8

Treating subjects with chronic migraine pain by intramuscular injection

Derek, 25 years old, was diagnosed with chronic migraine by his GP and treated with a chimeric clostridial neurotoxin of the invention comprising SEQ ID NO: 1 (converted into di-stranded form). The chimeric clostridial neurotoxin comprising SEQ ID NO: 1 (converted into di-stranded form) was administered in a unit dose of 2,500 pg and was administered by intramuscular injection as follows:

2 unit doses to the frontalis muscle on the left side of Derek's face, and 2 unit doses to the frontalis muscle on the right side of Derek's face;

1 unit dose to the procerus muscle;

1 unit dose to the corrugator supercilii muscle on the left side of Derek's face, and 1 unit dose to the corrugator supercilii muscle on the right side of Derek's face;

4 unit doses to the temporalis muscle on the left side of Derek's head, and 4 unit doses to the temporalis muscle on the right side of Derek's head;

3 unit doses to the occipitalis muscle on the left side of Derek's neck/head, and 3 unit doses to the occipitalis muscle on the right side of Derek's neck/head;

3 unit doses to the trapezius muscle on the left side of Derek's face, and 3 unit doses to the trapezius muscle on the right side of Derek's face; and

4 unit doses to the cervical paraspinal muscles on the left side of Derek's neck, and 4 unit doses to the cervical paraspinal muscles on the right side of Derek's neck;

A total of 35 unit doses (i.e., 87,500 pg) of the chimeric clostridial neurotoxin comprising SEQ ID NO: 1 (converted into a double-stranded form) were administered. 9 months later, when Derek received his next treatment, his pain was significantly reduced and he no longer had significant pain. The treatment was safely tolerated and no adverse events were observed.

Example 9

Treating subjects with episodic migraine headaches by intradermal injection

Tessa, 51 years old, was diagnosed with episodic migraine by his GP and treated with a chimeric clostridial neurotoxin of the invention comprising SEQ ID NO: 1 (converted into di-stranded form). The chimeric clostridial neurotoxin comprising SEQ ID NO: 1 (converted into di-stranded form) was administered in a unit dose of 5,000 pg and was administered by intradermal injection as follows:

1 unit dose to the supraorbital nerve area on the first side of Tessa's face and/or 1 unit dose to the supraorbital nerve area on the second side of Tessa's face;

1 unit dose to the supratrochlear nerve area on the first side of Tessa's face and/or 1 unit dose to the supratrochlear nerve area on the second side of Tessa's face;

1 unit dose to the intratrochlear nerve area on the first side of Tessa's face and/or 1 unit dose to the intratrochlear nerve area on the second side of Tessa's face;

1 unit dose to the zygomaticotemporal nerve area on the first side of Tessa's face and/or 1 unit dose to the zygomaticotemporal nerve area on the second side of Tessa's face;

1 unit dose to the zygomaticofacial nerve area on the first side of Tessa's face and/or 1 unit dose to the zygomaticofacial nerve area on the second side of Tessa's face;

2 unit doses to the auriculotemporal nerve area on the first side of Tessa's face and/or 2 unit doses to the auriculotemporal nerve area on the second side of Tessa's face;

2 unit doses to the Tessa cervical first lateral occipital nerve area and/or 2 unit doses to the Tessa cervical second lateral occipital nerve area; and/or

1 unit dose to the lesser occipital nerve area on the first side of Tessa's neck and/or 1 unit dose to the lesser occipital nerve area on the second side of Tessa's neck.

A total of 20 unit doses (i.e., 100,000 pg) of a chimeric clostridial neurotoxin comprising SEQ ID NO: 1 (converted into a double-stranded form) were administered. 9 months later, when Tessa received her next treatment, her pain was significantly reduced and she no longer had significant pain. The treatment was safely tolerated and no adverse events were observed.

Example 10

Chimeric Clostridial neurotoxin BoNT/AB is more effective than BoNT/A in inhibiting calcitonin gene-related peptide (CGRP) from three Release of forebrain neurons

The effects and efficacy of mrBoNT/AB were evaluated in rat primary neurons prepared from the trigeminal ganglion, a structure from which the three sensory branches of the trigeminal nerve emanate. The trigeminal ganglion is a key region rich in neurons (TGN) that is functionally involved in the pathophysiology of migraine. Briefly, primary rat TGN cultures were generated from 5- to 8-week-old rats (see, e.g., Sidders et al. (2018), J Mol Biol., 14; 430(18 Pt A): 3005-3015). After incubation of cells with toxins (rBoNT/A [SEQ ID NO: 6 converted to di-chain form] or mrBoNT/AB [SEQ ID NO: 1 converted to di-chain form]) at concentrations ranging from 1 pM to 100 nM for 24 hours, the cultures were stimulated with 65 mM KCl for 10 minutes to induce the release of CGRP, which is considered to be the main neurotransmitter responsible for the development and propagation of migraine. CGRP release was subsequently measured by ELISA (measured as described in Example 2; n=3, quadruplicate) and TGN were lysed for Western blot analysis of SNAP25.

As shown in FIG. 7 , mrBoNT/AB ( FIG. 7B ) was more effective than rBoNT/A in reducing CGRP release ( FIG. 7A ) and cleaving SNAP25 ( FIG. 8 ).

These data further demonstrate the improved efficacy of mrBoNT/AB (when compared to rBoNT/A) in targeting and inhibiting the release of pain mediators (e.g., CGRP) from neurons implicated in migraine pathophysiology. Thus, it is plausible that administration of a chimeric clostridial neurotoxin having a botulinum neurotoxin A (BoNT/A) light chain and translocation domain ( _HN domain) and a BoNT/B receptor binding domain ( _HC domain) constitutes an improved treatment for pain in general and migraine in particular.

Embodiment 11

Chimeric Clostridial neurotoxin BoNT/AB shows higher potency in human sensory neurons compared to BoNT/A

mrBoNT/AB was compared with rBoNT/A in a pain-relevant human setting, namely sensory neurons derived from human induced pluripotent stem cells (hiPSCs) (cell culture-related methods were as per the manufacturer's instructions: https://www.anatomic.tech/, see also, e.g., Walsh et al. (2020), Stem Cells, 38, 11, 1400–1408). Briefly, hiPSC-derived sensory neurons were cultured for 14 days and subsequently incubated for 24 hours with 3 fM to 1 nM rBoNT/A (SEQ ID NO: 6 converted to double-chain form) or mrBoNT/AB (SEQ ID NO: 1 converted to double-chain form), after which they were lysed and evaluated for SNAP25 cleavage by Western blotting (n=3, triplicate). Figure 9 illustrates that mrBoNT/AB cleaves SNAP25 more efficiently than rBoNT/A in such human cells. Furthermore, this further supports that administration of a chimeric clostridial neurotoxin having a botulinum neurotoxin A (BoNT/A) light chain and translocation domain ( _HN domain) and a BoNT/B receptor binding domain ( _HC domain) constitutes an improved treatment for human pain in general, and human migraine in particular.

Example 12

Chimeric Clostridial neurotoxin BoNT/AB cleaves SNAP25 in neurons involved in pain transmission in vivo

Materials and methods

Unused female Sprague Dawley rats were used for the study (180-220 g at the start of treatment, Janvier labs, France). The animals were placed in an opposite 12-h light/dark cycle (lights on from 18:00 to 06:00) and maintained in an enriched environment at a constant temperature (22 ± 2°C) and humidity (55 ± 5%), with food and water available ad libitum. The animals were adapted for at least 7 days before the experiment. The study was conducted in full compliance with the ARRIVE guidelines, the European Communities Commission Directive 2010/63/EU and the French National Council Decree 87/848.

Animals were administered vehicle (saline, 2 rats/group) or mrBoNT/AB (SEQ ID NO: 1 converted to the di-chain form) (300 pg/kg, 6 rats/group) by intramuscular (IM) or intradermal (ID) injection or animals were administered botulinum toxin (6 rats/group) using IM injection (for trigeminal ganglion analysis). IM administration was performed by distributing the total dose to 4 muscles of the head and neck (right and left temporalis, right and left occipital, 10 μL injection volume each). ID administration was performed by distributing the total dose to the dermis of the skin located above the 4 above muscles (10 μL injection volume each). Ten days after treatment administration, animals were euthanized and the following tissues were harvested: trigeminal ganglia, brainstem containing the trigeminal nucleus, and cervical spinal cord. Tissues were then fixed in isotonic buffered formalin 10% solution (VWR, France) for 48 hours, embedded in paraffin blocks and histological slides were prepared.

To evaluate the biological effects of both toxins in tissues, immunohistochemical staining was performed for the cleaved form of SNAP25 (c-SNAP25). After a heat-induced epitope retrieval step, endogenous peroxidase was blocked in a 3% H ₂ O ₂ solution in TBS buffer for 10 minutes. The sections were incubated with a non-commercial primary rabbit polyclonal antibody (EF14007, IpsenInnovation, France), which is specific only for the SNAP25 form cleaved by BoNT/A. The sections were then incubated with a biotinylated secondary antibody (anti-rabbit IgG, Vector Laboratories, USA) for 30 minutes, followed by an amplification system (avidin-biotin) coupled to horseradish peroxidase (Vector Laboratories, USA) for 30 minutes. Finally, the sections were incubated with a 0.02% diaminobenzidine (DAKO, USA) solution for 5 minutes, counterstained with hematoxylin (DAKO, USA), and the slides were observed under an optical microscope. For trigeminal ganglion samples, the amount of c-SNAP25 was quantified using the following 5-point scale scoring system: 0 (no staining), 1 (minimal staining intensity and density), 2 (moderate staining intensity and density), 3 (strong staining intensity and density) and 4 (very strong staining intensity and density). In the spinal cord, the intensity and density of c-SNAP25-positive nerve endings were graded as follows: 0 (no staining), 1 (minimal), 2 (mild), 3 (moderate), 4 (significant) on the 5 most intensely stained spinal cord sections, and then the cumulative score (0-20) was calculated for each animal. For brainstem samples, SNAP25 cleavage staining was quantified using a dedicated image analysis method that measures the proportion of c-SNAP25-stained nerve fibers.

result

As expected, no specific c-SNAP25 staining was observed in tissues from any vehicle-treated animals. Figure 10 shows that administration of mrBoNT/AB by either the IM or ID route resulted in cleavage of SNAP25 at the spinal trigeminal sensory nucleus of the brainstem, while less cleavage was observed in the trigeminal motor nucleus of the brainstem when administered by the ID route. Lower amounts of SNAP25 cleavage in the motor nucleus may result in reduced off-target effects, such as motor effects associated with treatment.

FIG. 11 shows that administration of mrBoNT/AB by IM or ID route resulted in cleavage of SNAP25 in the cervical spinal cord, specifically in the dorsal and ventral horns.

Figure 12 shows that administration of mrBoNT/AB by IM or ID route results in cleavage of SNAP25 in trigeminal ganglion axons. Surprisingly, botulinum toxin does not cleave SNAP25 in trigeminal ganglion axons, supporting a role for mrBoNT/AB in the treatment of pain, such as the improved efficacy of migraine.

Example 13

SNAP25 and chimeric Clostridial neurotoxin BoNT/AB receptors are present in various human tissues involved in pain transmission middle

Materials and methods

Human tissues were purchased from ProteoGenex (USA), Cureline (USA) and Clinisciences (France) and evaluated for the presence of SNAP25, SytII and SytI. The following tissues were evaluated, with n=3 to 5 donors each:

Pons and medulla oblongata (including parts of the brainstem and containing the central trigeminal motor and sensory nuclei); and

Cervical spinal cord (including dorsal horn).

To quantify protein expression, the same immunohistochemistry technique as described in Example 12 was used. Tissues were stained with antibodies against SNAP25 (using antibody 111 008), SytII (using antibody 105 123), and SytI (using antibody ab126253). Tissues were also stained with antibodies against β-3-tubulin (using antibody G7121), a pan-neuronal marker as a control.

Staining was quantified under light microscopy and the staining intensity was graded using a score of 0 to 4, where 0 = no staining; 1 = very weak/very sparse/infrequent staining; 2 = moderate staining; 3 = strong/frequent staining; 4 = very strong/intense/extremely frequent staining.

result

The results are shown in Table 3. β-3-tubulin control staining and SNAP25 staining were graded as the maximum value (4) in the pons, medulla oblongata, and cervical spinal cord. Meanwhile, SytII and SytI were found to be strongly expressed in the pons, medulla oblongata, and dorsal horn layers 1, 3, and 4. SytI was strongly expressed in dorsal horn layer 2, while SytII showed moderate staining in this structure.

Taken together, these results indicate that SNAP25, SytII, and SytI are highly expressed in several human tissues associated with pain transmission. Therefore, there are suitable SytII and SytI receptors for mrBoNT/AB to bind, internalize, and cleave SNAP25 in neurons present in these tissues, e.g., after mrBoNT/AB is transported (e.g., retrogradely) from neurons distal to the site of administration, thereby inhibiting pain transmission.

Table 3: Staining intensity of SNAP25, SytII and SytI in various human tissues related to pain transmission

Embodiment 14

Treatment for patients with migraine

Timothy, 33 years old, was diagnosed with migraine by his GP. He was treated by administering a unit dose (UD) of 2,500 pg of SEQ ID NO: 1 (converted to double-stranded form) as follows:

Intramuscular injection Total number of injection sites Dosage per injection site Dosage per course of treatment Frontalis 4 (2 pieces per side) 2.5ng 10ng Corrugator supercilii 2 (1 piece on each side) 2.5ng 5ng Nasal muscles 2 (1 piece on each side) 2.5ng 5ng Orbicularis oculi muscle 2 (1 piece on each side) 2.5ng 5ng Temporalis muscle 8 (4 pieces per side) 2.5ng 20ng Occipital muscle 6 (3 pieces per side) 2.5ng 15ng Trapezius 4 (2 pieces per side) 2.5ng 10ng total 28 70ng

He received a total dose of 70,000 pg of SEQ ID NO: 1 (converted to double-stranded form). The treatment was successful and his symptoms were relieved. He did not require treatment for more than 9 months.

Embodiment 15

Treatment for patients with migraine

Joseph, 31 years old, was diagnosed with migraine by his GP. He was treated by administering a unit dose (UD) of 4,000 pg of SEQ ID NO: 1 (converted to double-stranded form) as follows:

Intramuscular injection Total number of injection sites Dosage per injection site Dosage per course of treatment Frontalis 4 (2 pieces per side) 4ng 16ng Corrugator supercilii 2 (1 piece on each side) 4ng 8ng Nasal muscles 2 (1 piece on each side) 4ng 8ng Orbicularis oculi muscle 2 (1 piece on each side) 4ng 8ng Temporalis muscle 8 (4 pieces per side) 4ng 32ng Occipital muscle 6 (3 pieces per side) 4ng 24ng Trapezius 4 (2 pieces per side) 4ng 16ng total 28 112ng

He received a total dose of 112,000 pg of SEQ ID NO: 1 (converted to double-stranded form). The treatment was successful and his symptoms were relieved. He did not require treatment for more than 9 months.

All publications mentioned in the above description are incorporated herein by reference. Without departing from the scope and spirit of the present invention, various modifications and variations of the method and system of the present invention will be apparent to those skilled in the art. Although the present invention has been described in conjunction with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to these specific embodiments. In fact, various modifications of the modes for implementing the present invention that are apparent to those skilled in the art of biochemistry and biotechnology or related fields are intended to fall within the scope of the appended claims.

Claims (87)

1. A chimeric clostridial neurotoxin for use in the treatment of pain, wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin a (BoNT/a) light chain and translocation domain (H _N domain) and a BoNT/B receptor binding domain (H _C domain).

2. A method of treating pain, the method comprising administering to a subject a chimeric clostridial neurotoxin, wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin a (BoNT/a) light chain and translocation domain (H _N domain) and a BoNT/B receptor binding domain (H _C domain).

3. Use of a chimeric clostridial neurotoxin in the manufacture of a medicament for the treatment of pain, wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin a (BoNT/a) light chain and translocation domain (H _N domain) and a BoNT/B receptor binding domain (H _N domain).

4. A chimeric clostridial neurotoxin, method or use for said use according to any one of claims 1-3, wherein said chimeric clostridial neurotoxin treats pain by inhibiting release of a pain mediator from a neuron comprising an aδ or C nerve fiber, wherein said chimeric clostridial neurotoxin binds to a neuron comprising an aδ or C nerve fiber, respectively.

5. The chimeric clostridial neurotoxin, method or use for said use according to any one of the preceding claims, wherein said chimeric clostridial neurotoxin treats pain by inhibiting secretion of central nervous system neurons, preferably by inhibiting secretion of a mediator, more preferably a pain mediator, from central nervous system neurons.

6. A chimeric clostridial neurotoxin for use in the treatment of migraine, preferably migraine pain, wherein said chimeric clostridial neurotoxin comprises a botulinum neurotoxin a (BoNT/a) light chain and translocation domain (H _N domain) and a BoNT/B receptor binding domain (H _C domain).

7. A method of treating migraine (preferably migraine pain), the method comprising administering to a subject a chimeric clostridial neurotoxin, wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin a (BoNT/a) light chain and translocation domain (H _N domain) and a BoNT/B receptor binding domain (H _C domain).

8. Use of a chimeric clostridial neurotoxin in the manufacture of a medicament for the treatment of migraine (preferably migraine pain), wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin a (BoNT/a) light chain and translocation domain (H _N domain) and a BoNT/B receptor binding domain (H _C domain).

9. The chimeric clostridial neurotoxin, method or use for the use according to any one of claims 6 to 8, wherein the chimeric clostridial neurotoxin treats migraine by inhibiting release of a pain mediator from a neuron comprising an aδ or C nerve fiber, wherein the chimeric clostridial neurotoxin binds to a neuron comprising an aδ or C nerve fiber, respectively.

10. The chimeric clostridial neurotoxin, method or use for said use according to any one of claims 6 to 9, wherein said chimeric clostridial neurotoxin treats migraine by inhibiting secretion of central nervous system neurons, preferably by inhibiting secretion of mediators, more preferably pain mediators, from central nervous system neurons.

11. A chimeric clostridial neurotoxin for use in a method of treating a disorder in a subject, the method having a longer duration and/or a better therapeutic effect than treating the subject with BoNT/a, the method comprising administering to the subject a chimeric clostridial neurotoxin, wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin a (BoNT/a) light chain and translocation domain (H _N domain) and a BoNT/B receptor binding domain (H _C domain).

12. A method for treating a condition in a subject, the method treating the condition in the subject for a longer duration and/or with a better therapeutic effect than a subject treated with BoNT/a, the method comprising administering to the subject a chimeric clostridial neurotoxin, wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin a (BoNT/a) light chain and translocation domain (H _N domain) and a BoNT/B receptor binding domain (H _C domain).

13. Use of a chimeric clostridial neurotoxin in the manufacture of a medicament for treating a disorder in a subject, the medicament treating the disorder in the subject for a longer duration and/or with a better therapeutic effect than a subject treated with BoNT/a, wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin a (BoNT/a) light chain and translocation domain (H _N domain) and a BoNT/B receptor binding domain (H _C domain).

14. A chimeric clostridial neurotoxin for use in reducing pain in a subject, the method reducing pain in a subject to a greater extent than treating a subject with BoNT/a, the method comprising administering to a subject a chimeric clostridial neurotoxin, wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin a (BoNT/a) light chain and translocation domain (H _N domain) and a BoNT/B receptor binding domain (H _C domain).

15. A method of reducing pain in a subject to a greater extent than treating a subject with BoNT/a, the method comprising administering to the subject a chimeric clostridial neurotoxin, wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin a (BoNT/a) light chain and translocation domain (H _N domain) and a BoNT/B receptor binding domain (H _C domain).

16. Use of a chimeric clostridial neurotoxin in the manufacture of a medicament for reducing pain in a subject to a greater extent than a subject treated with BoNT/a, wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin a (BoNT/a) light chain and translocation domain (H _N domain) and a BoNT/B receptor binding domain (H _C domain).

17. A chimeric clostridial neurotoxin for use in reducing the amount of a pain mediator in a biofluid and/or brain of a subject, the method reducing the amount of a pain mediator in the biofluid and/or brain of a subject to a greater extent than the amount reduced by the same biofluid and/or brain of a BoNT/a treated subject, the method comprising administering to the subject a chimeric clostridial neurotoxin, wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin a (BoNT/a) light chain and translocation domain (H _N domain) and a BoNT/B receptor binding domain (H _C domain).

18. A method for reducing the amount of a pain mediator in a biological fluid and/or brain of a subject, the method comprising administering to the subject a chimeric clostridial neurotoxin, wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin a (BoNT/a) light chain and translocation domain (H _N domain) and a BoNT/B receptor binding domain (H _C domain) compared to the amount reduced in the same biological fluid and/or brain of a subject treated with BoNT/a.

19. Use of a chimeric clostridial neurotoxin in the manufacture of a medicament for reducing the amount of a pain mediator in a biological fluid and/or brain of a subject, the medicament reducing the amount of a pain mediator in the biological fluid and/or brain of a subject to a greater extent than the amount reduced by the same biological fluid and/or brain of a subject treated with BoNT/a, wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin a (BoNT/a) light chain and translocation domain (H _N domain) and a BoNT/B receptor binding domain (H _C domain).

20. The chimeric clostridial neurotoxin, method or use for the use according to any one of claims 11 to 19, wherein the chimeric clostridial neurotoxin inhibitory mediator (preferably pain mediator) is released from a neuron comprising an aδ or C nerve fiber, wherein the chimeric clostridial neurotoxin binds to a neuron comprising an aδ or C nerve fiber, respectively.

21. The chimeric clostridial neurotoxin, method or use for the use according to any one of claims 11 to 20, wherein said chimeric clostridial neurotoxin inhibits secretion of central nervous system neurons, preferably of inhibiting mediators, more preferably pain mediators, from central nervous system neurons.

22. The chimeric clostridial neurotoxin, method or use according to any of the preceding claims for said use, wherein said chimeric clostridial neurotoxin is transported by a neuron (e.g. retrograde) into a neuron of the central nervous system and cleaves a SNARE protein (e.g. SNAP 25) of said neuron.

23. A chimeric clostridial neurotoxin for use in a method of treating a sensory disorder by inhibiting release of a mediator from a neuron comprising an aδ or C nerve fiber, wherein the chimeric clostridial neurotoxin binds to a neuron comprising an aδ or C nerve fiber, respectively, and wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin a (BoNT/a) light chain and translocation domain (H _N domain) and a BoNT/B receptor binding domain (H _C domain).

24. A method of treating a sensory disorder by inhibiting release of a mediator from a neuron comprising an aδ or C nerve fiber, the method comprising administering to a subject the chimeric clostridial neurotoxin, wherein the chimeric clostridial neurotoxin binds to a neuron comprising an aδ or C nerve fiber, respectively, and wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin a (BoNT/a) light chain and translocation domain (H _N domain) and a BoNT/B receptor binding domain (H _C domain).

25. Use of a chimeric clostridial neurotoxin in the manufacture of a medicament for treating a sensory disorder by inhibiting the release of a mediator from a neuron comprising an aδ or C nerve fiber, wherein the chimeric clostridial neurotoxin binds to a neuron comprising an aδ or C nerve fiber, respectively, and wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin a (BoNT/a) light chain and translocation domain (H _N domain) and a BoNT/B receptor binding domain (H _C domain).

26. The chimeric clostridial neurotoxin for use according to any one of claims 1-5 or 11-25, method or use, wherein said pain or disorder is headache pain such as migraine pain or cluster headache pain, or bladder pain.

27. The chimeric clostridial neurotoxin for use according to any one of claims 1-5 or 11-26, method or use, wherein said pain or disorder is migraine pain.

28. The chimeric clostridial neurotoxin, method or use according to any one of claims 1-3, 5-8, 10-19, 21-22 or 26-27 for said use, wherein said chimeric clostridial neurotoxin binds to a neuron comprising an aδ or C nerve fiber.

29. The chimeric clostridial neurotoxin, method or use according to any one of claims 1-3, 5-8, 10-19, 21-22 or 26-27 for said use, wherein said chimeric clostridial neurotoxin inhibits release of a mediator from a neuron comprising an aδ or C nerve fiber.

30. The chimeric clostridial neurotoxin, method or use for the use according to any of the preceding claims, wherein the chimeric clostridial neurotoxin is used for treating pain, migraine or disorder by inhibiting the release of a mediator (e.g. a pain mediator) from neurons comprising aδ or C nerve fibers and by inhibiting the secretion (e.g. a mediator, preferably a pain mediator) from neurons of the central nervous system, wherein the chimeric clostridial neurotoxin binds to neurons comprising aδ or C nerve fibers, respectively.

31. Chimeric clostridial neurotoxin, method or use according to any of the preceding claims for said use, wherein said mediator (preferably pain mediator) is a neurotransmitter (preferably pain neurotransmitter).

32. The chimeric clostridial neurotoxin, method or use for said use according to any one of the preceding claims, wherein said (pain) mediator is one or more selected from the group consisting of: calcitonin gene-related peptide (CGRP); substance P; and neurokinins.

33. The chimeric clostridial neurotoxin, method or use for the use according to any of the preceding claims, wherein the (pain) mediator is CGRP and the pain is CGRP related pain or the disorder is CGRP related pain, or wherein CGRP related migraine pain is treated when migraine is treated.

34. The chimeric clostridial neurotoxin for use, method or use according to claim 33, wherein said CGRP related pain is CGRP related headache pain.

35. The chimeric clostridial neurotoxin for use, method or use according to claim 33 or 34, wherein said CGRP related pain is CGRP related migraine pain.

36. The chimeric clostridial neurotoxin, method or use for the use according to any one of claims 33 to 35, wherein the CGRP related pain is:

(a) CGRP related somatic pain is selected from: headache pain (e.g., post-traumatic headache, head injury headache or post-traumatic brain injury headache), arthritic pain (e.g., osteoarthritis pain and/or rheumatoid arthritis pain), motor pain, degenerative disc disease pain, carpal tunnel compression pain, soft tissue injury pain, temporomandibular joint pain, musculoskeletal pain, CGRP related somatic pain caused by or associated with vascular disorders (e.g., raynaud's syndrome, thromboangiitis obliterans, peripheral venous disease, peripheral arterial disease, varicose veins, venous thrombosis, coagulation disorders or lymphedema), facial pain, CGRP related somatic pain caused by or associated with trigeminal autonomic nerve headache; CGRP-related somatic pain caused by or associated with trigeminal neuralgia; pain caused by CGRP-related cancers (e.g., bone pain caused by CGRP-related cancers);

(b) CGRP related visceral pain is selected from: endometriosis pain, pancreatitis pain, gastrointestinal pain, and CGRP-related visceral pain caused by or associated with vascular disorders;

(c) CGRP related inflammatory pain is selected from: chronic pain, wound healing pain, itching pain, and burn pain; and/or

(D) CGRP related neuropathic pain is selected from: postherpetic neuralgia, diabetic pain, chronic neuropathic pain and Morton neuroma pain.

37. The chimeric clostridial neurotoxin, method or use for the use according to any one of the preceding claims, wherein the neuron is a neuron of a trigeminal ganglion.

38. The chimeric clostridial neurotoxin, method or use for the use according to any one of the preceding claims, wherein said chimeric clostridial neurotoxin is applied to the face, neck and/or skull.

39. The chimeric clostridial neurotoxin, method or use for the use according to any one of the preceding claims, wherein said chimeric clostridial neurotoxin is administered intradermally.

40. The chimeric clostridial neurotoxin, method or use for the use according to any one of the preceding claims, wherein said chimeric clostridial neurotoxin is administered by intradermal injection at up to 10 injection sites during each treatment.

41. The chimeric clostridial neurotoxin, method or use for the use according to any one of claims 1 to 39, wherein the chimeric clostridial neurotoxin is administered by intradermal injection at 10-40 injection sites during each treatment.

42. The chimeric clostridial neurotoxin, method or use for use according to any one of claims 1 to 39 or 41, wherein the chimeric clostridial neurotoxin is administered by intradermal injection at 25-35 injection sites during each treatment.

43. The chimeric clostridial neurotoxin, method or use for the use according to any one of claims 1 to 38, wherein the chimeric clostridial neurotoxin is administered intramuscularly.

44. The chimeric clostridial neurotoxin, method or use according to any one of claims 1 to 38 or 43 for said use, wherein the chimeric clostridial neurotoxin is administered by intramuscular injection at up to 10 injection sites during each treatment.

45. The chimeric clostridial neurotoxin, method or use according to any one of claims 1 to 38 or 43 for said use, wherein said chimeric clostridial neurotoxin is administered by intramuscular injection at 10-40 injection sites during each treatment.

46. The chimeric clostridial neurotoxin, method or use for use according to any one of claims 1 to 38, 43 or 45, wherein the chimeric clostridial neurotoxin is administered by intramuscular injection at 25-35 injection sites during each treatment.

47. The chimeric clostridial neurotoxin, method or use for the use according to any one of the preceding claims, comprising administering the chimeric clostridial neurotoxin to at least one of the following: the muscle of the forehead, the frown (e.g., frown (corrugator supercilii)), the frown (e.g., frown-nasal (procerus nasalis)), the occipital (e.g., upper or lower occipital), temporal (e.g., upper, middle or lower trapezius), biting, nasal, orbicularis oculi, cervical paraspinus, temporal fascia, supra-aural, pre-aural, post-aural, sternocleidomastoid, platykurtic, anterior nardostachycardia, posterior nardostachycardia, genius, oroorbicular, zygomatic, smile, buccinal, occipital, levator, lower labial, lowing, cantoneus, formar, shoulder, sternohyoid, cervical, semi-acanthus, semi-cephalic, biceps, levator, or levator muscles.

48. The chimeric clostridial neurotoxin, method or use for the use according to any one of the preceding claims, comprising administering a chimeric clostridial neurotoxin to at least one of: frontal muscle, frowning muscle (e.g., frowning muscle (corrugator supercilii)) muscle, nasal muscle, orbicularis oculi muscle, temporal muscle, occipital muscle, or trapezius muscle.

49. The chimeric clostridial neurotoxin, method or use for the use according to any one of the preceding claims, comprising administering a chimeric clostridial neurotoxin to: frontal muscle, frowning muscle (e.g., frowning muscle (corrugator supercilii)) muscle, nasal muscle, orbicularis oculi muscle, temporal muscle, occipital muscle, and trapezius muscle.

50. The chimeric clostridial neurotoxin, method or use for the use according to any one of the preceding claims, wherein the administration of the chimeric clostridial neurotoxin comprises:

(a) 2 injections into the frontal muscle (preferably 2 injections per frontal muscle);

(b) 1 injection to frowning muscle (preferably 1 intramuscular injection per frowning muscle);

(c) 1 injection to nasal muscle (preferably 1 intramuscular injection per nasal muscle);

(d) Injecting 1 time to orbicularis oculi muscle (preferably 1 time per orbicularis oculi muscle);

(e) 4 injections into the temporomyous muscle (preferably 4 injections per temporomyous muscle);

(f) 3 injections into the occipital muscle (preferably 3 injections per occipital muscle); and

(G) From 2 injections to trapezius muscle (preferably 2 injections per trapezius muscle).

51. The chimeric clostridial neurotoxin, method or use for the use according to any one of the preceding claims, wherein the administration of the chimeric clostridial neurotoxin comprises:

(a) 4 injections into the frontal muscle (preferably 2 injections into the frontal muscle on the first side of the face, and 2 injections into the frontal muscle on the second side of the face);

(b) 2 injections into the frowning muscle (preferably 1 injection into the frowning muscle on the first side of the face, and 1 injection into the frowning muscle on the second side of the face);

(c) Injecting 2 times to the nasal muscle (preferably 1 time to the nasal muscle on the first side of the face, and 1 time to the nasal muscle on the second side of the face);

(d) Injecting 2 times to orbicularis oculi muscles (preferably injecting 1 time to orbicularis oculi muscles on the first side of the face, and injecting 1 time to orbicularis oculi muscles on the second side of the face);

(e) Injecting 8 times to temporo-muscular muscles (preferably injecting 4 times to temporo-muscular muscles of the first side of the head, and injecting 4 times to temporo-muscular muscles of the second side of the head);

(f) Injecting 6 times to the occipital muscle (preferably 3 times to the occipital muscle on the first side of the head, and 3 times to the occipital muscle on the second side of the head); and

(G) 4 injections into the trapezius muscle (preferably 2 injections into the trapezius muscle on the first side of the neck and 2 injections into the trapezius muscle on the second side of the neck).

52. The chimeric clostridial neurotoxin, method or use for the use according to any one of the preceding claims, wherein said chimeric clostridial neurotoxin is administered by means of a unit dose per injection (e.g. per injection site).

53. The chimeric clostridial neurotoxin, method or use for the use according to any one of the preceding claims, wherein the administration of the chimeric clostridial neurotoxin comprises administration of:

(a) 2 unit doses to the frontal muscle (preferably 2 unit doses per frontal muscle);

(b) 1 unit dose to a frowning muscle (preferably 1 unit dose per frowning muscle);

(c) 1 unit dose to nasal muscle (preferably 1 unit dose per nasal muscle);

(d) 1 unit dose to orbicularis oculi muscle (preferably 1 unit dose per orbicularis oculi muscle);

(e) 4 unit doses to the temporo-muscular muscles (preferably 4 unit doses per temporo-muscular muscle);

(f) 3 unit doses to occipital muscle (preferably 3 unit doses per occipital muscle); and

(G) From 2 unit doses to trapezius muscle (preferably 2 unit doses per trapezius muscle).

54. The chimeric clostridial neurotoxin, method or use for the use according to any one of the preceding claims, wherein the administration of the chimeric clostridial neurotoxin comprises:

(a) 4 unit doses to the frontal muscle (preferably 2 unit doses to the facial first side frontal muscle, and 2 unit doses to the facial second side frontal muscle);

(b) 2 unit doses to the frowning muscle (preferably 1 unit dose to the frowning muscle on the first side of the face, and 1 unit dose to the frowning muscle on the second side of the face);

(c) 2 unit doses to the nasal muscle (preferably 1 unit dose to the nasal muscle on the first side of the face, and 1 unit dose to the nasal muscle on the second side of the face);

(d) 2 unit doses to orbicularis oculi muscles (preferably 1 unit dose to orbicularis oculi muscles on the first side of the face, and 1 unit dose to orbicularis oculi muscles on the second side of the face);

(e) 8 unit doses to the temporo-muscular muscle (preferably 4 unit doses to the temporo-muscular muscle on the first side of the head and 4 unit doses to the temporo-muscular muscle on the second side of the head);

(f) 6 unit doses to occipital muscle (preferably 3 unit doses to occipital muscle on the first side of the head, and 3 unit doses to occipital muscle on the second side of the head); and

(G) 4 unit doses to trapezius muscle (preferably 2 unit doses to trapezius muscle on the first side of the neck and 2 unit doses to trapezius muscle on the second side of the neck).

55. The chimeric clostridial neurotoxin, method or use for the use according to any one of the preceding claims, wherein the chimeric clostridial neurotoxin is administered in a unit dose of 5pg to 17,000pg chimeric clostridial neurotoxin.

56. The chimeric clostridial neurotoxin, method or use for the use according to any one of the preceding claims, wherein the chimeric clostridial neurotoxin is administered in a unit dose of 500pg to 17,000 pg.

57. The chimeric clostridial neurotoxin, method or use for the use according to any one of the preceding claims, wherein the chimeric clostridial neurotoxin is administered in a unit dose of 1,000pg to 17,000 pg.

58. The chimeric clostridial neurotoxin, method or use for use according to any of the preceding claims, wherein the total dose administered during each treatment is at most 255,000pg of chimeric clostridial neurotoxin, such as 3,640-255,000pg or at most 160,000pg (preferably at most 155,000 pg).

59. The chimeric clostridial neurotoxin, method or use for the use according to any of the preceding claims, wherein the total dose administered during each treatment is at most 120,000pg, preferably at most 112,000pg.

60. The chimeric clostridial neurotoxin, method or use for the use according to any of the preceding claims, wherein the total dose administered during each treatment is at most 100,000pg, preferably at most 70,000pg.

61. The chimeric clostridial neurotoxin, method or use for the use according to any one of the preceding claims, wherein the chimeric clostridial neurotoxin is administered in a unit dose of 3,640pg to 17,000 pg.

62. The chimeric clostridial neurotoxin, method or use for the use according to any one of the preceding claims, wherein the chimeric clostridial neurotoxin is administered in a unit dose of 1,000 to 5,500 pg.

63. The chimeric clostridial neurotoxin, method or use for the use according to any one of the preceding claims, wherein the chimeric clostridial neurotoxin is administered in a unit dose of 2,000 to 4,500 pg.

64. The chimeric clostridial neurotoxin, method or use for the use according to any one of the preceding claims, wherein the chimeric clostridial neurotoxin is administered in a unit dose of 3,500 to 4,500pg or 2,000 to 3,000pg (e.g. 2,500 pg).

65. The chimeric clostridial neurotoxin, method or use for the use according to any one of the preceding claims, wherein the chimeric clostridial neurotoxin is administered in a unit dose of 4,000 pg.

66. The chimeric clostridial neurotoxin, method or use for use according to any one of claims 47 to 65, wherein the administration to (e.g. injection to) a muscle is: by intramuscular injection or by intradermal injection in the intramuscular area; preferably by intramuscular injection.

67. The chimeric clostridial neurotoxin, method or use for the use according to any one of claims 1 to 38 or 47 to 65, wherein the chimeric clostridial neurotoxin is administered by intra-neural, peri-neural or by periganglionic administration.

68. The chimeric clostridial neurotoxin, method or use for the use according to any one of claims 1 to 38, 47 to 65 or 67, wherein the chimeric clostridial neurotoxin is administered to the trigeminal nerve, the semilunar ganglion, the interneuron, the glossopharyngeal nerve, the vagus nerve and/or via the occipital nerve to the upper cervical root.

69. The chimeric clostridial neurotoxin, method or use for the use according to any one of claims 1 to 38 or 47 to 65, wherein the chimeric clostridial neurotoxin is administered by perivascular administration.

70. Chimeric clostridial neurotoxin for use according to any of the preceding claims, method or use, wherein said treatment is a prophylactic treatment, preferably a prophylactic treatment of migraine.

71. A unit dosage form (e.g., for treating pain), the unit dosage form comprising:

5pg to 17,000pg of a chimeric clostridial neurotoxin; or (b)

0.2 Units up to 707 units of chimeric clostridial neurotoxin, wherein 1 unit is the amount of chimeric clostridial neurotoxin corresponding to the calculated half lethal dose (LD ₅₀) of the mouse; and

C. optionally a pharmaceutically acceptable carrier, excipient, adjuvant and/or salt;

Wherein the chimeric clostridial neurotoxin comprises a botulinum neurotoxin a (BoNT/a) light chain and translocation domain (H _N domain) and a BoNT/B receptor binding domain (H _C domain).

72. The unit dosage form of claim 71, wherein the unit dosage form comprises:

1,000 to 5,500pg of a chimeric clostridial neurotoxin; or (b)

42 Units to at most 229 units of chimeric clostridial neurotoxin, wherein 1 unit is the amount of chimeric clostridial neurotoxin corresponding to the calculated half lethal dose (LD ₅₀) of the mouse.

73. The unit dosage form of claim 71 or 72, wherein the unit dosage form comprises 2,000 to 4,500pg of chimeric clostridial neurotoxin.

74. The unit dosage form of any one of claims 71-73, wherein the unit dosage form comprises 3,500 to 4,500pg or 2,000 to 3,000pg (e.g., 2,500 pg) of chimeric clostridial neurotoxin.

75. The unit dosage form of any one of claims 71-74, wherein the unit dosage form comprises 4,000pg of the chimeric clostridial neurotoxin.

76. Chimeric clostridial neurotoxin, method, use or unit dosage form for use according to any of the preceding claims, wherein the chimeric clostridial neurotoxin has a safety ratio (preferably a safety ratio of at least 10) of more than 7, wherein the safety ratio is calculated as: the toxin dose required for-10% weight change measured in pg/mouse divided by DAS ED ₅₀ measured in pg/mouse, where ED ₅₀ = the dose required to produce a DAS score of 2.

77. The chimeric clostridial neurotoxin, method, use or unit dosage form for use according to any of the preceding claims, wherein the C-terminal amino acid residue of the H _N domain corresponds to the first amino acid residue of the 3 ₁₀ helix separating the H _N and H _C domains of BoNT/a, and wherein the N-terminal amino acid residue of the H _C domain corresponds to the second amino acid residue of the 3 ₁₀ helix separating the H _N and H _C domains of BoNT/B.

78. The chimeric clostridial neurotoxin, method, use or unit dosage form for use according to any of the preceding claims, wherein said chimeric clostridial neurotoxin comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID No. 1.

79. The chimeric clostridial neurotoxin, method, use or unit dosage form for use according to any of the preceding claims, wherein said chimeric clostridial neurotoxin is a double-stranded chimeric clostridial neurotoxin, wherein the light chain (L-chain) is linked to the heavy chain (H-chain) by a disulfide bond obtainable by a method comprising contacting a single-stranded chimeric clostridial neurotoxin comprising SEQ ID NO:1 with a protease that hydrolyses the peptide bond in its activation loop, thereby converting the single-stranded chimeric clostridial neurotoxin into the corresponding double-stranded chimeric clostridial neurotoxin.

80. The chimeric clostridial neurotoxin, method, use or unit dosage form for use according to any of the preceding claims, wherein said chimeric clostridial neurotoxin is a double-stranded chimeric clostridial neurotoxin, wherein the L-chain is linked to the H-chain by a disulfide bond obtainable by a method comprising contacting a single-stranded chimeric clostridial neurotoxin consisting of SEQ ID NO 1 with a protease that hydrolyses the peptide bond in its activation loop, thereby converting the single-stranded chimeric clostridial neurotoxin into the corresponding double-stranded chimeric clostridial neurotoxin.

81. The chimeric clostridial neurotoxin, method, use or unit dosage form for use according to any one of the preceding claims, wherein said BoNT/B H _C domain comprises one or more substitution mutations selected from the group consisting of: E1191M; S1199Y; V1118M; Y1183M; E1191I; E1191Q; E1191T; S1199F; S1199L; S1201V; and combinations thereof.

82. The chimeric clostridial neurotoxin, method, use or unit dosage form for use according to any of the preceding claims, wherein said BoNT/B H _C domain comprises substitution mutations at E1191M and S1199Y.

83. The chimeric clostridial neurotoxin, method, use or unit dosage form for use according to any of the preceding claims, wherein the starting methionine amino acid residue of the polypeptide sequence of the chimeric clostridial neurotoxin is optional.

84. The chimeric clostridial neurotoxin, method, use or unit dosage form for use according to any one of the preceding claims, wherein the starting methionine amino acid residue of the polypeptide sequence of the chimeric clostridial neurotoxin is absent.

85. Chimeric clostridial neurotoxin, method, use or unit dosage form for use according to any of the preceding claims, wherein said chimeric clostridial neurotoxin is a double-stranded chimeric clostridial neurotoxin comprising (or consisting of) a light chain comprising SEQ ID NO 17 or 18 (preferably SEQ ID NO 17) and a heavy chain comprising SEQ ID NO 19, wherein the light chain and heavy chain are linked together by a disulfide bond.

86. A kit, comprising:

(a) The unit dosage form of any one of claims 71-85; and

(B) Instructions for using the same, for example, to treat pain; and

(C) Optionally a diluent.

87. A method of determining whether a clostridial neurotoxin is suitable for treating pain, the method comprising:

(a) Comparing the level of a calcitonin gene-related peptide (CGRP) contained in the first sample to the level of CGRP contained in the second sample, wherein the first sample is obtained from the subject prior to administration of the clostridial neurotoxin, and wherein the second sample is obtained from the same subject after administration of the clostridial neurotoxin; and

(B) Determining that the clostridial neurotoxin is suitable for treating pain when the CGRP level in the second sample is lower than the CGRP level in the first sample; or alternatively

(C) When the CGRP level in the second sample is not lower (e.g., is higher than or equal to) the CGRP level in the first sample, it is determined that the clostridial neurotoxin is not suitable for treating pain.