WO2017081296A1 - Nouvelles approches thérapeutiques pour les affections démyélinisantes telles que la sclérose en plaques - Google Patents

Nouvelles approches thérapeutiques pour les affections démyélinisantes telles que la sclérose en plaques Download PDF

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WO2017081296A1
WO2017081296A1 PCT/EP2016/077501 EP2016077501W WO2017081296A1 WO 2017081296 A1 WO2017081296 A1 WO 2017081296A1 EP 2016077501 W EP2016077501 W EP 2016077501W WO 2017081296 A1 WO2017081296 A1 WO 2017081296A1
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trpa1
trpal
antagonist
ischaemia
subject
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Nicola Brenda HAMILTON-WHITAKER
David Ian ATTWELL
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Ucl Business Plc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A61K31/522—Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/15—Oximes (>C=N—O—); Hydrazines (>N—N<); Hydrazones (>N—N=) ; Imines (C—N=C)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/202—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having three or more double bonds, e.g. linolenic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4965—Non-condensed pyrazines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/59—Compounds containing 9, 10- seco- cyclopenta[a]hydrophenanthrene ring systems
    • A61K31/592—9,10-Secoergostane derivatives, e.g. ergocalciferol, i.e. vitamin D2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/59—Compounds containing 9, 10- seco- cyclopenta[a]hydrophenanthrene ring systems
    • A61K31/593—9,10-Secocholestane derivatives, e.g. cholecalciferol, i.e. vitamin D3
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/662—Phosphorus acids or esters thereof having P—C bonds, e.g. foscarnet, trichlorfon
    • A61K31/663—Compounds having two or more phosphorus acid groups or esters thereof, e.g. clodronic acid, pamidronic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/7036—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin having at least one amino group directly attached to the carbocyclic ring, e.g. streptomycin, gentamycin, amikacin, validamycin, fortimicins
    • 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

Definitions

  • This invention relates to a novel therapeutic approach for treating demyelinating diseases such as multiple sclerosis (MS) and for treating conditions associated with white matter
  • demyelinating diseases such as multiple sclerosis (MS)
  • MS multiple sclerosis
  • Oligodendrocytes create and maintain the myelin sheaths wrapped around the axons of neuronal cells. Myelination is needed to enable efficient transmission of electrical impulses (action potentials) along neuronal axons. Hence, oligodendrocytes are crucial for brain function. In demyelinating diseases such as MS, damage to the myelin sheath of neurons impairs the
  • Action potential propagation through myelinated axons is also impaired in ischaemia (1) . This had been thought to reflect a rundown of ion gradients across the axonal membrane, but impulse propagation only partly recovers on readmitting oxygen and glucose to the tissue to restore ion pumping (1) .
  • oligodendrocytes (3) show that ischaemia evokes Ca2+-dependent damage to the capacitance-reducing myelin sheaths, suggesting that irreversible myelin damage underlies much of the loss of action potential propagation in ischaemia.
  • Myelin is damaged in a Ca2+-dependent manner in ischaemia, abolishing action potential propagation (1), (2) .
  • MS is a demyelinating disease which involves both the loss of oligodendrocytes and damage to myelin sheaths. The aetiology of MS involves both genetic and environmental factors. In MS, the oligodendrocytes themselves demyelinate and die by immune attack. All current disease-modifying drugs target the immune system in order to decrease disease progression, but they are still relatively unsuccessful and have side effects caused by the diminished immune system.
  • the environmental factors known to increase the incidence or severity of MS include smoking (causing increases in CNS concentrations of nitric oxide, nicotine, carbon monoxide, hydrogen-cyanide) , surgery (the use of volatile anaesthetics such as sevofluorane) , viral infection (causing increases in CNS concentrations of TNF and INF- ⁇ ) and bacterial infections (causing increases in CNS concentrations of LPS, TNF and INF- ⁇ ) .
  • Vitamin D decreases brain nitric oxide concentrations.
  • TRPA1 agonists Many long chain fatty acids are TRPA1 agonists.
  • oligodendrocytes express TRP channels that are activated by intracellular protons, including TRPA1 channels, and have found that these channels are activated during pathology and lead to demyelination.
  • TRPA1 channels generate -70% of the ischaemia-evoked intracellular Ca2+ concentration ([Ca2+]i) rise, and that TRPA1 antagonists reduce ischaemic damage to myelin.
  • TRPA1 channels had not previously been implicated in oligodendrocyte pathology. Therefore this represents a
  • this invention provides an agent that blocks or desensitizes a Transient Receptor Potential (TRP) channel for use in a method of treatment or prophylaxis of a demyelinating or dysmyelinating disease or a failure of myelin to form in a subject, the method comprising administering the TRP blocker or desensitizer to the subject.
  • TRP Transient Receptor Potential
  • the TRP channel is activated by intracellular protons.
  • the TRP channel is Transient Receptor Potential cation channel of the subfamily Ankyrin, member 1 (TRPA1) .
  • TRPA1 Transient Receptor Potential cation channel of the subfamily Ankyrin, member 1
  • the invention provides a TRPA1 antagonist for use in a method of treatment or prophylaxis of a demyelinating or dysmyelinating disease or a failure of myelin to form in a subject, the method comprising administering the TRPA1 antagonist to the subject.
  • the demyelinating disease is multiple sclerosis (MS) .
  • the demyelinating disease or the MS is associated with brain hypoxia and/or white matter ischaemia and/or infection (including infection of a mother bearing a foetus, for example in conditions leading to
  • the demyelinating disease is associated with an autoimmune disease. In some embodiments, the demyelinating disease is caused by a genetic factor impairing fatty acid synthesis for myelin production. In some embodiments, the demyelinating disease is associated with fatty acid
  • the demyelinating disease a leukoencephalopathy, such as toxic leukoencephalopathy .
  • the demyelinating disease is caused by a disorder of fatty acid metabolism.
  • the demyelinating disease is Refsum disease.
  • the demyelinating disease is adrenoleukodystrophy (ALD) .
  • the disease is a dysmyelination disease, which is dysmyelinating leukodystrophy.
  • the disease is progressive multifocal leukoencephalopathy caused by virus or bacterial infection.
  • the demyelinating disease is acute disseminated encephalomyelitis involving inflammation or bacterial infection.
  • the demyelinating disease is osmotic demyelination syndrome.
  • this invention provides a Transient Receptor Potential cation channel of the subfamily Ankyrin, member 1
  • TRPA1 antagonist for use in a method of treatment or
  • Deleterious effects of white matter ischaemia include white matter damage, subcortical white matter lesions
  • oligodendrocytes block of action potential signalling within the brain and to the limbs due to loss of oligodendrocytes, which leads to chronic physical and/or mental disability (including paralysis and loss of speech) and sometimes death.
  • the white matter ischaemia is associated with a stroke. In other embodiments, the white matter ischaemia is a secondary ischaemia caused by spinal cord injury. In some embodiments, the white matter is damaged by hypoxia associated with multiple sclerosis (MS) . In some embodiments the white matter ischaemia and subsequent demyelination are associated with radiation treatment, chronic hypertensive encephalopathy or Leber's hyperintensive encephalopathy.
  • MS multiple sclerosis
  • TRPA1 antagonists TRPA1 blockers
  • ischaemia shows that block of oligodendrocyte TRPAl-containing channels may be useful for reducing myelin loss during the energy deprivation that follows stroke, secondary ischaemia caused by spinal cord injury, hypoxia associated with multiple sclerosis, hypoxia associated with radiation treatment, and ischaemia caused by chronic hypertensive encephalopathy and Leber's hyperintensive encephalopathy .
  • TRPAl antagonists target the
  • the TRPAl antagonist may reduce oligodendrocyte pathology in the subject.
  • the TRPAl antagonist may reduce oligodendrocyte loss in the subject.
  • the invention is therefore useful for treating, preventing and giving alleviation from demyelinating conditions such as MS; and for treating, preventing and giving alleviation from white matter ischaemia.
  • demyelinating conditions such as MS
  • white matter ischaemia white matter ischaemia
  • the TRP channel is Transient Receptor Potential cation channel of the subfamily Ankyrin, member 1 (TRPAl) .
  • TRPAl Transient Receptor Potential cation channel of the subfamily Ankyrin, member 1
  • the agent that blocks or desensitizes a Transient Receptor Potential (TRP) channel is an antagonist.
  • the agent that blocks or desensitizes a Transient Receptor Potential (TRP) channel is a TRPAl antagonist.
  • the invention provides a Transient Receptor Potential cation channel subfamily A, member 1 (TRPAl) antagonist for use in a method of treating or preventing an oligodendrocyte disease in a subject, the method comprising administering the TRPAl antagonist to the subj ect .
  • TRPAl Transient Receptor Potential cation channel subfamily A, member 1
  • the oligodendrocyte disease is associated with oligodendrocyte loss and/or oligodendrocyte demyelination and/or oligodendrocyte necrosis, apoptosis or other modes of death, and/or autoimmune attack directed at oligodendrocytes.
  • the oligodendrocyte disease is leukodystrophy.
  • the oligodendrocyte disease may be caused by an infection.
  • the infection may be a viral infection or the infection may be a bacterial infection. Alternatively or additionally, the oligodendrocyte disease may have a nutritional or environmental cause .
  • the subject may be a mammal, e.g. a human subject.
  • the human subject is a smoker.
  • a smoker can be regarded as a person who inhales tobacco smoke on one or more occasions per week.
  • a smoker may be an active smoker, who intentionally inhales tobacco smoke.
  • a smoker is a passive smoker who inhales tobacco smoke exhaled by an active smoker.
  • the TRPAl antagonist treats or is prophylactic against the harmful effects of nitric oxide (NO) , nicotine, hydrogen-cyanide (HCN) carbon monoxide (CO) and/or volatile anaesthetics such as sevofluorane .
  • the nitric oxide (NO), nicotine, hydrogen-cyanide (HCN) and/or carbon monoxide (CO) may be inhaled in tobacco smoke.
  • the smoker is a heroin (diamorphine) smoker.
  • the heroin smoker may inhale heroin on one or more occasions per week.
  • the TRPAl antagonist is used in a method of a method of preventing intracellular [Ca2+] rise and/or
  • the TRPAl antagonist is used in a method of reducing the intracellular concentration of divalent and trivalent cations including Ca2+, Mg2+, Zn2+, Fe2+ and Fe3+ and/or reducing changes in membrane K+ conductance .
  • the TRPA1 antagonist protects the subject from the harmful effects of nitric oxide (NO) and/or nitric acid produced by other sources, for example produced by microglia.
  • NO nitric oxide
  • the subject has undergone, is undergoing or is about to undergo surgery. The subject may have been treated with, may be being treated with or may be about to be treated with, a volatile anaesthetic, such as sevofluorane , e.g.
  • the volatile anaesthetic acts on TRPA1.
  • TRPA1 antagonist of the invention can be used as a preventative measure in association with surgery in order to stop the possibility of patients subsequently getting MS evoked by the anaesthesia.
  • the invention provides a method of treatment of demyelinating diseases, dysmyelinating diseases or diseases in which myelin fails to form, the method comprising administering a drug that blocks one or more downstream effectors of the calcium that enters through a Transient Receptor Potential (TRP) channel.
  • TRP Transient Receptor Potential
  • the TRP channel may be TRPA1.
  • the invention provides a method of treatment of a demyelinating diseases, dysmyelinating diseases or diseases in which myelin fails to form, the method comprising
  • the subject may be suffering from an autoimmune condition.
  • the subject has elevated estrogen levels.
  • the subject may be suffering from an infection.
  • the infection may be a viral infection or the infection may be a bacterial infection.
  • the bacterial infection may result in LPS-mediated stimulation of TRPAl-containing channels.
  • the infection may trigger an immune response.
  • the infection may trigger an autoimmune response.
  • the immune/autoimmune response may be mediated by Thl cells and/or mediated by Thl7 cells.
  • the immune/autoimmune response may be mediated by TNF, IFN (e.g. I N-gamma) , IL-l-beta and/or other immunostimulatory cytokines.
  • the subject may be suffering from an autoimmune response to myelin debris.
  • This autoimmune response may be mediated by Thl cells and/or mediated by Thl7 cells.
  • This autoimmune response may be mediated by TNF, IFN (e.g. IFN-gamma), IL-l-beta and/or other immunostimulatory cytokines.
  • a method of treating or preventing a demyelinating disease in a subject comprising administering an agent that blocks or desensitizes a Transient Receptor Potential (TRP) channel to the subject.
  • TRP Transient Receptor Potential
  • the TRP channel is activated by intracellular protons.
  • the TRP channel is Transient Receptor Potential cation channel of the subfamily Ankyrin, member 1 (TRPA1) .
  • TRPA1 Transient Receptor Potential
  • the agent that blocks or desensitizes a Transient Receptor Potential (TRP) channel is an antagonist.
  • the agent that blocks or desensitizes a Transient Receptor Potential (TRP) channel is a TRPA1 antagonist.
  • the TRP channel is a TRPA1 antagonist.
  • demyelinating disease is multiple sclerosis (MS) .
  • MS multiple sclerosis
  • the MS is associated with brain hypoxia and/or white matter ischaemia.
  • the demyelinating disease is associated with an autoimmune disease.
  • a method of treating or preventing white matter ischaemia in a subject comprising administering a TRPA1 antagonist to the subject.
  • the white matter ischaemia is associated with a stroke.
  • the white matter ischaemia is a secondary ischaemia caused by spinal cord injury.
  • the white matter ischaemia is caused by hypoxia associated with multiple sclerosis (MS) .
  • the method of treating or preventing white matter ischaemia is specifically a method of preventing damage to myelin .
  • Demyelination may be caused by inflammation; e.g. due to
  • Demyelination may be caused by viral or bacterial infection; e.g. due to progressive multifocal leukoencephalopathy, HIV, MS or ADEM associated with infection with the Gram-negative bacterium Chlamydia pneumoniae .
  • Demyelination may be caused by acquired metabolic changes; e.g. due to demyelination syndrome (also known as central pontine myelinolysis ) , toxic leukoencephalopathy (e.g. after smoking heroin), injesting paradichlorobenzene or glue sniffing.
  • Demyelination may be caused by hypoxia/ischaemia; e.g. due to radiotherapy, chronic hypertensive encephalopathy leading to subcortical leukoencephalopathy, stroke, spinal cord injury or possibly in MS (Davies et al . , 2013, which is hereby encorporated by reference) .
  • Demyelination may be caused by compression, e.g. due to trigeminal neuroalgia or spinal cord injury.
  • Demyelination may be caused by environmental toxicity which are linked to MS.
  • the present invention offers a novel therapeutic approach for these demyelination associated conditions.
  • a TRPA1 antagonist in the manufacture of a medicament for the treatment or prophylaxis of a demyelinating disease in a subj ect is provided.
  • use of a TRPA1 antagonist in the manufacture of a medicament for the treatment or prophylaxis of a demyelinating disease in a subj ect is provided.
  • use of a TRPA1 antagonist in the manufacture of a medicament for the treatment or prophylaxis of a demyelinating disease in a subj ect is provided.
  • use of a TRPA1 antagonist in the manufacture of a medicament for the treatment or prophylaxis of a demyelinating disease in a subj ect is provided.
  • use of a TRPA1 antagonist in the manufacture of a medicament for the treatment or prophylaxis of a demyelinating disease in a subj ect is provided.
  • the TRPA1 antagonist is specific for TRPA1 over other TRP channels. In other embodiments of this invention, the TRPA1 antagonist blocks TRPA1 as well as other TRP channels expressed by oligodendrocytes.
  • the TRPAl antagonist or TRPAl desensitiser is Hydra HC-030031, Abbott A967079, Glenmark GRC-17536, cannabinoids , gingerol, curcumin, cinnemaldehyde, Novartis compound 31, AMG0902, Novartis AP18, Amgen compound 10, Janssen Compound 43, CHEM-5861528 , resolving D2 , gentamicin, isopentenyl diphosphate (IPP) , TCS 5861528, a fatty acid, amiloride or analogues thereof.
  • IPP isopentenyl diphosphate
  • TRPAl channel blockade may be effected by an agent that has some action as a TRPAl agonist with reduced efficacy, such as a partial agonist or a mixed agonist/antagonist, that leads to subsequent blockade of TRPAl, e.g. by desensitization .
  • Diagnosis and prognosis In another aspect, a method of detecting or prognosing a
  • demyelinating disease in a subject comprising using a molecule that specifically binds to TRPAl or to its DNA or RNA to determine TRPAl expression level in a subject, the method further comprising comparing the TRPAl level to a control level.
  • a sample has been obtained from the subject and the TRPAl expression level is determined in said obtained sample.
  • the sample has been obtained from the CNS or CSF of the subject.
  • the sample comprises oligodendrocytes.
  • the method may comprise determining the TRPAl expression level in (or at the surface of) oligodendrocytes.
  • the method of detecting or prognosing a demyelinating disease comprises the detection of a labelled molecule that specifically binds TRPAl, which has been administered to the subject.
  • the invention provides an in vitro or in vivo method of screening for agents suitable for use in treating demyelinating diseases, dysmyelinating diseases and/or diseases in which myelin fails to form, the method comprising; providing an oligodendrocyte, applying a candidate agent to an oligodendrocyte, applying a candidate agent to an oligodendrocyte, applying a candidate agent to an oligodendrocyte, applying a candidate agent to an oligodendrocyte, applying a candidate agent to an oligodendrocyte, applying a candidate agent to an oligodendrocyte, applying a candidate agent to an oligodendrocyte, applying a candidate agent to an oligodendrocyte, applying a candidate agent to an oligodendrocyte, applying a candidate agent to an oligodendrocyte, applying a candidate agent to an oligodendrocyte, applying a candidate agent to an oligodendrocyte, applying a candidate agent to an oligo
  • the oligodendrocyte and measuring the effects of the candidate agent on expression levels of TRPAl in the oligodendrocyte and/or the rate of ion entry through TRPA1 channels in the oligodendrocyte.
  • the ion may be Ca2+, Mg2+ or any other permeant ion.
  • the TRPA1 antagonist can be combined with other agents and therapies, in which two or more agents or therapies are combined, for example, sequentially or simultaneously.
  • the agents may include nutritional supplementation and/or conventional medicines such as beta-interferon, or glatiramer acetate.
  • the therapy or therapies may include surgery.
  • the subject may be administered with the TRPA1 antagonist in combination with caffeine.
  • the caffeine may be consumed by the subject by drinking coffee.
  • the subject may be administered in combination with vitamin D.
  • the vitamin D may be consumed by the subject in the form of vitamin supplements.
  • the consumption of caffeine and/or vitamin D may be due to advice by a doctor or physician.
  • This invention also provides a pharmaceutical preparation or pharmaceutical composition comprising a TRPA1 antagonist, for use in a method of treating or preventing a demyelinating disease; for use in a method of treating or preventing white matter ischaemia; or for use in a method of treating or preventing an oligodendrocyte disease.
  • the pharmaceutical preparation or composition of the invention may be used according to the embodiments of the invention disclosed herein.
  • the pharmaceutical preparation or composition of the invention may comprise a pharmaceutically acceptable excipient .
  • the pharmaceutical compositions of the present invention are
  • kits may comprise a dry preparation (e.g. lyophilised preparation) of the TRPAl antagonist in a container, optionally with a buffer solution in a second container.
  • the kits may include instructions for dissolving the TRPAl antagonist, and/or mixing the TRPAl antagonist with the buffer, and/or administering the TRPAl antagonist to a subject.
  • a Whole-cell clamped oligodendrocyte shows Alexa dye in processes around an axon, b Ischaemia-evoked inward membrane current in a single cell, c Mean current in 179 control cells, 12 cells exposed to 25 ⁇ NBQX and 200 ⁇ D-AP5 from before the start of ischaemia, or preloaded (16) for 30 mins with 1 mM PDC .
  • NMDA does not elevate [Ca2+] i in oligodendrocytes a—b Oligodendrocyte membrane current (lower traces in a) and background-subtracted fluorescent dye ratio (R, see Methods, concentration increases are plotted upwards for all dyes) when measuring [Ca2+] i with Fura-2, [Na+] i with SBFI, and [K+]i with PBFI; 100 ⁇ NMDA was applied, or [K+]o was raised from 2.5 to 5 mM, with fluorescence measured in the soma (a) or myelinating processes (b) . Right panels show mean peak fluorescence change normalised to the evoked current (number of cells on bars) .
  • Ratiometric Fluo-4 /Alexa-Fluor-594 signals [Ca2+]i) and membrane potential (Vm) in oligodendrocytes exposed to normal ischaemia (starting at arrow), or ischaemia in zero [Ca2+]o, or with the drugs shown present at these concentrations ( ⁇ ) : AP5 50, MK-801 50, 7-chlorokynurenate (7CK to block the NMDAR glycine site) 100, NBQX 25, GABAzine (Gz, to block GABAARs) 20, TTX (to block Na+ channels) 1, Cd2+ (to block Ca2+ channels) 100.
  • Vm voltage potential with 2.5 or 0 mM [K+]bath, before (Resting) and during ischaemia (Isch peak), and the change produced by
  • ischaemia (01sch) .
  • h Effect of removing K+ from the bath solution on [H+] i in control conditions (relative to value at start of K+ removal), and the [H+] i increase evoked by ischaemia (data normalised to value at start of ischaemia) with 2.5 or 0 mM K+ in the bath, i High [HEPES] i blocks the ischaemia-evoked decrease of membrane conductance, j—k Ischaemia-evoked rise of [Ca2+]i (j) and [Mg2+]i (k) are inhibited with 50 mM internal
  • HEPES. 1 Applying light to uncage H+ (bars) raises [Ca2+]i (1, an effect reduced by 200 ⁇ IPP or 80 ⁇ HC-030031) and [Mg2+] i (m) , but not when the caged H+ is omitted from the pipette.
  • P values in 1 are from Mann-Whitney tests.
  • TRPAl-containing channels mediate ischaemic Ca2+ accumulation and myelin damage
  • j-m Mean data for lamella separations (j), g ratio (k) , axon diameter (1) and axon vacuoles (m: EM picture shows vacuoles (black arrows) forming within the axon and the periaxonal space during ischaemia) in control, ischaemia alone or ischaemia with RuR (10 ⁇ ) or with A967079 (10 ⁇ ) and HC-030031 (80 ⁇ ) (TRPA1 block) . Numbers on bars are 'images (axons)' . P values for j-m from Mann- Whitney test except 1 from Kolmogorov-Smirnov test.
  • oligodendrocyte TRPA1 channel described in the present application is linked with the environmental factors implicated in MS : 1: Oligodendrocytes express TRPA1 subunit containing channels, which in other organs are known to function as a molecular integrator of multiple irritants and inflammatory mediators. Synergistic effects of multiple agonists lead to larger currents, pore dilation, lowering of the activation threshold and upregulation of channel expression.
  • oestrogen have increased incidence of MS. 3: Multiple studies show smoking increases incidence, severity and progression of MS. 4: Smoke contains many harmful components including nitric oxide (NO) , nicotine, hydrogen cyanide (HCN) and carbon monoxide (CO) , that can enter the blood stream and cross the blood-brain barrier. 5: Nicotine reaches a plasma concentration ( ⁇ 300nM) that can activate TRPA1. 6: Nicotine administration to the CNS increases nitric oxide production. 7: The CSF concentrations of NO and its metabolites are greater in MS patients and increase during acute MS relapse. A persistent increase in NO metabolites is associated with increased MS progression. NO and its
  • Microglia contribute to the production of NO and its
  • TRPA1 can activate TRPA1.
  • Pre-incubation with TNF induces sensitisation and upregulation of TRPA1, increasing the maximum response activated whilst also decreasing the activation
  • Cannabinoids activate TRPA1 but also desensitise them to noxious stimuli .
  • Intracellular MK-801 (1 mM) has no effect on NMDA-evoked currents in oligodendrocytes (c) but blocks them in pyramidal cells (d) , while extracellular MK-801 (50 ⁇ ) blocks both, e Voltage- dependence of the current evoked in 16 oligodendrocytes by 100 ⁇ NMDA and by elevating [K+]o from 2.5 to 5 mM.
  • f Specimen plot of membrane current in an oligodendrocyte versus local [K+]o in response to applying 100 ⁇ NMDA or elevating [K+]o from 2.5 to 5 mM.
  • FIG. 10 In situ hybridization data on TRP channel expression a.
  • In situ data for TRPA1 and TRPV3 in the cerebellum of rats and mice show TRPA1 mRNA in white matter (WM) cells in rats and mice (with denser expression in the adjacent granule cell layer, GCL) , but TRPV3 mRNA only in white matter cells in rats.
  • Specimen cells are labelled with white circles, b Higher magnification views of white matter, combining in situ hybridization for TRPA1 and TRPV3 with immunocytochemistry for 01ig2 (to label oligodendrocyte lineage cells) and CC1 (to define myelinating oligodendrocytes) .
  • TRPA1 mRNA is present (in rats and mice) and TRPV3 mRNA is present (in rats but not mice) in myelinating oligodendrocytes (01ig2+, CC1+: arrowheads) and also in some presumed oligodendrocyte precursor cells (01ig2+, CC1-: arrows) .
  • [Ca2+] i increase (ratio signal from Fluo-4 and Alexa Fluor 594) in oligodendrocyte somata when ischaemia solution was applied with the following drugs present (data normalised to interleaved controls, shown as black bars) : RN-1734 (0.5 mM) , which
  • Figure 12 Schematic of how oligodendrocyte [Ca2+] i is raised in ischaemia
  • TRPAl mRNA and protein expression by CCl-expressing mature oligodendrocytes This decreases the membrane K+ conductance (either directly or via TRPAl-containing channels opening) which contributes to the inward current generated, and opens TRPAl-containing channels (7) that let Ca2+ and Mg2+ into the cell (8) .
  • Figure 13 TRPAl mRNA and protein expression by CCl-expressing mature oligodendrocytes
  • TRPAl agonists evoke currents in oligodendrocytes a, b. Increase in rat oligodendrocyte conductance is evoked when an electrophilic TRPAl agonist is bath-applied.
  • TRPAl agonist evoked increase in [Ca 2+ ]i is larger in the presence of an inflammatory cytokine.
  • Figure 15 TRPAl agonist propargyl isothiocyanate (PITC)
  • Negative staining for GFAP indicates that these cells are not astrocytes .
  • Arrows are oligodendrocytes identified by their location, morphology and GFAP negative status.
  • TRPAl antagonists can be readily identified and characterised using well-known methods in the art, including patch-clamping, ion imaging and binding assays.
  • TRPAl antagonists inhibit or block the flow of Ca2+ (or other divalent) ions through the TRPAl-containing ion channel.
  • inhibit or “block” is meant the ability to cause an overall decrease preferably of 20% or greater, more preferably of 50% or greater, and most preferably of 75%, 85%, 90%, 95%, or greater in Ca2+ ion flow.
  • the TRPAl antagonist may be specific for TRPAl (meaning that the extent of inhibition or blocking of TRPAl-containing channels is at least 50% greater than, at least 60% greater than, at least 70% greater than, at least 80% greater than, at least 90% greater than or most preferably at least 95% greater than the extent of inhibition or blocking of one or more other ion channels, e.g. those containing TRPV3, TRPC3, TRPC5, TRPC6, TRPC7, TRPV4, TRPV5, TRPV6, TRPM2, TRPM3, TRPM4 , TRPM5 or TRPM8 ) , or it may also act on other TRP channels.
  • other ion channels e.g. those containing TRPV3, TRPC3, TRPC5, TRPC6, TRPC7, TRPV4, TRPV5, TRPV6, TRPM2, TRPM3, TRPM4 , TRPM5 or TRPM8 .
  • the TRPAl antagonist inhibits the TRP channels expressed by oligodendrocytes. In some embodiments, the TRPAl antagonist inhibits TRP channels that are activated by intracellular protons, which are expressed by oligodendrocytes.
  • TRP channel blockade with agents that have some agonist action
  • the invention provides an agent that blocks or
  • TRP Transient Receptor Potential
  • an agonist that leads to subsequent desensitization of TRP channels such as TRPAl may be used to desensitize and thus block TRPAl channels.
  • TRPAl Transient Receptor Potential
  • a sufficient therapeutic plasma concentration is needed to effect desensitization.
  • the dose of the desensitizing agonist can be chosen accordingly.
  • a TRP channels agonist such as a TRPAl agonist
  • TRPA1 antagonist antibodies with reduced efficacy may be used as a partial agonist or a mixed agonist/antagonist to effect at least partial blockade of TRPAl channels.
  • the agonist may be electrophilic or non-electrophilic .
  • TRPA1 antagonist antibodies antibodies
  • blocking antibody or an antibody “antagonist” is one which inhibits or reduces biological activity of the antigen it binds.
  • Preferred blocking antibodies or antagonist antibodies completely inhibit the biological activity of the antigen.
  • Radiopharmaceuticals 4: 915-922) Specific dosages may be indicated herein or in the Physician's Desk Reference (2003) as appropriate for the type of medicament being administered may be used.
  • a therapeutically effective amount or suitable dose of an antibody molecule may be determined by comparing its in vitro activity and in vivo activity in an animal model. Methods for extrapolation of effective dosages in mice and other test animals to humans are known. The precise dose will depend upon a number of factors, including whether the antibody is for prevention or for treatment, the size and location of the area to be treated, the precise nature of the antibody (e.g. whole antibody,
  • a typical antibody dose will be in the range 100 ⁇ g to 1 g for systemic applications, and 1 ⁇ g to 1 mg for topical applications. An initial higher loading dose, followed by one or more lower doses, may be administered.
  • the antibody will be a whole antibody, e.g. the IgGl or IgG4 isotype. This is a dose for a single treatment of an adult patient, which may be
  • antibody is used in the broadest sense and includes monoclonal antibodies (including full length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (see below) so long as they exhibit the desired biological activity.
  • Antibody fragments comprise only a portion of an intact antibody, generally including an antigen binding site of the intact antibody and thus retaining the ability to bind antigen.
  • Oligodendrocyte TRPA1 channels and environmental factors in MS An extensive literature search links the oligodendrocyte TRPA1 channel (newly described in the present application) with the environmental factors implicated in MS. These links are shown in Fig. 5. 1: Oligodendrocytes express TRPA1 subunit containing channels (present disclosure) , which in other organs are known to function as a molecular integrator of multiple irritants and inflammatory mediators (Bautista et al . , 2006) . Synergistic effects of multiple agonists lead to larger currents (Hu et al . , 2010; Brierly et al . , 2011; Lennertz et al . , 2012; Meseguer et al .
  • Nicotine administration to the CNS increases nitric oxide production (Suemaru et al . , 1997; Smith et al . , 1998; Tonnessen et al., 2000) .
  • 7 The CSF concentrations of NO and its metabolites are greater in MS patients and increase during acute MS relapse (Yamashita et al . , 1997; Svenningsson et al . , 1999) . Whilst persistent increases in NO metabolites is associated with increased MS progression (Rejdak et al . , 2004) . NO and its metabolites can cause demyelination and oligodendrocyte necrosis (Mitrovic et al . , 1995, 1996; Smith et al . , 1999) . 8: Microglia contribute to the production of NO and its metabolites (Boje and Arora, 1992) . 9: NO and its metabolites can activate TRPA1
  • HCN and CO reduce the metabolic capacity of the brain making it more ischaemic (Lawther and Commins, 1970) .
  • Chronic low doses of cyanide cause demyelination (Smith et al . , 1963; Philbrick et al . , 1979) .
  • Ischaemia activates oligodendrocyte TRPA1 via intracellular acidification (Present disclosure) .
  • C02 can activate TRPA1 (Wang et al . , 2010), along with several oxidative stress-related substances (Andersson et al . , 2008) .
  • Vitamin D decreases the production of NO and pro-inflammatory cytokines by microglia (Lefebvre d' Hellencourt et al . , 2002) .
  • Caffeine consumed via 4-5 cups of coffee per day has been found to decrease the incidence of MS (paper in preparation by Dr Ellen Mowry, Johns Hopkins
  • oligodendrocyte TRPA1 subunit containing receptors is through the nasal cavity where the olfactory tract is susceptible to inhaled compounds that can circumvent the blood brain barrier to enter the CNS (Ilium, 2000) .
  • Neuropathological studies have shown that olfactory bulb/tract demyelination is frequent, can occur early, is highly inflammatory, and is specific to demyelinating disease, especially multiple sclerosis (DeLuca et al . , 2014) .
  • Demyelination can be caused by the following altered brain states: 1) inflamma ion, which occurs in MS (Lucchinetti et al . , 2000), neuromyelitis optica (Sharma et al . , 2010), acute
  • ADAM disseminated encephalomyelitis
  • bickerstaff brainstem encephalitis ; 2
  • viral or bacterial infection which occurs in progressive multifocal
  • leukoencephalopathy after smoking heroin (Wolters et al . , 1982) , injesting paradichlorobenzene (Weidman et al . , 2015) or glue sniffing (Davies et al . , 2000) ; 4) , hypoxia/ischaemia which occurs after radiotherapy (Greene-Schloesser et al . , 2012) , in chronic hypertensive encephalopathy leading to subcortical leukoencephalopathy (Stoner and Parker, 1991), in stroke
  • the present disclosure presents oligodendrocyte TRPAl as a new therapeutic target for demyelination in all of these instances because TRPAl elsewhere in the body, or in vitro, is known to be activated by inflammatory cytokines (Lowin et al . , 2015) , by bacterial infection, extracellular hypertonic solution (Zhang et al . , 2008) , morphine (Forster et al . , 2009) i.e. from heroin use, toluene (Taylor-Clark et al . , 2009) i.e. from glue sniffing, hypoxia (Takahashi et al . , 2011) , ischaemia (present disclosure), compression (as they are mechanosensors ; Brierley et al . , 2011), and by the aforementioned environmental factors implicated in MS.
  • inflammatory cytokines Lowin et al . , 2015
  • extracellular hypertonic solution Zhang
  • Leukodystrophies degeneration of white matter (oligodendrocytes) in the brain
  • Leukodystrophies include adrenomyeloneuropathy, Alexander disease, Cerebrotendineous xanthomatosis, hereditary CNS demyelinating disease, Krabbe disease, metachromatic leukodystrophy, Pelizaeus- Merzbacher disease, Canavan disease, leukoencephalopathy with vanishing white matter, adrenoleukodystrophy, Refsum disease and Xenobefantosis .
  • oligodendrocyte TRPAl is a new potential therapeutic target because of its newly found location and ability to be activated by many factors occurring in these diseases . Symptoms
  • Symptoms of demyelinating diseases that may be alleviated by the therapeutic approaches provided in include blurred (or double) vision, ataxia, clonus, dysarthria, fatigue, clumsiness, hand paralysis, hemiparesis, genital anaesthesia, incoordination, paresthesias, ocular paralysis, impaired muscle coordination, weakness (muscle) , loss of sensation, impaired vision,
  • Demyelination also occurs in toxic leukoencephalopathy which can be caused by smoking heroin (60) .
  • Morphine is a TRPAl agonist and sensitiser (62) and can sensitise TRPAl subunits to other TRPAl agonists in situ when it is applied at concentrations as low as 1 ⁇ (Kumazawa et al . , 1989) The peak saliva
  • the demyelinating disease is a leukoencephalopathy, such as toxic
  • the toxic compound in some embodiments, the toxic
  • TRPAl antagonists of the present invention may be comprised in pharmaceutical compositions with a pharmaceutically acceptable excipient .
  • a pharmaceutically acceptable excipient may be a compound or a combination of compounds entering into a pharmaceutical
  • composition which does not provoke secondary reactions and which allows, for example, facilitation of the administration of the TRPAl antagonist, an increase in its lifespan and/or in its efficacy in the body or an increase in its solubility in
  • TRPAl antagonists will usually be administered in the form of a pharmaceutical composition, which may comprise at least one component in addition to the TRPAl antagonist.
  • compositions may comprise, in addition to the TRPAl antagonist, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the TRPAl antagonist.
  • a pharmaceutically acceptable excipient such materials should be non-toxic and should not interfere with the efficacy of the TRPAl antagonist.
  • the precise nature of the carrier or other material will depend on the route of administration, which may be by bolus, infusion, injection or any other suitable route, as discussed below.
  • the pharmaceutical preparation will exclude water and/or exclude glycerol and/or exclude sodium azide and/or exclude bovine serum albumin (BSA) and/or exclude phosphates and/or exclude surfactants.
  • BSA bovine serum albumin
  • composition comprising the TRPAl antagonist may be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection.
  • Preservatives, stabilizers, buffers, antioxidants and/or other additives may be employed as required including buffers such as phosphate, citrate and other organic acids;
  • antioxidants such as ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol ; 3'-pentanol; and m- cresol); low molecular weight polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagines, histidine, arginine, or lysine;
  • chelating agents such as EDTA
  • sugars such as sucrose, mannitol, trehalose or sorbitol
  • salt- forming counter-ions such as sodium
  • metal complexes e.g. Zn- protein complexes
  • non-ionic surfactants such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG) .
  • a pharmaceutical composition comprising a TRPAl antagonist may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the
  • a TRPAl antagonist as described herein may be used in a method of treatment of the human or animal body, including prophylactic treatment (e.g. treatment before the onset of a condition in an individual to reduce the risk of the condition occurring in the individual; delay its onset; or reduce its severity after onset) .
  • the method of treatment may comprise administering a TRPAl antagonist to an individual in need thereof.
  • treatment refers generally to treatment and therapy of a human, in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, regression of the condition, amelioration of the condition, and cure of the condition.
  • prophylactic measure i.e., prophylaxis, prevention
  • prophylactic measure i.e., prophylaxis, prevention
  • therapeutically-effective amount pertains to that amount of a compound of the invention, or a material, composition or dosage from comprising said compound, which is effective for producing some desired therapeutic effe commensurate with a reasonable benefit/risk ratio, when
  • prophylactically effective amount refers to that amount of a compound of the invention, or a material, composition or dosage from comprising said compound, which is effective for producing some desired prophylactic effect, commensurate with a reasonable benefit/risk ratio, when administered in accordance with a desired treatment regimen.
  • prophylaxis in the context of the present specification should not be understood to circumscribe complete success i.e. complete protection or complete prevention. Rather prophylaxis in the present context refers to a measure which is administered in advance of detection of a symptomatic condition with the aim of preserving health by helping to delay, mitigate or avoid that particular condition.
  • treatment includes combination treatments and
  • agents i.e., a compound as described herein, plus one or more other agents
  • the agents when administered sequentially, can be administered at closely spaced intervals (e.g., over a period of 5-10 minutes) or at longer intervals (e.g., 1, 2, 3, 4 or more hours apart, or even longer periods apart where required) , the precise dosage regimen being commensurate with the properties of the therapeutic agent ( s ) .
  • agents i.e., a compound as described here, plus one or more other agents
  • the agents may be formulated together in a single dosage form, or alternatively, the individual agents may be formulated separately and presented together in the form of a kit,
  • Administration is normally in a "therapeutically effective amount", this being sufficient to show benefit to a patient.
  • Such benefit may be at least amelioration of at least one symptom.
  • the actual amount administered, and rate and time- course of administration, will depend on the nature and severity of what is being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the composition, the method of administration, the scheduling of administration and other factors known to medical practitioners. Prescription of
  • the therapeutic plasma concentration varies by TRPA1 agonist and antagonist and is estimated first by its in vitro EC50 or IC50 and later by its bioavailability, permeability across the blood-brain barrier, body clearance and unbound fraction, and resulting efficacy in the plasma.
  • the known IC50 of some TRPAl antagonists at human TRPAl are HC 030031 (IC50 : 6.2 ⁇ ) , AP18 (IC50 : 3. ⁇ ) , Jannssen Compound 43 (IC50 : 0.013 ⁇ ) , Amgen Compound 10 (IC50 : 0.17 ⁇ ) , Abbott A-967079 ( IC50 : 0.067 ⁇ ) ,
  • Administration can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell(s) being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician or clinician.
  • a suitable dose of the compound may be in the range of about 1 mg to about 200 mg per kilogram body weight of the subject per day e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, etc. mg, but this will vary according to the compound used.
  • the dosing may be split into loading and maintenance doses.
  • a loading dose is between and 5 and 500 mg, e.g.
  • a maintenance dose is between and 1 and 400 mg/kg/day.
  • a maximum daily maintenance dose is less than 1000 mg/day.
  • a TRPAl antagonist may be loaded using a dose divided into three equal doses given once daily on 3 consecutive days to achieve a therapeutic drug concentration gradually over the 3 days.
  • a loading dose of about 15 mg/kg (which may be rounded up to the nearest 100 mg) can be used.
  • a maintenance dose of about 4 mg/kg (which may be rounded up to the nearest 50 mg, with a maximum of 300 mg) can be used.
  • the compound is administered to a human patient according to the approved dosage regime for that drug, but which could be about 50mg, 3 times daily.
  • the compound is administered to a human patient according to the following dosage regime: about 100 mg, times daily.
  • the compound is administered to a human patient according to the following dosage regime: about 150 mg, times daily.
  • the compound is administered to a human patient according to the following dosage regime: about 200 mg, times daily.
  • Treatments may be repeated at daily, twice-weekly, weekly or monthly intervals, at the discretion of the physician.
  • TRPA1 antagonists may be delivered orally, through nasal inhalation or by injection.
  • Treatments may be every two to four weeks for subcutaneous administration and every four to eight weeks for intra-venous administration.
  • Treatment may be periodic, and the period between administrations is about two weeks or more, e.g. about three weeks or more, about four weeks or more, or about once a month.
  • Treatment may be given before, and/or after surgery, and/or may be administered or applied directly at the anatomical site of surgical treatment or invasive procedure. Suitable formulations and routes of administration are described above .
  • EXAMPLE 1 Proton-gated Ca2+-permeable TRP channels mediate myelin damage in ischaemia
  • [Ca2+] elevation is mediated by channels with characteristics of TRPA1, being inhibited by ruthenium red, isopentenyl
  • TRPA1 block reduces myelin damage in ischaemia.
  • Ischaemia blocks action potential propagation through myelinated axons (1) . This might reflect a rundown of ion gradients across the axonal membrane, but impulse propagation only partly recovers on readmitting oxygen and glucose to the tissue to restore ion pumping (1) . Electron microscopy (2) and imaging of dye-filled oligodendrocytes3 show ischaemia-evoked Ca2+-dependent damage to the capacitance-reducing myelin sheaths, suggesting that
  • Glutamate receptor block reduces myelin damage and loss of action potential propagation (2) -(7), and glutamate evokes a membrane current in oligodendrocytes mediated by AMPA/kainate and NMDA receptors (4), which have been
  • oligodendrocytes identified on oligodendrocytes using immunocytochemistry and electron microscopy (2) -(4) .
  • the damage occurring to oligodendrocytes is thought to be excitotoxic, resembling that occurring to neurons in ischaemia: a rise of extracellular glutamate concentration (8) caused by reversal of glutamate uptake carriers in oligodendrocytes and axons (9), (10) activates ionotropic receptors that raise (2) oligodendrocyte intracellular Ca2+ and thus damage the cells.
  • these receptors are down-regulated as the cells mature
  • Solution mimicking ischaemia (see ⁇ Methods', below) evoked a slowly increasing inward current in oligodendrocytes (Fig. la, b) , that often had a sharper increase superimposed upon it.
  • the timing of this sharper increase varied between cells, so that when responses in many cells were averaged (Fig. lc) it was not visible as a separate entity.
  • NBQX and D-AP5 reduced the ischaemia- evoked current by 66% (after 8-10 mins ischaemia: Fig. lc, d) , while mGluR block had no significant effect (Fig. 6a) .
  • Preloading for 30 mins with the glutamate transport blocker PDC to prevent ischaemia-evoked glutamate release by reversal of glutamate transporters in the white (9) and grey (16) matter, also greatly reduced the inward current (by 68%, Fig. lc, d) , while blocking other candidate release mechanisms had no significant effect (see Fig. 6a) .
  • glutamate release by reversed uptake plays a significant role in triggering the ischaemia-evoked current.
  • END Depolarization
  • ischaemia- evoked current in oligodendrocytes was not prevented by removal of external Ca2+ and chelation of trace Ca2+ with 50 ⁇ EGTA, or by gadolinium (100 ⁇ ) , which both block the END17 (Fig. Id, e) , indicating that a different mechanism maintains the inward current triggered by glutamate.
  • oligodendrocytes (Fig. If, g; decreased by 2.110.7 nS near -70 mV in 11 cells using 10 mM, and 2.310.6 nS in 10 cells using 0.5 mM, internal HEPES; see below for results with higher pH buffering) .
  • ischaemia which releases Mg2+ (although ATP was present in the pipette) , but the [Mg2+] i rise was abolished in the absence of extracellular Mg2+ (Fig. 3d) implying that Mg2+ enters across the cell membrane.
  • ischaemia did not raise [Na+]i (Fig. 3e) .
  • ischaemia activates a membrane conductance that allows the entry of divalent ions.
  • TRP channel activation may contribute to ischaemic damage to neurons (21) and astrocytes (22) .
  • TRP channel activation may contribute to ischaemic damage to neurons (21) and astrocytes (22) .
  • TRP channel activation may contribute to ischaemic damage to neurons (21) and astrocytes (22) .
  • TRP channel activation may contribute to ischaemic damage to neurons (21) and astrocytes (22) .
  • TRP channel activation may contribute to ischaemic damage to neurons (21) and astrocytes (22) .
  • TRP channel activation may contribute to ischaemic damage to neurons (21) and astrocytes (22) .
  • TRPA1 /TRPV3 blocker isopentenyl pyrophosphate (25) (IPP) and the specific26 TRPA1 blocker HC-030031 slowed and reduced the [Ca2+]i rise evoked by uncaging H+ in the cell (Fig. 31) .
  • the TRPA1 /TRPV3 agonists (20) , (27) menthol, vanillin, carvacrol (Cv) and 2-APB all evoked [Ca2+] i rises in
  • TRPAl and TRPV3 antibodies appeared to label the myelinating processes and somata of oligodendrocytes in rat, but the
  • TRPAl and TRPV3-expressing cells included myelinating oligodendrocytes expressing both 01ig2 and CC1 (Fig. lOb-d) .
  • decompaction (lamellar separation) per axon cross section (see Methods) .
  • oligodendrocytes is reported to be blocked by NMDA receptor antagonists (2), which contradicts our demonstration that NMDA evokes no [Ca2+]i rise in oligodendrocytes (Fig. 2) and that the ischaemia-evoked [Ca2+]i rise is unaffected by NMDA receptor blockers (Fig. 3) .
  • NMDA receptor antagonists (2) which contradicts our demonstration that NMDA evokes no [Ca2+]i rise in oligodendrocytes (Fig. 2) and that the ischaemia-evoked [Ca2+]i rise is unaffected by NMDA receptor blockers (Fig. 3) .
  • TRPA1 generates -70% of the ischaemia-evoked [Ca2+]i rise, and TRPA1 blockers reduce ischaemic damage to myelin (Fig. 4) .
  • Oligodendrocytes in cerebellar slices were patch-clamped, labelled using antibodies and in situ hybridization, and had their ion concentrations and morphology imaged, as described in the Methods.
  • mice were obtained from JAX (http://jaxmice.jax.org/strain/010773.html) . TRPA1 KO mice were obtained as a double knock-out with TRPV1 knocked out (kindly provided by John Wood and Jane Sexton) .
  • TRPV1 does not contribute to the ischaemia-evoked [Ca2+]i rise described here because the TRPV1 antagonist20 , 27 capsazepine did not reduce the ischaemia-evoked [Ca2+]I rise in rat oligodendrocytes (Fig. 11) and the TRPV1 agonists20 , 27 capsaicin (10 ⁇ ) and camphor (2 mM) did not evoke a [Ca2+]i rise (see Specificity of drugs acting on TRP channels and Fig. 4a) . Wild type and (double) KO mice were obtained from a colony obtained by breeding mice doubly
  • Slices were then incubated at room temperature (21- 24 °C) in the same solution until used in experiments.
  • Cerebellar slices from P10-17 mice were prepared in ice-cold solution containing (mM) 87 NaCl, 25
  • Oligodendrocytes, cerebellar granule cells and hippocampal pyramidal cells were identified by their location and morphology. All cells were whole-cell clamped with pipettes with a series resistance of 8-30 ⁇ . Electrode junction potentials were compensated. I-V relations were from responses to 200 msec voltage steps. Unless otherwise indicated, cells were voltage- clamped at -74 mV.
  • Cells were whole-cell clamped with electrodes containing either Cs- (to improve voltage uniformity) or K-gluconate-based
  • solution comprising (mM) 130 Cs-gluconate (or K- gluconate) , 2 NaCl, 0.5 CaC12, 10 HEPES, 10 BAPTA, 2 NaATP, 0.5 Na2GTP, 2 MgCl, 0.5
  • Fluo-4 and Alexa Fluor 594 were used in the internal solution to measure [Ca2+] i changes ratiometrically during H+-uncaging and most ischaemia experiments.
  • Mag-Fluo-4 was used instead of Fluo-4.
  • Fluo-4 (or Mag-Fluo-4) and Alexa Fluor 594 fluorescence were excited sequentially every 2, 10 or 30 seconds at 488 ⁇ 10nm and 585 ⁇ 10nm, and emission was collected using a triband filter cube (DAPI /FITC/Texas Red, 69002,
  • Caged-H+ were uncaged using 380120 nm light for 1 second every 2 seconds (repeated 30 times) interspersed with the above excitation wavelengths.
  • BCECF was imaged every 30 seconds at 400 and 480nm, with emission collected using the above triband filter.
  • X-Rhod-l-AM (38 ⁇ ) dye loading with the myelin marker DIOC6 into P12 cerebellar slices was performed as described previously for optic nerves2. Loading times ranged from 1-2 hrs and a de- esterification period of 30 mins at 36°C was allowed before imaging .
  • Potassium electrodes were made as described33. Electrodes were pulled with a resistance of 4-10 M . Electrode tips were
  • Cerebellar slices were fixed for 30 mins in 4% PFA, and incubated for 1 to 6 h in 0.1% Triton X-100, 10% goat serum in phosphate- buffered saline at 21°C, then with primary antibody at 4°C overnight with agitation, and then 2 hours or overnight at 24 °C with secondary antibody.
  • Primary antibodies were: anti-CCl
  • hybridization buffer [50% v/v deionized formamide (Sigma), 10% w/v dextran sulphate (Fluka) , 0.1 mg/ml yeast tRNA (Roche), lx Denhardt's solution (Sigma) and lx "salts" (200
  • Sections were then washed in lx MABT 5 times for 20 min each at room temperature, followed by two 5 min washes in staining buffer (100 mM NaCl, 50 mM MgC12, 100 mM Tris-HCl, pH 9.5, 0.1% Tween- 20) . Development was performed at 37 °C for 24-48 hours overnight with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate in freshly prepared staining solution (50% v/v staining buffer, 25 mM MgC12, 5% w/v polyvinyl alcohol) . Sections were washed in PBS and immunohistochemistry was performed as described above. The plasmids used to generate RNA probes were: IMAGE clone
  • Trpal linearized with Clal and transcribed with T3 RNA polymerase
  • IMAGE clone 40047664 for Trpv3 linearized with Xhol and transcribed with SP6 RNA polymerase
  • optic nerves were dissected from P28 Sprague- Dawley rats and incubated for 1 hour at 36°C in either control or ischaemic solution with and without the
  • TRPA1/V3 channel blocker ruthenium red (10 ⁇ ) or the combined presence of the TRPA1 blockers HC-030031 (80 ⁇ ) and A967079 (10 ⁇ ) .
  • the optic nerves were then immersion fixed in 2%
  • Axon diameter was calculated as (4. (axon area) /n) 0.5.
  • Axon vacuolisation was defined as the inclusion of one or more large (>0.1 ⁇ ) empty membrane bound (often circular) organelles within the axon or periaxonal space (Fig. 4m) which may reflect rearrangement of internal axonal membranes or be formed from inclusion of myelin membranes into the axon (Fig. 4m) .
  • FIG. 6a shows that there was no significant effect, when compared with interleaved control cells, on the peak ischaemia-evoked current of
  • release34 100 ⁇ CdC12 to prevent exocytotic glutamate release driven by Ca2+ entry through voltage-gated Ca2+ channels35, 36; 100 ⁇ lanthanum (LaC13) to block glutamate release through gap junctional hemichannels37 ; 10 ⁇ BBG to block P2X7 receptors38; 100 ⁇ amiloride to block ASIC channels which may be activated by the acid shift occurring in ischaemia39, 40 ; 1 ⁇ TTX to block action potential driven glutamate release35, 36; 100 ⁇
  • the ischaemia-evoked current reflects the sum of the current produced by a rise of [H+] i suppressing a mainly K+- specific ohmic conductance with a conductance of 2.3 nS (the slope of the 0.5 mM HEPES line in Fig. lg; the change of current this produces is schematised in Fig. 6b left panel), and a voltage-independent inward current of -58 pA (the mean value of the 50 mM HEPES data in Fig. lg) produced by the rise of [K+]o shifting the Nernst potential for K+ (the change of current this produces is schematised in Fig. 6b right panel) .
  • oligodendrocyte membrane is presumably closer than the K+-sensing electrode to the source of K+ when NMDA is applied than when the superfusate [K+] is raised.
  • TRPA1 and TRPV3 have previously been suggested to play a role in the response of the CNS to ischaemia (44), (45) .
  • TRPA1 is the major contributor to raising [Ca2+] i in ischaemia is based on the following logic.
  • TRPA1 and TRPV3 are currently known to be activated by intracellular protons (20), (23), (24) .
  • Drugs that activate TRPA1 but not TRPV3 (polygodial, AITC, FFA) , or that activate both TRPA1 and TRPV3 (menthol, vanillin, carvacrol, 2-APB) , but not drugs that activate TRPV3 and not TRPA1 (FPP, camphor), elevate [Ca2+] i (Fig. 4a, b) .
  • the most specific TRPA1 agonist is polygodial which does not act (46) on TRPV1, TRPV2, TRPV3, TRPV4 or TRPM8 , followed by AITC (allyl- isothiocyanate ) which also acts (20) on TRPV1.
  • Flufenamic acid activates TRPA1 but inhibits TRPV1 and TRPV347 and also acts on (20), (27), (47) -(49) TRPC3, TRPC5, TRPC6, TRPC7, TRPV4, TRPV5, TRPV6, TRPM2, TRPM3, TRPM4 , TRPM5 and TRPM8.
  • Carvacrol activates TRPA1 and TRPV3, and inhibits TRPM7 (20) , (27) .
  • pyrophosphate activates TRPV3 but does not activate TRPV1, TRPV2, TRPV4, TRPA1, and TRPM8 (25), (50) .
  • Camphor activates TRPV3, activates TRPV1 and TRPM8 , and inhibits TRPA1 (20), (27) .
  • Agonists for other TRPs did not raise [Ca2+]i detectably.
  • TRPAl /TRPV3 antagonist (25) isopentenyl pyrophosphate (IPP) , by the specific TRPAl antagonist (51) HC-030031, and by TRPAl knock-out (Fig. 4b) .
  • HC-030031 blocks TRPAl without affecting TRPVl, TRPV3, TRPV4, TRPM8 or 48 other receptors, transporters and enzymes (51) .
  • the blocker pharmacology matches that of the activator pharmacology.
  • A967079 greatly reduced the ischaemia-evoked [Ca2+]i rise (Fig. 4e) .
  • HC-030031 blocks TRPAl without affecting TRPVl, TRPV3, TRPV4, TRPM8 or 48 other receptors, transporters and enzymes (51) .
  • A967079 has minimal activity at 89 different G-protein- coupled receptors, enzymes, transporters, and ion channels including other TRP channels (52) .
  • TRPAl /TRPV3 blocker IPP in the TRPAl KO and (as expected) it had no effect
  • TRPAl blocker HC-030031 in the TRPV3 KO we applied the TRPAl blocker HC-030031 in the TRPV3 KO and (as expected) it greatly reduced the ischaemia- evoked [Ca2+]i rise.
  • TRP channels other than TRPAl are blocked by agents blocking TRP channels other than TRPAl, as follows: flufenamic acid (250 ⁇ ) which blocks (27), (54) TRPP2, TRPC3, TRPC5, TRPC7, TRPM2 , TRPM4 and TRPM5;
  • ML204 (20 ⁇ ) which blocks (20), (55) TRPC4 and TRPC5; SKF-96365 (100 ⁇ ) which blocks (27), (56), (57) TRPV2 , TRPC3, TRPC6, TRPC7 and TRPP1, as well as the store-operated calcium channel
  • capsazepine (CPZ, 100 ⁇ ) which blocks (20) TRPV1 and TRPM8;
  • oligodendrocytes are vulnerable to TRPAl agonists, and that TRPAl is a therapeutic target in neurodegerative diseases.
  • the present Example further characterises the expression of TRPAl by
  • oligodendrocytes the TRPAl-mediated current and the TRPAl evoked intracellular Ca 2+ concentration rise. TRPAl expression on oligodendrocytes
  • TRPAl protein expression is detected using labelled with antibodies to TRPAl (Abeam 58844) (Fig. 13) .
  • TRPAl appears on oligodendrocyte somata and myelin.
  • oligodendrocytes depending on the site at which the agonist activates TRPAl (Fig. 14) .
  • Electrophilic TRPAl agonists evoke an increase in oligodendrocyte conductance that can be inhibited by a TRPAl antagonist (Fig. 14a) .
  • non-electrophilic TRPAl agonists evokes an immediate inhibition of oligodendrocyte potassium conductance, such as occurs in ischaemia (Hamilton et al . , 2016) .
  • TRPAl agonists can evoke a sustained [Ca 2+ ]i increase or initiate oscillations that are inhibited by the TRPAl
  • Fig. 14d provides an indication that TRPAl-evoked Ca 2+ increases can be augmented by co-application of inflammatory cytokines.
  • the isothiocyanate based TRPAl agonist propargyl isothiocyanate preferentially binds to oligodendrocyte cell bodies within the corpus callosum (Fig. 15b) .
  • oligodendrocytes were identified by morphology, location in the white matter, and GFAP negative status (Fig. 15b and c) . This is further evidence that TRPAl agonists can preferentially act upon oligodendrocytes in the brain.
  • TRPAl contributes to specific mechanically activated currents and sensory neuron mechanical hypersensitivity. The Journal of Physiology 589, 3575-3593.
  • ADAM acute disseminated encephalomyelitis
  • Vitamin D3 inhibits proinflammatory
  • TRPAl channels mediate acute neurogenic inflammation and pain produced by bacterial endotoxins. Nat Commun 5, 3125.
  • TRPVl and TRPAl Mediate Peripheral Nitric Oxide- Induced Nociception in Mice. PLoS ONE 4, e7596.
  • TRPAl is a polyunsaturated fatty acid sensor in mammals. PLoS ONE 7, e38439.
  • Pendleburyab S.T.
  • Lee M.A.
  • Blamire A.M.
  • Styles P.
  • Nitric oxide metabolites in CSF of patients with MS are related to clinical disease course.
  • TRPA1 underlies a sensing mechanism for 02. Nat Chem Biol 7, 701-711.
  • Nicotine activates the chemosensory cation channel TRPA1. Nat Neurosci 12, 1293- 1299.
  • TRPV6 a putative epithelial calcium channel
  • Tonnessen B.H.
  • Severson S.R.
  • Hurt R.D.
  • Miller V.M.
  • oligodendrocytes and activated in ischaemia Nature 438, 1162-1168 (2005) .
  • oligodendrocytes PLoS Biol. 11, el001743 (2013) .
  • astrocytes, neurons, and oligodendrocytes a new resource for understanding brain development and function. J.
  • Attwell D. The electrical response of cerebellar Purkinje neurons to simulated ischaemia. Brain 128, 2408-2420
  • Isopentenyl pyrophosphate is a novel antinociceptive substance that inhibits TRPV3 and TRPA1 ion channels. Pain 152, 1156-1164 (2011) .
  • TRPM3 is expressed in sphingosine- responsive myelinating oligodendrocytes. J. Neurochem. 114, 654-665 (2010) .
  • TRPM7 transient receptor potential melastatin 7
  • Toxic encephalopathy due to paradichlorobenzene toxicity a case report and review of imaging characteristics.

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Abstract

Cette invention concerne une nouvelle approche thérapeutique pour le traitement des affections démyélinisantes telles que la sclérose en plaques (MS) et pour le traitement de troubles associés à l'ischémie de la substance blanche. L'invention porte également sur des modulateurs des canaux ioniques à utiliser dans lesdits traitements.
PCT/EP2016/077501 2015-11-13 2016-11-11 Nouvelles approches thérapeutiques pour les affections démyélinisantes telles que la sclérose en plaques WO2017081296A1 (fr)

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WO2023090313A1 (fr) * 2021-11-16 2023-05-25 京都府公立大学法人 Composition pour augmenter la quantité d'aliments ingérés et/ou améliorer l'anorexie, et composition pour activer une ankyrine 1 à potentiel de récepteur transitoire

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020018841A1 (fr) * 2018-07-18 2020-01-23 Duke University Compositions et méthodes de traitement du prurit aigu et chronique
WO2023090313A1 (fr) * 2021-11-16 2023-05-25 京都府公立大学法人 Composition pour augmenter la quantité d'aliments ingérés et/ou améliorer l'anorexie, et composition pour activer une ankyrine 1 à potentiel de récepteur transitoire

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