WO2023105289A1 - Méthodes et compositions pour le traitement de la douleur - Google Patents

Méthodes et compositions pour le traitement de la douleur Download PDF

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WO2023105289A1
WO2023105289A1 PCT/IB2022/000748 IB2022000748W WO2023105289A1 WO 2023105289 A1 WO2023105289 A1 WO 2023105289A1 IB 2022000748 W IB2022000748 W IB 2022000748W WO 2023105289 A1 WO2023105289 A1 WO 2023105289A1
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bont
derived
subtype
pain
neurons
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PCT/IB2022/000748
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J. Oliver Dolly
Gary Lawrence
Tomas ZURAWSKI
Jiafu Wang
Jianghui MENG
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Dublin City University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/4886—Metalloendopeptidases (3.4.24), e.g. collagenase
    • A61K38/4893—Botulinum neurotoxin (3.4.24.69)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/04—Centrally acting analgesics, e.g. opioids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/06—Antimigraine agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/24—Metalloendopeptidases (3.4.24)
    • C12Y304/24069—Bontoxilysin (3.4.24.69), i.e. botulinum neurotoxin
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • 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.
  • pain is usually regarded as a symptom of an underlying condition.
  • the existence of pain does not necessary correlate with the discovery of such an underlying condition.
  • Chronic pain such as that caused by rheumatoid arthritis, peripheral neuropathy, cancer and idiopathic pain, may persist for years.
  • 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.
  • 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.
  • nociception 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.
  • afferent sensory nerves nociceptors
  • 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%.
  • 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.
  • derive 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.
  • a nociceptive neural signal is meant at least one nociceptive signaling event associated with a noxious stimulus and normally resulting in the sensation of pain.
  • 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.
  • 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.
  • inhibitor or “inhibiting” is meant to prevent or reduce an amount or degree of the action that is inhibited.
  • 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.
  • 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.
  • 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.
  • CGRP calcitonin gene-related peptide
  • somatostatin important in central synaptic signaling and efferent signaling in the skin.
  • C-fibers unmyelinated axons
  • A-fiber nociceptors whose axons are myelinated and support conduction velocities of about 5-30 m/sec.
  • nociceptors are classified on the basis of their sensitivity and activation threshold responses to noxious mechanical (M), heat (H) and cold (C).
  • M noxious mechanical
  • H heat
  • C cold
  • 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.
  • TRP transient receptor potential
  • 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.
  • 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).
  • 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.
  • each of TRPA1 and TRPV1 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.
  • DRG dorsal root ganglia
  • 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.
  • DRG are active participants in peripheral processes, including injury caused by platelet-activating factor (RAF), inflammation, and chronic neuropathic pain development.
  • RAF platelet-activating factor
  • 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.
  • 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.
  • 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 are stimulated to release vasoactive neuropeptides such as substance P, neurokinin A, and calcitonin gene-regulated polypeptide (CGRP), causing a (sterile) inflammatory response.
  • vasoactive neuropeptides such as substance P, neurokinin A, and calcitonin gene-regulated polypeptide (CGRP)
  • CGRP calcitonin gene-regulated polypeptide
  • 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 Ca 2+ -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.
  • BoNT serotype A BoNT/A
  • 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.
  • LC light chain
  • HC heavy chain
  • 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.
  • H N amino-terminal half of the H chain
  • 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.
  • 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.
  • 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.
  • amino acid sequences may be encoded by a finite set of different DNA molecules having different, but defined, nucleotide sequences.
  • degenerate nucleotide sequences encoding a given peptide or protein may have different codons adapted or selected to favor expression in a particular host cell.
  • 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.
  • 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.
  • modified Clostridial neurotoxins having different or modified functional properties from the naturally- occurring toxin subtypes and strains thereof.
  • 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.
  • 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).
  • 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.
  • 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.
  • translocation domain “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.
  • 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.
  • H CN the amino-terminal portion of the domain, corresponding approximately to amino acids 871- 1091 of BoNT/A
  • H cc the carboxy-terminal portion of the H c domain, corresponding approximately to amino acids 1092-1296 of BoNT/A
  • Subdomain H CN is highly conserved among botulinum toxin subtypes, however, the H C c subdomain is less conserved.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • HC heavy chain
  • 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.
  • the BoNT molecule 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.
  • HN amino terminal half of HC
  • 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.
  • 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.
  • the light chain of BoNT/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.
  • the inhibition of neurotransmitter release by LC/A can usually be reversed by elevating Ca 2+ influx but this reversal is not seen in the case of LC/E, presumably due to the greater destruction of the SNAP- 25 substrate.
  • 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).
  • painstimulating peptides e.g. calcitonin gene-related peptide (CGRP) and substance P
  • TRPV1 transient receptor potential vallinoid 1
  • 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.
  • SVP2A glycosylated synaptic vesicle protein 2A
  • 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.
  • the LC/E protease removes 26 SNAP-25 amino acid residues, thus preventing the formation of a stable
  • LC/A also cleaves SNAP-25, it only cleaves 9 amino acid residues, and the blockage of exocytotic activity is less complete and stable.
  • 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.
  • HN aminoterminal half of the H chain
  • 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 C N and H C c subdomains cooperatively bind certain cell surface gangliosides and a protein receptor (synaptic vesicle glycoprotein receptor 2 (SV2)).
  • SV2 protein receptor
  • R is meant any serotype of botulinum toxin.
  • 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.
  • BoNT/A cooperative binding of the protein receptor and gangliosides results in an energy- and temperature-dependent endocytosis of the toxin within the cell.
  • 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.
  • CGRP calcitonin gene-related peptide
  • 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.
  • 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.
  • 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 inexpensively 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).
  • 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.
  • TGNs trigeminal ganglion neurons
  • TRPV1 nociceptive channel-transient receptor potential vanilloid 1
  • 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).
  • 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
  • rats pre-treated with the compositions of the present invention 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).
  • 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.
  • 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.
  • 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”
  • 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.
  • 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.
  • TNG trigeminal nucleus caudalis
  • 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.
  • TGNs trigeminal neurons
  • 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.
  • 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.
  • His6 hexahistadine
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • IMAC immobilised metal affinity chromatograph
  • 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 .
  • 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.
  • 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.
  • 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.
  • 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
  • LC/E-BoNT/A 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.
  • 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
  • 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 ⁇ g 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.
  • 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.
  • Figure 3A, D 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).
  • 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
  • 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.
  • Subject rats were euthanized, and the caudal brainstems containing the TNG were dissected and fixed. Cryosections were made and stained.
  • 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).
  • 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).
  • 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.
  • Example 7 Chimeras LC/E-BoNT/A and BoNT/EA Inhibit CGRP Release from TGNs Evoked by Strong Stimulation with Capsaicin
  • 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).
  • the SNARE protein syntaxin 1 remained uncleaved under these conditions, as revealed by the Western blotting.
  • 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.
  • 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.
  • AITC Allyl isothiocyanate
  • 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.
  • this accessible site was preferred herein for administering capsaicin, a TRPV1 agonist, or AITC, a TRPA1 agonist.
  • 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).
  • 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.
  • 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.
  • 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.

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Abstract

L'invention concerne des méthodes et des compositions permettant d'inhiber la sensation de douleur aiguë chez un mammifère. Les méthodes peuvent comprendre l'administration locale à un ou plusieurs neurones projetant aux ganglions trijumeaux dudit mammifère d'une composition qui tronque la protéine 25 associée à la synaptosomale intacte (SNAP-25) dans le cytoplasme dudit ou desdits neurones pour produire une pluralité de molécules SNAP-25 tronquées dépourvues de 26 acides aminés à partir de l'extrémité C-terminale associée. La méthode peut comprendre l'administration intracellulaire de ladite composition à un ou plusieurs neurones innervant ou projetant à un site de douleur aiguë potentielle. La composition peut comprendre une protéase, telle qu'une quantité efficace d'une endopeptidase à chaîne légère dérivée de neurotoxine Clostridium botulinum (BoNT) dérivée du sous-type E de BoNT. La composition peut comprendre une quantité efficace de protéine active dérivée de Clostridium botulinum (BoNT) présentant un domaine de liaison à la cellule, et de préférence un domaine de translocation, dérivé du sous-type A de BoNT et une endopeptidase de chaîne légère dérivée du sous-type E de BoNT. Dans certains modes de réalisation, la composition peut comprendre plus d'une protéase, telle qu'une endopeptidase de chaîne légère dérivée du sous-type E de BoNT et une endopeptidase de chaîne légère dérivée du sous-type A de BoNT. Dans des modes de réalisation actuellement préférés, la composition peut comprendre un polypeptide choisi parmi LC/E-BoNT/A et/ou BoNT/EA.
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