WO2024013052A1 - Nouvelle utilisation - Google Patents

Nouvelle utilisation Download PDF

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WO2024013052A1
WO2024013052A1 PCT/EP2023/068967 EP2023068967W WO2024013052A1 WO 2024013052 A1 WO2024013052 A1 WO 2024013052A1 EP 2023068967 W EP2023068967 W EP 2023068967W WO 2024013052 A1 WO2024013052 A1 WO 2024013052A1
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human
trpm3
inhibitor
use according
treatment
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PCT/EP2023/068967
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English (en)
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David Hall
Alexander PRESTON
Colin Macphee
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Glaxosmithkline Intellectual Property (No.3) Limited
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Priority claimed from US17/812,299 external-priority patent/US20230220037A1/en
Application filed by Glaxosmithkline Intellectual Property (No.3) Limited filed Critical Glaxosmithkline Intellectual Property (No.3) Limited
Publication of WO2024013052A1 publication Critical patent/WO2024013052A1/fr

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    • 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
    • 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
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A61K31/4045—Indole-alkylamines; Amides thereof, e.g. serotonin, melatonin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2807—Headache; Migraine

Definitions

  • the present disclosure relates to an inhibitor of human TRPM3 for use in the treatment or prevention of migraine or cluster headache in patients whose migraines/headaches are not responsive to CGRP inhibition or whose migraines/headaches are responsive to triptans.
  • the disclosure relates to an inhibitor of human TRPM3 for use in the treatment of medication overuse headache.
  • An inhibitor of human TRPM3 that inhibits the release of PACAP from trigeminal ganglion neurons, and a method for measuring PACAP release are also disclosed.
  • Migraine headaches are a common cause of disability in the United States, affecting approximately 27 million American adults, or 17.1% of women and 5.6% of men.
  • Chronic migraine which affects 3.2 million Americans (2%), is defined as having migraine symptoms for at least 15 days per month, lasting at least 4 hours, and for longer than 3 months in duration. This is in contrast to episodic migraine, which causes symptoms on fewer than 15 days per month.
  • Current treatment for migraine is divided into acute, abortive agents and medications that will prevent migraine onset.
  • Opioid analgesics and triptans are commonly prescribed for migraine. Whilst these types of drugs provide acute relief, regular use can result in increased headache severity, and progression of headache from an episodic to a chronic state. This form of medication overuse headache is difficult to treat and new treatment options are eagerly awaited.
  • Calcitonin gene-related peptide is a peptide that is released by peripheral neurons, including somatosensory neurons of the dorsal root, vagal and trigeminal ganglia where it is reported to act as a neurotransmitter, a vasodilator and as a local mediator of inflammation. Its short half-life (7 mins) normally results in localised effects.
  • CGRP levels are increased in the cranial circulation during migraine and cluster headache attacks, and intravenous administration of CGRP triggers migraine attacks in migraineurs suggesting that it has a prominent role in migraine.
  • PACAP pituitary adenylate cyclase activating polypeptide
  • PACAP has been implicated in spontaneous headache conditions. For example, elevated PACAP-38 levels have been observed during the attack phase in episodic cluster headache patients. It has also been demonstrated to relieve opioid induced hyperalgesia in animal models. For example, Pradhan and colleagues have demonstrated that PACAP blockade reduced periorbital allodynia in a mouse model of opioid induced hyperalgesia, and that PACAP blockade reduced cephalic allodynia in mice treated with combined morphine and nitroglycerin.
  • a PAC1 receptor monoclonal antibody AMG 301 was observed to offer no benefit over placebo for migraine prevention despite inhibiting nociceptive activity in the trigeminocervical complex to the same extent as sumatriptan in preclinical trials.
  • a monoclonal antibody targeting PACAP-38, ALD1910 remains in clinical development for the treatment of migraine patients.
  • the trigeminal ganglion has been examined to detect neuropeptides including calcitonin gene- related peptide (CGRP) and PACAP.
  • CGRP calcitonin gene- related peptide
  • PACAP-38 is present in the trigeminal ganglion, and plasma PACAP-38-like immunoreactivity is increased after electrical stimulation of the trigeminal ganglion.
  • Substance P is a mediator of the sterile inflammation of the dura, which may be a source of migraine pain. Triptans block this dural neurogenic inflammation dose-dependently in an animal model.
  • Substance P (neurokinin-1 receptor) antagonists are also highly effective in animal models of dural inflammation, but no NK-1 antagonist has yet demonstrated any clinical effect in acute migraine.
  • TRPM3 The human TRPM3 gene is comprised of 30 exons and maps to human chromosome 9q- 21.12.
  • TRPM3 isoforms vary from 1184 to 1744 amino acids in length and possess the characteristic six transmembrane domain of the TRP family. However, unlike some other TRPM family members, TRPM3 does not contain an enzyme domain in the C-terminal cytoplasmic region.
  • TRPM3 does not contain an enzyme domain in the C-terminal cytoplasmic region.
  • Several alternative spliced transcript variants encoding different isoforms have been identified in mice and humans. The isoforms appear to have very different physiological roles given that one mouse isoform, Trpm3al preferentially conducts monovalent cation influx, while another, Trpm3a2 strongly favours divalent ion entry. The mouse Trpm3a2 ⁇ om is the best studied.
  • TRPM3 expression is detectable in kidney, brain, ovary, and pancreas. Northern blotting of mouse tissues resulted in strong signals in brain, whereas in kidney no signal could be detected. Within brain subregions in the mouse, the highest levels of expression were found in the cerebellum, choroid plexus, the locus coeruleus, the posterior hypothalamus, and the substantia nigra, using a Trpm5-specific antisense RNA probe yielded a positive Trpm3 hybridization signal in 82% ⁇ 5% of mouse trigeminal neurons. The most abundant isoform in the human dorsal root ganglion as assessed by RNA expression has the UNIPROT ID: Q9HCF6-2.
  • the invention provides an inhibitor of human TRPM3 for use in the treatment or prevention of migraine in a human subject whose migraines are not responsive to CGRP inhibition.
  • the migraines are not responsive to CGRP therapy and are responsive to therapy with a triptan.
  • migraines that are "not responsive to CGRP inhibition” are migraines that are not adequately treated by CGRP inhibition.
  • Migraines that are "responsive to therapy with a triptan” are migraines that are adequately treated by a triptan.
  • the invention provides an inhibitor of human TRPM3 for use in the treatment or prevention of migraine in a human subject whose migraines are responsive to therapy with a triptan.
  • the invention provides an inhibitor of human TRPM3 for use in the treatment or prevention of cluster headache in a human subject whose headaches are not responsive to CGRP inhibition.
  • the headaches are not responsive to CGRP therapy and are responsive to therapy with a triptan.
  • the invention provides an inhibitor of human TRPM3 for use in the treatment of medication overuse headache in a human subject.
  • the invention provides a method for measuring PACAP in a sample comprising incubating a cell line expressing the PAC1 receptor with the sample and measuring cAMP signalling in the cell line.
  • the invention provides a method for identifying an inhibitor of human TRPM3, comprising measuring release of PACAP from dorsal root ganglia or trigeminal ganglia, or from primary cultures of cells isolated from dorsal root ganglia or trigeminal ganglia, following challenge with an agonist of human TRPM3 in the presence or absence of a test inhibitor, wherein the test inhibitor is identified as an inhibitor for human TRPM3 if PACAP production is reduced in the presence of the test inhibitor compared to PACAP production in the absence of the test inhibitor.
  • PACAP production is measured according to the method of the invention.
  • the invention provides (R)-2-(3,4-dihydroquinolin-l(2H)-yl)-N-(5- methylisoxazol-3-yl)propenamide or a salt thereof. DESCRIPTION OF DRAWINGS/FIGURES
  • FIGS. 1A and IB demonstrate that TRPM3 agonists, CIM0216 (0.37-10
  • FIG. 1A and IB demonstrate that TRPM3 agonists, CIM0216 (0.37-10
  • the CIM0216 response was inhibited by TRPM
  • FIGS 3A and 3B also show that CGRP responses were inhibited with isosakuranetin (10 ⁇ M).
  • FIG 4 shows a LocusZoom plot for the association between migraine defined using the 'migraine_diagnosis' classification and genetic variants at the TRPM3 locus.
  • the x-axis displays position on chromosome 9 using GRCh37/hgl9 as the human reference genome build.
  • the y-axis is the - logio(p-value) from a logistic regression testing for an association between migraine case-control status and genotype. Plus symbols (+) denote variants for which genotype was imputed; circle symbols (o) denote variants for which genotype calls were used; x symbols (x) denote imputed coding variants; diamond symbols (0) denote genotyped coding variants.
  • the horizontal line represents the genome-wide significance threshold of 5x10 -8 .
  • the credible set track displays the number and location of variants in the 99% credible set, which is likely to contain the causal variant.
  • the gene track displays genes in the locus with thick bars representing exons and thin lines representing introns.
  • PS and CIM0216 dose-dependent release of CGRP observed in WT neurons was lacking in Trpm3 deficient neurons. Capsaicin evoked CGRP release from Trpm3 deficient neurons.
  • FIG. 10A demonstrates that dural administration of TRPM3 agonists, Pregnenolone sulphate (5mM) and CIM0216 (215 ⁇ M), induce mechanical allodynia in the periorbital region of rats.
  • FIG. 11A demonstrates CIM0216 induces a concentration-dependent release of PACAP from trigeminal ganglia cultures and its inhibition by isosakuranetin.
  • FIG 13 shows the agonist activity of the isomers of CIM0216 in a calcium mobilisation assay.
  • R- CIM0126 is (R)-2-(3,4-dihydroquinolin-l(2H)-yl)-N-(5-methylisoxazol-3-yl)propenamide
  • S- CIM0216 is (S)-2-(3,4-dihydroquinolin-l(2H)-yl)-N-(5-methylisoxazol-3-yl)propenamide.
  • FIG 14A and 14B demonstrates that the TRPM3 agonists, CIM0216 and pregnenolone sulfate, stimulate release of substance P from trigeminal ganglia (TG) neurons in a concentration dependent manner.
  • FIGS 14A and 14B also show that substance P responses were inhibited with isosakuranetin (10 ⁇ M).
  • FIG. 17A and 17C demonstrates the concentration dependent response of pregnenolone sulfate in the presence of 2.5 ⁇ M /?-CIM0216), and
  • FIG. 17B and 17D demonstrates the concentration dependent response of R- CIM0216 in the presence of 25 ⁇ M pregnenolone sulfate.
  • Example 1 demonstrates that a SNP in the human TRPM3 gene, R1670Q shows a strong genetic association with migraine.
  • Example 3 ( Figure 9) shows that the potency of pregnenolone sulfate is 1.8-fold greater at the R1670Q variant than at canonical form, and the maximal fold change in fluorescence in a calcium mobilisation assay was 26% larger. Thus pregnenolone sulfate is more able to activate the TRPM3 variant associated with increased likelihood of migraine diagnosis than the canonical form of the channel.
  • TRPM3 activation is causative of migraine comes from the five-day rat dural infusion migraine model.
  • Historical data in this model using an inflammatory soup (2 mM histamine, bradykinin, serotonin, 0.2 mM prostaglandin E2) infusion shows mechanical nociception sensitivity (Oshinsky & Gomoncharconsiri, (2007).
  • Episodic dural stimulation in awake rats a model for recurrent headache. Headache: The Journal of Head and Face Pain, 47(7), 1026-1036).
  • This sensitivity is alleviated with sumatriptan and anti-CGRP therapies, current standard of care compounds for migraine, suggesting this model has clinical translation.
  • Example 4 shows that Pregnenolone sulphate and CIM0216 (TRPM3 agonists) also trigger mechanical allodynia in the periorbital region When comparing significant differences in sensitivity, both Pregnenolone sulphate and CIM0216 were effective in increasing sensitivity to periorbital von Frey (VF) stimulation as early as Day 5 (after 4 infusions) when compared to vehicle treated animals. In other words, Example 4 provides further evidence of the link between TRPM3 activation and migraine.
  • TRPM3 agonists TRPM3 agonists
  • Example 2 demonstrates that inhibition of human TRPM3 in sensory ganglion, including the trigeminal ganglion, reduces production of the neuropeptides CGRP, PACAP and substance P.
  • These neuropeptides are implicated in the pathogenesis of migraine and therapies inhibiting CGRP, PACAP and substance P (NK1 receptor antagonists) signalling have been developed although as of the date of filing, only CGRP blocking therapies have been approved as migraine therapies.
  • Trpm3 inhibitors have the ability to treat migraine mediated by CGRP, PACAP or substance P. It is known that there are classes of patients for whom the approved CGRP therapies are ineffective. It is believed trpm3 inhibition may be an effective therapeutic in such patients due to the impact of trpm3 inhibition upon PACAP and substance P.
  • Example 5 shows that this patient group is significant, comprising over 10% of migraine patients, and includes patients with and without mutations in trpm3. It is believed that trpm3 inhibition may be an effective therapeutic in patients that are not responsive to PACAP blockade due to the impact of trpm3 inhibition upon CGRP. Given that triptans may mediate their effects on migraine via PACAP and substance P, it is also plausible that trpm3 inhibition may be an effective therapy in a human subject whose migraines are responsive to therapy with triptans.
  • PACAP receptor antagonism has also been demonstrated to relieve opioid induced hyperalgesia in an animal model. Accordingly, the demonstration in the examples that trpm3 inhibition reduces PACAP shows that trpm3 inhibitors are suitable for the treatment of medication overuse headache, including opioid induced hyperalgesia. ..
  • migraine refers to a condition that satisfies the diagnostic criteria for migraine according to the International Classification of Headache Disorders (ICHD) of the HIS. This definition is periodically updated.
  • IBD International Classification of Headache Disorders
  • cluster headache refers to a condition that satisfies the diagnostic criteria for cluster headache according to the International Classification of Headache Disorders (ICHD) of the HIS. This definition is periodically updated.
  • ICHD International Classification of Headache Disorders
  • medication overuse headache refers to a condition that satisfies the diagnostic criteria for medication overuse headache according to the International Classification of Headache Disorders (ICHD) of the HIS. This definition is periodically updated.
  • ICHD International Classification of Headache Disorders
  • Human TRPM3 refers to a protein product of the TRPM3 gene present on chromosome 9q-21.12. Allelic variants including those encoded by SNPs associated with migraine are included within this definition. In addition, the definition covers all isoforms of human TRPM3 that may be generated from any allelic variant.
  • human TRPM3 refers to the hTRPM3 variant having the amino acid sequence set out in any one of sequences set out as: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO.5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17,
  • SEQ ID NO: 18 SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:
  • SEQ ID NO:31 SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36 and
  • SEQ ID NO: 37 or a processed version of any one of SEQ ID Nos. 1-37 lacking the initial methionine residue.
  • human TRPM3 refers to the TRPM3 variant having the amino acid sequence set out in SEQ ID NO: 2, or a processed version of this variant lacking the initial methionine residue.
  • human TRPM3 is a human TRPM3 having one or more mutations compared with the sequences set out in SEQ ID NO: 1 to SEQ ID NO:37.
  • human TRPM3 has an amino acid substitution at one or more of the following positions R1670, A1645, R1457, D602, K774, S1678, Y378, V990 and P1090 (numbering based on SEQ ID NO:2).
  • human TRPM3 has one or more of the following substitutions R1670Q, A1645V, R1457Q, D602V, K774R, S1678F, Y378C, V990M and P1090Q (numbering based on SEQ ID NO:2).
  • substitutions are described in relation to SEQ ID NO: 2, the substitution could occur in any isoform, and the invention is intended to encompass the corresponding variants with amino acid substitutions in each of SEQ ID NO:1 or SEQ ID NOS: 3-37 (or processed forms of these sequences lacking the initial methionine).
  • human TRPM3 is a human TRPM3 having a gain of function mutation.
  • a gain of function is one that results in constitutive activity (Ze., calcium influx in the absence of a stimulus), or increased sensitivity to stimuli (calcium influx at a lower concentration of agonist, or a larger calcium mobilisation at the same agonist concentration) as determined in a calcium mobilisation assay.
  • the human TRPM3 has one or more of the following amino acid substitutions R1670Q, A1645V, V990M and P1090Q (numbering based on SEQ ID NO: 2).
  • the human TRPM3 has the amino acid substitution R1670Q (numbering based on SEQ ID NO: 2).
  • nucleotide sequence of human TRPM3 is set out as SEQ ID NO: 38 to 69.
  • human TRPM3 channel encompasses a channel composed of at least one monomer of human TRPM3 as outlined above. The term therefore encompasses heterotetra meric channels formed from mixtures of human TRPM3 variants and homotetrameric channels formed from a single human TRPM3 variant. In one embodiment, the term human TRPM3 channel refers to a homotetrameric channel.
  • An inhibitor of human TRPM3 has one or more of the following properties:
  • Property 1 may be measured in a calcium mobilisation assay.
  • An inhibitor of human TRPM3 reduces fluorescence emissions in a calcium mobilisation assay compared to the negative control (agonist challenge/no inhibitor). In various embodiments, the fluorescence is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%. In one embodiment, an inhibitor of human TRPM3 reduces fluorescence by at least as much as a blocking concentration of isosakuranetin ((2S)-5,7-dihydroxy-2-(4-methoxyphenyl)-2,3- dihydrochromen-4-one).
  • Property 2 may be measured in an electrophysiological assay.
  • An inhibitor of human TRPM3 reduces current increases compared to the negative control (agonist/no inhibitor).
  • An inhibitor of human TRPM3 reduces current increases by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% compared to the negative control. In one embodiment, an inhibitor of human TRPM3 reduces current increases by at least as much as a blocking concentration of isosakuranetin ((2S)-5,7-dihydroxy-2-(4- methoxyphenyl)-2,3-dihydrochromen-4-one).
  • Property 3 may be measured by the CGRP release assay.
  • An inhibitor of human TRPM3 reduces CGRP levels in the media by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%.
  • an inhibitor of human TRPM3 reduces CGRP levels by at least as much as a blocking concentration of isosakuranetin ((2S)-5,7- dihydroxy-2-(4-methoxyphenyl)-2,3-dihydrochromen-4-one).
  • Property 4 may be measured by the PACAP release assay.
  • An inhibitor of human TRPM3 reduces PACAP levels in the media by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%.
  • an inhibitor of human TRPM3 reduces PACAP levels by at least as much as a blocking concentration of isosa kuranetin ((2S)- 5,7-dihydroxy-2-(4-methoxyphenyl)-2,3-dihydrochromen-4-one).
  • Property 5 may be measured by the substance P release assay.
  • An inhibitor of human TRPM3 reduces PACAP levels in the media by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%.
  • an inhibitor of human TRPM3 reduces substance P levels by at least as much as a blocking concentration of isosakuranetin ((2S)-5,7-dihydroxy-2-(4-methoxyphenyl)-2,3-dihydrochromen-4-one).
  • Property 6 is assessed in a suitable model, for example the five-day rat dural infusion migraine model described by Oshinsky et al., supra, using a TRPM3 agonist in place of inflammatory soup.
  • the level of facial allodynia is lower in the presence of an inhibitor of human TRPM3 compared to that observed in the absence of the inhibitor of human TRPM3.
  • the reduction in facial allodynia is measured using von Frey filaments.
  • an inhibitor of human TRPM3 reduces the von Frey threshold on a particular day following infusion by 0.5 g, 1 g, 1.5 g, or 2 g.
  • the reduction is measured from day 0 to day 14 post the completion of infusion.
  • the reduction is measured on day 0, day 3, day 6, day 9 or day 12.
  • any compound capable of promoting calcium ion influx in a cell line expressing human TRPM3 may be used as the agonist.
  • the calcium ion influx is mediated by human TRPM3.
  • the agonist is used at a concentration causing a response between 50% and 80% of the maximal response (i.e. between EC50 - EC80) for the assays used to assess properties 1 to 5.
  • the agonist is pregnenolone sulfate or CIM0216 (racemate of 2-(3,4-dihydroquinolin-l(2H)-yl)-N-(5-methylisoxazol-3-yl)-2-phenylacetamide).
  • the agonist is (R)-2-(3,4-dihydroquinolin-l(2H)-yl)-N-(5-methylisoxazol-3- yl)propenamide.
  • the agonist is (S)-2-(3,4-dihydroquinolin-l(2H)-yl)-N-(5- methylisoxazol-3-yl)propenamide.
  • Figure 13 shows that (R)-2-(3,4-dihydroquinolin-l(2H)-yl)-N-(5- methylisoxazol-3-yl)propenamide is a more potent agonist than the (S)-isomer. Further details of the conduct of these assays are set out in the section entitled "IDENTIFICATION OF INHIBITORS OF HUMAN TRPM3".
  • the invention provides (R)-2-(3,4-dihydroquinolin-l(2H)-yl)-N-(5- methylisoxazol-3-yl)propenamide or a salt thereof.
  • the invention provides (R)-2-(3,4-dihydroquinolin-l(2H)-yl)-N-(5-methylisoxazol-3-yl)propenamide (free base).
  • the nature of an inhibitor of human TRPM3 is not limited, and could be a chemical compound (i.e. a compound), an oligonucleotide, a peptide, a polypeptide, a protein, an antibody or an alternative antibody format.
  • the inhibitor of human TRPM3 is a compound.
  • antibody is used herein in the broadest sense to refer to molecules with an immunoglobulin-like domain (for example IgG, IgM, IgA, IgD or IgE) and includes monoclonal, recombinant, synthetic, polyclonal, chimeric, human, humanised, multispecific antibodies, including bispecific antibodies, and heteroconjugate antibodies; a single variable domain, antigen binding antibody fragments (e.g. Fab, F(ab')2, Fv, disulphide linked Fv, single chain Fv, disulphide-linked scFv, diabodies, TANDABTM, etc.) and modified versions of any of the foregoing.
  • immunoglobulin-like domain for example IgG, IgM, IgA, IgD or IgE
  • domain refers to a folded protein structure which retains its tertiary structure independent of the rest of the protein. Generally, domains are responsible for discrete functional properties of proteins and in many cases may be added, removed or transferred to other proteins without loss of function of the remainder of the protein and/or of the domain.
  • single variable domain refers to a folded polypeptide domain comprising sequences characteristic of antibody variable domains.
  • a single variable domain that is capable of binding an antigen or epitope independently of a different variable region or domain may be referred to as a "domain antibody” or "dAb(TM)".
  • a single variable domain may be a human single variable domain, but also includes single variable domains from other species such as rodent, nurse shark and Camelid VHH dAbsTM.
  • Camelid VHH are immunoglobulin single variable domain polypeptides that are derived from species including camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain antibodies naturally devoid of light chains. Such VHH domains may be humanised according to standard techniques available in the art, and such domains are considered to be "single variable domains". As used herein VH includes camelid VHH domains.
  • Non-immunoglobulin scaffold may be a derived from the group consisting of CTLA-4, lipocalin, Protein A derived molecules such as Z-domain of Protein A (Affibody, SpA), A-domain (Avimer/Maxibody); heat shock proteins such as GroEl and GroES; transferrin (trans-body); ankyrin repeat protein (DARPin); peptide aptamer; C-type lectin domain (Tetranectin); human y-crystallin and human ubiquitin (affilins); PDZ domains; LDL receptor class A domains; EGF domains; scorpion toxin kunitz type domains of human protease inhibitors; and fibronectin/adnectin.
  • Inhibitors of human TRPM3 are known in the art.
  • Inhibitors of human TRPM3 include those disclosed in WO2022112345 and WO2022112352.
  • an inhibitor of human TRPM3 is a compound of formula (I), or a pharmaceutically acceptable salt thereof: wherein:
  • R 1 is selected from the group consisting of: a 4-6 membered saturated heterocyclylic ring, which 4-6 membered heterocyclic ring is optionally substiiuted by one, two or three groups independently selected from oxo, fluoro, C1- 3fluoroalkyl and C 1-3 alkyl, which C 1-3 alkyl group may optionally by substituted by one hydroxy group; and a group of formula -CR 6 R 7 CONH2, wherein R 6 is optionally H or methyl and R 7 is methyl substituted by one hydroxy group or ethyl substituted by one hyroxy group;
  • R 2 is phenyl or a 5-6 membered nitrogen containing heteroaryl ring, which phenyl or 5-6 membered nitrogen containing heteroaryl ring is optionally substituted by one two or three substituents independently selected from the group consisting of halo, cyclopropyl, cyclopropyloxy, methoxy and C 1-3 alkyl, which C 1-3 alkyl group is optionally substituted by one, two or three substitutents selected from fluoro and cyclopropyl; and
  • R 3 is selected from cycloalkyl and methyl, which methyl group is optionally substituted by one, two or three fluoro groups.
  • R 4 and R 5 are independently selected from H, or methyl, which methyl group is optionally substituted with a group consisting of hydroxy, methoxy and N(CH 3 ) 2 .
  • R 1 is a group of formula -CR 6 R 7 CONH 2 , wherein R 6 is optionally H or methyl and R 7 is methyl substituted by one hydroxy group or ethyl substituted by one hydroxy group.
  • R 1 is a group of formula -CH(CH 2 OH)CONH 2 .
  • R 3 is methyl.
  • R 4 and R 5 are each H. In another embodiment, R 4 is H, and R 5 is methyl, which methyl group is optionally substituted with a group consisting of hydroxy and methoxy.
  • the inhibitor of human TRPM3 is a compound selected from the group consisting of: 2-(difluoromethyl)-N-(3,3-difluoropiperidin-4-yl)-5-((4-methylthiazol-5-yl)methoxy)benzofuran-3- carboxamide;
  • Inhibitors include, for example, primidone (5-ethyldihydro-5-phenyl-4,6(lH,5H)-pyrimidinedione), diclofenac, ononetin, econazole, the calmodulin antagonist W-7, the PPARy agonists rosiglitazone, troglitazone and pioglitazone, the flavonoid derivatives disclosed in Straub et al. (Mol Pharmacol, 2013, 84(5):736-50), the fenamate derivates disclosed in Klose et al.. (Br J Pharmacol., 2011, 162(8): 1757-1769), and the TRPM3-specific polyclonal antibody (TM3E3).
  • T3E3 TRPM3-specific polyclonal antibody
  • the inhibitor of human TRPM3 exhibits selectivity for inhibition of human TRPM3 channels in comparison to other TRP channels.
  • the inhibitor of human TRPM3 exhibits selectivity for inhibition of human TRPM3 over one or more of TRPM1, TRPV1, TRPV4 and TRPM8.
  • selectivity can be assessed in binding assays for example, an assay using a labelled ligand, or where the TRP channel also acts to increase intracellular calcium, in a calcium mobilisation assay.
  • the inhibitor of human TRPM3 may be an inhibitor of a mutated version of human TRPM3, such as those disclosed supra, for example a mutated version of human TRPM3 having a gain of function mutation.
  • an inhibitor of a mutated version of human TRPM3 is an inhibitor of the mutated version, and does not require selectivity over the corresponding wild type variant of TRPM3, although selectivity may be required in particular embodiments.
  • the inhibitor of a mutated version of human TRPM3 having a gain of function mutation is an inhibitor that is selective for the mutated version over the corresponding wild type TRPM3 variant.
  • the inhibitor of human TRPM3 exhibits selectivity for the TRPM3 having the sequence set out in SEQ ID NO: 2 (or a processed version of SEQ ID NO:2 lacking the initial methionine residue) over other TRPM3 variants.
  • selectivity for this isoform can be assessed in binding assays or using a calcium mobilisation assay using channels that are homotetrameric for SEQ ID NO:2 (or a processed version of SEQ ID NO:2 lacking the initial methionine residue) and channels that are homotetrameric for the reference TRPM3 variant.
  • the inhibitor of human TRPM3 selectively inhibits the response to a particular stimulus.
  • human TRPM3 is polymodally activated.
  • inhibition is selective to agonism by pregnenolone sulfate over heat.
  • inhibition is selective to agonism by pregnenolone sulfate over other agonists e.g., nifedipine, D-erythrosphingosine, CIM2016).
  • a functional assay such as a calcium mobilisation assay may be used to assess selectivity to particular stimuli.
  • the Kd for human TRPM3 channels or channels homotetrameric for a particular isoform of human TRPM3 is at least 10 fold lower compared to the reference channel. In a more particular embodiment, the Kd for the human TRPM3 channels or channels homotetrameric for a particular isoform of human TRPM3 is at least 100 fold lower compared to the reference channel. In embodiments above in which selectivity is assessed using a calcium mobilisation assay, the IC50 or Kd derived therefrom for human TRPM3 channels or channels homotetrameric for a particular isoform of human TRPM3 is at least 10 fold lower compared to the reference channel.
  • the IC50 or Kd derived therefrom for human TRPM3 channels or channels homotetrameric for a particular isoform of human TRPM3 is at least 100 fold lower compared to the reference channel.
  • the properties that define an inhibitor are determined experimentally in assays. These assays can be used to identify additional inhibitors of human TRPM3.
  • Property 1 is measured using a calcium mobilisation assay in which changes in intracellular calcium ion levels are detected by changes in calcium indicator compounds.
  • an inhibitor of human TRPM3 can be identified by a method comprising: a) contacting a cell line expressing human TRPM3 which cells contain an intracellular calcium indicator with an agonist of human TRPM3 in the presence and absence of a test inhibitor; and b) measuring a change in intracellular calcium concentrations by measuring a change in the intracellular calcium indicator; wherein the test inhibitor is identified as an inhibitor for human TRPM3 if intracellular calcium concentrations are reduced in the presence of the test inhibitor compared to those achieved in the absence of test inhibitor.
  • the intracellular calcium indicator is a synthetic calcium indicator
  • step a) is preceded by a step of loading the cells with the indicator.
  • synthetic calcium indicator compounds are available commercially, for example, the FLUO calcium indicators (Invitrogen).
  • Synthetic calcium indicators may be loaded into cells using methods known in the art.
  • water soluble salts of synthetic calcium indicators may be loaded into cells by well known methods including microinjection, by addition to patch pipette solutions, or by use of pinocytosis, for example using the INFLUX pinocytotic cell loading reagent.
  • Cell permeant AM esters of calcium indicators may be loaded into cells by addition to the media, typically in the presence of a non-ionic detergent such as PLURONIC F-127, and an anion transport inhibitor such as probenecid or sulfinpyrazone, and incubation at 20-37°C for a suitable period of time e.g., between 15 minutes and 4 hours). Following cell loading, cells may be washed and background fluorescence due to indicator leakage could be quenched by addition of, for example, an anti-fluorescein antibody, although this is not essential. Where cell permeant AM esters were used, incubation for a further 30 minutes after loading permits de-esterification of the intracellular AM esters prior to the assay being conducted.
  • a non-ionic detergent such as PLURONIC F-127
  • an anion transport inhibitor such as probenecid or sulfinpyrazone
  • the intracellular calcium indicator is a genetically encoded calcium indicator.
  • genetically encoded calcium indicators include the GCaMPs, pericams, GECOs, camgaroos. Constructs for transfecting cell lines with these genetically encoded calcium indicators are known in the art, for example, the GCaMP6s-P2A-Bsr construct (commercially available as Addgene plasmid # 40753). Clonal cell lines based upon this construct showed bright, uniform cytoplasmic staining. Transient and stable cell line production using this construct is described in Wu eta/., 2019 (2019, Sci Rep, 9: 12692).
  • DNA encoding the genetically encoded calcium indicator is transfected into a cell line expressing human TRPM3 before step a).
  • the cell line is a cell line stably expressing the genetically encoded calcium indicator.
  • calcium mobilisation assays using cells loaded with calcium indicators are well known in the art.
  • the calcium indicator is an indicator whose fluorescence changes in the presence of calcium ions.
  • calcium mobilisation assays involve measuring levels of fluorescence following excitation with an appropriate wavelength for the calcium indicator following the addition of an agonist and inhibitor.
  • the inhibitor may be added to the cells either before (e.g. 60 minutes before) or at the same time as the agonist. Fluoresence may be measured using a fluorescent imaging plate reader (several are commercially available, for example FLIPR TETRA), or by FACS analysis.
  • negative and positive controls and standards are included in each experiment. Negative controls lack agonist, positive controls lack inhibitor and a standard uses a blocking concentration of a known inhibitor, for example, isosakuranetin.
  • an inhibitor of human TRPM3 is a compound that reduces fluorescence emissions compared to the positive control.
  • the fluorescence is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%.
  • an inhibitor is a compound that reduces fluorescence by at least as much as a blocking concentration of isosakuranetin.
  • an inhibitor is a compound that reduces fluorescence by at least as much as a saturating concentration of isosakuranetin (i.e. maximal inhibition with isosakuranetin).
  • Property 2 is measured in an electrophysiological assay such as patch clamp or using an automated electrophysiology platform.
  • electrophysiological assays are well known in the art and several automated electrophysiology platforms are available commercially, for example the IONWORKS platforms, PATCHXPRESS, IONFLUX, QPATCH HT/HTX, PATCHLINER and SYNCHROPATCH platforms.
  • an inhibitor of human TRPM3 can be identified by a method comprising measuring the current increases in a cell line expressing human TRPM3 following challenge with an agonist of human TRPM3 in the presence and absence of a test inhibitor, wherein the test inhibitor is identified as an inhibitor for human TRPM3 if the increase in current is reduced in the presence of the test inhibitor compared to the increase achieved in the absence of the test inhibitor.
  • the change in current is measured at a membrane potential of between +/-80mV.
  • the change in current is measured at a membrane potential of -80mV, - 70mV, -60mV, -50mV, -40mV, -30mV, -20mV, -lOmV, OmV, +10mV, +20mV, +30mV, +40mV, +50mV, +60mV, +70mV or +80mV.
  • the change in current is measured at a membrane potential of -80mV.
  • the change in current is measured at a membrane potential of +80mV.
  • mean changes in current are measured.
  • the assays to assess properties 1 and 2 utilise cell lines expressing human TRPM3.
  • Primary or immortalised cell lines endogenously expressing human TRPM3 may be used.
  • the SHSY-5Y cell line may be used.
  • transient and stable cell lines expressing recombinant human TRPM3 may be prepared by conventional means.
  • the invention provides a cell line stably expressing recombinant human TRPM3.
  • TRPM3 variant i SEQ ID NO: 11
  • WO200526317 Further cell lines expressing TRPM3 variants are disclosed in WO200526317.
  • the assays to assess properties 1 and 2 may be conducted using a cell line expressing a recombinant human TRPM3 variant comprising one or more amino acid substitutions at residues selected from the group consisting of R1670, A1645, R1457, D602, K774, S1678, Y378, V990 and P1090 (numbering based on SEQ ID NO:2).
  • the cell line expresses a recombinant human TRPM3 variant having one, two or three of the following substitutions R1670Q, A1645V, R1457Q, D602V, K774R, S1678F, Y378C, V990M and P1090Q (numbering based on SEQ ID NO:2).
  • the cell line expresses a recombinant human TRPM3 variant comprising the amino acid substitution R1670Q (numbering based on SEQ ID NO:2).
  • the assays to assess properties 1 and 2 may be conducted using a cell line expressing recombinant human TRPM3 variant having the sequence set out in SEQ ID NO: 2, a processed version of SEQ ID NO:2 lacking the initial methionine residue, or a variant of SEQ ID NO: 2 or the processed version of SEQ ID NO: 2 comprising one two or three amino acid substitutions compared to the sequence set out in SEQ ID NO:2.
  • cell line expresses recombinant human TRPM3 variant having the sequence set out in SEQ ID NO: 2, a processed version of SEQ ID NO:2 lacking the initial methionine, or a variant of SEQ ID NO: 2 or the processed version of SEQ ID NO: 2 which has one, two or three amino acid substitutions at residues selected from the group consisting of R1670, A1645, R1457, D602, K774, S1678, Y378, V990 and P1090 (numbering based on SEQ ID NO:2).
  • the cell line expresses recombinant human TRPM3 varianthaving the sequence set out in SEQ ID NO: 2, a processed version of SEQ ID NO:2, or a variant of SEQ ID NO: 2 or the processed version of SEQ ID NO: 2 which has one, two or three of the following substitutions R1670Q, A1645V, R1457Q, D602V, K774R, S1678F, Y378C, V990M and P1090Q (numbering based on SEQ ID NO:2).
  • the cell line is formed by transient transfection of DNA encoding the human TRPM3 varianthaving the sequence set out in SEQ ID NO: 2, a processed version of SEQ ID NO:2 lacking the initial methionine, or a variant of SEQ ID NO:2 or the processed version of SEQ ID NO: 2 comprising the amino acid substitution R1670Q.
  • the cell line is formed by transient transfection of DNA encoding the human TRPM3 variant having the sequence set out in SEQ ID NO: 2.
  • the cell line is formed by transient transfection of DNA encoding the human TRPM3 varianthaving the sequence of SEQ ID NO:2 comprising the amino acid substitution R1670Q.
  • the cell line stably expresses the human TRPM3 varianthaving the sequence set out in SEQ ID NO: 2, a processed version of SEQ ID NO:2 lacking the initial methionine or a variant of SEQ ID NO:2 or the processed version of SEQ ID NO: 2 comprising the amino acid substitution R1670Q.
  • the cell line stably expresses the human TRPM3 varianthaving the sequence set out in SEQ ID NO: 2 or a processed version of SEQ ID NO: 2 lacking the initial methionine.
  • the cell line stably expresses the human TRPM3 variant that has the sequence of SEQ ID NO:2 or the processed form of SEQ ID NO:2 lacking the initial methionine, comprising the amino acid substitution R1670Q.
  • the cell line also stably expresses a genetically encoded calcium indicator.
  • the cell line is a human cell line.
  • the assays described above are conducted in this cell line.
  • the cell lines described herein may be used in the identification of an inhibitor of human TRPM3.
  • Property 3 utilises primary cell cultures derived from freshly extracted dorsal root ganglia or trigeminal ganglia from rats or mice, or samples of the brain stem containing the trigeminal nucleus caudalis itself isolated from rats or mice.
  • CGRP is measured in the incubation fluid.
  • the CGRP content of the incubation fluid may be measured by methods known in the art.
  • a suitable enzyme immunoassay kits with a detection threshold of 5 pg/mL is commercially available (Bertin Pharma) which permits the media to be photometrically analyzed.
  • Example 2 exemplifies a suitable assay.
  • TR-FRET time-resolved fluorescence resonance energy transfer
  • the invention provides a method for identifying an inhibitor of human TRPM3, comprising measuring release of PACAP from dorsal root ganglia, trigeminal ganglia or the trigeminal nucleus caudalis, or from primary cultures of cells isolated from dorsal root ganglia or trigeminal ganglia, following challenge with an agonist of human TRPM3 in the presence or absence of a test inhibitor, wherein the test inhibitor is identified as an inhibitor for human TRPM3 if PACAP production is reduced in the presence of the test inhibitor compared to PACAP production in the absence of the test inhibitor.
  • the method uses primary cultures of cells isolated from dorsal root ganglia or trigeminal ganglia (Ze., cells isolated from dorsal root ganglia or trigeminal ganglia and placed into culture). In one embodiment, the method uses primary cultures of cells isolated from trigeminal ganglia. In one embodiment, the method uses primary cultures of cells in multiwell plates. In another embodiment, the method uses samples of the brain stem containing the trigeminal nucleus caudalis.
  • an inhibitor of human TRPM3 reduces PACAP levels by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%. In one embodiment, an inhibitor of human TRPM3 reduces PACAP levels by at least as much as a blocking concentration of isosakuranetin.
  • Property 5 utilises primary cell cultures derived from freshly extracted dorsal root ganglia or trigeminal ganglia from rats or mice, or samples of the brain stem containing the trigeminal nucleus caudalis itself isolated from rats or mice.
  • Substance P is measured in the incubation fluid.
  • the substance P content of the incubation fluid may be measured by methods known in the art.
  • a suitable ELISA kit is commercially available from Cayman Chemicals.
  • Example 2 exemplifies a suitable assay.
  • Property 6 may be assessed in a suitable animal model, for example the five-day rat dural infusion migraine model described in Example 4.
  • the level of facial allodynia is lower in the presence of an inhibitor of human TRPM3 compared to that observed in the absence of the inhibitor of human TRPM3.
  • the reduction in facial allodynia is measured using von Frey filaments.
  • an inhibitor of humant TRPM3 reduces the von Frey threshold on a particular day following infusion by 0.5 g, 1 g, 1.5 g, or 2 g.
  • the reduction is measured from day 0 to day 14 post the completion of infusion.
  • the reduction is measured on day 0, day 3, day 6, day 9 or day 12.
  • any compound capable of promoting calcium ion influx in a cell line expressing human TRPM3 may be used as the agonist in the assays described supra.
  • the influx is mediated by human TRPM3.
  • the agonist is pregnenolone sulfate or CIM0216 (racemate of 2- (3,4-dihydroquinolin-l(2H)-yl)-/V-(5-methylisoxazol-3-yl)-2-phenylacetamide).
  • pregnenolone sulfate is used at a concentration in the range from 1 to 300 ⁇ M in the assays measuring properties 1-5.
  • pregnenolone sulfate is used at a concentration of about 100 ⁇ M in the assays measuring properties 1-5.
  • CIM0216 is used at a concentration in the range from 0.1 to 30 ⁇ M, more particularly in the range from 6 to 10 ⁇ M in the assays measuring properties 1-5.
  • CIM0216 or the R or S isomers thereof are used at a concentration of about 6 ⁇ M in the assays measuring properties 1-4.
  • CIM0216 or the R or S isomers thereof are used at a concentration of about 10 ⁇ M in the assays measuring properties 1-5.
  • pregnenolone sulfate is used at 5 mM/rat/day or CIM0216 or the R or S isomers thereof are used at 215 ⁇ M/rat/day. In another embodiment, CIM0216 or the R or S isomers thereof are used at a concentration of 215 ⁇ M/rat/day for dural infusion to measure property 6.
  • the invention provides an inhibitor of human TRPM3 as discussed herein for use in the treatment of migraine in a human subject whose migraines are not responsive to CGRP inhibition.
  • treatment of migraine refers to the symptomatic treatment of acute migraine.
  • Migraines may present with or without aura or visual disturbances.
  • the invention provides an inhibitor of human TRPM3 for use in the treatment of migraine with aura or visual disturbances in a human subject whose migraines are not responsive to CGRP inhibition.
  • the invention provides an inhibitor of human TRPM3 for use in the treatment of migraine without aura or visual disturbances in a human subject whose migraines are not responsive to CGRP inhibition.
  • human subjects whose migraines are not responsive to CGRP inhibition is not limited to human subjects that have previously been prescribed a CGRP inhibitor and found it to lack efficacy, and additionally encompasses individuals that have not previously been prescribed a CGRP inhibitor, but, whose migraines would nonetheless not respond to such treatment options.
  • Example 5 suggests that this includes over 10% of all migraineurs.
  • the invention comprises treating human subjects that have previously failed treatment with an antagonist of CGRP. Such patients can be readily by identified by a review of their clinical history. A suitable questionnaire to identify CGRP non responders is given in Example 5.
  • the invention provides an inhibitor of human TRPM3 as discussed herein for use in the treatment of migraine in a human subject whose migraines are responsive to therapy with a triptan.
  • treatment of migraine refers to the symptomatic treatment of acute migraine.
  • Migraines may present with or without aura or visual disturbances.
  • the invention provides an inhibitor of human TRPM3 for use in the treatment of migraine with aura or visual disturbances in a human subject whose migraines are responsive to therapy with a triptan.
  • the invention provides an inhibitor of human TRPM3 for use in the treatment of migraine without aura or visual disturbances in a human subject whose migraines are responsive to therapy with a triptan.
  • human subjects whose migraines are responsive to a triptan is not limited to human subjects that have previously been prescribed and responded to a triptan, but additionally encompasses individuals that have not previously been prescribed a triptan, but, whose migraines would nonetheless respond to such treatment.
  • the invention comprises treating human subjects that have previously been treated with a triptan and self reported a clinical response. Such patients can be readily by identified by a review of their clinical history.
  • the triptan is a triptan selected from the group consisting of: almotriptan, eletriptan, frovatriptan, naratriptan, rizatriptan, sumatriptan and zolmitriptan. In a more particular embodiment, the triptan is sumatriptan.
  • the invention provides an inhibitor of human TRPM3 as discussed herein for use in the treatment of migraine in a human subject whose migraines are not responsive to CGRP inhibition, but are responsive to therapy with a triptan.
  • a therapeutically effective amount of an inhibitor of human TRPM3 is administered to the human subject.
  • therapeutically effective amount refers to the quantity of the inhibitor of human TRPM3 that is required for symptomatic treatment of acute migraine. It may vary depending on the compound, migraine severity and the age and weight of the subject to be treated.
  • treatment of acute symptomatic migraine refers to the situation where the percentage of patients that are pain free 2 hours after administration of the inhibitor of human TRPM3 is higher for a population of patients receiving the inhibitor of human TRPM3 compared to a population of patients receiving placebo.
  • pain free is a patient reported measure.
  • the percentage of patients that are pain free 2 hours after administration of the inhibitor of human TRPM3 is 10% or higher (compared to placebo).
  • treatment of acute symptomatic migraine refers to the situation where the percentage of patients that have no headache pain 2 hours after administration of the inhibitor of human TRPM3 and have no relapse of headache pain within 24 hours after administration of the inhibitor of human TRPM3 is higher compared to a population of patients receiving placebo. Headache pain is a patient reported measure.
  • the percentage of patients that have no headache pain 2 hours after administration of the inhibitor of human TRPM3 and have no relapse of headache pain within 24 hours after administration of the inhibitor of human TRPM3 is 10% or higher (compared to placebo).
  • treatment of acute symptomatic migraine refers to the situation where the percentage of patients that have no headache pain 2 hours after administration of the inhibitor of human TRPM3 and have no relapse of headache pain within 48 hours in a population of patients receiving the the inhibitor of human TRPM3 is higher compared to a population of patients receiving placebo. Again, headache pain is a patient reported measure.
  • the percentage of patients that have no headache pain 2 hours after administration of the inhibitor of human TRPM3 and have no relapse of headache pain within 48 hours after administration of the inhibitor of human TRPM3 is 10% or higher (compared to placebo).
  • the inhibitor of human TRPM3 is administered in combination with at least one other therapeutic agent selected from: a triptan, an ergot, a non-steroidal anti-inflammatory drug, an acetaminophen containing product, a butalbital containing product, an anti-emetic, caffeine, dexamethasone, ubrogepant, rimegepant and lasmiditan.
  • a triptan an ergot
  • a non-steroidal anti-inflammatory drug an acetaminophen containing product
  • a butalbital containing product an anti-emetic
  • caffeine dexamethasone
  • ubrogepant ubrogepant
  • rimegepant and lasmiditan.
  • the inhibitor of human TRPM3 is administered in combination with a triptan selected from the group consisting of: almotriptan, eletriptan, frovatriptan, naratriptan, rizatriptan, sumatriptan and zolmitriptan.
  • a triptan selected from the group consisting of: almotriptan, eletriptan, frovatriptan, naratriptan, rizatriptan, sumatriptan and zolmitriptan.
  • the inhibitor of human TRPM3 is administered in combination with sumatriptan.
  • the inhibitor of human TRPM3 is administered in combination with oral sumatriptan at a maximum dose of 200 mg/day.
  • the inhibitor of human TRPM3 is administered in combination with a non-steroidal anti-inflammatory drug selected from the group consisting of diclofenac, ibuprofen, naproxen and ketorolac.
  • the inhibitor of human TRPM3 is administered in combination with an anti-emetic selected from the group consisting of: promethazine, prochlorperazine, metoclopramide, trimethobenzamide and ondansetron.
  • an anti-emetic selected from the group consisting of: promethazine, prochlorperazine, metoclopramide, trimethobenzamide and ondansetron.
  • the inhibitor of human TRPM3 and any other therapeutic agent(s) may be administered together or separately and, when administered separately, administration may occur simultaneously or sequentially, in any order.
  • the amounts of inhibitor of human TRPM3 of the present invention and the other therapeutic agent(s) and the relative timings of administration will be selected in order to achieve the desired combined therapeutic effect.
  • Simultaneous administration may be achieved by administration of (1) a unitary pharmaceutical composition including the therapeutic agents; or (2) simultaneous administration of separate pharmaceutical compositions each including one of the therapeutic agents.
  • the combination may be administered separately in a sequential manner wherein one treatment agent is administered first and the other second or vice versa. Such sequential administration may be close in time or remote in time.
  • the amounts of the inhibitor of human TRPM3 of the invention and the other therapeutic agent(s) and the relative timings of administration will be selected in order to achieve the desired combined therapeutic effect.
  • the invention provides a method of treating acute symptomatic migraine in a human subject comprising the steps of: a) identifying a a human subject whose migraines are not responsive to CGRP inhibition; and b) administering a therapeutically effective amount of an inhibitor of human TRPM3 as defined herein to the subject; whereby the acute symptomatic migraine in the subject is treated.
  • the invention provides a method of treating acute symptomatic migraine in a human subject comprising the steps of: a) identifying a a human subject whose migraines are responsive to therapy with a triptan; and b) administering a therapeutically effective amount of an inhibitor of human TRPM3 as defined herein to the subject; whereby the acute symptomatic migraine in the subject is treated.
  • the invention provides a method for decreasing the level of PACAP in cranial blood in a subject comprising the steps of: a) identifying a subject with migraine; and b) administering a therapeutically effective amount of an inhibitor of human TRPM3 as defined herein to the subject; whereby the level of PACAP in the cranial blood of the subject is decreased.
  • the invention provides a method for decreasing the level of PACAP in the systemic circulation of a subject comprising the steps of: a) identifying a subject with migraine; and b) administering a therapeutically effective amount of an inhibitor of human TRPM3 as defined herein to the subject; whereby the level of PACAP in the systemic circulation of the subject is decreased.
  • Certain embodiments of the methods for decreasing PACAP may further comprise the steps of: a) making a first measurement of the level of PACAP in a relevant blood sample; b) making a second measurement of the level of PACAP in a relevant blood sample after administering to the subject a therapeutically effective amount of the inhibitor of human TRPM3; and c) comparing the first measurement and second measurement.
  • the subject is a subject that carries a mutated version of human TRPM3 wherein the mutated verion of human TRPM3 has one or more of the following amino acid substitutions: R1670Q, A1645V, V990M and P1090Q (numbering based on SEQ ID NO: 2).
  • Certain embodiments of the methods for decreasing the level of PACAP may further comprise the steps of: determining whether the subject carries a mutated version of human TRPM3 wherein the mutated verion of human TRPM3 has one or more of the following amino acid substitutions: R1670Q, A1645V, V990M and P1090Q (numbering based on SEQ ID NO: 2). These steps may take place before step (a) in the above methods.
  • the invention provides an inhibitor of human TRPM3 for use in the prevention of migraine in a human subject whose migraines are not responsive to CGRP inhibition. In one embodiment, the invention provides an inhibitor of human TRPM3 for use in the prevention of chronic migraine in a human subject whose migraines are not responsive to CGRP inhibition.
  • human subjects whose migraines are not responsive to CGRP inhibition is not limited to human subjects that have previously been prescribed a CGRP inhibitor and found it to lack efficacy, and additionally encompasses individuals that have not previously been prescribed a CGRP inhibitor, but, whose migraines would nonetheless not respond to such treatment options.
  • Example 5 suggests that this includes over 10% of all migraineurs.
  • the invention comprises treating human subjects that have previously failed treatment with an antagonist of CGRP. Such patients can be readily by identified by a review of their clinical history. A suitable questionnaire to identify CGRP non responders is given in Example 5.
  • the invention provides an inhibitor of human TRPM3 as discussed herein for use in the prevention of migraine in a human subject whose migraines are responsive to therapy with a triptan. In one embodiment, the invention provides an inhibitor of human TRPM3 for use in the prevention of chronic migraine in a human subject whose migraines are responsive to therapy with a triptan.
  • human subjects whose migraines are responsive to a triptan is not limited to human subjects that have previously been prescribed and responded to a triptan, but additionally encompasses individuals that have not previously been prescribed a triptan, but, whose migraines would nonetheless respond to such treatment.
  • the invention comprises treating human subjects that have previously been treated with a triptan and self reported a clinical response. Such patients can be readily by identified by a review of their clinical history.
  • the triptan is a triptan selected from the group consisting of: almotriptan, eletriptan, frovatriptan, naratriptan, rizatriptan, sumatriptan and zolmitriptan. In a more particular embodiment, the triptan is sumatriptan.
  • the invention provides an inhibitor of human TRPM3 as discussed herein for use in the prevention of migraine in a human subject whose migraines are not responsive to CGRP inhibition, but are responsive to therapy with a triptan.
  • a therapeutically effective amount of an inhibitor of human TRPM3 is administered to the human subject.
  • the term "therapeutically effective amount” refers to the quantity of the inhibitor of human TRPM3 that is required for migraine prevention (e.g. prevention of chronic migraine). It may vary depending on the compound, the mean number of migraine days/month, and the age and weight of the subject to be treated.
  • prevention of migraine refers to the situation where the reduction in mean monthly migraine days is greater for a population of patients receiving the inhibitor of TRPM3 compared to placebo.
  • the reduction in mean monthly migraine days in a population of patients receiving the inhibitor of TRPM3 is at least 1, in a further embodiment, at least 2, in a further embodiment, at least 3 and in a further embodiment, at least 4.
  • prevention of migraine refers to the situation where the 50% responder rate is higher for a population of patients receiving the inhibitor of TRPM3 compared to placebo.
  • the 50% responder rate in patients receiving the inhibitor of TRPM3 is 20% higher for a population of patients receiving the inhibitor of TRPM3 compared to placebo. In a further embodiment, the 50% responder rate in patients receiving the inhibitor of TRPM3 is 25% higher for a population of patients receiving the inhibitor of TRPM3 compared to placebo.
  • the invention provides a method of preventing migraine in a human subject comprising the steps of: c) identifying a a human subject whose migraines are not responsive to CGRP inhibition; and d) administering a therapeutically effective amount of an inhibitor of human TRPM3 as defined herein to the subject; whereby migraine is prevented in the subject.
  • the invention provides a method of preventing migraine in a human subject comprising the steps of: c) identifying a a human subject whose migraines are responsive to therapy with a triptan; and d) administering a therapeutically effective amount of an inhibitor of human TRPM3 as defined herein to the subject; whereby migraine is prevented in the subject.
  • the inhibitor of human TRPM3 is administered in combination with at least one other therapeutic agent selected from: botulinum toxin A, a CGRP inhibitor, an anticonvulsant, a 0- blocker, an antidepressant and a non-steroidal anti-inflammatory drug.
  • the inhibitor of human TRPM3 is administered in combination with at least one other therapeutic agent selected from: valproate, divalproex sodium, amitriptyline, topiramate, venlafaxine, metoprolol, propranolol and timolol.
  • the invention provides a method for identifying whether a human subject that has migraines that are not responsive to CGRP inhibition and/or are responsive to triptans is a candidate for treatment with an inhibitor of human TRPM3, comprising: a) sequencing a human TRPM3 gene in the human subject; b) comparing the sequence with the sequences of the human TRPM3 exons set out in SEQ ID NOs: 38-69 and identifying whether changes would modify the amino acid sequence of any isoform; wherein, if a change in amino acid sequence is identified, the human subject is a candidate for treatment with an inhibitor of human TRPM3.
  • the human subject has migraines that are not responsive to CGRP inhibition.
  • the human subject has migraines that are responsive to a triptan.
  • the human subject has migraines that are not responsive to CGRP inhibition and that are responsive to a triptan.
  • the changes to the amino acid sequence are selected the group consisting of: R1670Q, A1645V, V990M and P1090Q (numbering based on SEQ ID NO: 2). In one embodiment, the change to the amino acid sequence is R1670Q (numbering based on SEQ ID NO: 2).
  • the treatment will be acute symptomatic treatment of migraine. In another embodiment, the treatment will be for the prevention of migraine.
  • the invention provides an inhibitor of human TRPM3 for use in the treatment or prevention of migraine in a candidate for treatment with an inhibitor of human TRPM3.
  • the invention also provides use of an inhibitor of human TRPM3 for use in the manufacture of a medicament for use in the treatment or prevention of migraine in a candidate for treatment with an inhibitor of human TRPM3.
  • the invention also provides a method for treating or preventing migraine in a patient in need thereof, comprising administering an inhibitor of human TRPM3 to a human subject that is identified as a candidate for treatment with an inhibitor of human TRPM3.
  • the invention provides a method for migraine prevention, which comprises the following steps: a) sequencing the human TRPM3 gene in a human subject whose migraines are not responsive to CGRP inhibition and/or responsive to triptans to obtain a sequence; b) comparing the sequence with the sequences of the human TRPM3 exons set out in SEQ ID NOs: 38-69 and identifying whether changes would modify the amino acid sequence of any isoform wherein, if a change in amino acid sequence is identified, the human subject is a candidate for treatment with an inhibitor of human TRPM3; c) administering to the human subject that is a candidate for treatment with an inhibitor of human TRPM3 a therapeutically effective amount of an inhibitor of human TRPM3.
  • the human subject has migraines that are not responsive to CGRP inhibition.
  • the human subject has migraines that are responsive to triptans.
  • the changes to the amino acid sequence are selected the group consisting of: R1670Q, A1645V, V990M and P1090Q (numbering based on SEQ ID NO: 2). In one embodiment, the change to the amino acid sequence is R1670Q (numbering based on SEQ ID NO: 2).
  • the inhibitor of human TRPM3 and any other therapeutic agent(s) may be administered together or separately and, when administered separately, administration may occur simultaneously or sequentially, in any order.
  • the amounts of inhibitor of human TRPM3 of the present invention and the other therapeutic agent(s) and the relative timings of administration will be selected in order to achieve the desired combined therapeutic effect.
  • Simultaneous administration may be achieved by administration of (1) a unitary pharmaceutical composition including the therapeutic agents; or (2) simultaneous administration of separate pharmaceutical compositions each including one of the therapeutic agents.
  • the combination may be administered separately in a sequential manner wherein one treatment agent is administered first and the other second or vice versa. Such sequential administration may be close in time or remote in time.
  • the amounts of the inhibitor of human TRPM3 of the invention and the other therapeutic agent(s) and the relative timings of administration will be selected in order to achieve the desired combined therapeutic effect.
  • the invention provides an inhibitor of human TRPM3 for use in the treatment or prevention of cluster headache in a human subject whose headaches are not responsive to CGRP inhibition and/or are responsive to therapy with a triptan.
  • human subjects whose headaches are not responsive to CGRP inhibition is not limited to human subjects that have previously been prescribed a CGRP inhibitor and found it to lack efficacy, and additionally encompasses individuals that have not previously been prescribed a CGRP inhibitor, but, whose headaches would nonetheless not respond to such treatment options.
  • the invention comprises treating human subjects that have previously failed treatment with an antagonist of CGRP. Such patients can be readily identified by a review of their clinical history.
  • human subjects whose headaches are responsive to therapy with a triptan is not limited to human subjects that have previously been prescribed and responded to a triptan, but additionally encompasses individuals that have not previously been prescribed a triptan, but, whose headaches would nonetheless respond to such treatment.
  • the invention comprises treating human subjects that have previously been treated with a triptan and self reported a clinical response. Such patients can be readily by identified by a review of their clinical history.
  • the headaches are not responsive to CGRP therapy and are responsive to therapy with a triptan.
  • the triptan is a triptan selected from the group consisting of: almotriptan, eletriptan, frovatriptan, naratriptan, rizatriptan, sumatriptan and zolmitriptan. In a more particular embodiment, the triptan is sumatriptan.
  • the inhibitor of human TRPM3 is for use in the treatment of cluster headache. In another embodiment, the inhibitor of human TRPM3 is for use in the prevention of cluster headache.
  • the human subject has a TRPM3 allele with a gain of function mutation.
  • the mutated version of human TRPM3 has one or more of the following amino acid substitutions is R1670Q, A1645V, V990M and P1090Q (numbering based on SEQ ID NO: 2).
  • the invention provides an inhibitor of human TRPM3 for use in the treatment of medication overuse headache in a human subject.
  • the medication overuse headache is opioid induced medication overuse headache. This condition may alternatively be referred to as opioid induced hyperalgesia.
  • the opioid induced medication overuse headache/opioid induced hyperalgesia is induced by opioids acting at the the mu opioid receptor.
  • the medication overuse headache is triptan induced medication overuse headache.
  • the triptan is sumatriptan.
  • the human subject has a TRPM3 allele with a gain of function mutation.
  • the mutated version of human TRPM3 has one or more of the following amino acid substitutions is R1670Q, A1645V, V990M and P1090Q (numbering based on SEQ ID NO: 2).
  • an inhibitor of human TRPM3 may be administered by any convenient route.
  • the inhibitor of human TRPM3 may be administered by orally, parenterally, intranasally or by inhalation.
  • the inhibitor of human TRPM3 is administered in a pharmaceutical composition.
  • the inhibitor of human TRPM3 is formulated in a pharmaceutical composition adapted for oral or parenteral administration, or for administration intranasally or by inhalation. Appropriate doses will readily be appreciated by those skilled in the art.
  • the invention provides a pharmaceutical composition comprising an inhibitor of human TRPM3 and a pharmaceutically acceptable excipient. According to another aspect, the invention provides a process for the preparation of a pharmaceutical composition comprising admixing an inhibitor of human TRPM3 with a pharmaceutically acceptable excipient.
  • compositions adapted for oral administration may be presented as discrete units such as capsules or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or whips; or oil-in-water liquid emulsions or water-in-oil liquid emulsions.
  • compositions adapted for nasal administration can comprise a coarse powder having a particle size for example in the range 20 to 500 microns which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose.
  • suitable formulations wherein the carrier is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oil solutions of the inhibitor of human TRPM3.
  • Pharmaceutical formulations adapted for administration by inhalation include fine particle dusts or mists, which may be generated by means of various types of metered, dose pressurized aerosols, nebulizers or insufflators.
  • compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.
  • formulations described herein may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents.
  • the present disclosure also provides unitary pharmaceutical compositions in which the inhibitor of human TRPM3 one or more other therapeutic agent(s) may be administered together.
  • the dose of each therapeutic agent may differ from the dose of that therapeutic agent when used alone.
  • the disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising an inhibitor of human TRPM3, at least one other therapeutic agent selected from: a triptan, an ergot, a nonsteroidal anti-inflammatory drug, an acetaminophen containing product, a butalbital containing product, an anti-emetic, caffeine, dexamethasone, ubrogepant and lasmiditan, and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition comprises an inhibitor of human TRPM3, a triptan selected from the group consisting of: almotriptan, eletriptan, frovatriptan, naratriptan, rizatriptan, sumatriptan and zolmitriptan and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition comprises an inhibitor of human TRPM3, sumatriptan and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition comprises an inhibitor of human TRPM3, a non-steroidal anti-inflammatory drug selected from the group consisting of diclofenac, ibuprofen, naproxen and ketorolac and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition comprises an inhibitor of human TRPM3, an anti-emetic selected from the group consisting of: promethazine, prochlorperazine, metoclopramide, trimethobenzamide and ondansetron and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition comprises an inhibitor of human TRPM3, at least one other therapeutic agent selected from: botulinum toxin A, a CGRP inhibitor, an anticonvulsant, a ⁇ -blocker, an antidepressant and a non-steroidal anti-inflammatory drug, and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition comprises an inhibitor of human TRPM3, at least one other therapeutic agent selected from: valproate, divalproex sodium, amitriptyline, topiramate, venlafaxine, metoprolol, propranolol and timolol, and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition comprises an inhibitor of human TRPM3, at least one other therapeutic agent selected from: carbamazepine and oxcarbazepine, and a pharmaceutically acceptable excipient.
  • GWAS Genome wide association study
  • association test results for the genotyped and the imputed SNPs.
  • For tests using imputed data we use the imputed dosages rather than best-guess genotypes.
  • the association test P value was computed using a likelihood ratio test, which in our experience is better behaved than a Wald test on the regression coefficient.
  • Genotyping and SNP Imputation DNA extraction and genotyping were performed on saliva samples by CLIA-certified and CAP- accredited clinical laboratories of Laboratory Corporation of America. Samples were genotyped on one of five genotyping platforms.
  • the VI and V2 platforms were variants of the Illumina HumanHap550 + BeadChip and contained a total of about 560,000 SNPs, including about 25,000 custom SNPs selected by 23andMe.
  • the V3 platform was based on the Illumina OmniExpress + BeadChip and contained a total of about 950,000 SNPs and custom content to improve the overlap with our V2 array.
  • the V4 platform is a fully custom array and includes a lower redundancy subset of V2 and V3 SNPs with additional coverage of lower-frequency coding variation, and about 570,000 SNPs.
  • the V5 platform in current use, is an Illumina Infinium Global Screening Array of about 640,000 SNPs supplemented with about 50,000 SNPs of custom content. Samples that failed to reach 98.5% call rate were re-analyzed. Individuals whose analyses failed repeatedly were re-contacted by 23andMe customer service to provide additional samples, as is done for all 23andMe customers.
  • Variants were imputed using two separate imputation reference panels. One included a larger number of samples, but did not include insertion or deletion variants. The other included a smaller number of individuals, but included insertion and deletion variants.
  • Phased participant data was generated using an internally-developed tool based on Beagle (Browning, S.R. & Browning, B.L., Rapid and accurate Haplotype Phasing and Missing Data Interference for Whole Genome Association Studies Using Localized Haplotype Clustering, Am. J. Hum. Genet., 81: 1084-1097 (2007)) or a new phasing algorithm, Eagle2 (Loh, P.r. et a.
  • Figure 4 is a regional association plot for migraine diagnosis on chromosome 9 where the x- axis shows physical positions on human genome build GRCh37/hgl9 and the y-axes shows the -loglO of the p-value for association with migraine diagnosis. Each point in the depicted plots represents a genetic variant tested for association in the region. The grey horizontal line represents the genome wide significance threshold of 5xl0 -8 . Human genes in the region are depicted on the lower panel. These GWAS data indicate that the locus (genetic region) shown is implicated in susceptibility to migraine diagnosis in humans. In Figure 4, the trait analyzed was "migraine diagnosis” where "the cases” are individuals with a migraine diagnosis and "the controls" are individuals who did not have a migraine diagnosis.
  • a common genetic variant changing the amino acid sequence of the TRPM3 protein was found to be the lead SNP for this locus.
  • This variant rs6560142 (dbSNP build 154 identifier) is located on chromosome 9 at position 73150984 (of the human genome build GRCh37/hgl9) and the observed frequency of the migraine diagnosis risk allele "T” is 0.56 in the research participant population with predominantly European ancestry (the protective allele "C” has a frequency of 0.44).
  • This variant can also be described at the amino acid level in TRPM3, for example: Argl670Gln, (numbering based on SEQ ID NO: 2).
  • the GWAS analysis identified the rs6560142 missense SNP in the TRPM3 coding region as the SNP in this locus with the lowest P value. Consequently, these data imply that TRPM3 has the highest probability of being the gene that is functionally responsible for the association between this locus and migraine diagnosis. Therefore, these novel GWAS data indicates that the TRPM3 protein and its functions contribute to migraine pathophysiology in humans.
  • DRG and TG were then incubated with 1 mg/ml trypsin solution (Sigma-Aldrich) for 30 - 45 min.
  • Digested ganglia were mechanically dissociated by trituration with a 1 ml pipette.
  • Dissociated DRG were centrifuged at 200g for 5 min. The cell pellet was resuspended in L15 culture media and plated onto poly-D Lysine 96 well plates (Greiner bio-one) coated with 10 ⁇ g/ml laminin (Sigma-Aldrich) and cultured at 37 °C, 5% CO2.
  • Dissociated TG were passed through a 70 ⁇ m cell strainer and overlaid onto 4% (w/v) BSA. Cells were collected after centrifugation and plated onto poly-D Lysine-laminin coated 96 well plates and cultured at 37°C, 5% CO 2 .
  • CGRP and PACAP release experiments were conducted after 17-24 h in culture.
  • TRPM3 agonist either CIM0216 or pregnenolone sulfate (PS) for 30 min at 37°C.
  • TRPM3 inhibitor isosa kuranetin for 30 min prior to addition of agonist.
  • conditioned medium was collected for the detection of CGRP, PACAP and substance P.
  • PACAP concentrations in conditioned medium collected from TG and DRG neuron cultures were determined indirectly.
  • Conditioned medium was incubated with Chinese Hamster Ovary cells expressing the pituitary adenylate cyclase-activating polypeptide type I (PAC1) receptor (generated in house).
  • PAC1 receptor pituitary adenylate cyclase-activating polypeptide type I (PAC1) receptor (generated in house).
  • PAC1 receptor pituitary adenylate cyclase-activating polypeptide type I
  • Binding of PACAP to the PAC1 receptor induced cAMP production was measured using a homogeneous time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassay (Perkin Elmer).
  • TR-FRET time-resolved fluorescence resonance energy transfer
  • Substance P concentrations in conditioned media collected from TG neuron cultures were determined using an ELISA (Cayman Chemicals).
  • TRPM3 neuropeptide release
  • CIM0216 induced a concentration dependent release of CGRP from DRG (Fig. 1A and B) and TG (Fig. 3A) neurons.
  • DRG and TG cultures were also responsive to pregnenolone sulfate, resulting in an increased release of CGRP; while a single concentration of PS (100 ⁇ M) was tested in DRG cultures (Fig. 1A), the response was demonstrated to be concentration dependent in TG cultures (Fig. 3B).
  • TRPM3 inhibitor isosakuranetin to antagonise CGRP release induced by CIM0216 and PS was evaluated.
  • Pre-incubation with isosakuranetin inhibited the response to both agonists in DRG (Fig. 1A, B and C), and TG neuron cultures (Fig. 3A, B and C).
  • TRPM3 activation in DRG neurons with either 6 ⁇ M or 10 ⁇ M CIM0216 (Fig. 2A, 2B respectively) was antagonised by isosakuranetin in a concentration dependent manner, resulting in reduced release of CGRP.
  • isosakuranetin 10 ⁇ M, reduced the release of CGRP to basal levels or below in the presence of 6 ⁇ M CIM0216 (Fig 2A) or 100 ⁇ M PS (Fig. 1A) in DRG cultures, or in the presence of CIM0216 ( ⁇ 100 ⁇ M) or PS ( ⁇ 100 ⁇ M) in TG cultures (Fig. 3A and B).
  • FIG. 11A CIM0216 induced a concentration-dependent release of PACAP from rat TG cells
  • FIG. 11B demonstrates concentration-dependent inhibition of the CIM0216 response by TRPM3 antagonist, isosakuranetin.
  • Figure 12 shows activation of TRPM3 results in the release of PACAP from mouse isolated DRG (Fig.l2A, 12B) and TG cells (Fig. 12C, 12D). These responses were absent from Trpm3 knock out (KO) mouse cells and inhibited by the TRPM3 antagonist isosakuranetin (Fig. 12 A-D).
  • Cells derived from Trpm3 knockout (Fig. 12, A-D) animals were still responsive to the TRPV1 agonist, capsaicin, showing that PACAP release to stimuli of other TRP channels is maintained.
  • Calcium mobilisation assays were performed in HEK MSR II cells loaded with the calcium indicator dye Fluo4.
  • the cells were induced to express TRPM3 (SEQ ID NO: 2) or mutants thereof by transducing them with Bacmam virus containing the codon optimised cDNA sequence for the required TRPM3 variant at a multiplicity of infection of approximately 40 for 48 hours prior to the experiment.
  • Cells were incubated in the presence of FLUO4-AM and TRPM3 inhibitors for approx 1.5 hrs prior to transfer to a FLIPR where the cells were treated with TRPM3 agonists to induce calcium mobilisation. Fluo4 fluorescence was monitored for 10 min. As a positive control the cells were then treated with the calcium ionophore ionomycin and the fluorescence monitored for a further 3 min.
  • Figures 5, 6, 7 show results in calcium mobilisation assays using pregnenelone sulfate (Figs 5 and 7) and CIM0216 (Fig 6) as TRPM3 agonists, and isosakuranetin as a TRPM3 inhibitor.
  • Figure 9 shows pregnenolone sulfate induced concentration-dependent increases in FLUO4 fluorescence in cells expressing canonical TRPM3 (SEQ NO: 2) and a variant of SEQID NO: 2 having the R1670Q mutation.
  • SEQ NO: 2 canonical TRPM3
  • SEQID NO: 2 having the R1670Q mutation.
  • the potency of pregnenolone sulfate was 1.8-fold greater at the R1670Q variant than at canonical and the maximal fold change in fluorescence was 26% larger.
  • pregnenolone sulfate is more able to activate the TRPM3 variant associated with increased likelihood of migraine diagnosis than the canonical form of the channel.
  • Figure 13 shows the agonist effect of the isomers of CIM0216 in a calcium mobilisation assay.
  • the results show that the R-isomer ((R)-2-(3,4-dihydroquinolin-l(2H)-yl)-N-(5-methylisoxazol-3- yl)propenamide ) is a more potent agonist compared to the S-isomer ((S)-2-(3,4-dihydroquinolin- l(2H)-yl)-N-(5-methylisoxazol-3-yl)propenamide).
  • the five-day rat Dural infusion migraine model is based on repeated inflammatory dural stimulation to mimic the repeated activation of dural afferents believed to occur in patients with recurrent migraine headache.
  • rats are tested in the periorbital region for mechanical allodynia (a pain response to stimuli which are not normally painful) using von Frey fibres, which are small calibrated fibres which deliver a calibrated amount of force.
  • von Frey fibres which are small calibrated fibres which deliver a calibrated amount of force.
  • Historical data in this model shows mechanical nociception sensitivity, which is alleviated with sumatriptan and anti-CGRP therapies, current standard of care compounds, suggesting this model has clinical translation.
  • a custom flange guide cannula (22GA, Plastics One) was inserted into the hole (cut 0.5 mm below pedestal). The cannula was fixed to the bone with small screws and dental cement. A dummy that extended just past the end of the cannula was inserted to prevent scar tissue from forming, and thus clogging the cannula. Animals were allowed 1-2 weeks of recovery before testing and infusions began.
  • Periorbital thresholds were monitored during the recovery period to ensure the thresholds returned to pre-surgery baselines. If animals did not return to baseline, they were excluded from the study. Extra animals were included to account for any post-surgery animal that needed to be excluded.
  • mice were infused supra-durally with treatments according to Table 1 for 5 consecutive days.
  • the animal's nose was place in the nose cone of the anaesthesia machine.
  • the base of the flange cannula was clasped, and the dummy cannula was removed.
  • a custom cannula injector was inserted into the flange cannula.
  • Animals were randomly assigned to a treatment group (A-C) using a software generated randomization scheme. From the first day of sensitization, each animal was tested routinely (every 2- 3 days) for changes in periorbital sensitivity by a blinded investigator.
  • Pre-infusion sensory testing occurred on Day 1 of the testing schedule to provide a point of comparison for subsequent testing.
  • Sensory testing occurred according to the testing schedule established in Table 1, and prior to infusion when applicable.
  • Sensory testing utilized von Frey filaments with reproducible calibrated buckling forces varying from 0.4 - 10g utilizing the Chaplan up and down method. Allodynia was tested by perpendicularly touching the periorbital region causing slight buckling of the filament for approximately 5 seconds. Based on the response pattern and the force of the final filament, the periorbital threshold (g) was calculated (Chaplan et al., 1994, Quantitative assessment of tactile allodynia in the rat paw. Journal of neuroscience methods, 53(1), 55-63).
  • the supra-dural infusions were above the dura in the right brain hemisphere; therefore, the right periorbital threshold data only was recorded.
  • a positive response was characterized by several behavioural criteria: stroking the face with a forepaw, head withdrawal from the stimulus, and head shaking.
  • TRPM3 agonists when administered durally for five days evoked an allodynic response to von Frey filaments in rats.
  • Rats treated with the TRPM3 agonists at lower thresholds than in vehicle treated rats (Figure 10A). This was sustained and did not return to baseline levels for the duration of the experiment (19 days). Animals showed no overt, lasting adverse effects to the agonists and were feeding and grooming normally. This can be seen in Figure 10B as TRPM3 agonist treated groups increase in body weight with the same trend as vehicle treated animals.
  • Sensitivity to non-noxious stimuli is referred to as allodynia and has been shown as a potential clinical correlate in migraine patients.
  • Patients with chronic or transformed migraine exhibit facial allodynia even on days when they do not have a headache (Cooke et al., (2007). Cutaneous allodynia in transformed migraine patients. Headache: The Journal of Head and Face Pain, 47(4), 531-539).
  • Example 4 is evidence that repeated activation by TRPM3 agonists can induce sensitisation in the rodent to a mechanical stimulus, that is similar to a pain assessment used in migraine patients.
  • Example 5 Patient Population (Non-Responders to CGRP Treatment)
  • response was one of the following: 'Slightly', 'moderately', 'very', 'extremely'
  • TRPM3 status Individuals were then stratified by TRPM3 status. The number of 'migraine high-risk' alleles in each individual (corresponding to the coding variant for 1670Gln) was scored as 0, 1 or 2 depending on allele dosage. Data were compiled in a pivot table, with aggregate data of CGRP responders stratified by TRPM3 status.
  • Example 5 is evidence that a population exists that does not respond to CGRP antagonism medication. These individuals could be a target population for a TRPM3 blocker therapy. Of the identified population, 50/58 have at least one migraine high-risk allele, suggesting a higher likelihood of responding to anti-TRPM3 migraine therapy.
  • TNC Trigeminal nucleus caudalis
  • the left and right TNC halves were separated using a scalpel and individual TNC halves were transferred to separate wells of a 48 well tissue culture plate containing 250 ⁇ L of L-15 Medium, GLUTAMAX containing 18 mM NaHCO 3 , 38 mM glucose (L15 assay buffer).
  • TNCs were washed 5 times in L15 assay buffer replacing the assay buffer every 5 min at room temperature, and then a further 5 times in a humidified incubator at 37°C, 5% CO2.
  • each TNC tissue was incubated sequentially with vehicle, and then TRPM3 agonist, either pregnenolone sulfate (PS) or R-CIM0216 (CIM0216*) or both combined, at increasing concentrations each for 10 mins at 37°C.
  • TRPM3 agonist either pregnenolone sulfate (PS) or R-CIM0216 (CIM0216*) or both combined, at increasing concentrations each for 10 mins at 37°C.
  • tissues were incubated with isosakuranetin for 10 mins following vehicle but prior to addition of agonist, and maintained in the presence of increasing agonist concentrations.
  • conditioned medium was collected to a 96 well polypropylene plate containing BSA on ice, such that the final BSA concentration is 1%, and any remaining liquid in the tissue well discarded prior to the next agonists/ antagonist addition.
  • the responsiveness of each tissue was verified by the addition of capsaicin (l ⁇ M) and/or KCI (40mM or 60mM) and measuring the neuropeptides released over 10 mins. In some experiments the treatment periods were 15 mins.
  • CGRP CGRP CGRP ELISA kit
  • Levels of CGRP were quantitated using a rat CGRP ELISA kit (Bertin Bioreagent, Cat. No. #A05482) according to manufacturer's instructions.
  • Levels of Substance P were quantitated using a Substance P ELISA kit (Cayman Chemical, Cat. number #583751). There is 100% sequence identity between rat and mouse CGRP, and between rat and mouse Substance P.
  • TNCs were incubated with TRPM3 agonists pregnenolone sulfate, R-CIM0216 (CIM0216*), or both combined, in the presence and absence of the TRPM3 inhibitor isosakuranetin.
  • Pregnenolone sulfate and CIM0216* in combination induced a concentration-dependent release of CGRP (FIG. 15A-B) and substance P (FIG. 15C-D) from mouse TNC. This concentration-dependent response was inhibited in the presence of the TRPM3 antagonist, isosakuranetin.
  • FIGURE 16 A-B The measured quantities of CGRP and substance P released from TNC over 15mins are shown in FIGURE 16 A-B.
  • KCI responses were observed in both Wild Type and knock out groups.
  • SEQ ID NO: 1 Amino acid sequence of human TRPM3 isoform XI 1
  • SEQ ID NO:2 Amino acid sequence of human TRPM3 isoform k
  • SEQ ID NO:3 Amino acid sequence of human TRPM3 isoform m MPEPWGTVYFLGIAQVFSFLFSWWNLEGVMNQADAPRPLNWTIRKLCHAAFLPSVRLLKAQKSWIERAFYKRE CVHIIPSTKDPHRCCCGRLIGQHVGLTPSISVLQNEKNESRLSRNDIQSEKWSISKHTQLSPTDAFGTIEFQGGG HSNKAMYVRVSFDTKPDLLLHLMTKEWQLELPKLLISVHGGLQNFELQPKLKQVFGKGLIKAAMTTGAWIFTGG VNTGVIRHVGDALKDHASKSRGKICTIGIAPWGIVENQEDLIGRDWRPYQTMSNPMSKLTVLNSMHSHFILAD NGTTGKYGAEVKLRRQLEKHISLQKINTRIGQGVPVVALIVEGGPNVISIVLEYLRDTPPVPVWCDGSGRASDIL AFGHKYSEEGGLINESLRDQLLVTIQKTFTYTRTQAQHLFIIL
  • SEQ ID N0:4 Amino acid sequence of human TRPM3 isoform X19
  • SEQ ID NO:5 Amino acid sequence of human TRPM3 isoform q
  • SEQ ID N0:6 Amino acid sequence of human TRPM3 isoform Q9HCF6-6
  • SEQ ID NO:7 Amino acid sequence of human TRPM3 isoform X12
  • SEQ ID N0:8 Amino acid sequence of human TRPM3 isoform Q9HCF6-8
  • SEQ ID NO:9 Amino acid sequence of human TRPM3 isoform s
  • SEQ ID NO: 10 Amino acid sequence of human TRPM3 isoform Q9HCF6-11
  • VGMILRLQDQPFRSD GRVIYCVNIIYWYIRLLDIFGVNKYLGPYVMMIGKMMIDMMYFVIIMLVVLMSFGVARQA
  • SEQ ID NO: 38 Nucleotide sequence of exon 1 of the human TRPM3 gene.
  • SEQ ID NO: 39 Nucleotide sequence of exon 2 of the human TRPM3 gene.
  • SEQ ID NO: 40 Nucleotide sequence of exon 3 of the human TRPM3 gene.
  • SEQ ID NO: 41 Nucleotide sequence of exon 4 of the human TRPM3 gene.
  • SEQ ID NO: 42 Nucleotide sequence of exon 5 of the human TRPM3 gene. GTGTTGCTGTGGGCGTCTGATAGGCCAGCATGTTGGCCTCACCCCCAGTATCTCCGTGCTTCAGAATGAAA
  • SEQ ID NO: 43 Nucleotide sequence of exon 6 of the human TRPM3 gene.
  • SEQ ID NO: 44 Nucleotide sequence of exon 7 of the human TRPM3 gene.
  • SEQ ID NO: 45 Nucleotide sequence of exon 8 of the human TRPM3 gene.
  • SEQ ID NO: 46 Nucleotide sequence of exon 9 of the human TRPM3 gene.
  • SEQ ID NO: 47 Nucleotide sequence of exon 10 of the human TRPM3gene.
  • SEQ ID NO: 48 Nucleotide sequence of exon 11 of the human TRPM3gene.
  • SEQ ID NO: 49 Nucleotide sequence of exon 11a of the human TRPM3gene.
  • SEQ ID NO: 50 Nucleotide sequence of exon 12 of the human TRPM3gene.
  • SEQ ID NO: 51 Nucleotide sequence of exon 13 of the human TRPM3gene.
  • SEQ ID NO: 52 Nucleotide sequence of exon 14 of the human TRPM3gene. GTGGGATCTCTGGAGCAAGCCATGTTGGATGCCTTAGTTCTGGACAGAGTGGATTTTGTGAAATTACTCATA GAGAATGGAGTAAGCATGCACCGTTTTCTCACCATCTCCAGACTAGAGGAATTGTACAATACG
  • SEQ ID NO: 53 Nucleotide sequence of exon 15 of the human TRPM3gene.
  • SEQ ID NO: 54 Nucleotide sequence of exon 16 of the human TRPM3gene.
  • SEQ ID NO: 55 Nucleotide sequence of exon 17 of the human TRPM3gene.
  • SEQ ID NO: 56 Nucleotide sequence of exon 18 of the human TRPM3gene.
  • SEQ ID NO: 57 Nucleotide sequence of exon 19 of the human TRPM3gene.
  • SEQ ID NO: 58 Nucleotide sequence of exon 20 of the human TRPM3gene.
  • SEQ ID NO: 59 Nucleotide sequence of exon 21 of the human TRPM3gene.
  • SEQ ID NO: 60 Nucleotide sequence of exon 22 of the human TRPM3gene.
  • SEQ ID NO: 61 Nucleotide sequence of exon 23 of the human TRPM3gene.
  • SEQ ID NO: 62 Nucleotide sequence of exon 24 of the human TRPM3gene.
  • SEQ ID NO: 63 Nucleotide sequence of exon 25 of the human TRPM3gene.
  • SEQ ID NO: 64 Nucleotide sequence of exon 26 of the human TRPM3gene.
  • SEQ ID NO: 65 Nucleotide sequence of exon 27 of the human TRPM3gene.
  • SEQ ID NO: 66 Nucleotide sequence of exon 28 of the human TRPM3gene.
  • SEQ ID NO: 67 Nucleotide sequence of exon 29 of the human TRPM3gene.
  • SEQ ID NO: 68 Nucleotide sequence of exon 29 of the human TRPM3gene where exon 30 is used GGTGGAGAACATGTCTATGCGGCTGGAGGAAGTCAACGAGAGAGCACTCCATGAAGGCTTCACTCCAGA CCGTGGACATCCGGCTGGCGCAGCTGGAAGACCTTATCGGGCGCATGGCCACGGCCCTGGAGCGCCTGACA GGTCTGGAGCGGGCCGAGTCCAACAAAATCCGCTCGAGGACCTCGTCAGACTGCACGGACGCCGCCTACATT GTCCGTCAGAGCAGCTTCAACAGCCAGGAAGGGAACACCTTCAAGCTCCAAGAGAGTATAGACCCTGCAG
  • SEQ ID NO: 69 Nucleotide sequence of exon 30 of the human TRPM3gene.

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Abstract

La présente invention concerne un inhibiteur de TRPM3 humain destiné à être utilisé dans le traitement ou la prévention de la migraine ou de la céphalée vasculaire de Horton chez des patients dont les migraines/céphalées ne réagissent pas à l'inhibition du CGRP ou dont les migraines/céphalées sont sensibles aux triptans. Selon un autre aspect, l'invention concerne un inhibiteur de TRPM3 humain destiné à être utilisé dans le traitement d'une céphalée liée à une surconsommation de médicaments. L'invention concerne également un inhibiteur de TRPM3 humain qui inhibe la libération de PACAP à partir de neurones ganglionnaires trijumeaux, et un procédé de mesure de libération de PACAP.
PCT/EP2023/068967 2022-07-13 2023-07-10 Nouvelle utilisation WO2024013052A1 (fr)

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US17/812,299 US20230220037A1 (en) 2022-01-12 2022-07-13 Novel use
US17/812,299 2022-07-13
US63/388,720 2022-07-13

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005026317A2 (fr) 2003-05-12 2005-03-24 Bristol-Myers Squibb Company Polynucleotides codant de nouveaux variants du membre ltrpc3 du canal de la famille trp
WO2022112352A1 (fr) 2020-11-24 2022-06-02 Katholieke Universiteit Leuven Dérivés hétérocycliques pour le traitement de troubles à médiation par trpm3
WO2022112345A1 (fr) 2020-11-24 2022-06-02 Katholieke Universiteit Leuven Dérivés d'aryle pour le traitement de troubles à médiation par trpm3
WO2022152715A1 (fr) * 2021-01-14 2022-07-21 Glaxosmithkline Intellectual Property (No.3) Limited Inhibiteurs de trpm3 et leurs utilisations

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005026317A2 (fr) 2003-05-12 2005-03-24 Bristol-Myers Squibb Company Polynucleotides codant de nouveaux variants du membre ltrpc3 du canal de la famille trp
WO2022112352A1 (fr) 2020-11-24 2022-06-02 Katholieke Universiteit Leuven Dérivés hétérocycliques pour le traitement de troubles à médiation par trpm3
WO2022112345A1 (fr) 2020-11-24 2022-06-02 Katholieke Universiteit Leuven Dérivés d'aryle pour le traitement de troubles à médiation par trpm3
WO2022152715A1 (fr) * 2021-01-14 2022-07-21 Glaxosmithkline Intellectual Property (No.3) Limited Inhibiteurs de trpm3 et leurs utilisations

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