WO2023105289A1 - Methods and compositions for the treatment of pain - Google Patents

Abstract

Methods and compostions for inhibiting the sensation of acute pain in a mammal. The methods may include locally administering to one or more neurons proj ecting to the trigeminal ganglia of said mammal a composition that truncates intact synaptosomal-associated protein 25 ( SNAP-25 ) in the cytoplasm of said at least one or more neurons to yield a plurality of truncated SNAP-25 molecules lacking 26 amino acids from the C-terminal end thereof. The method may comprising intracellularly administering said composition to one or more neurons innervating or proj ecting to a site of potential acute pain. The composition may comprise a protease, such as an ef fective amount of a Cl ostridi um botulinum neurotoxin (BoNT ) - derived light chain endopeptidase derived from BoNT subtype E. The compostion may comprise an ef fective amount of a Cl ostridi um botulinum neurotoxin (BoNT ) -derived active protein having a cell-binding domain, and preferably a translocation domain, derived from BoNT subtype A and a light chain endopeptidase derived from BoNT subtype E. In some embodiments the compostion may comprise more than one protease, such as a light chain endopeptidase derived from BoNT subtype E and a light chain endopeptidase derived from BoNT subtype A. In currently preferred embodiments, the composition may comprise a polypeptide selected from LC/E-BoNT/A and/or BoNT/EA.

Description

METHODS AND COMPOSITIONS FOR THE TREAMENT OF PAIN

Cross-Reference to Related Applications

This application claims the benefit of U.S. provisional patent application Serial No. 63/286,189, filed December 6, 2021, which is hereby incorporated by reference herein in its entirety.

Field of the Invention

The present invention is drawn to methods and compositions for the treatment of pain in a mammal, particularly head, neck and facial pain; preferably acute pain such as acute headache pain and migraine pain.

Background of the Invention

Pain is a distressing feeling often caused by intense or damaging stimuli; it has been defined as "an unpleasant sensory and emotional experience associated with, or resembling that associated with, actual or potential tissue damage". In medical diagnosis, pain is usually regarded as a symptom of an underlying condition. However, the existence of pain does not necessary correlate with the discovery of such an underlying condition.

Pain motivates the individual to withdraw from damaging situations, to protect a damaged body part while it heals, and to avoid similar experiences in the future. When a painful stimulus is removed, most types of pain tend to decrease in intensity as the body is healed. However, in other cases pain may persist after all indicia of, and damage caused by, the stimulus appears to have subsided. Additionally, sometimes pain arises in the absence of any detectable stimulus, damage or disease.

Chronic pain, such as that caused by rheumatoid arthritis, peripheral neuropathy, cancer and idiopathic pain, may persist for years. By contrast, pain that is capable of being resolved more quickly, such as upon removal of the painful stimulus or healing of the affected body part is termed "acute" pain. Traditionally, the distinction between acute and chronic pain has relied upon the interval of time between onset and resolution of the pain. The most commonly used time markers to differentiate chronic and acute pain has been to determine pain levels at 3 months and 6 months (and/or 1 year) after the onset of pain. Others apply the term "acute" to pain that lasts less than 30 days and "chronic" to pain of more than six months' duration.

In contrast to the unpleasant sensory and emotional subjective experience of pain, the physiological process associated with pain is termed "nociception". Nociception deals with the series of physiological and biochemical events and processes required for an organism to receive a painful stimulus, convert it to a molecular signal, and recognize and characterize the signal in order, for example, to trigger an appropriate defense response. Thus, in nociception noxious chemical, mechanical and/or thermal stimulation of afferent sensory nerves (nociceptors) produces one or more signals that travel along a chain of nerve fibers (often via the spinal cord or brain stem) to one or more regions of the brain.

As used herein, the following terms have the following meanings, unless clearly intended otherwise by the context:

By "derive", "derived from" and "derivative" a Clostridial botulinum neurotoxin (BoNT) or part thereof is meant having the identical amino acid sequence of a wild type of the referenced BoNT or part thereof, or a substantially identical amino acid sequence differing from the wild type of the referenced BoNT or part thereof by no more than 10%, or 8%, or 6%, or 4%, or 2%. Additionally, those of ordinary skill are aware that a BoNT derivative or part thereof may comprise minor differences in amino acid sequence as artifacts of molecular biological techniques related to its production as a recombinantly produced protein caused by techniques involving, without limitation, the insertion of restriction endonuclease sites and or the insertion of alteration of nucleotide residues to encode additional peptidase sites. When used with reference to a nucleotide sequence encoding a BoNT or part thereof, the terms derive", "derived from" and "derivative" means nucleotide sequence identical to a nucleotide sequence encoding a wild type BoNT or part thereof, or any other nucleotide sequence which encodes a BoNT derivative, as defined herein.

By "chemical activation", "nociceptior activation", a "nocicpetive neural signal" (or terms similar to these terms) is meant at least one nociceptive signaling event associated with a noxious stimulus and normally resulting in the sensation of pain. Thus, a nociceptive neural signal may refer to, without limitation, CGRP release from sensory neurons into the synapse, substance P release from sensory neurons, somatostatin release from sensory neurons, neural depolarization, and/or increased Ca⁺⁺ ion influx.

Unless otherwise clear from the context of usage, by "at least one" neuron or nociceptor is meant to include one or more individual neurons, a nerve comprising a bundle of neurons or neural processses, ganglia comprising the cell bodies of first order neurons, and spinal cord and brain structures including the trigeminal nucleus caudalis, the brainstem, the thalamus, and/or the cerebral cortex.

By "inhibit" or "inhibiting" is meant to prevent or reduce an amount or degree of the action that is inhibited.

By a "noxious chemical" is meant a chemical which, when contacted with a efferent nociceptor elicits a nociceptive activation or a nocicpetive neural signal in at least one nociceptor.

Cutaneous nociceptors are a homogenous group of neurons responding to noxious stimuli and having nerve endings in the skin. These neurons can be resolved into distinct classes based upon their activation profiles. Stimuli adequate for such nociceptor activation may include temperature extremes, intense pressure, and chemicals that signal actual or potential tissue damage. Nociceptors are generally silent and transmit action potentials only when stimulated.

However, nociceptor activation does not lead to the perception of pain per se without the transmission of peripheral information to higher centers, and normally depends on the frequency of action potentials in primary afferents and central nervous system influences. Furthermore, nociceptive activation stimulates the exocytosis of various neurotransmitters such as glutamate and various peptides, which may include substance P, calcitonin gene-related peptide (CGRP) and somatostatin, important in central synaptic signaling and efferent signaling in the skin.

Most nociceptors have small diameter, unmyelinated axons (C-fibers), and conduct action potentials at relatively low velocies of about 0.4-1.4 m/sec. Initial fast-onset pain is mediated by A-fiber nociceptors whose axons are myelinated and support conduction velocities of about 5-30 m/sec.

Additionally, nociceptors are classified on the basis of their sensitivity and activation threshold responses to noxious mechanical (M), heat (H) and cold (C). The most common nociceptors are polymodal, responding to thermal, mechanical and chemical stimuli. The expression of different combinations of transduction moleucles (particularly chemical sensors) confers a large functional hetergeneity to polymodal nociceptors.

Nociceptive sensors include integral membrane ion channels involved in sensation, such as those of the transient receptor potential (TRP) superfamily. The TRP superfamily is subdivided into seven subfamilies: TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPP (polycystin), TRPML (mucolipin), TRPA (ankyrin). Nociceptive stimuli activate TRP channels located on nerve endings, which cause first-order neurons to depolarize and fire action potentials. Action potential frequency determines stimulus intensity. A delta fibers release glutamate onto second-order neurons, while C fibers release neuropeptide neurotransmitters.

Two TRP subfamilies are particularly important with respect to their ability to respond to a noxious chemical stimulus.

TRPV1, a nonselective cation channel found in the trigeminal ganglia that may be activated by a wide variety of exogenous and endogenous physical and chemical stimuli, is selectively activated by capsaicin (the irritating compound in chili peppers).

The TRPA1 channel, while structurally and functionally related to TRPV1, is expressed in adult mammals in a distinct (although overlapping) subset of sensory neurons and has a different chemo- and thermo-sensitivity profile from TRPV1. TRPA1 is a non-selective integral cation channel implicated in both acute and chronic head and facial pain such as migraine due to its expression in trigeminal sensory nerves and activation by a range of chemicals recognised to be initiators or sensitisers of this type of pain. Allyl isothiocyanate (AITC), a pungent noxious substance produced by plants such as mustard, wasabi and horseradish, selectively activates TRPA1 and is a useful tool for the study of TRPA1-mediated pain behavior. When activated by their respective chemical activators, each of TRPA1 and TRPV1 generate an action potential through depolarization of the neuronal cell membrane and stimulate the cellular release of neurotransmitters. These neurotransmitters may perpetuate the signal along the neural pathway, and/or may directly or indirectly stimulate the body to generate a defense response, such as by generating an efferant neural signal to muscles or other cells involved in the body response.

Most of the body's sensory neurons are contained in the dorsal root ganglia (DRG), which emerge from the intervertebral neural foramina. The DRG are groups of neural cell bodies responsible for the transmission of sensory messages from afferent receptors such as thermoreceptors, nociceptors, proprioceptors, and chemoreceptors, to the central nervous system (CNS) for a response. DRG neurons are considered pseudounipolar neurons, with one axon that bifurcates into two separate branches resulting in a distal process and proximal process. Action potentials generated by impulses from the periphery do not always need to go through the DRG; they may also bypass the cell bodies of the DRG and continue through to the proximal process and spinal cord. Studies have suggested that the DRG are active participants in peripheral processes, including injury caused by platelet-activating factor (RAF), inflammation, and chronic neuropathic pain development.

A different grouping of neural cells make up the trigeminal system, a neural pathway distinct from the DRG. The trigeminal ganglia (TG) are part of the trigeminal nerves, which gather sensory stimuli from the head and face and transmits this information directly to the brainstem, where the stimulus is then relayed to the thalamus and cerebral cortex. These sensory signals bypass the DRG and spinal cord.

Migraine is a debilitating neurological disease having symptoms that are distinct from other headache conditions. Researchers currently believe that migraine is the result of fundamental neurological abnormalities caused by genetic mutations at work in the brain. New models are aiding scientists in studying the basic science involved in the biological cascade, genetic components and mechanisms of migraine.

Migraine is characterized in part by moderate to acute severe head pain which may be hard to endure and may last from 7 to 72 hours if untreated. Migraine is often an episodic condition, with some migraineurs suffering 14 or more migraine headaches per month.

In migraine, impulses from the cortex, thalamus, and hypothalamus activate the so-called "migraine center" responsible for the generation of migraine attacks, putatively located in the brain stem (serotonergic raphe nuclei, locus ceruleus). The migraine center triggers cortical spreading depression (suppression of brain activity across the cortex) accompanied by oligemia, often resulting in an aura. Trigeminovascular input from meningeal vessels is relayed to the brain stem, via projecting fibers to the thalamus and then, by the parasympathetic efferent pathway, back to the meningeal vessels (trigeminal autonomic reflex circuit). Perivascular trigeminal C-fiber endings (trigeminovascular system) are stimulated to release vasoactive neuropeptides such as substance P, neurokinin A, and calcitonin gene-regulated polypeptide (CGRP), causing a (sterile) inflammatory response. Vasoconstriction and vascular hyperesthesia with subsequent vasodilatation spread via trigeminal axon reflexes. The perception of pain is mediated by the pathway from the trigeminal nerve to the trigeminal nucleus caudalis, thalamus and cortex. Trigeminal impulses also reach autonomic centers.

Botulinum neurotoxin (BoNT) serotypes A-G, produced by Clostridium botulinum, are the most potent poisons known. These toxins function by specifically blocking the release of acetylcholine from peripheral nerves by proteolytically cleaving SNARE ("Soluble NSF Attachment Protein Receptors") proteins, which mediate the fusion of the synaptic vesicle with the cell membrane. Fusion of synaptic vesicles in the axon of neurons are essential for Ca²⁺-stimulated exocytosis of neurotransmitters from the neuron. Due to its persistent activity, BoNT serotype A (BoNT/A) has achieved great success as a therapeutic agent in the treatment of various neurological conditions caused by activity of cholinergic nerves supplying various muscles and glands.

The botulinum toxins possess a minimum of approximately 35% amino acid identity with each other and share the same general functional domain organization and overall structural architecture. The naturally-occurring Clostridial toxins are each translated as a single chain polypeptide of approximately 150 kDa that is subsequently cleaved by proteolytic scission within a disulfide loop by a naturally occurring protease, such as, e.g., an endogenous Clostridial toxin protease or a naturally occurring protease produced in the environment. This post-translational processing yields a mature di-chain molecule comprising an approximately 50 kDa light chain (LC) and an approximately 100 kDa heavy chain (HC) held together by a single inter-chain disulfide bond and noncovalent interactions.

Each mature di-chain Clostridial toxin molecule comprises three functionally distinct domains: 1) an enzymatic domain located in the LC that includes a metalloprotease region containing a zinc-dependent endopeptidase activity which specifically targets specific peptide bonds in one or more SNARE proteins that mediate the fusion of the synaptic vesicle with the cell membrane; 2) a translocation domain contained within the amino-terminal half of the H chain (termed "H_N") that facilitates release of at least the LC chain of the toxin from an endosome into the cytoplasm of the target cell; and 3) a binding domain found within the carboxyl-terminal half of the H chain (H_c) that determines the binding activity and binding specificity of the toxin.

It will be understood that there exist strains or subtypes of each serotype of these toxins; these may vary somewhat in their amino acid sequences, particularly (but not exclusively) in non-critical regions (so called "variable" regions) without a substantial change in the identity or activity characteristic of the indicated toxin or toxin domain.

In Table 1 below, the one-letter and three letter amino acid codes are provided:

Table 2

Those of ordinary skill in the art recognize that naturally-occurring Clostridial domain variants having variations in the amino acid shown above (or in the nucleotide sequences encoding these amino acid sequences) may occur in nature. As used herein, the term "naturally-occurring Clostridial domain variant" means any Clostridial domain (endopeptidase, translocation, and/or binding domains) produced by a naturally-occurring process, including, without limitation, Clostridial domain isoforms produced from alternatively-spliced transcripts, Clostridial domain isoforms produced by spontaneous mutations and Clostridial domain subtypes. As used herein, a naturally-occurring Clostridial domain variant functions in substantially the same manner as the reference Clostridial domain on which the naturally-occurring Clostridial domain variant is based, and can be substituted for the reference Clostridial domain in any aspect of the present invention.

A naturally-occurring Clostridial domain variant may contain substitutions in one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, ten or more amino acids, 20 or more amino acids, 30 or more amino acids, 40 or more amino acids, 50 or more amino acids or 100 or more amino acids from the reference Clostridial domain on which the naturally- occurring Clostridial domain variant is based. A naturally- occurring Clostridial domain variant can also contain substitutions in at least 10 contiguous amino acids, at least 15 contiguous amino acids, at least 20 contiguous amino acids, or at least 25 contiguous amino acids from the reference Clostridial domain on which the naturally-occurring Clostridial domain variant is based, and may possess at least 50% amino acid identity, 65% amino acid identity, 75% amino acid identity, 85% amino acid identity or 95% amino acid identity to the reference Clostridial domain on which the naturally-occurring Clostridial domain variant is based. It will also be understood that conservative amino acid insertions and deletions can also be made so long as the characteristic function and identity of the domain is not substantially altered.

Due to the degeneracy of the genetic code, one of ordinary skill in the art will recognize that these amino acid sequences may be encoded by a finite set of different DNA molecules having different, but defined, nucleotide sequences. For example, degenerate nucleotide sequences encoding a given peptide or protein may have different codons adapted or selected to favor expression in a particular host cell. Using this information one can construct an expressible open nucleic acid reading frame for assembly of a nucleic acid molecule comprising any combination of these amino acid domain-encoding regions, either alone or with additional nucleic acid sequences, inserted into a suitable expression vector and subsequent expression within a chosen host cell. For example, International Patent Publication W001/14570 discloses methods of making single-chain, protease- cleavable recombinant modified or unmodified Clostridial neurotoxin derivatives and chimeric and hybrid forms thereof using such methods. Additional publications disclosing methods of making expressible recombinant neurotoxins and derivatives thereof include U.S. Patents 5,989,545; 6,203,794; 6,395,513; 9,216,210 and 10,457,927; U.S. Publication Numbers U.S. 2003/0166238; U.S. 2002/169942; U.S. 2004/176299; U.S. 2004/126397; U.S. 2005/035730; U.S. 2005/068494; and U.S. 2006/011966; International Patent Applications W095/32738; WO 99/55359; W096/33273; W098/07864; W099/17806; WO98/07864; WO02/44199; W002/40506, and U.S. Patent Application Serial No. 13/644,386, filed October 4, 2012. These and all other patents, patent publications, and non-patent publications cited in this patent application, whether or not specifically indicated as such, are hereby individually incorporated by reference as part of this specification.

The use of recombinant DNA techniques permits the construction of modified Clostridial neurotoxins having different or modified functional properties from the naturally- occurring toxin subtypes and strains thereof. For example, altering the naturally-occurring amino acid sequence of the native neurotoxin light chain and/or adding a different therapeutic moiety permits the construction of transport proteins designed to carry a therapeutic agent within a neuron. See U.S. Patent No. 6,203,794. Altering the targeting (cellbinding) domain permits the toxin to be transported within pancreatic cells, such as acinar cells, thereby preventing secretion of activated digestive enzymes by such cells, See U.S. Patent No. 6,843,998, or sensory afferent neurons, see U.S. Patent No. 6,395,513.

In addition, US Patent No. 7,422,877 discloses the creation of chimeric neurotoxin derivatives comprising, for example, the binding domain and the translocation domain (or modified versions thereof) of one neurotoxin subtype (for example, BoNT/A) and the light chain region of another neurotoxin subtype, for example, BoNT/E. It will be seen that given the general structural homology between the neurotoxin subtypes, any combination of the three basic Clostridial neurotoxin domains, may be made in a single amino acid chain (or in cleaved di-chain molecules). Therefore, for example, a binding domain from any of neurotoxin subtypes A, B, Cl, D, E, F, G, or TeTX may be independently combined with a translocation domain from neurotoxin subtypes A, B, Cl, D, E, F, G, or TeTX, and further independently combined with a endopeptidase domain from any of neurotoxin subtypes A, B, Cl, D, E, F, G or TeTX. This can be done, for example, by recombinant construction and expression of a single chimeric chain which is subsequently cleaved to yield the dichain toxin, or by separate expression of single H and L chains, which are then combined by, for example, creation of an interchain disulfide bond and subsequently purified. Furthermore, using such techniques, the activity of various domains may be altered (for example, mutations can be introduced in an LC domain to destroy the protease activity of the LC), or the naturally-occurring domains may be replaced with other moieties, as described elsewhere herein, where for example, the HC domain of BoNT/A (or a portion thereof) is mutated or deleted and a targeting ligand (TL) appended.

When discussing the three general neurotoxin domains of each Clostridial neurotoxin subtype (binding, translocation and endopeptidase), it will be understood that Clostridial neurotoxin research is a well-developed field, and the correlation of the amino acid sequences comprising each of these domains with their functions is well known. Additionally, the subdivision of these general domains into subdomains is also known. Reference to each of these terms ("translocation domain", "binding domain", and "protease", "endopeptidase", "LC" or "light chain" domain) shall be understood to include the corresponding domains contained in any of the amino acid sequences of Clostridial neurotoxin subtypes listed in SEQ ID NO: 7-14 as appearing in Table 2, as well as conservatively modified and optimized variants of these sequences or domains within these sequences.

Additionally, the subdivision of these general domains into subdomains is also known. For example, the subdivision of binding domain H_c into subdomains H_CN (the amino-terminal portion of the domain, corresponding approximately to amino acids 871- 1091 of BoNT/A) and H_cc (the carboxy-terminal portion of the H_c domain, corresponding approximately to amino acids 1092-1296 of BoNT/A) is also well known. See e.g., Lacy DB and Stevens RC, Sequence Homology and Structural Analysis of the Clostridial Neurotoxins, 1999, J. Mol. Biol, 291:1091-1104. Subdomain H_CN is highly conserved among botulinum toxin subtypes, however, the H_Cc subdomain is less conserved.

Additionally, the nucleotide and amino acid sequences of each of these domains and subdomains are known and have been disclosed in this specification, and therefore by using this disclosure in combination with knowledge of the genetic code, nucleotide sequences encoding a protein to be expressed can be made. It would, of course, be a matter of routine for a person of ordinary skill in the art to immediately envision other nucleotide sequences encoding the indicated polypeptides. Also, due to the redundancy of the genetic code, a finite number of nucleotide sequences are possible for each polypeptide. Further, it is clear that nucleic acids can be synthesized that comprise conservatively modified variants of these nucleotide sequences (or unique portions of them) in the region of homology containing no more than 10%, 8% or 5% base pair differences from a reference sequence.

Further, it will be understood that the amino acid sequences set forth in Table 2 and elsewhere in this specification or the associated sequence listing provide a full disclosure of any and all nucleotide sequences encoding these amino acid sequences and indicated regions thereof. A nucleotide sequence encoding an endopeptidase domain, translocation domain, or binding domain (including any subdomain) of a given neurotoxin subtype may respectively have 60% or greater, or 65% or greater, or 70% or greater, or 75% or greater, or 80% or greater, or 85% or greater, or 90% or greater, or 95% or greater, or 100% identity to any of such reference amino acid sequence regions listed in Table 2 and/or elsewhere in this specification.

Botulinum neurotoxins are expressed by Clostridial cells which also produce one or more non-toxin "neurotoxin associated proteins" or NAPs that non-covalently associate with the neurotoxin to form hemagglutinin complexes, also known as progenitor complexes. These NAPS help the neurotoxin resist protease degradation in the intestine when it is ingested in contaminated food.

The NAP proteins include three hemagglutinin (HA) proteins (HA1, HA2 and HA3), and a non-toxic, nonhemagglutinin protein (NTNH). BoNT types A2, E and F do not have the HA genes, and only produce a 12S (about 300 kDa) complex comprising BoNT and NTNH. "S" stands for Svedberg unit, a unit of centrifugal sedimentation rate. Types B, Cl and D produce 12S and 16S (about 500 kDa) complexes; the 16S complex includes BoNT, NTNH, HA1, HA2 and HA3. Type Al has the 12S and 16S complexes plus a 19S complex of about 900 kDA, which may represent a dimer of 16S complexes.

Currently, BoNT/Al- and /B-hemagglutinin complexes have been approved for such clinical uses. The therapeutic benefits of BoNT/Al complex are more persistent than that of BoNT/B due to its protease having a longer life-time in neurons. As indicated above, BoNTs consist of a light chain- associated protease domain (LC) which is linked to a heavy chain (HC) through a single covalent disulphide bond and additional non-covalent bonds. A carboxy terminal (C-terminal) moiety of HC (HC) binds to its specific acceptors expressed on various nerve types, including motor, autonomic and sensory neurons. When bound to a target cell the BoNT molecule is transported into vesicles by endocytosis; the amino terminal (N-terminal) half of HC (HN) forms a channel that allows the LC to translocate from 'endosomal-like' membrane vesicles into the cytosol. Thereafter, the LC cleaves a specific SNARE protein substrate, thereby destroying the SNARE's ability to mediate vesicle-membrane fusion, and thus neurotransmitter, cytokine and pain peptide exocytotic release from the cell.

The LCs of the various BoNT serotypes are similar, but not identical, and two different LCs may cleave different SNARE proteins, or cleave the same SNARE protein differently. For example, LC/A, LC/C1, and LC/E cleave SNAP-25; LC/B, LC/D, LC/F, and LC/G cleave synaptobrevin-2 (VAMP-2); additionally, LC/C1 cleaves syntaxin, another SNARE protein which has been reported to be required for cell division. The LC of TeTx cleaves VAMP- 2. The LCs of each serotype cleave their substrate at unique position in the molecule.

For example, the light chain of BoNT/A (LC/A) removes 9 amino acids from the C-terminus of SNAP-25, whereas the LC/E deletes a further 17 C-terminal residues and, thus, gives a more disruptive blockade of neuro-exocytosis by destabilising stable SNARE complexes. To illustrate, the inhibition of neurotransmitter release by LC/A can usually be reversed by elevating Ca²⁺ influx but this reversal is not seen in the case of LC/E, presumably due to the greater destruction of the SNAP- 25 substrate. However, despite this greater "robustness" of activity, LC/E induces only short transient neuromuscular paralysis and therefore the clinical applications of the use of BoNT/E as a therapeutic agent are limited.

BoNT/A is unable to block the exocytotic release of painstimulating peptides [e.g. calcitonin gene-related peptide (CGRP) and substance P] from sensory neurons when elicited by activating TRPV1 (transient receptor potential vallinoid 1), a cation channel involved in the signalling of most forms of pain (Meng et al., 2007; Meng et al., 2009).

Similarly, BoNT/E also fails to inhibit the capsasin- stimulated, TRPVl-mediated release of CGRP and substance P from sensory neurons. This inability may be due to its cognate cell surface acceptor (glycosylated synaptic vesicle protein 2A (SVP2A) and glycosylated SVP2B), present on motor neurons, being sparse or absent from sensory neurons. A chimeric protein in which the He (receptor-binding domain) of BoNT/E is replaced by its counterpart from BoNT/A is able to block the release of these pain-mediating peptides, indicating that the BoNT/A cell surface receptor facilitates the endocytosis and delivery of LC/E into nociceptive C-fibres.

Once inside the neuron, the LC/E protease, removes 26 SNAP-25 amino acid residues, thus preventing the formation of a stable

SNARE complex required for neuro-exocytosis (Meng et al., 2009).

Although LC/A also cleaves SNAP-25, it only cleaves 9 amino acid residues, and the blockage of exocytotic activity is less complete and stable.

In order to make it practical to clinically exploit such an advantageous feature of the LC/E protease, it is desirable to greatly extend the duration of action of LC/E.

Each mature di-chain Clostridial toxin molecule comprises three functionally distinct domains: 1) an enzymatic domain located in the LC that includes a metalloprotease region containing a zinc-dependent endopeptidase activity which specifically targets peptide bonds in one or more SNARE proteins that mediate the fusion of the synaptic vesicle with the cell membrane; 2) a translocation domain contained within the aminoterminal half of the H chain (termed "HN") that facilitates release of at least the LC chain of the toxin from an endosome into the cytoplasm of the target cell; and 3) a binding domain found within the carboxyl-terminal half of the H chain (He) that determines the cellular binding activity and binding specificity of the toxin.

The He region comprises H_CN and Hee sub-domains (the N- and C-terminal portions of H_c, respectively). Most or all BoNT/R toxins bind a target cell using a "dual receptor", wherein the H_c portion of the toxin comprising both H_CN and H_Cc subdomains cooperatively bind certain cell surface gangliosides and a protein receptor (synaptic vesicle glycoprotein receptor 2 (SV2)). See e.g., Dong et al., SCIENCE, 2006 Apr 28;312(5773):592-6. By "R" is meant any serotype of botulinum toxin. Although the term "BoNT/R" is generally used to indicate subtypes of botulinum toxin, the term may also be used herein to include the related Clostridium tetani toxin (TeTX) regions thereof.

Using BoNT/A as an example, cooperative binding of the protein receptor and gangliosides results in an energy- and temperature-dependent endocytosis of the toxin within the cell. The N-terminal portion of HC (H_N) is responsible for translocation of the metalloprotease LC to the cytosol of the neuron, where the enzyme specifically cleaves 9 C-terminal residues from the synaptosomal-associated protein having a Mr = 25 k (SNAP-25) to yield the truncated SNAP-25 fragment SNAP-25_A. Because intact SNAP-25 is required for fusion of synaptic vesicles with the neuronal cell membrane and since neurotransmitters are exported from the cell by fusion of synaptic vesicles with the cell membrane, this proteolysis results in the blockade of neurotransmitter release by the neuron

BoNT/A has been found to inhibit the chemically stimulated release of the pain-associated peptides substance P and calcitonin gene-related peptide (CGRP) from peripheral rat sensory neurons in models of neuropathic pain. BoNT/A has also been tested by injection around the forehead for use in treating certain cases of chronic (but not episodic or acute) migraine for patients experiencing migraine for more than 15 days per month. The frequency of migraine attacks was reduced in some, but not all patients.

A common feature of migraine has been shown to be an increased extracellular level of CGRP. Furthermore, the intravenous infusion of CGRP produces migraine-like symptoms in volunteers. BoNT/A was shown to lower the blood levels of CGRP in blood samples from those suffering from migraine, but this effect was seen only in those that responded to the therapy. There therefore remains a need for a more efficacious and universal treatment for alleviating pain symptoms, particularly those associated with migraine.

The present invention is directed to improved methods and compositions for the prevention, treatment and alleviation of symptoms of migraine and acute pain using protein-based biological agents related to Clostridial botulinum toxin. Unlike many treatments for migraine pain such as opioids, triptans, lasmiditan and similar drugs, the specificity of the present compositions and methods results in much lower risk of serious side effects such as stroke, heart attack, ulcers or drug dependency. Also, unlike chemical agents such as GCRP antagonists and GCRP receptor antagonists, which are used as oral agents in the microgram range and may have systemic effects, the present compositions and methods are exquisitely specific, cause no discernable neurological or systemic side effects, and are administered by local injection in tiny amounts (in the pico- to nano-gram range).

Furthermore, the compositions of the present invention provide a substantial advantage over the use of BoNT/A in the treatment of migraine and intense pain signals. Tn vitro experiments show that BoNT/A reduces the amount of CGRP release evoked from cultured trigeminal ganglion neurons (TGNs) when relatively low levels (0.02-0.1 pM) of the pain proxy stimulant capsaicin, an activator of the nociceptive channel-transient receptor potential vanilloid 1 (TRPV1)- is applied. By contrast, the present compositions reduce the release of CGRP in cultured TGNs upon strong pain stimulus to these neurons (0.3- 1.0 pM capsaicin).

Equally importantly, these compositions have now been shown to exhibit long-lasting analgesic properties in vivo when applied directly to rat whisker pad (implicating the trigeminal system and bypassing the dorsal root ganglia) induced by a noxious chemical stimulus (2.5 pg of capsaicin, a selective activator of TRPV1 or 20 pl of 100 nM of allyl isothiocyanate (AITC), a selective activator of TRPA1).

Furthermore, rats pre-treated with the compositions of the present invention, and then provided the noxious chemical stimulus failed to display a significant increase in typical indicia of a nocifensive response (an increase in grooming, an increasing "freezing" (non-motile) time, and a decrease in distance walked in the cage). By contrast, rats given only the vehicle (an aqueous solution of 0.05% human serum albumin in 0.9% NaCl) as a pre-treatment without the added compositions of the invention showed significant increases in each of these nociceptive behaviors when provided with the chemical stimulus. These data thus show that administration of the compositions of the present invention have a prophylactic effect which suppresses the perception of pain even when the nociceptor, (i.e., TRPA1 or TRPV1) is specifically activated with a chemical activator. The in vivo experiments also show that the compositions appear to be devoid of toxicity as reflected by the absence of disruption to traditional indicia of pain (food uptake, grooming, and mobility) and the absence of other discernable changes from normal behavior in animals treated with the compositions of the present invention but not administered the chemical irritant. These data also suggested the reduction of such nocifensive indicia of pain in mammals when administered after the nociceptive stimulus is applied.

Detailed Description of the Invention

The polypeptides used in the present methods and compositions comprise chimeric BoNT-derived constructs combining features of BoNT serotypes A (having a high level of sensory neurotropism) and E (whose LC provides a more extensive disruption to SNAP-25 than does LC/A). The active agent of one such composition, termed "BoNT/EA", comprises a fusion of the LC and translocation domain (HN) regions of BoNT/E with the neuronal acceptor region (H_c) of BoNT/A. See Meng, et al., J. NEUROsci. (April 152009) 29(15):4981-4992. The active agent of the other composition comprises a fusion of the BoNT/E LC to whole BoNT/A, which stabilizes the LC /E protease rendering it longer-lasting, and is termed "LC/E-BoNT/A". See e.g., United States Patents No. US 9,216,210 B2 and 10,457,927.

A pre-print of the paper "Botulinum Neurotoxin Chimeras Suppress Stimulation by Capsaicin of Rat Trigeminal Sensory Neurons in vivo and in vitro", was filed herewith as part of U.S. provisional patent application Serial No. 63/286,189, filed December 6, 2021, to which the present application claims priority. This paper is now published as Toxins (Basel,)2022 Feb 4;14(2):116.

Brief Description of the Drawings

Fig. 1A is a graph showing the increase in weight of rats treated with the peptide LC/E-BoNT/A (triangles) as compared to vehicle-treated (control) rats (circles) over a 15 day period following administration of the peptide.

Fig. IB is a graph showing the effect upon grooming behavior of rats treated with LC/E-BoNT/A (downward hatching, left to right) as compared to vehicle treated (control) rats (downward hatching, left to right).

Fig. 1C is a graph showing the effect upon the distance traveled of rats treated with LC/E-BoNT/A (downward hatching, left to right) as compared to vehicle treated (control) rats (downward hatching, left to right).

Fig. ID is a graph showing the effect upon "freezing time" of rats treated with LC/E-BoNT/A (downward hatching, left to right) as compared to vehicle treated (control) rats (downward hatching, left to right).

Fig. 2A is a graph showing the increase in weight of rats treated with the peptide LC/E-BoNT/A and then injected with capsaicin, as compared to positive and negative control groups of rats, over a 30 day period following administration of the capsaicin.

Fig. 2B is a graph showing the effect upon grooming behavior of rats treated with the peptide LC/E-BoNT/A and then injected with capsaicin, as compared to positive and negative control groups of rats, over a 30 day period following administration of the capsaicin.

Fig. 2C is a graph showing the effect upon the distance traveled of rats treated with the peptide LC/E-BoNT/A and then injected with capsaicin, as compared to positive and negative control groups of rats, over a 30 day period following administration of the capsaicin.

Fig. 2D is a graph showing the effect upon the "freezing time" of rats treated with the peptide LC/E-BoNT/A and then injected with capsaicin, as compared to positive and negative control groups of rats, over a 30 day period following administration of the capsaicin.

Fig. 3A showing the effect upon grooming behavior of male rats treated with the peptide LC/E-BoNT/A and then injected with capsaicin, as compared to positive and negative control groups of male rats, over a 30 day period following administration of the capsaicin.

Fig. 3B is a graph showing the effect upon the distance traveled of male rats treated with the peptide LC/E-BoNT/A and then injected with capsaicin, as compared to positive and negative control groups of male rats, over a 30 day period following administration of the capsaicin.

Fig. 3C is a graph showing the effect upon the "freezing time" of male rats treated with the peptide LC/E-BoNT/A and then injected with capsaicin, as compared to positive and negative control groups of male rats, over a 30 day period following administration of the capsaicin.

Fig. 3D showing the effect upon grooming behavior of female rats treated with the peptide LC/E-BoNT/A and then injected with capsaicin, as compared to positive and negative control groups of female rats, over a 30 day period following administration of the capsaicin.

Fig. 3E is a graph showing the effect upon the distance traveled of female rats treated with the peptide LC/E-BoNT/A and then injected with capsaicin, as compared to positive and negative control groups of female rats, over a 30 day period following administration of the capsaicin.

Fig. 3F is a graph showing the effect upon the "freezing time" of female rats treated with the peptide LC/E-BoNT/A and then injected with capsaicin, as compared to positive and negative control groups of female rats, over a 30 day period following administration of the capsaicin.

Fig. 4A is a depiction of a coronal section of the rat brainstem showing the loci of the trigeminal nucleus caudalis (TNG) or the sides of the brainstem that are contralateral and ipsilateral to the site of injection of capsaicin and LC/E-BoNT/A.

Fig. 4B shows a photomicrogram of the ipsilateral TNG (bordered by dashed line), following neutral activation by capsaicin and staining for CGRP (shown as a faint light line at the top and right borders of the TNG.)

Fig. 4C is a grid showing photomicrograms of sections of the ipsilaterial TNG (bordered by dashed lines) in which the tissue was stained for c-fos expression (bright localized spots); rats were administered (clockwise from top left): a) control Vehicle 1, followed by control Vehicle 2; b) control Vehicle 1, followed by capsaicin; c) LC/E-BoNT/A, followed by capsaicin; and d) LC/E-BoNT/A, followed by control Vehicle 2.

Fig. 4D is a graph quantitating the number of c-fos positive cells detected in the stained TNG sections of the experiment of Fig. 4G in each the ipsilateral and the contralateral TNG of the treated rats.

Fig. 5A is a graph showing the % of capsaicin-evoked CGRP released from trigeminal neurons (TGNs) in culture. The amounts of peptide exocytosed and retained by the cells, respectively, was quantified upon increasing concentrations of capsaicin.

Fig. 5B shows an SDS-PAGE Western blot from lysates of cultured TGN cells incubated with 100 nM BoNT/A, BoNT/EA, or LC/E-BoNT/A for 48 hrs, so that the bulk (75%) of their total SNAP-25 content was truncated, consistent with the neurotoxins' substrate specificity.

Fig. 5C is a graph showing the amount (expressed as a percent of the control) of capsaicin-evoked CGRP release from trigeminal neurons (TGNs) in culture that had been preincubated for 48 hours with BoNT/A, BoNT/EA, or LC/E-BoNT/A. The amounts of the CGRP peptide exocytosed and retained by the cells, respectively, was quantified upon exposure of the cells to increasing concentrations of capsaicin.

Fig. 5D shows the spontaneous CGRP release in TGN cell culture that had been preincubated for 48 hours with BoNT/A, BoNT/EA, or LC/E-BoNT/A (and a control) over a 30 minute exposure to HEPES buffered saline lacking capsaicin.

Fig. 5E is a graph showing the mean amounts of total CGRP in TGN cells incubated with BoNT/A or BoNT/EA (and a control) lacking either peptide).

Fig. 5F is a graph showing the mean amounts of total CGRP in TGN cells incubated with LC/E-BoNT/A (and a control lacking the peptide). As a lower seeding density of cells was used for the experiments in this experiment compared to the experiment of Fig. 4E, smaller CGRP amounts were detected in this experiment.

Fig. 6A is a graph showing the increase in weight of rats treated with the peptide LC/E-BoNT/A and then injected with AITC, as compared to positive and negative control groups of rats, over a 15 day period following administration of the AITC. Fig. 6B is a graph showing the effect upon grooming behavior of rats treated with the peptide LC/E-BoNT/A and then injected with AITC, as compared to positive and negative control groups of rats, over a 15 day period following administration of the AITC.

Fig. 6C is a graph showing the effect upon the distance traveled of rats treated with the peptide LC/E-BoNT/A and then injected with AITC, as compared to positive and negative control groups of rats, over a 15 day period following administration of the AITC.

Fig. 6D is a graph showing the effect upon the "freezing time" of rats treated with the peptide LC/E-BoNT/A and then injected with AITC, as compared to positive and negative control groups of rats, over a 15 day period following administration of the AITC.

Examples

Example 1: Construction of LC/E-BoNT/A Open Reading Frame

A synthetic BoNT/A gene, having its codons optimised for enhanced expression in E. coli and three extra nucleotides (AAA) encoding a Lys residue, was cloned into Nde I and Sal I sites of a prokaryotic expression vector pET29a(+) to yield pET-29a- BoNT/A. Plasmid pET-29a-BoNT/A was then further modified in order to provide scission sites for controlled specific nicking and simultaneous removal of the hexahistadine (His6) tag (SEQ ID NO: 15) encoded by the pET-29a cloning vector. A nucleotide sequence encoding a thrombin cleavage sites was engineered into the nucleic acid region encoding the HC/LC loop of the toxin. This is shown below in both nucleic acid and amino acid form, as SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

Modified BoNT/A loop nucleotide sequence (SEQ ID NO: 1) and its encoded amino acid sequence (SEQ ID NO: 2)

An additional thrombin site was inserted between the regions encoding the HC/A and His6 (SEQ ID NO: 15) regions of the expressed protein. This is shown below in both nucleic acid and amino acid form, as SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

Nucleotide sequence fused to the 3' end of BoNT/A gene (SEQ ID NO: 3) and its encoded amino acid sequence (SEQ ID NO: 4)

The nucleotide sequence provided above contains the following regions, from left to right, respectively: a) nucleotides 1-3: AAA codon inserted encoding additional Lys residue, as described above, to provide an optional trypsin cleavage site in the expressed protein, in order to remove the C-terminal His6 (SEQ ID NO: 15); b) single underline: Sal I restriction endonuclease site; double underline: Hind III restriction endonuclease site d) bold: thrombin recognition sequence; single underline: Pst I restriction endonuclease site; f) double underline: Xho I restriction endonuclease site; g) nucleotides 49-66: nucleotide region encoding a His6 tag (SEQ ID NO: 15). The aligned amino acid sequences are displayed above the corresponding nucleotides. The arrow indicates the thrombin cleavage site, and the asterisk denotes the translational "stop" codon.

This nucleic acid construct, comprising the BoNT/A open reading frame described above, and comprising both SEQ ID NO: 1 and SEQ ID NO: 3, was termed pET29a-BoNT/A-2T.

A PCR product (amplicon) was amplified from a synthetic nucleic acid encoding the LC/E protease (residues 1-411), and two restriction sites (Nde I and Eco RV) were incorporated during the amplification at the 5' and 3' ends of the nucleic acid amplicon, respectively. This PCR amplicon was then digested by Nde I and Eco RV and cloned into pET29a(+) vector, also digested with Nde I and Eco RV. The resultant intermediate vector construct was named pET29a-LC/E.

The above-noted intact "single chain" open reading frame BoNT gene region of BoNT/A-2T was amplified by PCR using pET29a- BoNT/A-2T as a template with a pair of primers (a bacteriophage T7 terminal reverse primer and a forward primer containing an EcoRV restriction sequence upstream of the BoNT/A 5' coding sequence). The resulting PCR amplicon was digested by EcoRV and Xho I enzymes, purified, and inserted into Eco RV- and Xho I- cleaved pET29a-LC/E plasmid. This final construct was called pET29a-LC/E-BoNT/A, and the open nucleic acid reading frame is disclosed as SEQ ID NO: 5, while the corresponding amino acid sequence is disclosed herein as SEQ ID NO: 6.

Example 2: Expression and and Purification of LC/E-BoNT/A

For expression of LC/E-BoNT/A, the sequence-verified construct was transformed into E. coli strain BL21(DE3), and expression of the target protein was induced using Studier's auto-induction medium (Studier, F.W., 41 Protein Expr. Purif. 207 (2005)). Partial purification (~60%) of the His6 (SEQ ID NO: 15) tagged protein in the bacteria lysate was achieved with immobilised metal (Co⁺⁺) affinity chromatograph (IMAC), using Talon superflow resin. A major protein of Mr~200 kDa is eluted by greater than or equal to 50 mM imidazole. The pooled IMAC eluted fractions were buffer-exchanged into 0.02 M sodium phosphate buffer (pH 6.5), and then further purified by loading onto a UNO-SI cation exchange column, followed by washing with up to 150 mM NaCl, and then elution with a NaCl gradient; the toxin was eluted at NaCl concentrations of equal to or greater than 220 m .

After buffer-exchanging the eluted intact toxin into 25 mM HEPES/145 mM NaCl (pH 7.4), the purified single chain ("SC") protein was stored at -80°C, and the single chain nature of the toxin was confirmed by reducing and non-reducing SDS-PAGE analysis. The purified protein was indeed expressed in a SC form, as revealed by a single band migrating with an apparent molecular weight of about 200 kDa.

Nicking of this SC polypeptide into two disulphide-linked chains was performed by incubation of the purified SC toxin with biotinylated thrombin (1 unit/mg of protein) at 22°C for 3 hours; the thrombin protease was then removed by treating the sample with streptavidin agarose. A band having an apparent molecular weight of about 100 kDa appears after thrombin treatment of the protein in samples run on an SDS-PAGE gel under reducing conditions; the ~200 kDa band is not seen under these conditions, but is present in gels run under non-reducing conditions, while the ~100 kDa band is absent in these latter samples.

The ~100 kDa band seen under reducing conditions represents two distinct polypeptide chains: the LC/E-LC/A and the HC/A chains, which have similar sizes. The identities of the polypetides in this band are confirmed by Western blotting of SDS-PAGE gels run on nicked and unnicked LC/E-BoNT/A using antibodies specific against each of the postulated single chain polypeptides LC/E and BoNT/A.

The nicked sample continues to migrate at ~200 kDa in the absence of reducing agent, indicating that the inter-chain disulphide bond between LC/E-LC/A and HC/A was formed, and persists, in all of the samples as shown in the lanes of the Western blots marked (-) and developed using either anti-LC/E or anti-BoNT/A antibodies. Thus, SDS-PAGE and Western blotting under redicing and non-reducing conditions highlight the specific nicking at the loop region that occurs without degradation of the composite toxin. A slight difference in the mobility of the un-nicked and nicked protein is due to removal of the His6 tag (SEQ ID NO: 15) in the thrombin-treated samples; this experiment therefore also demonstrated that thrombin protease can simultaneously nick the toxin between the linked cysteine residues of the disulphide bond between the HC and the first LG, and remove the His6 (SEQ ID NO: 15).

Purified, thrombin-nicked toxin prepared as described above is used as the active LC/E-BoNT/A preparation in the subsequently described in vivo experiments.

Example 3: Injection of LC/E-BoNT/A into the Right Whisker Pad of Rats does not Alter their Grooming, Exploratory or Locomotor Behavior

As an initial test of the safety of the preparation in vivo, a single injection of 75 mLDso units/kg of LC/E-BoNT/A in Vehicle 1 (0.05% human serum albumin (HSA) in 0.9% NaCl) into the right whisker pad of male and female rats was made, and the spontaneous grooming and exploring behavior and locomotor activity of the test animals was recorded at time periods following administration of the LC/E-BoNT/A preparation of 1, 2, 3, 4, 8, and 15 days. A mLD50 unit is defined as the minimal dose causing death in 50% of mice according to the methods of Maisey, E. A., et al., 177 EUR. J. BIOCHEM. 683-691 (1988), hereby incorporated by reference. The mLD50 of an LC/E-BoNT/A preparation is generally about 0.7 x 10^-8 g.

Assessment of weight gain, grooming, motility and locomotor activity factors provides a measure of the general welfare of the animals, as these are factors that are commonly altered when the animal experiences pain or discomfort. Control animals

(grey bars and circles) were provided the same vehicle (Vehicle 1) as used in the experimental animals but without added LC/E- BoNT/A. As shown in Fig. 1A-1D, the test animals treated with LC/E-

BoNT/A (triangles and upward cross-hatched bars, left to right) do not exhibit any significant differences in weight gain, grooming, motility and locomotor activity as compared to the control animals which were injected with Vehicle 1 alone, and not injected with the LC/E-BoNT/A molecule (circles and downward cross-hatched bars, left to right). Thus, administration of LC/E-BoNT/A alone does not hinder the test animals' behavior or elict discernable nocifensive behavior. Example 4: LC/E-BoNT/A Causes Long-Lasting Preventative Alleviation of Acute Nocifensive Behaviour fnduced by Capsaicin in Rats

As in Example 3, rats were given a single injection in their whisker pads of either LC/E-BoNT/A (75 units/kg) in "Vehicle 1", or the same volume of Vehicle 1 alone. Each of these two sets of rats were divided into two subgroups; in one such subgroup the whisker pad was injected with 2.5 μg of capsaicin in "Vehicle 2" (an aqueous solution of 5% ethanol/5% Tween 80/0.9% NaCl) in a volume of 20 pl on specified days following the set of first injections, the whisker pads of the other subgroup was injected only with 20 pl of Vehicle 2.

Injection of capsaicin into the whisker pad of rats increases attention of the animals to the injected area, which is reflected by intensification of grooming; this usually arises from pain or discomfort (Deuis et al. FRONT. MOL. NEUROSCI. 10:284 (2017), hereby incorporated by reference herein). Acute nociception was assessed on days 4, 8, 15, and 30.

As in the previous experiment, the rodents' grooming, exploration, weight gain, and locomotor activity were monitored over the course of the experiment. As shown in Fig. 2A, injection of the LC/E-BoNT/A neurotoxin did not affect weight gain compared to the other groups.

Among the control animals pre-injected with Vehicle 1, the subsequent administration of capsaicin triggered nocifensive behavior, represented by a significant increase in grooming (Figure 2B) and freezing time (Figure 2D), and a decrease in the distance (Figure 2C) walked in the testing cage, compared to those animals injected with Vehicle 1 and then by Vehicle 2 and no capsaicin.

By contrast, animals pre-treated with LC/E-BoNT/A showed a significant reduction in grooming behavior after injecting capsaicin on days 4, 8, and 15 (Figure 2B); after 30 days, no significant effect was apparent. As a positive control, the subcutaneous injection of the opioid analgesic buprenorphine (0.2 mg/kg) 30 min before the administration of capsaicin also prevented the grooming intensification (Figure 2B), verifying the latter as a nocifensive behavior.

Locomotor activity in Vehicle 1/capsaicin-treated rats was substantially decreased, reflected in shorter total distances walked on days 4, 8, and 15 when compared to the Vehicle 2- treated control group; see Fig. 2C. However, this effect was largely abrogated in animals pre-treated with LC/E-BoNT/A. The freezing time (periods of immobility) was also significantly increased after capsaicin administration in comparison with the Vehicle 2 controls. Notably, in those animals pre-treated with LC/E-BoNT/A this pain-like effect was completely reversed on days 4, 8 and, to a lesser extent, on the subsequent days (Figure 2D). As expected, administration of the buprenorphine control also prevented the impairments in the locomotor activity evoked by capsaicin (Figure 2C, D).

Example 5: LC/E-BoNT/A Equally Diminishes Nocifensive Behaviour Evoked by Capsaicin in Both Male and Female Rats

Evidence was sought for similarities or differences on the effectiveness of LC/E-BoNT/A in both sexes for lowering the capsaicin-induced increase in the acute nociceptive behavior. Another experimental set including male and female rats was injected with LC/E-BoNT/A into the right whisker pad, and the acute nociceptive behavior evoked by capsaicin assessed as above. Again, LC/E-BoNT/A did not influence the weight gain in either sex (data not shown). Capsaicin administration evoked the nocifensive response indicated by increased grooming (Figure 3A, D) accompanied by a decrease in the exploratory and locomotor behavior (Figure 3B, C, E and F) in males and females (previously injected with Vehicle 1); although females appeared more responsive to capsaicin on day 4, there was no significant difference from males so this reflects random variation. For both sexes, the substantial influence of LC/E-BoNT/A in decreasing grooming occurred predominantly between days 4 and 15 with no significant change on day 30 (Figure 3A, D). In each case, the capsaicin-induced reduction in total distance walked was diminished by pre-treatment with the neurotoxin (Figure 3B, E), and the immobility resulting from capsaicin administration was considerably reduced in rats pre-treated with LC/E-BoNT/A compared to the Vehicle 1

capsaicin-injected controls (Figure 3C, F). Additionally, administration of buprenorphine prevented the elevation of grooming and impairments in locomotor activity evoked by capsaicin in both male and female rats (Figure 3A-F).

Example 6: LC/E-BoNT/A Precludes the Induction of c-Fos Expression in the TNC after Capsaicin Injection into the Whisker Pad, a Biochemical Indication of Reduced Nociceptor Activation

The effect of the LC/E-BoNT/A neurotoxin on neural activation evoked by capsaicin was assessed by quantifying the expression of c-Fos in the trigeminal nucleus caudalis (TNC) in the brainstem of rats; this was relevant because afferents from the whisker pad project to the TNG, which is considered to be the site of the second order neurons of the nociceptive pathways of the face, and which, as described above, is a critical part of the pathway of migraine pain perception. c-Fos expression is commonly used as a valuable tool to identify subpopulations of neurons activated in response to noxious stimuli and related nociceptive pathways (Bergerot et al., EUR. J. NEUROSCI. 2 (6):1517-1534 (2006); Harriott et al., J. HEADACHE PAIN 20:91 (2019). As the inhibition of nocifensive behaviour was observed 4 days after LC/E-BoNT/A administration into the right whisker pad (see e.g., Fig. 2B-2D), samples were collected at this time for c-Fos detection.

Subject rats were euthanized, and the caudal brainstems containing the TNG were dissected and fixed. Cryosections were made and stained.

In Fig. 4B and Fig. 4C the TNC is highlighted by the dashed line, and in Figure 4B the outer border of the ipsilateral TNG is delineated by CGRP staining (fuzzy light line on top and right sides). In Fig. 4G c-Fos positive cells are shown as light irregular dots. The number of c-Fos positive cells found in TNG was significantly higher in ipsilateral sections of the group injected with capsaicin, compared to the ipsilateral vehicle 2-injected (control) and contralateral vehicle 1

capsaicin groups (Figs. 4G and 4D).

Consistent with the in vivo observation that LC/E-BoNT/A alleviates nocifensive behavior in capsaicin-treated animals, brainstem sections from the animals pre-treated with a single injection of LC/E-BoNT/A showed a substantial reduction in the number of cells expressing c-Fos in the ipsilateral side where capsaicin was injected, with 77 ± 4% inhibition of this marker on day 4 after administration of the neurotoxin.

By contrast, in the brainstem sections from these animals no substantial differences were observed in the c-Fos expression in cells residing on the contralateral side to the injection sites of the animals tested, regardless of whether they had been given capsaicin and/or LC/E-BoNT/A or not (Figure 4D).

Example 7: Chimeras LC/E-BoNT/A and BoNT/EA Inhibit CGRP Release from TGNs Evoked by Strong Stimulation with Capsaicin

The successful treatment of chronic migraines by BoNT/A (reviewed by Burstein et al., HEADACHE 60(7) :1259-1272 (July 2020) is assumed to relate to it lowering the exocytosis of neurotransmitters, particularly CGRP (Cernuda-Morollon et al., PAIN 156(5): 820-824 (May 2015). This therapy has proved most effective in certain (but not all) cohorts of patients; reports indicate that the level of improvement varies between individuals or with the exact nature of the condition (s). An earlier observation (Meng et al. 2009, cited above) of only feeble inhibition by BoNT/A of CGRP release from rat cultured TGNs when triggered by activating TRPV1 with 1 pM capsaicin raised the possibility that such limitations might arise from different levels of excitation of the pertinent transmitter- secreting neurons.

This notion was investigated herein by evoking CGRP release from rat TGNs in culture with a range of capsaicin concentrations and measuring the amount of the peptide by ELISA (Figure 5A). A concentration-dependent increase in release of CGRP was obtained upon treatment of the cultured TGN cells with 0.001-0.1 pM of capsaicin representing 0.5-37% of the total CGRP, but lower levels were seen upon treatment with higher amounts (0.25-10 pM) of capsaicin.

For assaying the susceptibility of the release of CGRP to BoNT/A, the cultured TGN cells were incubated with 100 nM BoNT/A for 48 hours so that the bulk (79%) of their total SNAP-25 content was truncated (See Figure 5B). As expected, consistent with the substrate specificity of BoNT/A, the SNARE protein syntaxin 1 remained uncleaved under these conditions, as revealed by the Western blotting.

Additionally, cultured TGNs were also similarly incubated with either 100 nM BoNT/EA or 100 nM LC/E-BoNT/A for 48 hours, with BoNT/EA cleaving SNAP-25 to yield the cleavage product SNAP-25_E (having 26 amino acids removed from its C-terminus end), and LC/E-BoNT/A yielding a majority of the SNAP-25_E cleavage product and a minority of the SNAP-25A cleavage product, the latter having 9 amino acids removed from its C-terminus end. See Fig. 5B.

48 hours' incubation with BoNT/A failed to block CGRP exocytosis elicited by 0.3 or 1 pM capsaicin (Figure 5C), as reported previously for treatment with 1 pM capsaicin (Meng et al., 2007). However, upon lowering the amounts of capsaicin used for the stimulation, the release of CGRP in the BoNT/A-treated cells became progressively inhibited (Figure 5C).

To examine the possibility of SNAP-25A mediating exocytosis when evoked by the higher capsaicin concentrations, the TGNs incubated for 48 hours with 100 nM chimeric BoNT/EA or LC/E- BoNT/A, which truncated a similar majority (77 and ~63%) of SNAP-25 to SNAP-25E (Figure 5B) were compared. For the cells incubated with BoNT/EA or LC/E-BoNT/A a pronounced reduction in CGRP release was elicited at all of the capsaicin concentrations applied (Figure 5C). It is noteworthy that each of the neurotoxins caused a limited but significant suppression of the spontaneous release of the peptide (Figure 5D); only a slight effect was expected because the majority of spontaneous CGRP release from TGNs is Ca²⁺-independent (Meng et al., J. CELL SCI.15:120(Pt.16) 2864-2874 (Aug. 2007). The total cellular content of CGRP was not altered by incubation with BoNT/A or BoNT/EA, but increased significantly by incubation with LC/E- BoNT/A (Figs. 5E and 5F).

Example 8: LC/E-BoNT/A Causes Long-Lasting Preventative Alleviation of Acute Nocifensive Behaviour Induced by AITC in Rats

Rats were anesthetised with 3.5% isoflurane and given a unilateral single subcutaneous injection of 30 pL of LC/E-BoNT/A (75 units/kg), diluted in Vehicle 1(0.05% human serum albumin in 0.9% NaCl) into their right whisker pad (perinasal area), using a Hamilton syringe (50 pL) fitted with a 30-gauge needle. Controls received 30 pL of Vehicle 1 alone. Starting 4 days later, behavioural assessments were performed between 11 a.m. and 4 p.m. by an operator unaware of the treatments given to the animals. Allyl isothiocyanate (AITC) was freshly diluted to 100 nM in Vehicle 3 (2.5% DMSO in 0.9% NaCl) before injection into rats; control animals were injected with Vehicle 3 alone.

Testing for indicia of nocifensive behavior was performed in a quiet room with a temperature of 20 ± 1 °C. After ten minutes acclimatisation, the rats were briefly restrained and injected with AITC (100 nM/20 pL) or Vehicle 3 into the right whisker pad. Immediately after injection, the animals were placed into a recording cage and their behavior was videorecorded for 30 min. The acute nocifensive response was taken as the cumulative amount of time each animal spent grooming (face-wash strokes, chin/cheek rubs, hind paw face scratching) the injected facial area with its paws, and by assessments of their exploratory behaviour (distance travelled and periods of immobility (freezing time)). Recordings were analysed by an observer blinded to the experimental conditions.

After pre-treatment with LC/E-BoNT/A (75 units/kg) or Vehicle 1 into the right whisker pad, the rats' behavior was assessed following the administration of 20 μL Vehicle 3, either alone or containing 100 nM AITC, on days 4, 8, and 15 following the day of injection.

As shown in Fig. 6A-6D, results indicate that the neurotoxin did not affect weight gain compared to the other groups (Figure 6A). In animals pre-injected with Vehicle 1, the subsequent administration of AITC triggered nocifensive behaviour (Group 2), represented by a significant increase in grooming (Figure 6B) and freezing time (Figure 6D), while it decreased the distance walked in the testing cage (Figure 6C), compared to the Vehicle 1

Vehicle 3-injected control (Group 1). On the other hand, rats pretreated with LC/E-BoNT/A before injecting AITC (Group 4), showed a significant reduction in acute grooming behaviour on days 4, 8, and 15 (Figure 6B) compared to vehicle 1→AITC (Group 2). Locomotor (Figure 6C) and freezing time (Figure 6D) were also restored to near control values. The analgesic action of LC/E-BoNT/A was specific for AITC-induced pain behavior because there were no differences between control Group 1 (Vehicle 1

Vehicle 3- injected group) and control Group 3 (LC/E-BoNT/A

Vehicle 3- injected group) for any of the baseline behavior levels (Figure 6B-D).

Thus, these results show that, similar to the in vivo experiments described in Examples 3-7 hereof specifically targeting capsaicin-sensitive TRPVl-containing neurons, nocifensive behavior in rats administered AITC, a noxious chemical stimulus specific for neurons containing the TRPA1 nociceptor, is greatly reduced by administration of LC/E-BoNT/A to neurons projecting to the trigeminal ganglia. Moreover, this analgesia is long-lasting and prevents the sequelae of pain development when used prophylactically.

Migraine is a good model for the study of acute or episodic head or facial pain because of the prevalence of this debilitating condition, which involves the trigeminal sensory system. The trigeminal system conveys sensory information from the craniofacial region, which is composed of peripheral structures such as the trigeminal nerve and associated ganglia, as well as central structures like the dorsal brainstem region which includes the TNG (reviewed by Gambeta et al., MOL. PAIN 16:1744806920901860(2020). Sensory inputs from the periphery are relayed by afferent fibres that make connections with second- order neurons in the TNC, so sensory information gets propagated to the thalamus where sensory stimuli are processed. Third-order neuronal projections conduct the stimulus to the somatosensory cortex and insula; there, signals are interpreted with respect to location, intensity and duration (Roper and Brown, Pain and Other Disorders of Somatic Sensation, Headache, and Backache, in Adams and Victor's Principles of Neurology (8^th ed. (Roper A. Brown R, eds) at 109-168 (McGraw-Hill 2005); Ossipov et al., J. CLIN. INVEST. 120(11):3779-87 (November 2010); Chichorro et al., CEPHALALGIA 37(7) 613-626 (June 2017). Considering that one of the 3 branches of the trigeminal nerve, the infraorbital branch, innervates the rats' whisker pad, this accessible site was preferred herein for administering capsaicin, a TRPV1 agonist, or AITC, a TRPA1 agonist.

The examples above utilize capsaicin and AITC as means to trigger acute pain because each noxious chemical recruits pathophysiological mechanisms that are distinct from those involved in neuropathic models (Percie du Sert et al., BR. J. PHARMACOL. 171(12):2951-63 (June 2014).

To the extent that a plurality of inventions may be disclosed herein, any such invention shall be understood to have been disclosed herein alone, in combination with other features or inventions disclosed herein, or lacking any feature or features not explicitly disclosed as essential for that invention. For example, the inventions described in this specification can be practiced within elements of, or in combination with, any other features, elements, methods or structures described herein. Additionally, Applicants intend that a feature illustrated herein as being present in a particular embodiment or example may, in other examples of the present invention, be explicitly lacking from the invention, or combinable with features described in other examples or embodiments in this patent application, in a manner not otherwise illustrated in this patent application or present in that particular example.

Claims

Claims

We claim:

1)A method of inhibiting the sensation of acute pain in a mammal comprising locally administering to one or more nociceptors projecting to the trigeminal ganglia of said mammal an effective amount of a Clostridium botulinum neurotoxin (BoNT)-derived active protein having a cellbinding domain derived from BoNT subtype A and a light chain endopeptidase derived from BoNT subtype E, said one or more nociceptors being chemically activatable by exposure to a noxious chemical.

2)The method of claim 1 wherein said acute pain is associated with a migraine episode.

3)The method of any preceding claim in which said effective amount of a Clostridium botulinum neurotoxin (BoNT)-derived active protein is administered in advance of exposure of said one or more nociceptors to said noxious chemical, thereby prophylactically inhibiting the sensation of acute pain in said mammal.

4)The method of any preceding claim in which the noxious chemical agent is selected from the group consisting of capsaicin and allyl isothiocyanate (AITC).

5)The method of any preceding claim in which said one or more nociceptors contain a Transient Receptor Potential integral cation channel selected from the group consisting of TRPV1 and TRPA1. 6)The method of any preceding claim in which said one or more nociceptors contain both TRPV1 and TRPA1 Transient Receptor Potential integral cation channels.

7)A method of inhibiting the release of a neuropeptide selected from the group consisting of: substance P, calcitonin gene-related peptide (CGRP), somatostatin and glutamate from one or more neurons projecting to or contained within the trigeminal ganglia of a mammal when said neuron is stimulated by a noxious chemical, comprising; locally administering to said one or more neurons an effective amount of a Clostridium botulinum neurotoxin (BoNT)-derived active protein having a cell-binding domain derived from BoNT subtype A and a light chain endopeptidase derived from BoNT subtype E.

8)The method of claim 7 in which said noxious chemical is selected from capsaicin and allyl isothiocyanate (AITC).

9)The method of claims 7 or 8 in which said one or more neurons comprise a Transient Receptor Potential integral cation channel selected from the group consisting of TRPV1 and TRPA1.

10)The method of any of claims 7-9 in which said one or more nociceptors contain both TRPV1 and TRPA1 Transient Receptor Potential integral cation channels.

11)The method of any of claims 7-10 in which said effective amount of a Clostridium botulinum neurotoxin (BoNT)-derived active protein is administered to the forehead or face of said mammal.

12) method of inhibiting the release of a neuropeptide selected from the group consisting of substance P, calcitonin gene-related peptide (CGRP) from one or more neuron projecting to or contained in the trigeminal ganglia of a mammal, said one or more neuron containing an integral cation channel selected from the group consisting of TRPV1 and TRPA1, comprising; administering to a location on the head, face and/or neck of said mammal an effective amount of a Clostridium botulinum neurotoxin (BoNT)-derived active protein having a cellbinding domain derived from BoNT subtype A and a light chain endopeptidase derived from BoNT subtype E.

13)The method of claim 12 wherein said neuropeptide release is associated with a migraine episode.

14)The method of claims 12-13 in which said effective amount of a Clostridium botulinum neurotoxin (BoNT)-derived active protein is administered in advance of activation of said one or more integral cation channel by a noxious chemical, thereby prophylactically inhibiting the sensation of pain in said mammal.

15)The method of any of claims 12-14 in which said one or more neurons contain both TRPV1 and TRPA1 Transient Receptor Potential integral cation channels. 16)A method of treating acute pain in a mammal, comprising locally administering to one or more site of acute pain a composition comprising an effective amount of a Clostridium botulinum neurotoxin (BoNT)-derived active protein having a cell-binding domain derived from BoNT subtype A and a light chain endopeptidase derived from BoNT subtype E.

17)The method of claim 16 wherein the acute pain is migraine pain and said one or more site of acute pain is the head, including the face, neck and/or forehead.

18)The method of either claim 16 or claim 17 wherein the BoNT- derived active protein comprises LC/E-BoNT/A.

19)The method of any of claims 16-18 wherein the step of locally administering said composition comprises injecting said composition into innervated tissue.

20)The method of claim 19 wherein said innervated tissue is the forehead.

21)The method of claim 19 wherein said innervated tissue comprises neurons that connect with the trigeminal nucleus caudalis.

22)A method of prophylactically treating acute pain in a mammal, intracellularly administering to one or more neurons innervating or projecting to a site of potential acute pain a composition comprising an effective amount of a Clostridium botulinum neurotoxin (BoNT)-derived light chain endopeptidase derived from BoNT subtype E. )A method for inhibiting a nociceptive neural signal comprising administering to at least one sensory neuron located and capable of transmitting said nociceptive signal a composition that truncates intact synaptosomal-associated protein 25 (SNAP-25) in the cytoplasm of said at least one sensory neuron to yield a plurality of truncated SNAP-25 molecules lacking 26 amino acids from the C-terminal end thereof. )The method of claim 23 in which said composition selectively binds to neural cells. )The method of claim 23-24 in which said composition comprises a protease. )The method of claim 25 in which said protease is an endopeptidase. ) The method of claim 26 in which said endopeptidase is derived from the light chain (LC) of Clostridium botulinum neurotoxin (BoNT) subtype E. )The method of any of claims 23-27 in which said composition comprises a cell binding domain derived from the H_c domain of Clostridium botulinum neurotoxin (BoNT) subtype A. )The method of any of claims 23-28 in which said at least one sensory neuron transmits nociceptive signals via the trigeminal system. )The method any of claims 23-29 resulting in the inhibition of CGRP release within the trigeminal nucleus caudalis.