WO2024118515A1 - Pikfyve inhibitor combination therapy - Google Patents

Pikfyve inhibitor combination therapy Download PDF

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Publication number
WO2024118515A1
WO2024118515A1 PCT/US2023/081184 US2023081184W WO2024118515A1 WO 2024118515 A1 WO2024118515 A1 WO 2024118515A1 US 2023081184 W US2023081184 W US 2023081184W WO 2024118515 A1 WO2024118515 A1 WO 2024118515A1
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subject
antioxidant
pharmaceutically acceptable
edaravone
pikfyve
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PCT/US2023/081184
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French (fr)
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Peter Ronald Young
Carlo SPIRLI
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Orphai Therapeutics Inc.
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Publication of WO2024118515A1 publication Critical patent/WO2024118515A1/en

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds

Definitions

  • the invention relates to compositions and methods comprising a PIKfyve inhibitor for use in combination with other therapeutic agents in the treatment of neurological disease and disorders.
  • Neurodegenerative diseases and disorders cause progressive loss of neuronal function, and the societal burden of neurodegenerative diseases is increasing.
  • Neurodegenerative diseases may include amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Parkinson Disease (PD), Alzheimer’s Disease and Huntington disease (HD).
  • ALS amyotrophic lateral sclerosis
  • FDD frontotemporal dementia
  • PD Parkinson Disease
  • AD Alzheimer’s Disease
  • Huntington disease Huntington disease
  • ALS Amyotrophic lateral sclerosis
  • Apilimod also referred to as STA-5326, hereinafter “apilimod”
  • apilimod is recognized as a potent inhibitor of IL- 12 and IL-23.
  • IL- 12 and IL-23 are inflammatory cytokines normally produced by immune cells, such as B-cells and macrophages, in response to antigenic stimulation.
  • PIKfyve phosphatidylinositol-3- phosphate 5 -kinase
  • PIKfyve inhibitor YM201636 with a morpholino-pyrimidine (diazine) core which and was originally identified in a screen of PI3K class IA inhibitors was reported to inhibit the proliferation of cancer cells in vitro as well as the growth of transplanted tumors in mice. Subsequently, these three compounds were all shown to be highly selective inhibitors of PIKfyve despite very different chemical scaffolds and likely off-target activities, suggesting that they can be used together to identify PIKfyve dependent activities.
  • PIKfyve inhibitors, including apilimod have been shown to increase the survival of motor neurons.
  • compositions and methods relating to the use of a PIKfyve inhibitor for treating neurological diseases and disorders, particularly in combination an antioxidant (e.g., edaravone).
  • an antioxidant e.g., edaravone
  • the combination of a PIKfyve inhibitor (e.g., any PIKfyve inhibitors known or described herein,) and an antioxidant achieved synergistic effects in improving ALS motor neuron viability.
  • a method described herein further comprises using additional therapeutic agents for treating neurological diseases (e.g., ALS), including glutamatergic agents (e.g., riluzole).
  • additional therapeutic agents for treating neurological diseases e.g., ALS
  • glutamatergic agents e.g., riluzole
  • the PIKfyve inhibitor used in a method described in is not apilimod or pharmaceutically acceptable salt thereof.
  • Some aspects of the present disclosure provide methods of treating a neurological disease or disorder in a subject in need thereof, the method comprising administering to the subject a PIKfyve inhibitor and an antioxidant, wherein the PIKfyve inhibitor is not apilimod or a pharmaceutically acceptable salt thereof.
  • the PIKfyve inhibitor or a pharmaceutically acceptable salt thereof and the antioxidant are in one composition.
  • the PIKfyve inhibitor or a pharmaceutically acceptable salt thereof and the antioxidant are in different compositions.
  • Some aspects of the present disclosure provide methods of treating a neurological disease or disorder in a subject in need thereof, the method comprising administering to the subject the antioxidant, wherein the subject is receiving or has received treatment with a PIKfyve inhibitor or a pharmaceutically acceptable salt thereof, wherein the PIKfyve inhibitor is not apilimod or a pharmaceutically acceptable salt thereof.
  • Some aspects of the present disclosure provide methods of treating a neurological disease or disorder in a subject in need thereof, the method comprising administering to the subject a PIKfyve inhibitor or a pharmaceutically acceptable salt thereof, wherein the subject is receiving or has received treatment with an antioxidant, wherein the PIKfyve inhibitor is not apilimod or a pharmaceutically acceptable salt thereof.
  • the PIKfyve inhibitor is YM-201636, MOMIPP, MF4, APY0201, AS2677131, AS2795440, VRG101, Vacuolin-1, WX8, NDF, WWL, XB6, AS-201, AS-202, VRG50635, VRG50648, XBA, or analogs or salts thereof.
  • the PIKfyve inhibitor is YM-201636, APY0201, or a pharmaceutically acceptable salt thereof.
  • the antioxidant is ascorbic acid, cysteine, cysteamine, edaravone, glutathione and bilirubin, amifostine (WR-2721), vitamin A, vitamin C, vitamin E, and flavonoids such as Indian holy basil (Ocimum sanctum), orientin or vicenin.
  • the antioxidant is administered orally.
  • the antioxidant is edaravone.
  • edaravone is administered at a daily dose of 60 mg for 14 days, followed by 14 days of no administration, further followed by a daily dose of 60 mg for 10 days in a 14-day period, further followed by 14 days of no administration.
  • the antioxidant is administered intravenously.
  • edaravone is administered at a daily dose of 105 mg.
  • edaravone is administered orally.
  • the PIKfyve inhibitor is administered orally. In some embodiments, the composition is administered orally.
  • the method further comprises administering to the subject a glutamatergic agent.
  • the glutamatergic agent is riluzole.
  • riluzole is administered to the subject twice daily and 50 mg of riluzole is administered in each administration.
  • the glutamatergic agent is administered orally.
  • the subject is not receiving or has not received treatment with a glutamatergic agent.
  • the glutamatergic agent is riluzole.
  • the neurological disease of disorder is selected from Alzheimer's disease, amyotrophic lateral sclerosis (ALS), attention deficit hyperactivity disorder, autism, cerebellar ataxia, Charcot-Marie-Tooth disease, Creutzfeldt- Jakob disease, dementia, epilepsy, Friedreich's ataxia, Huntington's disease, multiple sclerosis, obsessive compulsive disorder (OCD), Parkinson's disease, Rett syndrome, senile chorea, spinal ataxia, spinal cord injury, supranuclear palsy, traumatic brain injury.
  • the neurological disease or disorder is dementia.
  • the dementia is selected from AIDS dementia complex (ADC), dementia associated with Alzheimer’s disease (AD), dementia pugilistica, diffuse Lewy body disease, frontotemporal dementia, mixed dementia, senile dementia of Lewy body type, and vascular dementia.
  • ADC AIDS dementia complex
  • AD dementia associated with Alzheimer’s disease
  • AD dementia pugilistica
  • diffuse Lewy body disease dementia pugilistica
  • frontotemporal dementia mixed dementia
  • senile dementia of Lewy body type vascular dementia
  • vascular dementia vascular dementia
  • the neurological disease or disorder is amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD).
  • ALS amyotrophic lateral sclerosis
  • FTD frontotemporal dementia
  • the subject is in need of treatment is one having repeat expansions of the C9ORF72 gene. In some embodiments, the subject in need of treatment is one having a mutation in the SOD1 gene. In some embodiments, the subject in need of treatment is one having accumulation of TDP-34 aggregates. In some embodiments, the subject in need of treatment is one having a mutation in the TDP-43 gene. In some embodiments, the subject is human. In some embodiments, the subject is not receiving or has not received treatment with AMX0035, PBA, TUDCA, methylcobalamin, or any combinations thereof.
  • PIKfyve inhibitor and an antioxidant in a method of treating a neurological disease or disorder in a subject in need thereof, wherein the PIKfyve inhibitor is not apilimod or a pharmaceutically acceptable salt thereof.
  • PIKfyve inhibitor in a method of treating a neurological disease or disorder in a subject in need thereof, wherein the subject is receiving or has received treatment with an antioxidant, wherein the PIKfyve inhibitor is not apilimod or a pharmaceutically acceptable salt thereof.
  • an antioxidant in a method of treating a neurological disease or disorder in a subject in need thereof, wherein the subject is receiving or has received treatment with a PIKfyve inhibitor, and wherein the PIKfyve inhibitor is not apilimod or a pharmaceutically acceptable salt thereof.
  • the PIKfyve inhibitor is YM-201636, MOMIPP, MF4, APY0201, AS2677131, AS2795440, VRG101, Vacuolin-1, WX8, NDF, WWL, XB6, AS-201, AS-202, VRG50635 , VRG50648, XBA, or analogs or salts thereof.
  • the PIKfyve inhibitor is YM-201636, APY0201 or a pharmaceutically acceptable salt thereof.
  • the antioxidant is ascorbic acid, cysteine, cysteamine, edaravone, glutathione and bilirubin, amifostine (WR-2721), vitamin A, vitamin C, vitamin E, and flavonoids such as Indian holy basil (Ocimum sanctum), orientin or vicenin.
  • the antioxidant is edaravone.
  • the subject is not receiving or has not received treatment with AMX0035, PBA, TUDCA, methylcobalamin, or any combinations thereof.
  • control bar indicates untreated cells while the DMSO bar indicates cells treated with 0.01% DMSO which is the buffer in which all the compounds have been dissolved.
  • FIGURE 1 is a graph demonstrating the ability of Edaravone (1 pM) and apilimod (6.25 nM) in combination to increase cell viability synergistically after glutamate exposure in iPSC- derived motoneurons from a C9orf72-ALS patient. *** denotes p ⁇ 0.001; * denotes significance ofp ⁇ 0.05.
  • FIGURE 2 is a graph demonstrating the ability of Edaravone (1 pM) and apilimod (6.25 nM) in combination to increase cell viability synergistically after glutamate exposure in iPSC- derived motoneurons from a SOD1-ALS patient. ** denotes significance of p ⁇ 0.01; * denotes significance ofp ⁇ 0.05.
  • FIGURE 3 is a graph demonstrating the ability of Edaravone (1 pM) and apilimod (6.25 nM) in combination to increase cell viability synergistically after growth factors deprivation (- GF) in iPSC-derived motoneurons from a TDP-43-ALS patient. *** denotes p ⁇ 0.001; ** denotes significance ofp ⁇ 0.01.
  • FIGURE 4 is a graph demonstrating the ability of Edaravone (1 pM) and apilimod (6.25 nM) in combination to increase cell viability synergistically after glutamate exposure in iPSC- derived motoneurons from a Sporadic-ALS patient. *** denotes significance of p ⁇ 0.001; * denotes significance ofp ⁇ 0.05.
  • FIGURE 5 is a graph demonstrating the ability of Edaravone (IpM) and YM201636 (12.5 nM) in combination to increase cell viability synergistically after glutamate exposure in iPSC-derived motoneurons from a C9orf72-ALS patient. *** denotes significance of p ⁇ 0.001; ** denotes significance ofp ⁇ 0.01.
  • FIGURE 6 is a graph demonstrating the ability of Edaravone (IpM) and YM201636 (12.5 nM) in combination to increase cell viability synergistically after glutamate exposure in iPSC-derived motoneurons from a SOD1-ALS patient. *** denotes significance of p ⁇ 0.001.
  • FIGURE 7 is a graph demonstrating the ability of Edaravone (IpM) and YM201636 (12.5 nM) in combination to increase cell viability synergistically after growth factors deprivation (-GF) in iPSC-derived motoneurons from a TDP-43-ALS patient. *** denotes significance ofp ⁇ 0.001; * denotes significance of p ⁇ 0.05.
  • FIGURE 8 is a graph demonstrating the ability of Edaravone (IpM) and YM201636 (12.5 nM) in combination to increase cell viability synergistically after glutamate exposure in iPSC-derived motoneurons from a Sporadic-ALS patient. **** denotes significance of p ⁇ 0.0001; *** denotes significance ofp ⁇ 0.001; * denotes significance ofp ⁇ 0.05.
  • FIGURE 9 is a graph demonstrating the ability of Edaravone (I pM) and APY0201 (6.25 nM) in combination to increase cell viability synergistically after glutamate exposure in iPSC-derived motoneurons from a C9orf72-ALS patient. **** denotes p ⁇ 0.0001; *** denotes significance ofp ⁇ 0.001** denotes significance ofp ⁇ 0.01.
  • FIGURE 10 is a graph demonstrating the ability of Edaravone (I pM) and APY0201 (6.25 nM) in combination to increase cell viability synergistically after glutamate exposure in iPSC-derived motoneurons from a SOD1-ALS patient. **** denotes p ⁇ 0.0001; ** denotes significance ofp ⁇ 0.01.
  • FIGURE 11 is a graph demonstrating the ability of Edaravone (I pM) and APY0201 (6.25 nM) in combination to increase cell viability synergistically after growth factors deprivation (-GF) in iPSC-derived motoneurons from a TDP-43-ALS patient. ** denotes significance ofp ⁇ 0.01; * denotes significance of p ⁇ 0.05.
  • FIGURE 12 is a graph demonstrating the ability of Edaravone (IpM) and APY0201 (6.25 nM) in combination to increase cell viability synergistically after glutamate exposure in iPSC-derived motoneurons from a Sporadic-ALS patient. ** denotes significance of p ⁇ 0.01; * denotes p ⁇ 0.05.
  • the term “pharmaceutically acceptable salt,” may be a salt formed from, for example, an acid and a basic group of a compound described herein (e.g. , any PIKfyve inhibitors known or described herein).
  • salts may be selected from, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, mesylate, dimesylate, lactate (e.g., D-lactate or L- lactate), salicylate, citrate, tartrate (e.g., L-tartrate), malonate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, besylate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, pamoate (e.g, l,l’
  • the salt of apilimod comprises methanesulfonate.
  • pharmaceutically acceptable salt may also refer to a salt prepared from a compound described herein (e.g., any PIKfyve inhibitors known or described herein), having an acidic functional group, such as a carboxylic acid functional group, and a pharmaceutically acceptable inorganic or organic base.
  • pharmaceutically acceptable salt may also refer to a bis-salt, e.g., an apilimod dimesylate salt.
  • Suitable bases include, but are not limited to, hydroxides of alkali metals such as sodium, potassium, and lithium; hydroxides of alkaline earth metal such as calcium and magnesium; hydroxides of other metals, such as aluminum and zinc; ammonia, and organic amines, such as unsubstituted or hydroxy-substituted mono-, di-, or trialkylamines; dicyclohexylamine; tributyl amine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, bis-, or tris-(2 -hydroxy-lower alkyl amines), such as mono-, bis-, or tris- (2 -hydroxy ethyl)amine, 2-hydroxy-tert-butyl amine, or tris-(hydroxymethyl)methylamine, N, N,-di-lower alkyl-N-(hydroxy lower alkyl)-amines, such as N,N-di
  • pharmaceutically acceptable salt may also refer to a salt prepared from any one of the PIKfyve inhibitors described herein (e.g., YM- 201636, APY0201) or known in the art, having a basic functional group, such as an amino functional group, and a pharmaceutically acceptable inorganic or organic acid.
  • Suitable acids include hydrogen sulfate, citric acid, acetic acid, oxalic acid, hydrochloric acid (HC1), hydrogen bromide (HBr), hydrogen iodide (HI), nitric acid, hydrogen bisulfide, phosphoric acid, lactic acid, salicylic acid, tartaric acid, bitartratic acid, ascorbic acid, succinic acid, maleic acid, besylic acid, fumaric acid, gluconic acid, glucuronic acid, formic acid, benzoic acid, glutamic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid.
  • the salts of a compound may be synthesized from the parent compound by conventional chemical methods. Generally, such salts can be prepared by reacting the parent compound with the appropriate acid in water or in an organic solvent, or in a mixture of the two.
  • One salt form of a compound described herein may be converted to the free base and optionally to another salt form by methods well known to the skilled person.
  • the free base may be formed by passing the salt solution through a column containing an amine stationary phase (e.g., a Strata-NH2 column).
  • a solution of the salt in water may be treated with sodium bicarbonate and a base (e.g., hydroxide or carbonate bases) to decompose the salt.
  • the free base may then be combined with another acid using routine methods.
  • polymorph may refer to solid crystalline forms of a compound of the present disclosure (e.g., any PIKfyve inhibitors known or described herein) or complex thereof. Different polymorphs of the same compound may exhibit different physical, chemical and/or spectroscopic properties. Different physical properties include, but are not limited to, stability (e.g., to heat or light), compressibility and density (important in formulation and product manufacturing), and dissolution rates (which may affect bioavailability).
  • Differences in stability may result from changes in chemical reactivity (e.g., differential oxidation, such that a dosage form discolors more rapidly when comprised of one polymorph than when comprised of another polymorph) or mechanical characteristics (e.g., tablets crumble on storage as a kinetically favored polymorph converts to thermodynamically more stable polymorph) or both (e.g., tablets of one polymorph are more susceptible to breakdown at high humidity).
  • chemical reactivity e.g., differential oxidation, such that a dosage form discolors more rapidly when comprised of one polymorph than when comprised of another polymorph
  • mechanical characteristics e.g., tablets crumble on storage as a kinetically favored polymorph converts to thermodynamically more stable polymorph
  • both e.g., tablets of one polymorph are more susceptible to breakdown at high humidity.
  • Different physical properties of polymorphs may affect their processing. For example, one polymorph may be more likely to form solvates or may be more difficult to filter or wash free of impurities than another
  • hydrate may describe a compound of the present disclosure (e.g., any PIKfyve inhibitors known or described herein) or a salt thereof, which further includes a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces.
  • the term “clathrate” may refer to a compound of the present disclosure (e.g., any PIKfyve inhibitors known or described herein) or a salt thereof in the form of a crystal lattice that may contain spaces (e.g., channels) that may have a guest molecule (e.g., a solvent or water) trapped within.
  • a compound of the present disclosure e.g., any PIKfyve inhibitors known or described herein
  • a salt thereof in the form of a crystal lattice may contain spaces (e.g., channels) that may have a guest molecule (e.g., a solvent or water) trapped within.
  • prodrug may refer to a derivative of a compound described herein (e.g., any PIKfyve inhibitors known or described herein) that may hydrolyze, oxidize, or otherwise react under biological conditions (in vitro or in vivo) to provide a compound of the disclosure.
  • Prodrugs may only become active upon such reaction under biological conditions, or they may have activity in their unreacted forms.
  • prodrugs contemplated in this disclosure include, but are not limited to, analogs or derivatives that may comprise biohydrolyzable moieties such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzable ureides, and biohydrolyzable phosphate analogues.
  • Other examples of prodrugs may include, but are not limited to, derivatives of compounds of any one of the formulae disclosed herein that comprise -NO, -NO2, -ONO, or -ONO2 moieties.
  • Some of the compounds suitable for use in the methods described in this disclosure may have one or more double bonds, or one or more asymmetric centers. Such compounds may occur as racemates, racemic mixtures, single enantiomers, individual diastereomers, diastereomeric mixtures, and cis- or trans- or E- or Z-double isomeric forms. All such isomeric forms of these compounds are expressly included in the present disclosure.
  • the compounds of this disclosure may also be represented in multiple tautomeric forms, in such instances, the disclosure expressly includes all tautomeric forms of the compounds described herein (e.g, there may be a rapid equilibrium of multiple structural forms of a compound), the disclosure expressly includes all such reaction products). All such isomeric forms of such compounds are expressly included in the present disclosure. All crystal forms of compounds described herein (e.g., any PIKfyve inhibitors known or described herein) are expressly included in the present disclosure.
  • solvate or “pharmaceutically acceptable solvate,” may refer to a solvate formed from the association of one or more solvent molecules to one of the compounds disclosed herein (e.g., any PIKfyve inhibitors known or described herein).
  • solvate may include hydrates (e.g., hemi -hydrate, mono-hydrate, dihydrate, trihydrate, tetrahydrate, and the like).
  • analog may refer to a chemical compound that is structurally similar to another chemical compound but differs slightly in composition (e.g, as in the replacement of one atom by an atom of a different element or in the presence of a particular functional group, or the replacement of one functional group by another functional group).
  • an analog may be a compound that is similar or comparable in function and appearance, but not in structure or origin to the reference compound.
  • derivative may refer to compounds that have a common core structure and are substituted with various groups as described herein.
  • ND Neurodegenerative diseases
  • ALS amyotrophic lateral sclerosis
  • FDD frontotemporal dementia
  • PD Parkinson’s disease
  • AD Alzheimer’s disease
  • HD Huntington’s disease
  • the present disclosure in some aspects, demonstrates that administration of a PIKfyve inhibitor and an antioxidant in (iPSC)-derived motoneurons from ALS patients has synergistic results in cell viability.
  • the combination therapy of a PIKfyve inhibitor and an antioxidant e.g., edaravone
  • the present disclosure in some aspects, provides compositions and methods related to the use of a PIKfyve inhibitor (e.g., any PIKfyve inhibitors known or described herein), in combination with an antioxidant (e.g., edaravone) for treating neurological diseases and disorders.
  • a method described herein comprises administering to a subject in need thereof a PIKfyve inhibitor (e.g., any PIKfyve inhibitors known or described herein), and an antioxidant (e.g., edaravone).
  • a PIKfyve inhibitor e.g., any PIKfyve inhibitors known or described herein
  • an antioxidant e.g., edaravone
  • the PIKfyve inhibitor is not apilimod or a pharmaceutically acceptable salt thereof.
  • a method described herein comprises administering a PIKfyve inhibitor (e.g., any PIKfyve inhibitors known or described herein), or a pharmaceutically active salt thereof, to a subject in need thereof, wherein the subject is receiving or has received treatment with an antioxidant (e.g., edaravone).
  • a method described herein comprises administering an antioxidant (e.g., edaravone) to a subject in need thereof, wherein the subject is receiving or has received treatment with a PIKfyve inhibitor (e.g., any PIKfyve inhibitors known or described herein), or a pharmaceutically acceptable salt thereof.
  • the PIKfyve inhibitor is not apilimod or a pharmaceutically acceptable salt thereof.
  • PIKfyve inhibitor may refer to a molecule that inhibits the expression and/or activity (e.g., kinase activity) of PIKfyve (phosphoinositide kinase, FYVE-type zinc finger containing).
  • PIKfyve phosphoinositide kinase is an enzyme that has evolutionarily conserved lipid and protein kinase that has pleiotropic cellular functions.
  • Phosphorylation of the phosphatidylinositol-3-phosphate (PI3P) by PIKfyve generates two phosphoinositide (PI) derivatives (1) phosphatidylinositol 3, 5 -bisphosphate [PtdIns(3,5)P2] or (2) phosphatidylinositol 5-phosphate (PtdIns5P).
  • PI phosphoinositide
  • the two phosphoinositide (PI) derivatives may govern various cellular processes, including, but not limited to, cytoskeleton rearrangements, remodeling the actin cytoskeleton, intracellular membrane trafficking pathways, endocytosis, epidermal growth factor receptor (EGFR) signaling, translocation of the glucose transporter GLUT4 after insulin stimulation, regulation of synapse strength and cell proliferation.
  • EGFR epidermal growth factor receptor
  • the FYVE finger domain of PIKfyve has been shown to play a vital role in localizing the protein to the cytosolic leaflet of endosomes through directly binding to membrane PtdIns3P and is thereby involved in multiple processes of endosome dynamics.
  • PIKfyve inhibition significantly increased the survival of ALS patient-derived motor neurons by converting PtdIns3P into PtdIns(3,5)P2, which enhanced the fusion of lysosomes with both endosomes and autophagosomes under cell stress.
  • PIKfyve inhibition can also lead to activation of transcription factor TFEB, which drives the clearance of toxic protein aggregates in ALS patients.
  • a PIKfyve inhibitor that may be used in accordance with the methods described herein is a small molecule.
  • Nonlimiting examples of small molecule PIKfyve inhibitors include: apilimod and analogs or salts thereof, YM-201636 (CAS Number 371942-69-7), MOMIPP (CAS Number 1363421-46-8), MF4, APY0201 (CAS Number 1232221-74-7), AS2677131 (CAS Number 2171502-44-4), AS2795440, VRG101 (by Verge Genomics), Vacuolin-1 (CAS Number 351986-85-1), WX8 (MLS000543798; PubChem CID 135510930), NDF (MLS000699212; PubChem CID 9629709), WWL (MLS000703078), XB6 (MLS001167897), VRG50635 (by Verge Genomics; prodrug of VRG50648), VRG50648 (by Verge Genomics; active form of VRG50635) and XBA (MLS001167909).
  • PIKfyve inhibitors that may be used in any one of the methods described herein include, without limitation, small molecule PIKfyve inhibitors described in WO18175906, W0202009971, WO19046316, WO21163727, WO21113633, WO17218815, WO22169882, WO21247841, WO21247859, WO21247862, WO19113523, WO20243457, WO21146192, WO21183439, WO18138106, WO22086993, US2021/0122752, US2020/0165246. US11352354, and WO21252895, the entire contents of each of which are incorporated herein by reference.
  • a PIKfyve inhibitor used in accordance with the present disclosure is an RNAi agent or an antisense oligonucleotide (e.g., AS-201 or AS-202 by Acurastem) that targets an RNA (e.g., mRNA) encoded by the PIKfyve gene, e.g., as described in International Patent Application Publication No. WO2021155067, the entire contents of which and the sequences disclosed in the specification and the associated sequence listing are incorporated herein by reference.
  • an RNAi agent or an antisense oligonucleotide e.g., AS-201 or AS-202 by Acurastem
  • RNA e.g., mRNA encoded by the PIKfyve gene
  • the PIKfyve inhibitor used in a method described herein is not apilimod or a pharmaceutically acceptable salt thereof.
  • apilimod refers to 2-[2-Pyridin-2-yl)-ethoxy]-4-N’-(3-methyl-benzilidene)- hydrazino]-6-(morpholin-4-yl)-pyrimidine (IUPAC name: (E)-4-(6-(2-(3-methylbenzylidene) hydrazinyl)-2-(2-(pyridin-2-yl)ethoxy)pyrimidin-4-yl)morpholine), represented by Formula I: or any pharmaceutically acceptable salt, solvate, clathrate, hydrate, polymorph, metabolite, prodrug, analog, or derivative thereof.
  • apilimod used in accordance with a method described herein is apilimod dimesylate.
  • Apilimod may be prepared, for example, according to the methods described in U.S. Pat. Nos. 7,923,557, and 7,863,270, and WO 2006/128129, the entire contents of each of which are incorporated herein by reference.
  • APY0201 refers to 2-[7-(4-morpholinyl)-2-(4-pyridinyl)pyrazolo[l,5- a]pyrimidin-5-yl]hydrazone, 3-methyl-benzaldehyde, represented by Formula II: or any pharmaceutically acceptable salt, solvate, clathrate, hydrate, polymorph, metabolite, prodrug, analog, or derivative thereof.
  • APY0201 is a PIKfyve inhibitor that inhibits IL- 12/23 production in vitro and ex vivo in murine plasma (e.g., as described in Hayakawa et al., Bioorg Med Chem. 2014 Jun 1;22(11):3021-9 and Drewry et al., J Med. Chem. 2022 Oct 13;65(19): 12860-12882, incorporated herein by reference).
  • YM201636 refers to a compound with CAS number of 371942-69-7 and a structure represented by Formula III: or any pharmaceutically acceptable salt, solvate, clathrate, hydrate, polymorph, metabolite, prodrug, analog, or derivative thereof.
  • YM201636 is a PIKfyve inhibitor with a morpholinopyrimidine (diazine) core and has been identified in a screen of PI3K class IA inhibitors.
  • YM201636 has been reported to inhibit the proliferation of cancer cells in vitro as well as the growth of transplanted tumors in mice (Ikomonov et al., Toxicology and Applied Pharmacology, Volume 383, 15 November 2019, 114771 and Drewry et al., J Med. Chem. 2022 Oct 13;65(19): 12860- 12882, incorporated herein by reference).
  • MOMIPP refers to a compound with CAS number of 1363421-46-8 and a structure represented by Formula IV: or any pharmaceutically acceptable salt, solvate, clathrate, hydrate, polymorph, metabolite, prodrug, analog, or derivative thereof.
  • MF4 refers to a compound with a structure represented by Formula (V):
  • AS2677131 refers to a compound with CAS number of 2171502-44-4 and a structure represented by Formula (VI): or any pharmaceutically acceptable salt, solvate, clathrate, hydrate, polymorph, metabolite, prodrug, analog, or derivative thereof.
  • AS2677131 has been reported to inhibit the production of IL-12p40, IL-6, and IL-1
  • AS2795440 refers to 2,3-Dimethyl-lH-pyrrolo[3,2-b]pyridine-5- carboxylic acid (l"-isopropyl-6-methyl-l",2",3",4",5",6"-hexahydro-[3,3';6',4"]terpyridin-5- yl)-amide, represented by Formula (VII): or any pharmaceutically acceptable salt, solvate, clathrate, hydrate, polymorph, metabolite, prodrug, analog, or derivative thereof.
  • AS2795440 has been reported to inhibit the production of IL-12p40, IL-6, and IL- 1 , potent pro-inflammatory cytokines in vitro, and block the development of arthritis in rats (Terajima M, et al. Eur J Pharmacol. 2016 Jun 5;780:93- 105, incorporated herein by reference).
  • Vacuolin-1 refers to a compound with CAS number of 351986-85-1 and a structure represented by Formula (VIII): or any pharmaceutically acceptable salt, solvate, clathrate, hydrate, polymorph, metabolite, prodrug, analog, or derivative thereof.
  • Vacuolin-1 has been reported to inhibit infection by chimeric vesicular stomatitis virus (VSV) that contains an envelope protein for either Zaire ebolavirus (VSV-ZEBOV) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2) (VSVSARS-CoV-2) in vitro ( Kang Y, et al. PNAS. 2020 Aug 25;117(34):20803-20813 tint incorporated herein by reference).
  • VSV chimeric vesicular stomatitis virus
  • SARS-CoV- 2 severe acute respiratory syndrome coronavirus 2
  • WX8 (MLS000543798; PubChem CID 135510930) refers to lH-indole-3- carbaldehyde [4-anilino-6-(4-morpholinyl)-l,3,5-triazin-2-yl]hydrazine, a compound represented by Formula (IX): or any pharmaceutically acceptable salt, solvate, clathrate, hydrate, polymorph, metabolite, prodrug, analog, or derivative thereof. WX8 has been reported to kill autophagy-dependent cancer cells with no effect on non-malignant human cells (Sharma G, et al. Autophagy. 2019;
  • NDF 3- methylbenzaldehyde (2,6-dimorpholin-4-ylpyrimidin-4-yl)hydrazine, represented by Formula (X): or any pharmaceutically acceptable salt, solvate, clathrate, hydrate, polymorph, metabolite, prodrug, analog, or derivative thereof.
  • Formula (X) or any pharmaceutically acceptable salt, solvate, clathrate, hydrate, polymorph, metabolite, prodrug, analog, or derivative thereof.
  • NDF is described in Sharma G, et al. Autophagy. 2019;
  • WWL benzaldehyde [2,6-di(4-morpholinyl)-4- pyrimidinyl]hydrazone, represented by Formula (XI): or any pharmaceutically acceptable salt, solvate, clathrate, hydrate, polymorph, metabolite, prodrug, analog, or derivative thereof.
  • WWL is described in Sharma G, et al. Autophagy. 2019; 15(10): 1694-1718, incorporated herein by reference.
  • XBA N-(3-chloro-4-fluorophenyl)-4,6- dimorpholino-l,3,5-triazin-2-amine hydrochloride, represented by Formula (XII): or any pharmaceutically acceptable salt, solvate, clathrate, hydrate, polymorph, metabolite, prodrug, analog, or derivative thereof.
  • Formula (XII) or any pharmaceutically acceptable salt, solvate, clathrate, hydrate, polymorph, metabolite, prodrug, analog, or derivative thereof.
  • XBA is described in Sharma G, et al. Autophagy. 2019; 15(10): 1694-1718, incorporated herein by reference.
  • XB6 refers to N-(4-ethylphenyl)- 4,6-dimorpholino- l,3,5-triazin-2-amine hydrochloride, represented by Formula (XIII): or any pharmaceutically acceptable salt, solvate, clathrate, hydrate, polymorph, metabolite, prodrug, analog, or derivative thereof.
  • XBA is described in Sharma G, et al. Autophagy. 2019; 15(10): 1694-1718, incorporated herein by reference.
  • a PIKfyve inhibitor or a pharmaceutically acceptable salt thereof is used in combination with an antioxidant (e.g., edaravone) for treating neurological diseases and disorders.
  • an antioxidant e.g., edaravone
  • a PIKfyve inhibitor is any described herein or known in the art (e.g., apilimod, YM-201636, MOMIPP, MF4, APY0201, AS2677131 , AS2795440, VRG101, Vacuolin-1 , WX8, NDF, WWL, XB6, VRG50635, VRG50648, XBA, and analogs or salts thereof,)
  • an “antioxidant” may refer to substances that may prevent or slow damage to cells caused by free radicals, and unstable molecules that the body produces as a reaction to environmental and other pressures.
  • the terms “antioxidant” and “free-radical scavengers” may be used interchangeably.
  • the sources of antioxidants may be natural or artificial.
  • Antioxidants may include, but are not limited to, ascorbic acid, cysteine, cysteamine, edaravone, glutathione and bilirubin, amifostine (WR-2721), vitamin A, vitamin C, vitamin E, and flavonoids such as Indian holy basil (Ocimum sanctum), orientin and vicenin.
  • the antioxidant is edaravone (RadicavaTM, RadicutTM).
  • about 25-80 e.g., 25-80, 25-75, 25-60, 25-50, 25-40, 25-30, 30-80, 30-75, 30-60, 30-50, 30-40, 40-80, 40-75, 40- 60, 40-50, 50-80, 50-75, 50-60, 60-80, 60-75, or 75-80
  • milligrams of edaravone is administered to the subject.
  • about 60 milligrams of edaravone is administered to the subject.
  • edaravone is administered intravenously to the subject.
  • about 75-125 e.g., 75-125, 75-115, 75-105, 75-95, 75-85, 85-125, 85-115, 85-105, 85-95, 95-125, 95-115, 95- 105, 105-125, 105-115, 105-125, 105-115, or 115-125) milligrams of edaravone is administered to the subject.
  • about 75, 85, 95, 105, 115, or 125 milligrams of edaravone administered to the subject.
  • about 105 milligrams of edaravone is administered to the subject.
  • edaravone is administered orally to the subject.
  • edaravone is administered to the subject 1-3 (e.g., 1-3, 1-2, 2-3) times daily. In some embodiments, in any one of the methods described herein, edaravone is administered to the subject 1, 2, 3 times daily. In some embodiments, in any one of the methods described herein, wherein edaravone is administered to the subject daily.
  • edaravone is administered to the subject intermittently over a period of time.
  • the period of time may be about 1-12 (e.g., 1-12, 1-10, 1-8, 1-6, 1-4, 1-2, 2-12, 2-10, 2-8, 2-6, 2-4, 4-12, 4-10, 4-8, 4-6, 6-12, 6-10, 6-8, 8-12, 8-10, 10-12) weeks.
  • the period of time is about 1, 2, 4, 6, 8, 10, 12 weeks.
  • the period of time is about 8 weeks.
  • edaravone is administered to the subject for about 14 days, followed by about 14 days of no administration, further followed by administration for about 10 days in about a 14-day period, further followed by about 14 days of no administration.
  • edaravone is administered to the subject at a daily dose of about 60 mg for about 14 days, followed by about 14 days of no administration, further followed by administration of a daily dose of about 60 mg for about 10 days in about a 14-day period, further followed by about 14 days of no administration.
  • any one of the methods described herein may further comprise administering to the subject a glutamatergic agent.
  • a “glutamatergic agent” may be described as a composition, or a drug, or a chemical that directly modulates the excitatory amino acid (glutamate/ aspartate) system in the body or brain.
  • the glutamatergic agent may be selected from, but is not limited to, a glutamate transporter modulating agent and a glutamate receptor antagonist.
  • the glutamate transporter modulating agent may be an excitatory amino acid reuptake inhibitor.
  • the glutamate receptor antagonist may be an N-methyl-D-aspartate (NMDA) receptor antagonist.
  • the glutamate receptor antagonist may be an antagonist of the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMP A) receptor, or the kainite receptor.
  • AMP A a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor
  • the glutamatergic agent may be a glutamate receptor antagonist selected from, but not limited to, AP5 (R-2-amino-5-phosphonopentanoate), AP7 (2-amino-7- phosphonoheptanoic acid), CNQX (6-cyano-7-nitroquinoxaline-2,3-dione), CPPene (3-[(R)-2- carboxypiperazin-4-yl]-prop-2-enyl-l-phosphonic acid), NBQX (2,3-dihydroxy-6-nitro-7- sulfamoyl-benzo[f]quinoxaline-2, 3-dione), and selfotel (CGS-19755).
  • AP5 R-2-amino-5-phosphonopentanoate
  • AP7 (2-amino-7- phosphonoheptanoic acid
  • CNQX (6-cyano-7-nitroquinoxaline-2,3-dione)
  • CPPene (3-[(R)
  • the glutamatergic agent may be a glutamate receptor antagonist selected from, but not limited to, amantadine, atomoxetine, AZD6765, agmatine, gacyclidine, ketamine, memantine, eliprodil, delucemin.
  • the glutamatergic agent may be selected from, but not limited to, BHV-5000, lamotrigine, lanicemine, riluzole, trigriluzole, and topiramate.
  • the glutamatergic agent is riluzole.
  • a method of treating a neurological disorder comprises administering a PIKfyve inhibitor or a pharmaceutically acceptable salt thereof, and an antioxidant (e.g., edaravone), wherein the subject is receiving or has received treatment with a glutamatergic agent.
  • a method of treating a neurological disorder comprises administering a PIKfyve inhibitor, or a pharmaceutically acceptable salt thereof, and an antioxidant (e.g., edaravone), wherein the subject is receiving or has received treatment with riluzole.
  • a method of treating a neurological disorder comprises administering a PIKfyve inhibitor, or a pharmaceutically acceptable salt thereof, and an antioxidant (e.g., edaravone), wherein the subject is not receiving or has not received treatment with a glutamatergic agent (e.g., riluzole).
  • an antioxidant e.g., edaravone
  • a PIKfyve inhibitor is any described herein or known in the art (e.g., apilimod, YM-201636, MOMIPP, MF4, APY0201, AS2677131 , AS2795440, VRG101, Vacuolin-1 , WX8, NDF, WWL, XB6, VRG50635, VRG50648, XBA, and analogs or salts thereof,).
  • any one of the methods described herein about 25-75 (e.g., 25-75, 25-60, 25-50, 25-40, 25-30, 30-75, 30-60, 30-40, 30-50, 40-75, 40-60, 40-50, 50-75, 50- 60, 60-75) milligrams of riluzole is administered to the subject. In some embodiments, in any one of the methods described herein, about 25, 30, 40, 50, 60, 75 milligrams of riluzole are administered to the subject. In some embodiments, in any one of the methods described herein, about 50 milligrams of riluzole is administered to the subject.
  • riluzole is administered to the subject 1-3 (e.g., 1-3, 1-2, 2-3) times daily. In some embodiments, in any one of the methods described herein, riluzole is administered to the subject 1, 2, 3 times daily. In some embodiments, in any one of the methods described herein, riluzole is administered to the subject twice daily. In some embodiments, in any one of the methods described herein, riluzole is administered to the subject twice daily and about 50 mg of riluzole is administered in each administration.
  • the subject in any one of the methods described herein, is not receiving treatment or has not received treatment with AMX0035, 4-phenylbutyric acid (PBA), tauroursodeoxycholic acid (TUDCA), methylcobalamin, or any combinations thereof.
  • AMX0035 a fixed combination of PBA and TUDCA is an approved medication for ALS marketed under the brand name Relyvrio.
  • methylcobalamin refers to carbanide;cobalt(2+);[(2R,3S,4R,5S)-5-(5,6-dimethylbenzimidazol-l-yl)-4-hydroxy-2- (hydroxymethyl)oxolan-3-yl]-l-[3-[(lR,2R,3R,5Z,7S,10Z,12S,13S,15Z,17S,18S,19R)- 2,13,18-tris(2-amino-2-oxoethyl)-7,12,17-tris(3-amino-3-oxopropyl)-3,5,8,8,13,15,18,19- octamethyl-2,7, 12,17 -tetrahydro- 1 H-corrin-24-id-3-yl]propanoylamino]propan-2-yl hydrogen phosphate.
  • the molecular formula of methylcobalamin is C63H92CoN13O14P, with
  • the different therapeutic agents may be formulated in compositions (e.g., different, or same compositions) for administration to a subject.
  • the composition(s) may take any suitable form (e.g., liquids, aerosols, solutions, inhalants, mists, sprays; or solids, powders, ointments, pastes, creams, lotions, gels, patches, and the like) for administration by any desired route (e.g., pulmonary, inhalation, intranasal, oral, buccal, sublingual, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, intrapleural, intrathecal, transdermal, transmucosal, rectal, and the like).
  • a pharmaceutical composition of the disclosure may be in the form of an aqueous solution or powder for aerosol administration by inhalation or insufflation (either through the mouth or the nose), in the form of a tablet or capsule for oral administration; in the form of a sterile aqueous solution or dispersion suitable for administration by either direct injection or by addition to sterile infusion fluids for intravenous infusion; or in the form of a lotion, cream, foam, patch, suspension, solution, or suppository for transdermal or transmucosal administration.
  • the administration route of the composition comprising an antioxidant e.g., edaravone
  • a PIKfyve inhibitor e.g., riluzole
  • the administration route of the antioxidant e.g., edaravone
  • a PIKfyve inhibitor is any described herein or known in the art (e.g., apilimod, YM-201636, MOMIPP, MF4, APY0201, AS2677131 , AS2795440, VRG101, Vacuolin-1 , WX8, NDF, WWL, XB6, VRG50635, VRG50648, XBA, and analogs or salts thereof).
  • a “neurological disease or disorder,” as used herein, refers to a disease of the nervous system characterized by progressive loss of neuronal structure and function.
  • the neurological disease or disorder may be selected from a neurodegenerative disease or disorder, epilepsy, a neuromuscular disorder, or a neurodevelopmental disorder.
  • Neurodegenerative diseases and disorders that may be treated according to the methods described may be selected from, but are not limited to, Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), diffuse Lewy body disease, motor neuron diseases, multiple sclerosis (MS), Parkinson’s disease (PD), Friedreich’s ataxia, prion disease, spinocerebellar ataxia (SCA), cerebellar ataxia, and spinal muscular atrophy (SMA).
  • AD Alzheimer’s disease
  • ALS amyotrophic lateral sclerosis
  • MS multiple sclerosis
  • PD Parkinson’s disease
  • Friedreich’s ataxia prion disease
  • SCA spinocerebellar ataxia
  • cerebellar ataxia cerebellar ataxia
  • SMA spinal muscular atrophy
  • neurodegenerative diseases and disorders that may be treated according to the methods described herein may be selected from, but are not limited to, Charcot-Marie-Tooth disease, Creutzfeldt-Jakob disease (CJD), progressive supranuclear palsy (PSP, Steele-Richardson- Olszewski syndrome), senile chorea, Huntington’s Chorea, spinal ataxia including spinocerebellar ataxia (SCA), Friedreich’s ataxia, Subacute sclerosing panencephalitis, frontotemporal lobar degeneration, and Hallerrorden-Spatz disease (Pantothenate kinaseassociated neurodegeneration, PKAN).
  • CJD Creutzfeldt-Jakob disease
  • PSP Progressive supranuclear palsy
  • SEP Spinocerebellar ataxia
  • Friedreich s ataxia
  • Subacute sclerosing panencephalitis frontotemporal lobar degeneration
  • Hallerrorden-Spatz disease P
  • the methods described herein may be used for the treatment of ALS or frontotemporal dementia, where the patient in need of treatment may be a patient having repeat expansions in the C9ORF72 gene.
  • the GGGGCC repeat expansion in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS), accounting for about 10% of all ALS cases worldwide and 10% familial frontotemporal dementia (FTD).
  • the repeat expansion generates neurotoxic species including dipeptide repeat proteins (DPRs), nuclear RNA foci, and RNA/DNA G-quadruplexes.
  • DPRs dipeptide repeat proteins
  • nuclear RNA foci nuclear RNA foci
  • RNA/DNA G-quadruplexes RNA/DNA G-quadruplexes.
  • the repeat expansion also suppresses the production of C9ORF72 protein, a protein that normally regulates vesicle trafficking and lysosomal biogenesis.
  • C9ORF72 protein a protein that normally regulates vesicle trafficking and lysosomal biogenesis.
  • the repeat expansion in C9ORF72 triggered neurodegeneration through two mechanisms: accumulation of glutamate receptors and impaired clearance of neurotoxic dipeptide repeat proteins.
  • the methods described herein may be used for the treatment of ALS or frontotemporal dementia, where the patient in need of treatment may be a patient having repeat expansions in the C9ORF72 gene.
  • the methods described herein may be used for the treatment of ALS or frontotemporal dementia, where the patient in need of treatment may be a patient having mutations in the SOD1 gene.
  • the methods described herein may be used for the treatment of ALS or frontotemporal dementia, where the patient in need of treatment may be a patient having mutations in the TDP-43 gene.
  • the neurological disease or disorder may be dementia.
  • the patient in need of treatment of is one having a mutation in TDP-43 and/or an accumulation of TDP-43 aggregates, a product of the TARDBP gene, found in many sporadic and familial ALS.
  • the patient has ALS that arose spontaneously and does not have one of the previous mutations.
  • dementia may also be considered neurodegenerative diseases.
  • the term ‘dementia’ may describe a group of symptoms affecting memory, thinking and social abilities severely enough to interfere with daily functioning.
  • the present disclosure may provide methods of treating dementia, including AIDS dementia complex (ADC), dementia associated with Alzheimer’s disease (AD), dementia pugilistica, diffuse Lewy body disease, frontotemporal dementia, mixed dementia, senile dementia of Lewy body type, and vascular dementia.
  • ADC AIDS dementia complex
  • AD dementia associated with Alzheimer’s disease
  • dementia pugilistica dementia associated with Alzheimer’s disease
  • diffuse Lewy body disease dementia pugilistica
  • diffuse Lewy body disease dementia pugilistica
  • frontotemporal dementia mixed dementia
  • senile dementia of Lewy body type vascular dementia.
  • the neurological disease or disorder may also be selected from, but not limited to, bipolar disorder, treatment resistant and major depression, general anxiety disorder, panic disorder, social anxiety, mood disorders, cognitive disorders, agitation, apathy, psychoses, post-traumatic stress disorders, irritability, disinhibition, learning disorders, memory loss, personality disorders, bipolar disorders, eating disorders, conduct disorder, pain disorders, delirium, drug addiction, tinnitus, mental retardation, cervical spondylotic myelopathy, spinal cord injury, hereditary cerebellar ataxia, Tourette syndrome, autism spectrum disorder, attention deficit hyperactivity disorder, obsessive compulsive disorder (OCD), traumatic brain injury, schizophrenia, fragile X syndrome, Parkinson’s Disease and Huntington’s disease.
  • bipolar disorder treatment resistant and major depression
  • general anxiety disorder panic disorder
  • social anxiety anxiety
  • mood disorders cognitive disorders
  • agitation apathy
  • psychoses post-traumatic stress disorders
  • irritability disinhibition
  • learning disorders memory loss
  • personality disorders bipolar disorders
  • neuromuscular disorders that may be treated according to the methods described herein may be selected from, but are not limited to, infantile spinal muscular atrophy (SMA1, Werdnig-Hoffmann disease), and juvenile spinal muscular atrophy (SMA3, Kugelberg- Welander disease).
  • SMA1 infantile spinal muscular atrophy
  • SMA3 juvenile spinal muscular atrophy
  • SMA3 Kugelberg- Welander disease
  • Neurodevelopmental disorders that may be treated according to the methods described herein may include but is not limit to Rett syndrome.
  • a “subject in need thereof’ refers to a subject in need of treatment for a neurological disease or disorder (e.g., a neurological disease or disorder as provided herein).
  • the subject in need may be one that is “non-responsive” or “refractory” to a standard therapy for the neurological disease or disorder.
  • the terms “non-responsive” and “refractory” may refer to the subject’s response to therapy as not clinically adequate to relieve one or more symptoms associated with the neurological disease” or disorder.
  • a “subject” may refer generally to a mammal.
  • the mammal may be e.g., a human, primate, mouse, rat, dog, cat, cow, horse, goat, camel, sheep, or a pig.
  • the subject may be a human.
  • the terms “subject” and “patient” may be used interchangeably herein.
  • treatment may describe the management and care of a subject having a neurological disease or disorder, and may include the administration of a therapeutic agent, or combination thereof, to slow the progression of the disease or disorder and/or to alleviate one or more symptoms of the neurological disease or disorder.
  • treating may include administering an amount of the therapeutic agent, or combination of agents, effective to alleviate one or more symptoms of the neurological disease or disorder.
  • alleviate may refer to a process by which the severity of a symptom may be reduced or decreased, but it may not necessarily be eliminated, although it may be eliminated for a period of time, or temporarily. While elimination of the symptom may be preferred, it is not required.
  • prevention refers to reducing or eliminating the onset of a symptom, especially in the context of preventing the progression of the disease or disorder, where progression may be defined by the onset one or more symptoms.
  • combination therapy or “co-therapy” may include the administration therapeutic agents as part of a specific treatment regimen intended to provide a beneficial effect from the co-action of these compounds.
  • the beneficial effect may result in the slowing of the progression of the neurological disease or disorder, and/or the alleviation of one or more symptoms of the neurological disease or disorder.
  • the beneficial effect of the combination may include, but is not limited to, pharmacokinetic or pharmacodynamic co-action resulting from the combination.
  • Combination therapy may not be intended to encompass the administration of two or more of these therapeutic compounds as part of separate monotherapy regimens that incidentally and arbitrarily result in the combinations of the present disclosure.
  • administration of a PIKfyve inhibitor, or a pharmaceutically acceptable salt thereof may be simultaneous with or sequential to the administration of one or more additional therapeutic agents (e.g., the antioxidant and/or the glutamatergic agent).
  • administration of the different components of a combination therapy may be at different frequencies.
  • the one or more additional therapeutic agents may be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a PIKfyve inhibitor , or a pharmaceutically acceptable salt thereof.
  • a PIKfyve inhibitor is any described herein or known in the art (e.g., apilimod, YM-201636, MOMIPP, MF4, APY0201, AS2677131 , AS2795440, VRG101, Vacuolin-1 , WX8, NDF, WWL, XB6, VRG50635, VRG50648, XBA, and analogs or salts thereof,)
  • the therapeutic agents may be administered in a composition.
  • the compositions may be a pharmaceutical composition.
  • a “pharmaceutical composition” is a formulation containing one or more therapeutic agents in a pharmaceutically acceptable form suitable for administration to a subject.
  • pharmaceutically acceptable may refer to those compounds, materials, compositions, carriers, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • a pharmaceutical composition may be prepared using a pharmaceutically acceptable excipient.
  • a “pharmaceutically acceptable excipient” may describe an excipient that may be useful in preparing a pharmaceutical composition that may be generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes excipient that may be acceptable for veterinary use as well as human pharmaceutical use.
  • Pharmaceutically acceptable excipients may be selected from, but are not limited to, sterile liquids, water, buffered saline, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), oils, detergents, suspending agents, carbohydrates (e.g., glucose, lactose, sucrose or dextran), antioxidants (e.g., ascorbic acid or glutathione), chelating agents, low molecular weight proteins, or suitable mixtures thereof.
  • polyol for example, glycerol, propylene glycol, liquid polyethylene glycol and the like
  • oils for example, glycerol, propylene glycol, liquid polyethylene glycol and the like
  • detergents for example, glycerol, propylene glycol, liquid polyethylene glycol and the like
  • suspending agents e.g., carbohydrates (e.g., glucose, lactose, sucrose or dextran), antioxidants (e.g., ascor
  • a pharmaceutical composition may be provided in bulk or in dosage unit form. It may be advantageous to formulate pharmaceutical compositions in dosage unit form for ease of administration and uniformity of dosage.
  • dosage unit form as used herein may refer to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the disclosure may be dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved.
  • a dosage unit form may be an ampoule, a vial, a suppository, a dragee, a tablet, a capsule, an IV bag, or a single pump on an aerosol inhaler.
  • the dosages may vary depending on the agent, the age, weight, and clinical condition of the recipient patient, and the experience and judgment of the clinician or practitioner administering the therapy, among other factors affecting the selected dosage.
  • the dose may be a therapeutically effective amount. Dosages may be provided in mg/kg/day units of measurement (which dose may be adjusted for the patient’s weight in kg, body surface area in m 2 , and age in years).
  • a “therapeutically effective amount” of a pharmaceutical composition may be that which provides an objectively identifiable improvement as noted by the clinician or other qualified observer. In one non-limiting example, alleviating a symptom of a disorder, disease, or condition.
  • a pharmaceutical composition may be in the form of an orally acceptable dosage form including, but not limited to, capsules, tablets, buccal forms, troches, lozenges, and oral liquids in the form of emulsions, aqueous suspensions, dispersions or solutions.
  • Capsules may contain mixtures of a compound of the present disclosure with inert fdlers and/or diluents such as the pharmaceutically acceptable starches (e.g., com, potato or tapioca starch), sugars, artificial sweetening agents, powdered celluloses, such as crystalline and microcrystalline celluloses, flours, gelatins, gums, etc.
  • inert fdlers and/or diluents such as the pharmaceutically acceptable starches (e.g., com, potato or tapioca starch), sugars, artificial sweetening agents, powdered celluloses, such as crystalline and microcrystalline celluloses, flours, gelatins, gums, etc.
  • carriers which are commonly used include lactose and com starch.
  • Lubricating agents such as magnesium stearate, can also be added.
  • oral administration in a capsule form useful diluents include lactose and dried com starch.
  • the compound of the present disclosure when aqueous suspensions and/or emulsions may be administered orally, the compound of the present disclosure may be suspended or dissolved in an oily phase is combined with emulsifying and/or suspending agents. In further embodiments, certain sweetening and/or flavoring and/or coloring agents may be added.
  • a pharmaceutical composition may be in a solid oral dosage form.
  • a pharmaceutical composition may be in the form of a tablet.
  • the solid oral dosage form is an orally disintegrating tablet.
  • the tablet may comprise a unit dosage of a compound of the present disclosure together with an inert diluent or carrier such as a sugar or sugar alcohol, for example lactose, sucrose, sorbitol or mannitol.
  • the tablet may further comprise a non-sugar derived diluent such as sodium carbonate, calcium phosphate, calcium carbonate, or a cellulose or derivative thereof such as methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, and starches such as com starch.
  • the tablet may further comprise binding and granulating agents such as polyvinylpyrrolidone, disintegrants (e.g., swellable crosslinked polymers such as crosslinked carboxymethylcellulose), lubricating agents (e.g., stearates), preservatives (e.g., parabens), antioxidants (e.g., BHT), buffering agents (for example phosphate or citrate buffers), and effervescent agents such as citrate/bicarbonate mixtures.
  • binding and granulating agents such as polyvinylpyrrolidone, disintegrants (e.g., swellable crosslinked polymers such as crosslinked carboxymethylcellulose), lubricating agents (e.g., stearates), preservatives (e.g., parabens), antioxidants (e.g., BHT), buffering agents (for example phosphate or citrate buffers), and effervescent agents such as citrate/bicarbonate mixtures.
  • the tablet may be a coated tablet.
  • the coating may be a protective fdm coating (e.g., a wax or varnish) or a coating designed to control the release of the active agent, for example a delayed release (release of the active after a predetermined lag time following ingestion) or release at a particular location in the gastrointestinal tract.
  • a protective fdm coating e.g., a wax or varnish
  • a coating designed to control the release of the active agent for example a delayed release (release of the active after a predetermined lag time following ingestion) or release at a particular location in the gastrointestinal tract.
  • the latter may be achieved, for example, using enteric fdm coatings such as those sold under the brand name EudragitTM.
  • tablet formulations may be made by conventional compression, wet granulation or dry granulation methods and utilize pharmaceutically acceptable diluents, binding agents, lubricants, disintegrants, surface modifying agents (including surfactants), suspending or stabilizing agents, including, but not limited to, magnesium stearate, stearic acid, talc, sodium lauryl sulfate, microcrystalline cellulose, carboxymethylcellulose calcium, polyvinylpyrrolidone, gelatin, alginic acid, acacia gum, xanthan gum, sodium citrate, complex silicates, calcium carbonate, glycine, dextrin, sucrose, sorbitol, dicalcium phosphate, calcium sulfate, lactose, kaolin, mannitol, sodium chloride, talc, dry starches and powdered sugar.
  • pharmaceutically acceptable diluents including, but not limited to, magnesium stearate, stearic acid, talc, sodium lauryl
  • surface modifying agents include nonionic and anionic surface modifying agents.
  • Non-limiting examples of surface modifying agents may include, but are not limited to, poloxamer 188, benzalkonium chloride, calcium stearate, cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters, colloidal silicon dioxide, phosphates, sodium dodecylsulfate, magnesium aluminum silicate, and triethanolamine.
  • a pharmaceutical composition may be in the form of a hard or soft gelatin capsule.
  • the compound of the present disclosure may be in a solid, semi-solid, or liquid form.
  • a pharmaceutical composition may be in the form of a sterile aqueous solution or dispersion suitable for parenteral administration.
  • parenteral includes, but is not limited to, subcutaneous, intracutaneous, intravenous, intramuscular, intra-articular, intraarterial, intrasynovial, intrastemal, intrathecal, intralesional and intracranial injection or infusion techniques.
  • a pharmaceutical composition may be in the form of a sterile aqueous solution or dispersion suitable for administration by either direct injection or by addition to sterile infusion fluids for intravenous infusion, and comprises a solvent or dispersion medium containing, water, ethanol, a polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), suitable mixtures thereof, or one or more vegetable oils.
  • solutions or suspensions of the compound of the present disclosure as a free base or pharmacologically acceptable salt may be prepared in water suitably mixed with a surfactant.
  • suitable surfactants are given below.
  • Dispersions may also be prepared, non-limiting examples include, in glycerol, liquid polyethylene glycols and mixtures of the same in oils.
  • the pharmaceutical compositions for use in the methods of the present disclosure may further comprise one or more additives in addition to any carrier or diluent (such as lactose or mannitol) that may be present in the formulation.
  • the one or more additives may comprise or consist of one or more surfactants.
  • surfactants may have one or more long aliphatic chains such as fatty acids which enables them to insert directly into the lipid structures of cells to enhance drug penetration and absorption.
  • HLB hydrophilic-lipophilic balance
  • HLB values may be more hydrophobic, and may have greater solubility in oils, while surfactants with higher HLB values may be more hydrophilic and may have greater solubility in aqueous solutions.
  • hydrophilic surfactants may generally be considered to be those compounds having an HLB value greater than about 10
  • hydrophobic surfactants may be generally those having an HLB value less than about 10.
  • these HLB values are merely a guide since for many surfactants, the HLB values may differ by as much as about 8 HLB units, depending upon the empirical method chosen to determine the HLB value.
  • surfactants for use in the compositions of the disclosure are polyethylene glycol (PEG)-fatty acids and PEG-fatty acid mono and diesters, PEG glycerol esters, alcoholoil transesterification products, polyglyceryl fatty acids, propylene glycol fatty acid esters, sterol and sterol derivatives, polyethylene glycol sorbitan fatty acid esters, polyethylene glycol alkyl ethers, sugar and its derivatives, polyethylene glycol alkyl phenols, polyoxyethylenepolyoxypropylene (POE-POP) block copolymers, sorbitan fatty acid esters, ionic surfactants, fat-soluble vitamins and their salts, water-soluble vitamins and their amphiphilic derivatives, amino acids and their salts, and organic acids and their esters and anhydrides.
  • PEG polyethylene glycol
  • PEG-fatty acid mono and diesters PEG glycerol esters
  • alcoholoil transesterification products polyglyceryl fatty acids
  • the therapeutic agents may be formulated for co-administration in a single dosage form, or they can be administered separately in different dosage forms. When administered separately, administration may be by the same or a different route of administration for each of the components of the combination therapy.
  • a PIKfyve inhibitor may be administered at the same time or at a different time, the context of the combination therapy with the antioxidant and/ or the glutamatergic agent.
  • a PIKfyve inhibitor, and/or the antioxidant, and/or the glutamatergic agent are administered in a single dosage form, or in separate dosage forms.
  • a PIKfyve inhibitor is any described herein or known in the art (e.g., apilimod, YM-201636, MOMIPP, MF4, APY0201, AS2677131 , AS2795440, VRG101, Vacuolin-1 , WX8, NDF, WWL, XB6, VRG50635, VRG50648, XBA, and analogs or salts thereof,)
  • the term “therapeutically effective amount” refers to an amount sufficient to treat, ameliorate a symptom of, reduce the severity of, or reduce the duration of the neurological disease or disorder, or to enhance or improve the therapeutic effect of another therapy.
  • the precise effective amount for a subject may depend upon the subject’s body weight, size, and health; the nature and extent of the condition; and the therapeutic or combination of therapeutics selected for administration.
  • the combination therapy methods described herein provide a synergistic response.
  • the term “synergistic” refers to the efficacy of the combination being more than the additive effects of either single therapy alone.
  • the synergistic effect of combination therapy may permit the use of lower dosages and/or less frequent administration of at least one agent in the combination compared to its dose and/or frequency outside of the combination.
  • the synergistic effect may also be manifested in the avoidance or reduction of adverse or unwanted side effects associated with the use of either therapy in the combination alone.
  • Neurodegenerative diseases (ND) of the central nervous system (CNS) cause progressive loss of neuronal structure and function and their impact in our societies is increasing.
  • ND central nervous system
  • ALS amyotrophic lateral sclerosis
  • FDD frontotemporal dementia
  • PD Parkinson Disease
  • HD Huntington disease
  • ALS Amyotrophic lateral sclerosis
  • ALS often has a focal onset but subsequently spreads to different body regions, where failure of respiratory muscles typically limits survival to 2-5 years after disease onset (Goutman 2022).
  • the clinical presentation of ALS typically consists of adult-onset focal muscle weakness and wasting, which tends to spread with disease progression. The weakness most commonly starts in the limb muscles, more often in distal muscles than in proximal muscles.
  • FTD is characterized by the degeneration of frontal and anterior temporal lobes and presents clinically by behavioral changes, impairment of executive functioning and/or language impairment.
  • ALS and FTD are now considered to be two ends of a spectrum due to the overlap in molecular mechanisms underlying both neurodegenerative disorders (Goutman 2022).
  • Apilimod dimesylate capsule is a first-in-class inhibitor of phosphatidylinosito 1-3 -phosphate 5 -kinase (PIKfyve) with an IC50 of 14nM and Kd of approximately 75 pM (range 69-81 pM) (Gayle 2017; Cai 2013).
  • PIKfyve phosphatidylinosito 1-3 -phosphate 5 -kinase
  • PIKfyve is a 240-kDa endosomal phosphatidylinositol (PI) lipid kinase that catalyzes the phosphorylation of phosphatidylinositol 3-phosphate (PI(3)P) to phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2).
  • PIKfyve normally synthesizes PI(3,5)P2 on endosomes and lysosomes, supporting lysosomal homeostasis (Rutherford 2006).
  • TFEB transcription factor EB
  • ALS amyotrophic lateral sclerosis
  • iMN patient-derived induced motor neurons
  • DPR dipeptide repeat
  • apilimod also potently inhibits the activation of microglial cells in vitro as demonstrated by the inhibition of the release of IL-12/IL-23 p40 production in response to toll-like receptor (TLR) activation. Coupled to its ability to inhibit inflammatory cytokine induction, apilimod therefore holds promise as a therapeutic for ALS.
  • Edaravone is a member of the substitute 2-pyrazolin-5-one class, has the chemical name 3-methyl-l-phenyl-2-pyrazolin-5-one.
  • Goutman 2022 the antioxidant properties
  • Edaravone protects neurons in the brain and spinal cord by eliminating the reactive oxygen species (ROS) such as hydroxyl radical, peroxyl radical, hydrogen peroxide, peroxynitrite, and many others that quicken and exacerbate the progression of neuronal degeneration, thus protecting from neurologic damage that leads to the death of motor neurons (Goutman 2022).
  • ROS reactive oxygen species
  • Edaravone also has anti-inflammatory properties against activated microglial cells which may contribute to its activity. Both oral and IV doses of Edaravone have been approved by the FDA.
  • iPSC Neuron Differentiation Peripheral blood mononuclear cell (PBMC)-derived iPSC lines from ALS patients were obtained from the Cedars-Sinai Answer ALS repository.
  • PBMC Peripheral blood mononuclear cell
  • iPSCs were generated using episomal plasmid-based reprogramming methods.
  • iPSCs were maintained in MTeSR medium according to standard Cedars Sinai protocols and differentiated into spinal neurons according to the direct induced motor neurons (diMNs) protocol, which generates a mixed population consisting of 20%-30% islet- 1 positive motor neurons (Coyne 2020). Briefly, iPSC colonies were maintained on Matrigel coated 10 cm dishes for three weeks before passaging for differentiation.
  • diMNs direct induced motor neurons
  • stage 1 media consisting of 47.5% IMDM (GIBCO), 47.5% F12 (GIBCO), 1% NEAA (GIBCO), 1% Pen/Strep (GIBCO), 2% B27 (GIBCO), 1% N2 (GIBCO), 0.2 mM LDN193189 (Stemgent), 10 mM SB431542 (StemCell Technologies), and 3 mM CHIR99021 (Sigma Aldrich) was added and exchanged daily until day 6. On day 6 of differentiation, cells were incubated in StemPro Accutase (GIBCO) for 5 minutes at 37°C.
  • Cells were collected from plates and centrifuged at 500 x g for 1.5 minutes. Cells were plated at 1 x 10 6 cells per well of a 6 well plate or 3 x 10 6 cells per T25 flask in stage 2 media consisting of 47.5% IMDM (GIBCO), 47.5% F12 (GIBCO), 1% NEAA (GIBCO), 1% Penicillin/Streptomycin (GIBCO), 2% B27 (GIBCO), 1% N2 (GIBCO), 0.2 mM LDN193189 (Stemgent), 10 mM SB431542 (StemCell Technologies), 3 mM CHIR99021 (Sigma Aldrich), 0.1 mM all-trans Retinoic Acid (RA) (Sigma Aldrich), and 1 mM Smoothened agonist (SAG) (Cayman Chemicals).
  • TDP-43 whole cell imaging
  • cells were trypsinized and plated in 24 well optical bottom plates (Cellvis) at a density of 250,000 cells per well. Stage 3 media was exchanged every 3 days for the duration of the experiment. All cells were maintained at 37°C with 5% CO2 until [0119]
  • iMNs were plated in 24 well optical bottom plates (Cellvis) at a density of 250,000 neurons per well. Neurons were rinsed with IX PBS and fed with fresh stage 3 media daily to remove dead cells and debris.
  • iMNs derived from TDP-43 mutated iPSC were cultured in medium without growth factors (BDNF, GDNF and db-cAMP) for the last 48 hours.
  • iMNs derived from c9orf72, SOD1 and sporadic iPSCs were then treated with glutamate by replacing media with artificial cerebrospinal fluid (ACSF) (Tocris) containing 0-, or 10-mM glutamate (Sigma Aldrich and incubated at 37°C with 5% CO2 for 4 hours. After 3.5 hours (or after the 48h growth factor depletion for the TDP-43 iMNs), one drop of NucBlueTM Live ReadyProbes (Thermo Fisher Scientific) and 1 mM propidium iodide (Thermo Fisher Scientific) and returned to the incubator for 30 minutes.
  • ACSF cerebrospinal fluid
  • iMNs were imaged with a Zeiss Axiovert microscope equipped with a Axiocam 503 videocamera, 2 images were taken (magnification 4x) and analyzed per well. PI and DAPIspots were counted using the FIJI software. Cell viability for each iMNs line and following each treatment is reported in Tables 1-3 (see also FIGs. 1-12).
  • the combination of apilimod and edaravone provided an increase in viability of 39.22% with respect to glutamate only treated cells while apilimod and edaravone alone provide an increase of 7.5% and 7.65%, respectively leading to a predicted additive response of 15.5% (Table 4).
  • the combination of apilimod with edaravone showed a synergistic effect (i.e., more than the sum of the improved viability with each compound alone) in any line tested.
  • the combination of YM201636 with edaravone showed a synergistic effect (i.e., more than the sum of the improved viability with each compound alone) in any line tested.

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Abstract

Provided herein are methods of treating a neurological disease or disorder (e.g., ALS) comprising administering PIKfyve inhibitor (e.g., any PIKfyve inhibitors known or described herein, including but not limited to APY0201, YM201636, or a pharmaceutically acceptable salt thereof), or a pharmaceutically acceptable salt thereof and an antioxidant (e.g., edaravone). In some embodiments, further therapeutic agents (e.g., glutamatergic agents) are administered to the subject.

Description

PIKFYVE INHIBITOR COMBINATION THERAPY
CROSS-REFERENCE TO RELATED APPLICATOINS
[0001] This application claims the benefit of priority under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63/428,365 filed November 28, 2022. The disclosure of the prior application is considered part of and is herein incorporated by reference in the disclosure of this application in its entirety.
FIELD OF THE INVENTION
[0002] The invention relates to compositions and methods comprising a PIKfyve inhibitor for use in combination with other therapeutic agents in the treatment of neurological disease and disorders.
BACKGROUND OF INVENTION
[0003] Neurodegenerative diseases and disorders cause progressive loss of neuronal function, and the societal burden of neurodegenerative diseases is increasing. Neurodegenerative diseases may include amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Parkinson Disease (PD), Alzheimer’s Disease and Huntington disease (HD).
[0004] Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder affecting primarily the motor system characterized by muscle weakness and wasting. However, extra-motor manifestations are increasingly recognized. ALS often has a focal onset and subsequently spreads to different body regions, where failure of respiratory muscles typically limits survival to 2-5 years after disease onset. Apilimod, also referred to as STA-5326, hereinafter “apilimod”, is recognized as a potent inhibitor of IL- 12 and IL-23. IL- 12 and IL-23 are inflammatory cytokines normally produced by immune cells, such as B-cells and macrophages, in response to antigenic stimulation. Autoimmune disorders and other disorders characterized by chronic inflammation are characterized in part by inappropriate production of these cytokines. In immune cells, the selective inhibition of IL-12/IL-23 transcription by apilimod was recently shown to be mediated by apilimod’s direct binding to phosphatidylinositol-3- phosphate 5 -kinase (PIKfyve). PIKfyve plays a role in Toll-like receptor signaling, which is important in innate immunity. The PIKfyve inhibitor APY0201 also selectively inhibits IL- 12/IL-23 production in vitro and ex vivo in murine plasma. Additionally, the PIKfyve inhibitor YM201636 with a morpholino-pyrimidine (diazine) core which and was originally identified in a screen of PI3K class IA inhibitors was reported to inhibit the proliferation of cancer cells in vitro as well as the growth of transplanted tumors in mice. Subsequently, these three compounds were all shown to be highly selective inhibitors of PIKfyve despite very different chemical scaffolds and likely off-target activities, suggesting that they can be used together to identify PIKfyve dependent activities. PIKfyve inhibitors, including apilimod, have been shown to increase the survival of motor neurons.
SUMMARY OF INVENTION
[0005] The present disclosure, in some aspects, provides compositions and methods relating to the use of a PIKfyve inhibitor (e.g., any PIKfyve inhibitors known or described herein,) for treating neurological diseases and disorders, particularly in combination an antioxidant (e.g., edaravone). As demonstrated herein, the combination of a PIKfyve inhibitor (e.g., any PIKfyve inhibitors known or described herein,) and an antioxidant (e.g., edaravone) achieved synergistic effects in improving ALS motor neuron viability. In some embodiments, a method described herein further comprises using additional therapeutic agents for treating neurological diseases (e.g., ALS), including glutamatergic agents (e.g., riluzole). In some embodiments, the PIKfyve inhibitor used in a method described in is not apilimod or pharmaceutically acceptable salt thereof.
[0006] Some aspects of the present disclosure provide methods of treating a neurological disease or disorder in a subject in need thereof, the method comprising administering to the subject a PIKfyve inhibitor and an antioxidant, wherein the PIKfyve inhibitor is not apilimod or a pharmaceutically acceptable salt thereof. In some embodiments, the PIKfyve inhibitor or a pharmaceutically acceptable salt thereof and the antioxidant are in one composition. In some embodiments, the PIKfyve inhibitor or a pharmaceutically acceptable salt thereof and the antioxidant are in different compositions.
[0007] Some aspects of the present disclosure provide methods of treating a neurological disease or disorder in a subject in need thereof, the method comprising administering to the subject the antioxidant, wherein the subject is receiving or has received treatment with a PIKfyve inhibitor or a pharmaceutically acceptable salt thereof, wherein the PIKfyve inhibitor is not apilimod or a pharmaceutically acceptable salt thereof.
[0008] Some aspects of the present disclosure provide methods of treating a neurological disease or disorder in a subject in need thereof, the method comprising administering to the subject a PIKfyve inhibitor or a pharmaceutically acceptable salt thereof, wherein the subject is receiving or has received treatment with an antioxidant, wherein the PIKfyve inhibitor is not apilimod or a pharmaceutically acceptable salt thereof.
[0009] In some embodiments, the PIKfyve inhibitor is YM-201636, MOMIPP, MF4, APY0201, AS2677131, AS2795440, VRG101, Vacuolin-1, WX8, NDF, WWL, XB6, AS-201, AS-202, VRG50635, VRG50648, XBA, or analogs or salts thereof. In some embodiments, the PIKfyve inhibitor is YM-201636, APY0201, or a pharmaceutically acceptable salt thereof.
[0010] In some embodiments, the antioxidant is ascorbic acid, cysteine, cysteamine, edaravone, glutathione and bilirubin, amifostine (WR-2721), vitamin A, vitamin C, vitamin E, and flavonoids such as Indian holy basil (Ocimum sanctum), orientin or vicenin. In some embodiments, the antioxidant is administered orally. In some embodiments, the antioxidant is edaravone. In some embodiments, edaravone is administered at a daily dose of 60 mg for 14 days, followed by 14 days of no administration, further followed by a daily dose of 60 mg for 10 days in a 14-day period, further followed by 14 days of no administration.
[0011] In some embodiments, the antioxidant is administered intravenously. In some embodiments, edaravone is administered at a daily dose of 105 mg. In some embodiments, edaravone is administered orally.
[0012] In some embodiments, the PIKfyve inhibitor is administered orally. In some embodiments, the composition is administered orally.
[0013] In some embodiments, the method further comprises administering to the subject a glutamatergic agent. In some embodiments, the glutamatergic agent is riluzole. In some embodiments, riluzole is administered to the subject twice daily and 50 mg of riluzole is administered in each administration. In some embodiments, the glutamatergic agent is administered orally.
[0014] In some embodiments, the subject is not receiving or has not received treatment with a glutamatergic agent. In some embodiments, the glutamatergic agent is riluzole.
[0015] In some embodiments, the neurological disease of disorder is selected from Alzheimer's disease, amyotrophic lateral sclerosis (ALS), attention deficit hyperactivity disorder, autism, cerebellar ataxia, Charcot-Marie-Tooth disease, Creutzfeldt- Jakob disease, dementia, epilepsy, Friedreich's ataxia, Huntington's disease, multiple sclerosis, obsessive compulsive disorder (OCD), Parkinson's disease, Rett syndrome, senile chorea, spinal ataxia, spinal cord injury, supranuclear palsy, traumatic brain injury. In some embodiments, the neurological disease or disorder is dementia. In some embodiments, the dementia is selected from AIDS dementia complex (ADC), dementia associated with Alzheimer’s disease (AD), dementia pugilistica, diffuse Lewy body disease, frontotemporal dementia, mixed dementia, senile dementia of Lewy body type, and vascular dementia. In some embodiments, the neurological disease or disorder is amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD).
[0016] In some embodiments, the subject is in need of treatment is one having repeat expansions of the C9ORF72 gene. In some embodiments, the subject in need of treatment is one having a mutation in the SOD1 gene. In some embodiments, the subject in need of treatment is one having accumulation of TDP-34 aggregates. In some embodiments, the subject in need of treatment is one having a mutation in the TDP-43 gene. In some embodiments, the subject is human. In some embodiments, the subject is not receiving or has not received treatment with AMX0035, PBA, TUDCA, methylcobalamin, or any combinations thereof.
[0017] Further provided herein are uses of a PIKfyve inhibitor and an antioxidant in a method of treating a neurological disease or disorder in a subject in need thereof, wherein the PIKfyve inhibitor is not apilimod or a pharmaceutically acceptable salt thereof.
[0018] Further provided herein are uses of a PIKfyve inhibitor in a method of treating a neurological disease or disorder in a subject in need thereof, wherein the subject is receiving or has received treatment with an antioxidant, wherein the PIKfyve inhibitor is not apilimod or a pharmaceutically acceptable salt thereof.
[0019] Further provided herein, are uses of an antioxidant in a method of treating a neurological disease or disorder in a subject in need thereof, wherein the subject is receiving or has received treatment with a PIKfyve inhibitor, and wherein the PIKfyve inhibitor is not apilimod or a pharmaceutically acceptable salt thereof.
[0020] In some embodiments, the PIKfyve inhibitor is YM-201636, MOMIPP, MF4, APY0201, AS2677131, AS2795440, VRG101, Vacuolin-1, WX8, NDF, WWL, XB6, AS-201, AS-202, VRG50635 , VRG50648, XBA, or analogs or salts thereof. In some embodiments, the PIKfyve inhibitor is YM-201636, APY0201 or a pharmaceutically acceptable salt thereof. [0021] In some embodiments, the antioxidant is ascorbic acid, cysteine, cysteamine, edaravone, glutathione and bilirubin, amifostine (WR-2721), vitamin A, vitamin C, vitamin E, and flavonoids such as Indian holy basil (Ocimum sanctum), orientin or vicenin. In some embodiments, the antioxidant is edaravone. In some embodiments, the subject is not receiving or has not received treatment with AMX0035, PBA, TUDCA, methylcobalamin, or any combinations thereof. BRIEF DESCRIPTION OF DRAWINGS
[0022] In each figure, the control bar indicates untreated cells while the DMSO bar indicates cells treated with 0.01% DMSO which is the buffer in which all the compounds have been dissolved.
[0023] FIGURE 1 is a graph demonstrating the ability of Edaravone (1 pM) and apilimod (6.25 nM) in combination to increase cell viability synergistically after glutamate exposure in iPSC- derived motoneurons from a C9orf72-ALS patient. *** denotes p<0.001; * denotes significance ofp<0.05.
[0024] FIGURE 2 is a graph demonstrating the ability of Edaravone (1 pM) and apilimod (6.25 nM) in combination to increase cell viability synergistically after glutamate exposure in iPSC- derived motoneurons from a SOD1-ALS patient. ** denotes significance of p<0.01; * denotes significance ofp<0.05.
[0025] FIGURE 3 is a graph demonstrating the ability of Edaravone (1 pM) and apilimod (6.25 nM) in combination to increase cell viability synergistically after growth factors deprivation (- GF) in iPSC-derived motoneurons from a TDP-43-ALS patient. *** denotes p<0.001; ** denotes significance ofp<0.01.
[0026] FIGURE 4 is a graph demonstrating the ability of Edaravone (1 pM) and apilimod (6.25 nM) in combination to increase cell viability synergistically after glutamate exposure in iPSC- derived motoneurons from a Sporadic-ALS patient. *** denotes significance of p<0.001; * denotes significance ofp<0.05.
[0027] FIGURE 5 is a graph demonstrating the ability of Edaravone (IpM) and YM201636 (12.5 nM) in combination to increase cell viability synergistically after glutamate exposure in iPSC-derived motoneurons from a C9orf72-ALS patient. *** denotes significance of p<0.001; ** denotes significance ofp<0.01.
[0028] FIGURE 6 is a graph demonstrating the ability of Edaravone (IpM) and YM201636 (12.5 nM) in combination to increase cell viability synergistically after glutamate exposure in iPSC-derived motoneurons from a SOD1-ALS patient. *** denotes significance of p<0.001.
[0029] FIGURE 7 is a graph demonstrating the ability of Edaravone (IpM) and YM201636 (12.5 nM) in combination to increase cell viability synergistically after growth factors deprivation (-GF) in iPSC-derived motoneurons from a TDP-43-ALS patient. *** denotes significance ofp<0.001; * denotes significance of p<0.05.
[0030] FIGURE 8 is a graph demonstrating the ability of Edaravone (IpM) and YM201636 (12.5 nM) in combination to increase cell viability synergistically after glutamate exposure in iPSC-derived motoneurons from a Sporadic-ALS patient. **** denotes significance of p<0.0001; *** denotes significance ofp<0.001; * denotes significance ofp<0.05.
[0031] FIGURE 9 is a graph demonstrating the ability of Edaravone (I pM) and APY0201 (6.25 nM) in combination to increase cell viability synergistically after glutamate exposure in iPSC-derived motoneurons from a C9orf72-ALS patient. **** denotes p<0.0001; *** denotes significance ofp<0.001** denotes significance ofp<0.01.
[0032] FIGURE 10 is a graph demonstrating the ability of Edaravone (I pM) and APY0201 (6.25 nM) in combination to increase cell viability synergistically after glutamate exposure in iPSC-derived motoneurons from a SOD1-ALS patient. **** denotes p<0.0001; ** denotes significance ofp<0.01.
[0033] FIGURE 11 is a graph demonstrating the ability of Edaravone (I pM) and APY0201 (6.25 nM) in combination to increase cell viability synergistically after growth factors deprivation (-GF) in iPSC-derived motoneurons from a TDP-43-ALS patient. ** denotes significance ofp<0.01; * denotes significance of p<0.05.
[0034] FIGURE 12 is a graph demonstrating the ability of Edaravone (IpM) and APY0201 (6.25 nM) in combination to increase cell viability synergistically after glutamate exposure in iPSC-derived motoneurons from a Sporadic-ALS patient. ** denotes significance of p<0.01; * denotes p<0.05.
DEFINITIONS
[0035] As described herein, the term “pharmaceutically acceptable salt,” may be a salt formed from, for example, an acid and a basic group of a compound described herein (e.g. , any PIKfyve inhibitors known or described herein). For example, salts may be selected from, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, mesylate, dimesylate, lactate (e.g., D-lactate or L- lactate), salicylate, citrate, tartrate (e.g., L-tartrate), malonate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, besylate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, pamoate (e.g, l,l’-methylene-bis-(2-hydroxy-3- naphthoate)), sulfuric, maleic, L-ascorbic, phosphoric, malonic, L-tartaric, glycolic, L-malic, L-sapartic, L-gflutamic, 1 -hydroxy-2 -naphthoic, and pyruvic salts. In some embodiments, the salt of apilimod comprises methanesulfonate. The term “pharmaceutically acceptable salt” may also refer to a salt prepared from a compound described herein (e.g., any PIKfyve inhibitors known or described herein), having an acidic functional group, such as a carboxylic acid functional group, and a pharmaceutically acceptable inorganic or organic base. The term “pharmaceutically acceptable salt” may also refer to a bis-salt, e.g., an apilimod dimesylate salt.
[0036] Suitable bases include, but are not limited to, hydroxides of alkali metals such as sodium, potassium, and lithium; hydroxides of alkaline earth metal such as calcium and magnesium; hydroxides of other metals, such as aluminum and zinc; ammonia, and organic amines, such as unsubstituted or hydroxy-substituted mono-, di-, or trialkylamines; dicyclohexylamine; tributyl amine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, bis-, or tris-(2 -hydroxy-lower alkyl amines), such as mono-, bis-, or tris- (2 -hydroxy ethyl)amine, 2-hydroxy-tert-butyl amine, or tris-(hydroxymethyl)methylamine, N, N,-di-lower alkyl-N-(hydroxy lower alkyl)-amines, such as N,N-dimethyl-N-(2- hydroxyethyl)amine, or tri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; and amino acids such as arginine, lysine, and the like. The term “pharmaceutically acceptable salt” may also refer to a salt prepared from any one of the PIKfyve inhibitors described herein (e.g., YM- 201636, APY0201) or known in the art, having a basic functional group, such as an amino functional group, and a pharmaceutically acceptable inorganic or organic acid. Suitable acids include hydrogen sulfate, citric acid, acetic acid, oxalic acid, hydrochloric acid (HC1), hydrogen bromide (HBr), hydrogen iodide (HI), nitric acid, hydrogen bisulfide, phosphoric acid, lactic acid, salicylic acid, tartaric acid, bitartratic acid, ascorbic acid, succinic acid, maleic acid, besylic acid, fumaric acid, gluconic acid, glucuronic acid, formic acid, benzoic acid, glutamic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid.
[0037] The salts of a compound (e.g., any PIKfyve inhibitors known or described herein) may be synthesized from the parent compound by conventional chemical methods. Generally, such salts can be prepared by reacting the parent compound with the appropriate acid in water or in an organic solvent, or in a mixture of the two.
[0038] One salt form of a compound described herein (e.g., any PIKfyve inhibitors known or described herein) may be converted to the free base and optionally to another salt form by methods well known to the skilled person. For example, the free base may be formed by passing the salt solution through a column containing an amine stationary phase (e.g., a Strata-NH2 column). Alternatively, a solution of the salt in water may be treated with sodium bicarbonate and a base (e.g., hydroxide or carbonate bases) to decompose the salt. The free base may then be combined with another acid using routine methods.
[0039] As described herein, the term “polymorph” may refer to solid crystalline forms of a compound of the present disclosure (e.g., any PIKfyve inhibitors known or described herein) or complex thereof. Different polymorphs of the same compound may exhibit different physical, chemical and/or spectroscopic properties. Different physical properties include, but are not limited to, stability (e.g., to heat or light), compressibility and density (important in formulation and product manufacturing), and dissolution rates (which may affect bioavailability). Differences in stability may result from changes in chemical reactivity (e.g., differential oxidation, such that a dosage form discolors more rapidly when comprised of one polymorph than when comprised of another polymorph) or mechanical characteristics (e.g., tablets crumble on storage as a kinetically favored polymorph converts to thermodynamically more stable polymorph) or both (e.g., tablets of one polymorph are more susceptible to breakdown at high humidity). Different physical properties of polymorphs may affect their processing. For example, one polymorph may be more likely to form solvates or may be more difficult to filter or wash free of impurities than another due to, for example, the shape or size distribution of particles of it.
[0040] As described herein, the term “hydrate” may describe a compound of the present disclosure (e.g., any PIKfyve inhibitors known or described herein) or a salt thereof, which further includes a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces.
[0041] As described herein, the term “clathrate” may refer to a compound of the present disclosure (e.g., any PIKfyve inhibitors known or described herein) or a salt thereof in the form of a crystal lattice that may contain spaces (e.g., channels) that may have a guest molecule (e.g., a solvent or water) trapped within.
[0042] As described herein, the term “prodrug” may refer to a derivative of a compound described herein (e.g., any PIKfyve inhibitors known or described herein) that may hydrolyze, oxidize, or otherwise react under biological conditions (in vitro or in vivo) to provide a compound of the disclosure. Prodrugs may only become active upon such reaction under biological conditions, or they may have activity in their unreacted forms. Examples of prodrugs contemplated in this disclosure include, but are not limited to, analogs or derivatives that may comprise biohydrolyzable moieties such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzable ureides, and biohydrolyzable phosphate analogues. Other examples of prodrugs may include, but are not limited to, derivatives of compounds of any one of the formulae disclosed herein that comprise -NO, -NO2, -ONO, or -ONO2 moieties.
[0043] Some of the compounds suitable for use in the methods described in this disclosure (e.g, any PIKfyve inhibitors known or described herein) may have one or more double bonds, or one or more asymmetric centers. Such compounds may occur as racemates, racemic mixtures, single enantiomers, individual diastereomers, diastereomeric mixtures, and cis- or trans- or E- or Z-double isomeric forms. All such isomeric forms of these compounds are expressly included in the present disclosure. The compounds of this disclosure (e.g., any PIKfyve inhibitors known or described herein) may also be represented in multiple tautomeric forms, in such instances, the disclosure expressly includes all tautomeric forms of the compounds described herein (e.g, there may be a rapid equilibrium of multiple structural forms of a compound), the disclosure expressly includes all such reaction products). All such isomeric forms of such compounds are expressly included in the present disclosure. All crystal forms of compounds described herein (e.g., any PIKfyve inhibitors known or described herein) are expressly included in the present disclosure.
[0044] As described herein, the term “solvate” or “pharmaceutically acceptable solvate,” may refer to a solvate formed from the association of one or more solvent molecules to one of the compounds disclosed herein (e.g., any PIKfyve inhibitors known or described herein). The term solvate may include hydrates (e.g., hemi -hydrate, mono-hydrate, dihydrate, trihydrate, tetrahydrate, and the like).
[0045] As described herein, the term “analog” may refer to a chemical compound that is structurally similar to another chemical compound but differs slightly in composition (e.g, as in the replacement of one atom by an atom of a different element or in the presence of a particular functional group, or the replacement of one functional group by another functional group). Thus, an analog may be a compound that is similar or comparable in function and appearance, but not in structure or origin to the reference compound. As used herein, the term “derivative” may refer to compounds that have a common core structure and are substituted with various groups as described herein.
DETAILED DESCRIPTION
[0046] Neurodegenerative diseases (ND) cause progressive loss of neuronal structure and function. Because of neuronal loss, patients with ND have decreased nervous system function, resulting in behavioral and physiological deficits. Common ND include, amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Parkinson’s disease (PD), Alzheimer’s disease (AD) and Huntington’s disease (HD). Worldwide, about 50 million people have ND, a figure that is projected to increase to 115 million people by 2050. There are no cures for ND and relatively few therapies exist. To date, the antioxidant, edaravone, is one of only three drugs approved for use in the treatment of ALS. The present disclosure, in some aspects, demonstrates that administration of a PIKfyve inhibitor and an antioxidant in (iPSC)-derived motoneurons from ALS patients has synergistic results in cell viability. The combination therapy of a PIKfyve inhibitor and an antioxidant (e.g., edaravone) provides unexpected benefits on neuronal survival that are greater than the application of either therapy individually. [0047] The present disclosure, in some aspects, provides compositions and methods related to the use of a PIKfyve inhibitor (e.g., any PIKfyve inhibitors known or described herein), in combination with an antioxidant (e.g., edaravone) for treating neurological diseases and disorders. In some embodiments, a method described herein comprises administering to a subject in need thereof a PIKfyve inhibitor (e.g., any PIKfyve inhibitors known or described herein), and an antioxidant (e.g., edaravone). In some embodiments, the PIKfyve inhibitor is not apilimod or a pharmaceutically acceptable salt thereof.
[0048] In some embodiments, a method described herein comprises administering a PIKfyve inhibitor (e.g., any PIKfyve inhibitors known or described herein), or a pharmaceutically active salt thereof, to a subject in need thereof, wherein the subject is receiving or has received treatment with an antioxidant (e.g., edaravone). In some embodiments a method described herein comprises administering an antioxidant (e.g., edaravone) to a subject in need thereof, wherein the subject is receiving or has received treatment with a PIKfyve inhibitor (e.g., any PIKfyve inhibitors known or described herein), or a pharmaceutically acceptable salt thereof. In some embodiments, the PIKfyve inhibitor is not apilimod or a pharmaceutically acceptable salt thereof.
[0049] As described herein, the term “PIKfyve inhibitor” may refer to a molecule that inhibits the expression and/or activity (e.g., kinase activity) of PIKfyve (phosphoinositide kinase, FYVE-type zinc finger containing). PIKfyve phosphoinositide kinase is an enzyme that has evolutionarily conserved lipid and protein kinase that has pleiotropic cellular functions. Phosphorylation of the phosphatidylinositol-3-phosphate (PI3P) by PIKfyve generates two phosphoinositide (PI) derivatives (1) phosphatidylinositol 3, 5 -bisphosphate [PtdIns(3,5)P2] or (2) phosphatidylinositol 5-phosphate (PtdIns5P). The two phosphoinositide (PI) derivatives may govern various cellular processes, including, but not limited to, cytoskeleton rearrangements, remodeling the actin cytoskeleton, intracellular membrane trafficking pathways, endocytosis, epidermal growth factor receptor (EGFR) signaling, translocation of the glucose transporter GLUT4 after insulin stimulation, regulation of synapse strength and cell proliferation. The FYVE finger domain of PIKfyve has been shown to play a vital role in localizing the protein to the cytosolic leaflet of endosomes through directly binding to membrane PtdIns3P and is thereby involved in multiple processes of endosome dynamics. It has also been demonstrated that, PIKfyve inhibition significantly increased the survival of ALS patient-derived motor neurons by converting PtdIns3P into PtdIns(3,5)P2, which enhanced the fusion of lysosomes with both endosomes and autophagosomes under cell stress. PIKfyve inhibition can also lead to activation of transcription factor TFEB, which drives the clearance of toxic protein aggregates in ALS patients. In some embodiments, a PIKfyve inhibitor that may be used in accordance with the methods described herein is a small molecule. Nonlimiting examples of small molecule PIKfyve inhibitors include: apilimod and analogs or salts thereof, YM-201636 (CAS Number 371942-69-7), MOMIPP (CAS Number 1363421-46-8), MF4, APY0201 (CAS Number 1232221-74-7), AS2677131 (CAS Number 2171502-44-4), AS2795440, VRG101 (by Verge Genomics), Vacuolin-1 (CAS Number 351986-85-1), WX8 (MLS000543798; PubChem CID 135510930), NDF (MLS000699212; PubChem CID 9629709), WWL (MLS000703078), XB6 (MLS001167897), VRG50635 (by Verge Genomics; prodrug of VRG50648), VRG50648 (by Verge Genomics; active form of VRG50635) and XBA (MLS001167909).
[0050] Other PIKfyve inhibitors that may be used in any one of the methods described herein include, without limitation, small molecule PIKfyve inhibitors described in WO18175906, W0202009971, WO19046316, WO21163727, WO21113633, WO17218815, WO22169882, WO21247841, WO21247859, WO21247862, WO19113523, WO20243457, WO21146192, WO21183439, WO18138106, WO22086993, US2021/0122752, US2020/0165246. US11352354, and WO21252895, the entire contents of each of which are incorporated herein by reference.
[0051] In some embodiments, a PIKfyve inhibitor used in accordance with the present disclosure is an RNAi agent or an antisense oligonucleotide (e.g., AS-201 or AS-202 by Acurastem) that targets an RNA (e.g., mRNA) encoded by the PIKfyve gene, e.g., as described in International Patent Application Publication No. WO2021155067, the entire contents of which and the sequences disclosed in the specification and the associated sequence listing are incorporated herein by reference.
[0052] In some embodiments, the PIKfyve inhibitor used in a method described herein is not apilimod or a pharmaceutically acceptable salt thereof.
[0053] The term “apilimod” refers to 2-[2-Pyridin-2-yl)-ethoxy]-4-N’-(3-methyl-benzilidene)- hydrazino]-6-(morpholin-4-yl)-pyrimidine (IUPAC name: (E)-4-(6-(2-(3-methylbenzylidene) hydrazinyl)-2-(2-(pyridin-2-yl)ethoxy)pyrimidin-4-yl)morpholine), represented by Formula I:
Figure imgf000014_0001
or any pharmaceutically acceptable salt, solvate, clathrate, hydrate, polymorph, metabolite, prodrug, analog, or derivative thereof. The CAS number of apilimod (Formula I) is 541550- 19-0 (CAS name: Benzaldehyde, 3-methyl-, 2-[6-(4-morpholinyl)-2-[2-(2-pyridinyl)ethoxy]- 4-pyrimidinyl]hydrazone). In some embodiments, apilimod used in accordance with a method described herein is apilimod dimesylate. Apilimod may be prepared, for example, according to the methods described in U.S. Pat. Nos. 7,923,557, and 7,863,270, and WO 2006/128129, the entire contents of each of which are incorporated herein by reference.
[0054] The term “APY0201” refers to 2-[7-(4-morpholinyl)-2-(4-pyridinyl)pyrazolo[l,5- a]pyrimidin-5-yl]hydrazone, 3-methyl-benzaldehyde, represented by Formula II:
Figure imgf000014_0002
or any pharmaceutically acceptable salt, solvate, clathrate, hydrate, polymorph, metabolite, prodrug, analog, or derivative thereof. APY0201 is a PIKfyve inhibitor that inhibits IL- 12/23 production in vitro and ex vivo in murine plasma (e.g., as described in Hayakawa et al., Bioorg Med Chem. 2014 Jun 1;22(11):3021-9 and Drewry et al., J Med. Chem. 2022 Oct 13;65(19): 12860-12882, incorporated herein by reference).
[0055] The term “YM201636” refers to a compound with CAS number of 371942-69-7 and a structure represented by Formula III:
Figure imgf000015_0001
or any pharmaceutically acceptable salt, solvate, clathrate, hydrate, polymorph, metabolite, prodrug, analog, or derivative thereof. YM201636 is a PIKfyve inhibitor with a morpholinopyrimidine (diazine) core and has been identified in a screen of PI3K class IA inhibitors. YM201636 has been reported to inhibit the proliferation of cancer cells in vitro as well as the growth of transplanted tumors in mice (Ikomonov et al., Toxicology and Applied Pharmacology, Volume 383, 15 November 2019, 114771 and Drewry et al., J Med. Chem. 2022 Oct 13;65(19): 12860- 12882, incorporated herein by reference).
[0056] The term “MOMIPP” refers to a compound with CAS number of 1363421-46-8 and a structure represented by Formula IV:
Figure imgf000015_0002
or any pharmaceutically acceptable salt, solvate, clathrate, hydrate, polymorph, metabolite, prodrug, analog, or derivative thereof.
[0057] The term “MF4” refers to a compound with a structure represented by Formula (V):
Figure imgf000016_0001
(V), wherein R is O, or any pharmaceutically acceptable salt, solvate, clathrate, hydrate, polymorph, metabolite, prodrug, analog, or derivative thereof. MF4 is described in, e.g., Lartigue et al., Traffic. 2009;10(7):883-893, incorporated herein by reference.
[0058] The term “AS2677131” refers to a compound with CAS number of 2171502-44-4 and a structure represented by Formula (VI):
Figure imgf000016_0002
or any pharmaceutically acceptable salt, solvate, clathrate, hydrate, polymorph, metabolite, prodrug, analog, or derivative thereof. AS2677131 has been reported to inhibit the production of IL-12p40, IL-6, and IL-1|3, potent pro-inflammatory cytokines in vitro, and block the development of arthritis in rats (Terajima M, et al. Eur J Pharmacol. 2016 Jun 5;780:93- 105, incorporated herein by reference).
[0059] The term “AS2795440” refers to 2,3-Dimethyl-lH-pyrrolo[3,2-b]pyridine-5- carboxylic acid (l"-isopropyl-6-methyl-l",2",3",4",5",6"-hexahydro-[3,3';6',4"]terpyridin-5- yl)-amide, represented by Formula (VII):
Figure imgf000016_0003
or any pharmaceutically acceptable salt, solvate, clathrate, hydrate, polymorph, metabolite, prodrug, analog, or derivative thereof. AS2795440 has been reported to inhibit the production of IL-12p40, IL-6, and IL- 1 , potent pro-inflammatory cytokines in vitro, and block the development of arthritis in rats (Terajima M, et al. Eur J Pharmacol. 2016 Jun 5;780:93- 105, incorporated herein by reference).
[0060] The term “Vacuolin-1” refers to a compound with CAS number of 351986-85-1 and a structure represented by Formula (VIII):
Figure imgf000017_0001
or any pharmaceutically acceptable salt, solvate, clathrate, hydrate, polymorph, metabolite, prodrug, analog, or derivative thereof. Vacuolin-1 has been reported to inhibit infection by chimeric vesicular stomatitis virus (VSV) that contains an envelope protein for either Zaire ebolavirus (VSV-ZEBOV) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2) (VSVSARS-CoV-2) in vitro ( Kang Y, et al. PNAS. 2020 Aug 25;117(34):20803-20813„ incorporated herein by reference).
[0061] The term “WX8” (MLS000543798; PubChem CID 135510930) refers to lH-indole-3- carbaldehyde [4-anilino-6-(4-morpholinyl)-l,3,5-triazin-2-yl]hydrazine, a compound represented by Formula (IX):
Figure imgf000017_0002
or any pharmaceutically acceptable salt, solvate, clathrate, hydrate, polymorph, metabolite, prodrug, analog, or derivative thereof. WX8 has been reported to kill autophagy-dependent cancer cells with no effect on non-malignant human cells (Sharma G, et al. Autophagy. 2019;
15(10): 1694-1718, incorporated herein by reference). [0062] The term “NDF” (MLS000699212; PubChem CID 9629709) refers to 3- methylbenzaldehyde (2,6-dimorpholin-4-ylpyrimidin-4-yl)hydrazine, represented by Formula (X):
Figure imgf000018_0001
or any pharmaceutically acceptable salt, solvate, clathrate, hydrate, polymorph, metabolite, prodrug, analog, or derivative thereof. NDF is described in Sharma G, et al. Autophagy. 2019;
15(10): 1694-1718, incorporated herein by reference.
[0063] The term “WWL” (MLS000703078) refers to benzaldehyde [2,6-di(4-morpholinyl)-4- pyrimidinyl]hydrazone, represented by Formula (XI):
Figure imgf000018_0002
or any pharmaceutically acceptable salt, solvate, clathrate, hydrate, polymorph, metabolite, prodrug, analog, or derivative thereof. WWL is described in Sharma G, et al. Autophagy. 2019; 15(10): 1694-1718, incorporated herein by reference.
[0064] The term “XBA” (MLS001167909) refers to N-(3-chloro-4-fluorophenyl)-4,6- dimorpholino-l,3,5-triazin-2-amine hydrochloride, represented by Formula (XII):
Figure imgf000018_0003
or any pharmaceutically acceptable salt, solvate, clathrate, hydrate, polymorph, metabolite, prodrug, analog, or derivative thereof. XBA is described in Sharma G, et al. Autophagy. 2019; 15(10): 1694-1718, incorporated herein by reference.
[0065] The term “XB6” (MLS001167897), refers to N-(4-ethylphenyl)- 4,6-dimorpholino- l,3,5-triazin-2-amine hydrochloride, represented by Formula (XIII):
Figure imgf000019_0001
or any pharmaceutically acceptable salt, solvate, clathrate, hydrate, polymorph, metabolite, prodrug, analog, or derivative thereof. XBA is described in Sharma G, et al. Autophagy. 2019; 15(10): 1694-1718, incorporated herein by reference.
[0066] According to the present disclosure, a PIKfyve inhibitor or a pharmaceutically acceptable salt thereof is used in combination with an antioxidant (e.g., edaravone) for treating neurological diseases and disorders. In some embodiments, a PIKfyve inhibitor is any described herein or known in the art (e.g., apilimod, YM-201636, MOMIPP, MF4, APY0201, AS2677131 , AS2795440, VRG101, Vacuolin-1 , WX8, NDF, WWL, XB6, VRG50635, VRG50648, XBA, and analogs or salts thereof,)
[0067] As described herein, an “antioxidant” may refer to substances that may prevent or slow damage to cells caused by free radicals, and unstable molecules that the body produces as a reaction to environmental and other pressures. The terms “antioxidant” and “free-radical scavengers” may be used interchangeably. In some embodiments, the sources of antioxidants may be natural or artificial. Antioxidants may include, but are not limited to, ascorbic acid, cysteine, cysteamine, edaravone, glutathione and bilirubin, amifostine (WR-2721), vitamin A, vitamin C, vitamin E, and flavonoids such as Indian holy basil (Ocimum sanctum), orientin and vicenin. In a preferred embodiment, the antioxidant is edaravone (Radicava™, Radicut™). [0068] In some embodiments, in any one of the methods described herein, about 25-80 (e.g., 25-80, 25-75, 25-60, 25-50, 25-40, 25-30, 30-80, 30-75, 30-60, 30-50, 30-40, 40-80, 40-75, 40- 60, 40-50, 50-80, 50-75, 50-60, 60-80, 60-75, or 75-80) milligrams of edaravone is administered to the subject. In some embodiments, in any one of the methods described herein, about 25, 30, 40, 50, 60, 75, or 80 milligrams of edaravone administered to the subject. In some embodiments, in any one of the methods described herein, about 60 milligrams of edaravone is administered to the subject. In some embodiments, edaravone is administered intravenously to the subject.
[0069] In some embodiments, in any one of the methods described herein, about 75-125 (e.g., 75-125, 75-115, 75-105, 75-95, 75-85, 85-125, 85-115, 85-105, 85-95, 95-125, 95-115, 95- 105, 105-125, 105-115, 105-125, 105-115, or 115-125) milligrams of edaravone is administered to the subject. In some embodiments, in any one of the methods described herein, about 75, 85, 95, 105, 115, or 125 milligrams of edaravone administered to the subject. In some embodiments, in any one of the methods described herein, about 105 milligrams of edaravone is administered to the subject. In some embodiments, edaravone is administered orally to the subject.
[0070] In some embodiments, in any one of the methods described herein, edaravone is administered to the subject 1-3 (e.g., 1-3, 1-2, 2-3) times daily. In some embodiments, in any one of the methods described herein, edaravone is administered to the subject 1, 2, 3 times daily. In some embodiments, in any one of the methods described herein, wherein edaravone is administered to the subject daily.
[0071] In some embodiments, in any one of the methods described herein, edaravone is administered to the subject intermittently over a period of time. In some embodiments, in anyone of the methods described herein, the period of time may be about 1-12 (e.g., 1-12, 1-10, 1-8, 1-6, 1-4, 1-2, 2-12, 2-10, 2-8, 2-6, 2-4, 4-12, 4-10, 4-8, 4-6, 6-12, 6-10, 6-8, 8-12, 8-10, 10-12) weeks. In some embodiments, in any one of the methods described herein, the period of time is about 1, 2, 4, 6, 8, 10, 12 weeks. In some embodiments, in any one of the methods described herein, the period of time is about 8 weeks. In a preferred embodiment, in any one of the methods described herein, edaravone is administered to the subject for about 14 days, followed by about 14 days of no administration, further followed by administration for about 10 days in about a 14-day period, further followed by about 14 days of no administration. In a preferred embodiment, in any one of the methods described herein, edaravone is administered to the subject at a daily dose of about 60 mg for about 14 days, followed by about 14 days of no administration, further followed by administration of a daily dose of about 60 mg for about 10 days in about a 14-day period, further followed by about 14 days of no administration.
[0072] In some embodiments, any one of the methods described herein may further comprise administering to the subject a glutamatergic agent. As described herein, a “glutamatergic agent” may be described as a composition, or a drug, or a chemical that directly modulates the excitatory amino acid (glutamate/ aspartate) system in the body or brain. In some embodiments, the glutamatergic agent may be selected from, but is not limited to, a glutamate transporter modulating agent and a glutamate receptor antagonist. In embodiments, the glutamate transporter modulating agent may be an excitatory amino acid reuptake inhibitor. In some embodiments, the glutamate receptor antagonist may be an N-methyl-D-aspartate (NMDA) receptor antagonist. In embodiments, the glutamate receptor antagonist may be an antagonist of the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMP A) receptor, or the kainite receptor.
[0073] In some embodiments, the glutamatergic agent may be a glutamate receptor antagonist selected from, but not limited to, AP5 (R-2-amino-5-phosphonopentanoate), AP7 (2-amino-7- phosphonoheptanoic acid), CNQX (6-cyano-7-nitroquinoxaline-2,3-dione), CPPene (3-[(R)-2- carboxypiperazin-4-yl]-prop-2-enyl-l-phosphonic acid), NBQX (2,3-dihydroxy-6-nitro-7- sulfamoyl-benzo[f]quinoxaline-2, 3-dione), and selfotel (CGS-19755).
[0074] In some embodiments, the glutamatergic agent may be a glutamate receptor antagonist selected from, but not limited to, amantadine, atomoxetine, AZD6765, agmatine, gacyclidine, ketamine, memantine, eliprodil, delucemin.
[0075] In some embodiments, the glutamatergic agent may be selected from, but not limited to, BHV-5000, lamotrigine, lanicemine, riluzole, trigriluzole, and topiramate. In a preferred embodiment, the glutamatergic agent is riluzole.
[0076] In some embodiments, a method of treating a neurological disorder comprises administering a PIKfyve inhibitor or a pharmaceutically acceptable salt thereof, and an antioxidant (e.g., edaravone), wherein the subject is receiving or has received treatment with a glutamatergic agent. In some embodiments, a method of treating a neurological disorder comprises administering a PIKfyve inhibitor, or a pharmaceutically acceptable salt thereof, and an antioxidant (e.g., edaravone), wherein the subject is receiving or has received treatment with riluzole. In some embodiments, a method of treating a neurological disorder comprises administering a PIKfyve inhibitor, or a pharmaceutically acceptable salt thereof, and an antioxidant (e.g., edaravone), wherein the subject is not receiving or has not received treatment with a glutamatergic agent (e.g., riluzole). In some embodiments a PIKfyve inhibitor is any described herein or known in the art (e.g., apilimod, YM-201636, MOMIPP, MF4, APY0201, AS2677131 , AS2795440, VRG101, Vacuolin-1 , WX8, NDF, WWL, XB6, VRG50635, VRG50648, XBA, and analogs or salts thereof,). [0077] In some embodiments, in any one of the methods described herein, about 25-75 (e.g., 25-75, 25-60, 25-50, 25-40, 25-30, 30-75, 30-60, 30-40, 30-50, 40-75, 40-60, 40-50, 50-75, 50- 60, 60-75) milligrams of riluzole is administered to the subject. In some embodiments, in any one of the methods described herein, about 25, 30, 40, 50, 60, 75 milligrams of riluzole are administered to the subject. In some embodiments, in any one of the methods described herein, about 50 milligrams of riluzole is administered to the subject.
[0078] In some embodiments, in any one of the methods described herein, riluzole is administered to the subject 1-3 (e.g., 1-3, 1-2, 2-3) times daily. In some embodiments, in any one of the methods described herein, riluzole is administered to the subject 1, 2, 3 times daily. In some embodiments, in any one of the methods described herein, riluzole is administered to the subject twice daily. In some embodiments, in any one of the methods described herein, riluzole is administered to the subject twice daily and about 50 mg of riluzole is administered in each administration.
[0079] In some embodiments, in any one of the methods described herein, the subject is not receiving treatment or has not received treatment with AMX0035, 4-phenylbutyric acid (PBA), tauroursodeoxycholic acid (TUDCA), methylcobalamin, or any combinations thereof. “AMX0035” (a fixed combination of PBA and TUDCA) is an approved medication for ALS marketed under the brand name Relyvrio. The term “methylcobalamin” refers to carbanide;cobalt(2+);[(2R,3S,4R,5S)-5-(5,6-dimethylbenzimidazol-l-yl)-4-hydroxy-2- (hydroxymethyl)oxolan-3-yl]-l-[3-[(lR,2R,3R,5Z,7S,10Z,12S,13S,15Z,17S,18S,19R)- 2,13,18-tris(2-amino-2-oxoethyl)-7,12,17-tris(3-amino-3-oxopropyl)-3,5,8,8,13,15,18,19- octamethyl-2,7, 12,17 -tetrahydro- 1 H-corrin-24-id-3-yl]propanoylamino]propan-2-yl hydrogen phosphate. The molecular formula of methylcobalamin is C63H92CoN13O14P, with a CAS number of 13422-55-4.
[0080] In some embodiments, in any one of the methods described herein, the different therapeutic agents may be formulated in compositions (e.g., different, or same compositions) for administration to a subject. The composition(s) may take any suitable form (e.g., liquids, aerosols, solutions, inhalants, mists, sprays; or solids, powders, ointments, pastes, creams, lotions, gels, patches, and the like) for administration by any desired route (e.g., pulmonary, inhalation, intranasal, oral, buccal, sublingual, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, intrapleural, intrathecal, transdermal, transmucosal, rectal, and the like). In some embodiments, a pharmaceutical composition of the disclosure may be in the form of an aqueous solution or powder for aerosol administration by inhalation or insufflation (either through the mouth or the nose), in the form of a tablet or capsule for oral administration; in the form of a sterile aqueous solution or dispersion suitable for administration by either direct injection or by addition to sterile infusion fluids for intravenous infusion; or in the form of a lotion, cream, foam, patch, suspension, solution, or suppository for transdermal or transmucosal administration. In some embodiments, the administration route of the composition comprising an antioxidant (e.g., edaravone), and/or a PIKfyve inhibitor, and/or the glutamatergic agent (e.g., riluzole) may be orally. In some embodiments, the administration route of the antioxidant (e.g., edaravone) may be intravenously or orally. In some embodiments a PIKfyve inhibitor is any described herein or known in the art (e.g., apilimod, YM-201636, MOMIPP, MF4, APY0201, AS2677131 , AS2795440, VRG101, Vacuolin-1 , WX8, NDF, WWL, XB6, VRG50635, VRG50648, XBA, and analogs or salts thereof).
[0081] Methods described herein are for treating neurological diseases or disorders. A “neurological disease or disorder,” as used herein, refers to a disease of the nervous system characterized by progressive loss of neuronal structure and function. In accordance with the methods described herein, the neurological disease or disorder may be selected from a neurodegenerative disease or disorder, epilepsy, a neuromuscular disorder, or a neurodevelopmental disorder.
[0082] Neurodegenerative diseases and disorders that may be treated according to the methods described may be selected from, but are not limited to, Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), diffuse Lewy body disease, motor neuron diseases, multiple sclerosis (MS), Parkinson’s disease (PD), Friedreich’s ataxia, prion disease, spinocerebellar ataxia (SCA), cerebellar ataxia, and spinal muscular atrophy (SMA). Other, less common neurodegenerative diseases and disorders that may be treated according to the methods described herein may be selected from, but are not limited to, Charcot-Marie-Tooth disease, Creutzfeldt-Jakob disease (CJD), progressive supranuclear palsy (PSP, Steele-Richardson- Olszewski syndrome), senile chorea, Huntington’s Chorea, spinal ataxia including spinocerebellar ataxia (SCA), Friedreich’s ataxia, Subacute sclerosing panencephalitis, frontotemporal lobar degeneration, and Hallerrorden-Spatz disease (Pantothenate kinaseassociated neurodegeneration, PKAN).
[0083] In one non-limiting example, the methods described herein may be used for the treatment of ALS or frontotemporal dementia, where the patient in need of treatment may be a patient having repeat expansions in the C9ORF72 gene. The GGGGCC repeat expansion in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS), accounting for about 10% of all ALS cases worldwide and 10% familial frontotemporal dementia (FTD). The repeat expansion generates neurotoxic species including dipeptide repeat proteins (DPRs), nuclear RNA foci, and RNA/DNA G-quadruplexes. The repeat expansion also suppresses the production of C9ORF72 protein, a protein that normally regulates vesicle trafficking and lysosomal biogenesis. In human induced motor neurons, the repeat expansion in C9ORF72 triggered neurodegeneration through two mechanisms: accumulation of glutamate receptors and impaired clearance of neurotoxic dipeptide repeat proteins. In one nonlimiting example, the methods described herein may be used for the treatment of ALS or frontotemporal dementia, where the patient in need of treatment may be a patient having repeat expansions in the C9ORF72 gene. In another example, the methods described herein may be used for the treatment of ALS or frontotemporal dementia, where the patient in need of treatment may be a patient having mutations in the SOD1 gene. In another example, the methods described herein may be used for the treatment of ALS or frontotemporal dementia, where the patient in need of treatment may be a patient having mutations in the TDP-43 gene. In some embodiments, the neurological disease or disorder may be dementia. In an embodiment for the treatment of ALS or frontotemporal dementia, the patient in need of treatment of is one having a mutation in TDP-43 and/or an accumulation of TDP-43 aggregates, a product of the TARDBP gene, found in many sporadic and familial ALS. In another example, the patient has ALS that arose spontaneously and does not have one of the previous mutations.
[0084] Various forms of dementia may also be considered neurodegenerative diseases. In general, the term ‘dementia’ may describe a group of symptoms affecting memory, thinking and social abilities severely enough to interfere with daily functioning. Accordingly, in some aspects, the present disclosure may provide methods of treating dementia, including AIDS dementia complex (ADC), dementia associated with Alzheimer’s disease (AD), dementia pugilistica, diffuse Lewy body disease, frontotemporal dementia, mixed dementia, senile dementia of Lewy body type, and vascular dementia.
[0085] In some embodiments, in any one of the methods described herein, the neurological disease or disorder may also be selected from, but not limited to, bipolar disorder, treatment resistant and major depression, general anxiety disorder, panic disorder, social anxiety, mood disorders, cognitive disorders, agitation, apathy, psychoses, post-traumatic stress disorders, irritability, disinhibition, learning disorders, memory loss, personality disorders, bipolar disorders, eating disorders, conduct disorder, pain disorders, delirium, drug addiction, tinnitus, mental retardation, cervical spondylotic myelopathy, spinal cord injury, hereditary cerebellar ataxia, Tourette syndrome, autism spectrum disorder, attention deficit hyperactivity disorder, obsessive compulsive disorder (OCD), traumatic brain injury, schizophrenia, fragile X syndrome, Parkinson’s Disease and Huntington’s disease.
[0086] Further neuromuscular disorders that may be treated according to the methods described herein may be selected from, but are not limited to, infantile spinal muscular atrophy (SMA1, Werdnig-Hoffmann disease), and juvenile spinal muscular atrophy (SMA3, Kugelberg- Welander disease).
[0087] Neurodevelopmental disorders that may be treated according to the methods described herein may include but is not limit to Rett syndrome.
[0088] As described herein, a “subject in need thereof’ refers to a subject in need of treatment for a neurological disease or disorder (e.g., a neurological disease or disorder as provided herein). In some embodiments, the subject in need may be one that is “non-responsive” or “refractory” to a standard therapy for the neurological disease or disorder. In some embodiments, the terms “non-responsive” and “refractory” may refer to the subject’s response to therapy as not clinically adequate to relieve one or more symptoms associated with the neurological disease” or disorder.
[0089] As described herein a “subject” may refer generally to a mammal. The mammal may be e.g., a human, primate, mouse, rat, dog, cat, cow, horse, goat, camel, sheep, or a pig. In some preferred embodiments, the subject may be a human. The terms “subject” and “patient” may be used interchangeably herein.
[0090] As described herein, the terms, “treatment”, “treating” or “treat” may describe the management and care of a subject having a neurological disease or disorder, and may include the administration of a therapeutic agent, or combination thereof, to slow the progression of the disease or disorder and/or to alleviate one or more symptoms of the neurological disease or disorder. In some embodiments, treating may include administering an amount of the therapeutic agent, or combination of agents, effective to alleviate one or more symptoms of the neurological disease or disorder. As described herein “alleviate” may refer to a process by which the severity of a symptom may be reduced or decreased, but it may not necessarily be eliminated, although it may be eliminated for a period of time, or temporarily. While elimination of the symptom may be preferred, it is not required. As described herein the terms, “prevention”, “preventing” or “prevent” refer to reducing or eliminating the onset of a symptom, especially in the context of preventing the progression of the disease or disorder, where progression may be defined by the onset one or more symptoms. [0091] The terms, “combination therapy” or “co-therapy” may include the administration therapeutic agents as part of a specific treatment regimen intended to provide a beneficial effect from the co-action of these compounds. The beneficial effect may result in the slowing of the progression of the neurological disease or disorder, and/or the alleviation of one or more symptoms of the neurological disease or disorder. The beneficial effect of the combination may include, but is not limited to, pharmacokinetic or pharmacodynamic co-action resulting from the combination. The beneficial effect of the combination may also relate to the mitigation of a toxicity, side effect, or adverse event associated with another agent in the combination. “Combination therapy” may not be intended to encompass the administration of two or more of these therapeutic compounds as part of separate monotherapy regimens that incidentally and arbitrarily result in the combinations of the present disclosure.
[0092] In the context of combination therapy, administration of a PIKfyve inhibitor, or a pharmaceutically acceptable salt thereof, may be simultaneous with or sequential to the administration of one or more additional therapeutic agents (e.g., the antioxidant and/or the glutamatergic agent). In another aspect, administration of the different components of a combination therapy may be at different frequencies. The one or more additional therapeutic agents may be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a PIKfyve inhibitor , or a pharmaceutically acceptable salt thereof. In some embodiments a PIKfyve inhibitor is any described herein or known in the art (e.g., apilimod, YM-201636, MOMIPP, MF4, APY0201, AS2677131 , AS2795440, VRG101, Vacuolin-1 , WX8, NDF, WWL, XB6, VRG50635, VRG50648, XBA, and analogs or salts thereof,)
[0093] In some embodiments, in any one of the methods described herein, the therapeutic agents may be administered in a composition. In some embodiments, the compositions may be a pharmaceutical composition. As described herein, a “pharmaceutical composition” is a formulation containing one or more therapeutic agents in a pharmaceutically acceptable form suitable for administration to a subject. The term “pharmaceutically acceptable” may refer to those compounds, materials, compositions, carriers, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
[0094] In further embodiments, a pharmaceutical composition may be prepared using a pharmaceutically acceptable excipient. As described herein, a “pharmaceutically acceptable excipient” may describe an excipient that may be useful in preparing a pharmaceutical composition that may be generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes excipient that may be acceptable for veterinary use as well as human pharmaceutical use. Pharmaceutically acceptable excipients may be selected from, but are not limited to, sterile liquids, water, buffered saline, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), oils, detergents, suspending agents, carbohydrates (e.g., glucose, lactose, sucrose or dextran), antioxidants (e.g., ascorbic acid or glutathione), chelating agents, low molecular weight proteins, or suitable mixtures thereof.
[0095] In some embodiments, a pharmaceutical composition may be provided in bulk or in dosage unit form. It may be advantageous to formulate pharmaceutical compositions in dosage unit form for ease of administration and uniformity of dosage. The term “dosage unit form” as used herein may refer to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the disclosure may be dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved. A dosage unit form may be an ampoule, a vial, a suppository, a dragee, a tablet, a capsule, an IV bag, or a single pump on an aerosol inhaler.
[0096] In some embodiments, for example in therapeutic applications, the dosages may vary depending on the agent, the age, weight, and clinical condition of the recipient patient, and the experience and judgment of the clinician or practitioner administering the therapy, among other factors affecting the selected dosage. Generally, the dose may be a therapeutically effective amount. Dosages may be provided in mg/kg/day units of measurement (which dose may be adjusted for the patient’s weight in kg, body surface area in m2, and age in years). As described herein, a “therapeutically effective amount” of a pharmaceutical composition may be that which provides an objectively identifiable improvement as noted by the clinician or other qualified observer. In one non-limiting example, alleviating a symptom of a disorder, disease, or condition. As described herein, the term “dosage effective manner” may refer to an amount of a pharmaceutical composition to produce the desired biological effect in a subject or cell. [0097] In some embodiments, a pharmaceutical composition may be in the form of an orally acceptable dosage form including, but not limited to, capsules, tablets, buccal forms, troches, lozenges, and oral liquids in the form of emulsions, aqueous suspensions, dispersions or solutions. Capsules may contain mixtures of a compound of the present disclosure with inert fdlers and/or diluents such as the pharmaceutically acceptable starches (e.g., com, potato or tapioca starch), sugars, artificial sweetening agents, powdered celluloses, such as crystalline and microcrystalline celluloses, flours, gelatins, gums, etc. In some embodiments, for example in the use of tablets for oral use, carriers which are commonly used include lactose and com starch. Lubricating agents, such as magnesium stearate, can also be added. In some embodiments, oral administration in a capsule form, useful diluents include lactose and dried com starch. In some embodiments, when aqueous suspensions and/or emulsions may be administered orally, the compound of the present disclosure may be suspended or dissolved in an oily phase is combined with emulsifying and/or suspending agents. In further embodiments, certain sweetening and/or flavoring and/or coloring agents may be added.
[0098] A pharmaceutical composition may be in a solid oral dosage form. In some embodiments, a pharmaceutical composition may be in the form of a tablet. In some embodiments, the solid oral dosage form is an orally disintegrating tablet. The tablet may comprise a unit dosage of a compound of the present disclosure together with an inert diluent or carrier such as a sugar or sugar alcohol, for example lactose, sucrose, sorbitol or mannitol. The tablet may further comprise a non-sugar derived diluent such as sodium carbonate, calcium phosphate, calcium carbonate, or a cellulose or derivative thereof such as methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, and starches such as com starch. In some embodiments, the tablet may further comprise binding and granulating agents such as polyvinylpyrrolidone, disintegrants (e.g., swellable crosslinked polymers such as crosslinked carboxymethylcellulose), lubricating agents (e.g., stearates), preservatives (e.g., parabens), antioxidants (e.g., BHT), buffering agents (for example phosphate or citrate buffers), and effervescent agents such as citrate/bicarbonate mixtures.
[0099] In some embodiments, the tablet may be a coated tablet. In further embodiments, the coating may be a protective fdm coating (e.g., a wax or varnish) or a coating designed to control the release of the active agent, for example a delayed release (release of the active after a predetermined lag time following ingestion) or release at a particular location in the gastrointestinal tract. The latter may be achieved, for example, using enteric fdm coatings such as those sold under the brand name Eudragit™. [0100] In some embodiments, tablet formulations may be made by conventional compression, wet granulation or dry granulation methods and utilize pharmaceutically acceptable diluents, binding agents, lubricants, disintegrants, surface modifying agents (including surfactants), suspending or stabilizing agents, including, but not limited to, magnesium stearate, stearic acid, talc, sodium lauryl sulfate, microcrystalline cellulose, carboxymethylcellulose calcium, polyvinylpyrrolidone, gelatin, alginic acid, acacia gum, xanthan gum, sodium citrate, complex silicates, calcium carbonate, glycine, dextrin, sucrose, sorbitol, dicalcium phosphate, calcium sulfate, lactose, kaolin, mannitol, sodium chloride, talc, dry starches and powdered sugar. In preferred embodiments, surface modifying agents include nonionic and anionic surface modifying agents. Non-limiting examples of surface modifying agents may include, but are not limited to, poloxamer 188, benzalkonium chloride, calcium stearate, cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters, colloidal silicon dioxide, phosphates, sodium dodecylsulfate, magnesium aluminum silicate, and triethanolamine.
[0101] In some embodiments, a pharmaceutical composition may be in the form of a hard or soft gelatin capsule. In accordance with this formulation, the compound of the present disclosure may be in a solid, semi-solid, or liquid form.
[0102] In some embodiments, a pharmaceutical composition may be in the form of a sterile aqueous solution or dispersion suitable for parenteral administration. As described herein, term “parenteral” includes, but is not limited to, subcutaneous, intracutaneous, intravenous, intramuscular, intra-articular, intraarterial, intrasynovial, intrastemal, intrathecal, intralesional and intracranial injection or infusion techniques.
[0103] In some embodiments, a pharmaceutical composition may be in the form of a sterile aqueous solution or dispersion suitable for administration by either direct injection or by addition to sterile infusion fluids for intravenous infusion, and comprises a solvent or dispersion medium containing, water, ethanol, a polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), suitable mixtures thereof, or one or more vegetable oils. In further embodiments, solutions or suspensions of the compound of the present disclosure as a free base or pharmacologically acceptable salt may be prepared in water suitably mixed with a surfactant. Non-limiting examples of suitable surfactants are given below. Dispersions may also be prepared, non-limiting examples include, in glycerol, liquid polyethylene glycols and mixtures of the same in oils.
[0104] In some embodiments, the pharmaceutical compositions for use in the methods of the present disclosure may further comprise one or more additives in addition to any carrier or diluent (such as lactose or mannitol) that may be present in the formulation. The one or more additives may comprise or consist of one or more surfactants. As described herein, “surfactants” may have one or more long aliphatic chains such as fatty acids which enables them to insert directly into the lipid structures of cells to enhance drug penetration and absorption. An empirical parameter commonly used to characterize the relative hydrophilicity and hydrophobicity of surfactants is the hydrophilic-lipophilic balance (“HLB” value). Surfactants with lower HLB values may be more hydrophobic, and may have greater solubility in oils, while surfactants with higher HLB values may be more hydrophilic and may have greater solubility in aqueous solutions. Thus, hydrophilic surfactants may generally be considered to be those compounds having an HLB value greater than about 10, and hydrophobic surfactants may be generally those having an HLB value less than about 10. However, these HLB values are merely a guide since for many surfactants, the HLB values may differ by as much as about 8 HLB units, depending upon the empirical method chosen to determine the HLB value.
[0105] Among the surfactants for use in the compositions of the disclosure are polyethylene glycol (PEG)-fatty acids and PEG-fatty acid mono and diesters, PEG glycerol esters, alcoholoil transesterification products, polyglyceryl fatty acids, propylene glycol fatty acid esters, sterol and sterol derivatives, polyethylene glycol sorbitan fatty acid esters, polyethylene glycol alkyl ethers, sugar and its derivatives, polyethylene glycol alkyl phenols, polyoxyethylenepolyoxypropylene (POE-POP) block copolymers, sorbitan fatty acid esters, ionic surfactants, fat-soluble vitamins and their salts, water-soluble vitamins and their amphiphilic derivatives, amino acids and their salts, and organic acids and their esters and anhydrides.
[0106] The therapeutic agents may be formulated for co-administration in a single dosage form, or they can be administered separately in different dosage forms. When administered separately, administration may be by the same or a different route of administration for each of the components of the combination therapy. In some embodiments of the methods described herein, a PIKfyve inhibitor, may be administered at the same time or at a different time, the context of the combination therapy with the antioxidant and/ or the glutamatergic agent. In some embodiments, a PIKfyve inhibitor, and/or the antioxidant, and/or the glutamatergic agent are administered in a single dosage form, or in separate dosage forms. In some embodiments a PIKfyve inhibitor is any described herein or known in the art (e.g., apilimod, YM-201636, MOMIPP, MF4, APY0201, AS2677131 , AS2795440, VRG101, Vacuolin-1 , WX8, NDF, WWL, XB6, VRG50635, VRG50648, XBA, and analogs or salts thereof,)
[0107] As described herein, the term “therapeutically effective amount” refers to an amount sufficient to treat, ameliorate a symptom of, reduce the severity of, or reduce the duration of the neurological disease or disorder, or to enhance or improve the therapeutic effect of another therapy. The precise effective amount for a subject may depend upon the subject’s body weight, size, and health; the nature and extent of the condition; and the therapeutic or combination of therapeutics selected for administration.
[0108] The combination therapy methods described herein provide a synergistic response. As described herein, the term “synergistic” refers to the efficacy of the combination being more than the additive effects of either single therapy alone. The synergistic effect of combination therapy may permit the use of lower dosages and/or less frequent administration of at least one agent in the combination compared to its dose and/or frequency outside of the combination. The synergistic effect may also be manifested in the avoidance or reduction of adverse or unwanted side effects associated with the use of either therapy in the combination alone.
[0109] All percentages and ratios used herein, unless otherwise indicated, are by weight. Other features and advantages of the present disclosure are apparent from the different examples. The provided examples illustrate different components and methodology useful in practicing the present disclosure. The examples do not limit the claimed disclosure. Based on the present disclosure the skilled artisan can identify and employ other components and methodology useful for practicing the present disclosure.
EXAMPLES
[0110] Neurodegenerative diseases (ND) of the central nervous system (CNS) cause progressive loss of neuronal structure and function and their impact in our societies is increasing. Among these neurodegenerative diseases are amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Parkinson Disease (PD), and Huntington disease (HD) (Gonzales 2022).
[0111] Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder affecting primarily the motor system, but in which extra-motor manifestations are increasingly recognized. The loss of upper and lower motor neurons in the motor cortex, the brain stem nuclei and the anterior horn of the spinal cord gives rise to progressive muscle weakness and wasting. ALS often has a focal onset but subsequently spreads to different body regions, where failure of respiratory muscles typically limits survival to 2-5 years after disease onset (Goutman 2022). [0112] The clinical presentation of ALS typically consists of adult-onset focal muscle weakness and wasting, which tends to spread with disease progression. The weakness most commonly starts in the limb muscles, more often in distal muscles than in proximal muscles. In about 25%-30% of cases there is a bulbar onset of the disease, presenting with dysarthria, dysphagia, dysphonia, or more rarely with masseter weakness. There is a high degree of variability in the age at onset, the site of onset and the disease progression rate of ALS. The disease is relentlessly progressive in most patients, with a median survival of about 3 years after symptom onset, where death is mostly attributed to respiratory failure. About 50% of patients will suffer from extra-motor manifestations to some degree in addition to their motor problems. In 10%— 15% of cases, an additional diagnosis of frontotemporal dementia (FTD) can be made, whilst 35%-40% of patients will have mild behavioral and/or cognitive changes. FTD is characterized by the degeneration of frontal and anterior temporal lobes and presents clinically by behavioral changes, impairment of executive functioning and/or language impairment. ALS and FTD are now considered to be two ends of a spectrum due to the overlap in molecular mechanisms underlying both neurodegenerative disorders (Goutman 2022).
[0113] So far, at least 20 genes have been associated with ALS. The five most common genetic causes are hexanucleotide expansions in chromosome 9 open reading frame 72 (C9orf72) and mutations in superoxide dismutase 1 (SOD1), TAR DNA-b inding protein 43 (TARDBP), fused in sarcoma (FUS) and TANK-binding kinase 1 (TBK1). Together, they explain about 15% of all patients (Goutman 2022). The rest of the patients are sporadic.
[0114] Apilimod dimesylate capsule (Apilimod) is a first-in-class inhibitor of phosphatidylinosito 1-3 -phosphate 5 -kinase (PIKfyve) with an IC50 of 14nM and Kd of approximately 75 pM (range 69-81 pM) (Gayle 2017; Cai 2013). PIKfyve, is a 240-kDa endosomal phosphatidylinositol (PI) lipid kinase that catalyzes the phosphorylation of phosphatidylinositol 3-phosphate (PI(3)P) to phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2). PIKfyve normally synthesizes PI(3,5)P2 on endosomes and lysosomes, supporting lysosomal homeostasis (Rutherford 2006). Pharmacological inhibition of PIKfyve with apilimod increases the activity of transcription factor EB (TFEB) (Gayle 2017), a master gene for lysosomal biogenesis that coordinates expression of lysosomal hydrolases, membrane proteins and genes involved in autophagy (Settembre 2011). Active, nuclear TFEB levels have been found to be decreased in the brains of patients with ALS and AD (Wang 2016). Furthermore, published work has shown that activating TFEB through viral overexpression in neurodegenerative models of Parkinson’s disease (PD) and Alzheimer’s disease (AD) can clear protein aggregates, protect neurons, and ameliorate behavioral phenotypes (Decressac 2013, Polito 2014; Xiao 2014). Together these data support the hypothesis that Apilimod, through activation of TFEB, can clear pathogenic protein aggregates and protect neurons.
[0115] Like other neurodegenerative diseases, amyotrophic lateral sclerosis (ALS) is characterized by autophagy and lysosomal disfunction and pathogenic protein aggregate formation (Bertram 2005). Primary pharmacodynamics data in patient-derived induced motor neurons (iMN) and mouse models of ALS show that C9ORF72 mutation confers lysosomal deficiency and associated vulnerability to degenerative stimuli in neurons, including neurotrophic growth factor withdrawal, glutamate-induced excitotoxicity, and dipeptide repeat (DPR) aggregates. Consistent with its ability to activate TFEB, apilimod treatment ameliorated these deficits, improving neuron survival and reversing the accumulation of DPR aggregates (Shi 2018). Furthermore, neuroinflammation is known to be an early signal and driver of progression in many neurodegenerative diseases (Geloso 2017; Bertram 2005). Similar to its effects on PBMCs (Wada 2007), apilimod also potently inhibits the activation of microglial cells in vitro as demonstrated by the inhibition of the release of IL-12/IL-23 p40 production in response to toll-like receptor (TLR) activation. Coupled to its ability to inhibit inflammatory cytokine induction, apilimod therefore holds promise as a therapeutic for ALS.
[0116] Edaravone is a member of the substitute 2-pyrazolin-5-one class, has the chemical name 3-methyl-l-phenyl-2-pyrazolin-5-one. Although the exact mechanism of action of edaravone in the treatment of ALS is unknown, its therapeutic effect may be linked to its known antioxidant properties (Goutman 2022) in which it plays a role as a free radical scavenger of peroxyl radical and peroxynitrite, which has a cytoprotective and neuroprotective mechanism against oxidative stress conditions. Edaravone protects neurons in the brain and spinal cord by eliminating the reactive oxygen species (ROS) such as hydroxyl radical, peroxyl radical, hydrogen peroxide, peroxynitrite, and many others that quicken and exacerbate the progression of neuronal degeneration, thus protecting from neurologic damage that leads to the death of motor neurons (Goutman 2022). Edaravone also has anti-inflammatory properties against activated microglial cells which may contribute to its activity. Both oral and IV doses of Edaravone have been approved by the FDA. [0117] Results
[0118] Compounds were tested in glutamate exposed iPSC-derived motor neurons, or growth factors-deprived iPSC-derived motor neurons, in the case of iPSC derived from ALS patients with TDP-43 mutations. iPSC Neuron Differentiation Peripheral blood mononuclear cell (PBMC)-derived iPSC lines from ALS patients were obtained from the Cedars-Sinai Answer ALS repository. iPSCs were generated using episomal plasmid-based reprogramming methods. iPSCs were maintained in MTeSR medium according to standard Cedars Sinai protocols and differentiated into spinal neurons according to the direct induced motor neurons (diMNs) protocol, which generates a mixed population consisting of 20%-30% islet- 1 positive motor neurons (Coyne 2020). Briefly, iPSC colonies were maintained on Matrigel coated 10 cm dishes for three weeks before passaging for differentiation. Once iPSC colonies reached 30%- 40% confluence (about 4-5 days after passaging), stage 1 media consisting of 47.5% IMDM (GIBCO), 47.5% F12 (GIBCO), 1% NEAA (GIBCO), 1% Pen/Strep (GIBCO), 2% B27 (GIBCO), 1% N2 (GIBCO), 0.2 mM LDN193189 (Stemgent), 10 mM SB431542 (StemCell Technologies), and 3 mM CHIR99021 (Sigma Aldrich) was added and exchanged daily until day 6. On day 6 of differentiation, cells were incubated in StemPro Accutase (GIBCO) for 5 minutes at 37°C. Cells were collected from plates and centrifuged at 500 x g for 1.5 minutes. Cells were plated at 1 x 106 cells per well of a 6 well plate or 3 x 106 cells per T25 flask in stage 2 media consisting of 47.5% IMDM (GIBCO), 47.5% F12 (GIBCO), 1% NEAA (GIBCO), 1% Penicillin/Streptomycin (GIBCO), 2% B27 (GIBCO), 1% N2 (GIBCO), 0.2 mM LDN193189 (Stemgent), 10 mM SB431542 (StemCell Technologies), 3 mM CHIR99021 (Sigma Aldrich), 0.1 mM all-trans Retinoic Acid (RA) (Sigma Aldrich), and 1 mM Smoothened agonist (SAG) (Cayman Chemicals). Media was exchanged daily until day 12. For the majority of experiments, on day 12 of differentiation, cells were switched to stage media consisting of 47.5% IMDM (GIBCO), 47.5% F12 (GIBCO), 1% NEAA (GIBCO), l%Penicillin/Streptomycin (GIBCO), 2% B27 (GIBCO), 1% N2 (GIBCO), 0.1 mM Compound E(Millipore), 2.5 mM DAPT (Sigma Aldrich), 0.1 mM db-cAMP (Millipore), 0.5 mM all-transRA (Sigma Aldrich), 0.1 mM SAG (Cayman Chemicals), 200 ng/mL Ascorbic Acid (Sigma Aldrich), 10 ng/mL BDNF (PeproTech), 10 ng/mL GDNF (PeproTech). For all whole cell imaging (TDP-43, Ran), cells were trypsinized and plated in 24 well optical bottom plates (Cellvis) at a density of 250,000 cells per well. Stage 3 media was exchanged every 3 days for the duration of the experiment. All cells were maintained at 37°C with 5% CO2 until
Figure imgf000034_0001
[0119] On day 12 of differentiation, iMNs were plated in 24 well optical bottom plates (Cellvis) at a density of 250,000 neurons per well. Neurons were rinsed with IX PBS and fed with fresh stage 3 media daily to remove dead cells and debris. iMNs derived from TDP-43 mutated iPSC were cultured in medium without growth factors (BDNF, GDNF and db-cAMP) for the last 48 hours. iMNs derived from c9orf72, SOD1 and sporadic iPSCs were then treated with glutamate by replacing media with artificial cerebrospinal fluid (ACSF) (Tocris) containing 0-, or 10-mM glutamate (Sigma Aldrich and incubated at 37°C with 5% CO2 for 4 hours. After 3.5 hours (or after the 48h growth factor depletion for the TDP-43 iMNs), one drop of NucBlue™ Live ReadyProbes (Thermo Fisher Scientific) and 1 mM propidium iodide (Thermo Fisher Scientific) and returned to the incubator for 30 minutes. iMNs were imaged with a Zeiss Axiovert microscope equipped with a Axiocam 503 videocamera, 2 images were taken (magnification 4x) and analyzed per well. PI and DAPIspots were counted using the FIJI software. Cell viability for each iMNs line and following each treatment is reported in Tables 1-3 (see also FIGs. 1-12).
[0120] For the C9orf72 mutated line, the combination of apilimod and edaravone provided an increase in viability of 23.3% with respect to glutamate only treated, cells (p<0.05) while apilimod and edaravone alone provide an increase of 4.1% (p=n.s.) and 2.2% (p=n.s.), respectively leading to a predicted additive response of 6.3% (Table 4). For the SOD1 mutated line, the combination of apilimod and edaravone provided an increase in viability of 41.33% with respect to glutamate only treated cells (p<0.001) while apilimod and edaravone alone provide an increase of 7.82% (p=n.s.) and 3.56% (p= n.s.), respectively leading to a predicted additive response of 11.38% (Table 4). For the TDP-43 line, the combination of apilimod and edaravone provided an increase in viability of 40.77% with respect to untreated growth factor deprived cells (p<0.001) while apilimod and edaravone alone provide an increase of 5.32% (p=n.s.) and 4.55% (p= n.s.), respectively leading to a predicted additive response of 9.87% (Table 4). For the Sporadic line, the combination of apilimod and edaravone provided an increase in viability of 39.22% with respect to glutamate only treated cells while apilimod and edaravone alone provide an increase of 7.5% and 7.65%, respectively leading to a predicted additive response of 15.5% (Table 4). Hence the combination of apilimod with edaravone showed a synergistic effect (i.e., more than the sum of the improved viability with each compound alone) in any line tested.
[0121] For the C9orf72 mutated line, the combination of YM201636 and edaravone provided an increase in viability of 31.8% (p<0.001) with respect to glutamate only treated cells while YM201636 and edaravone alone provide an increase of 5.6% (p=n.s.) and 6.7% (p=n.s.), respectively leading to a predicted additive response of 12.3% (Table 5). For the SOD1 mutated line, the combination of YM201636 and edaravone provided an increase in viability of 39.54% (p<0.0001) with respect to glutamate only treated cells while YM201636 and edaravone alone provide an increase of 4% (p=n.s.) and 8.81% (p=n.s.), respectively leading to a predicted additive response of 12.81% (Table 5). For the TDP-43 mutated line, the combination provided an increase in viability of 32.34% (p<0.001) with respect to glutamate only treated cells while YM201636 and edaravone alone provide an increase of 10.28% (p=n.s.) and 11.42% (p=n.s.), respectively leading to a predicted additive response of 21.7% (Table 5). For the Sporadic mutated line, the combination provided an increase in viability of 32.05% (p<0.001) with respect to glutamate only treated cells while YM201636 and edaravone alone provide an increase of 8.52% (p=n.s.) and 9.49% (p=n.s.), respectively leading to a predicted additive response of 18.01% (Table 5). Hence the combination of YM201636 with edaravone showed a synergistic effect (i.e., more than the sum of the improved viability with each compound alone) in any line tested.
[0122] For the C9orf72 mutated line, the combination of APY0201 and edaravone provided an increase in viability of 27.54% (p<0.001) with respect to glutamate only treated cells while APY0201 and edaravone alone provide an increase of 6.12% (p=n.s.) and 8.11% (p=n.s.), respectively, leading to a predicted additive response of 14.23% (Table 6). For the SOD1 mutated line, the combination of APY0201 and edaravone provided an increase in viability of 25.63% (p<0.001) with respect to glutamate only treated cells while APY0201 and edaravone alone provide an increase of 4.86 % (p=n.s.) and 3.74% (p=n.s.), respectively leading to a predicted additive response of 8.6% (Table 6). For the TDP-43 mutated line, the combination provided an increase in viability of 31.53% (p<0.001) with respect to the untreated growth factor deprived cells while APY0201 and edaravone alone provide an increase of 3.72% (p=n.s.) and 2.83% (p=n.s.), respectively leading to a predicted additive response of 6.55% (Table 6). For the Sporadic mutated line, the combination provided an increase in viability of 28.56% (p<0.001) with respect to glutamate only treated cells while APY0201 and edaravone alone provide an increase of 6.96% (p=n.s.) and 7.29% (p=n.s.), respectively leading to a predicted additive response of 14.25% (Table 6). Hence the combination of APY0201 with edaravone showed a synergistic effect (i.e., more than the sum of the improved viability with each compound alone) in any line tested. [0123] Table 1. Cell viability (Apilimod+Edaravone) with 10 gM Glutamate or growth factor depletion (TDP-43) treatment
Figure imgf000037_0001
(-GF = Growth factor deprived)
[0124] Table 2. Cell viability (YM201636+Edaravone) with 10 pM Glutamate or growth factor depletion (TDP-43) treatment
Figure imgf000037_0002
Figure imgf000038_0001
(-GF = Growth factor deprived)
[0125] Table 3. Cell viability (APY0201+Edaravone) with 10 pM Glutamate or growth factor depletion (TDP-43) treatment
Figure imgf000038_0002
(-GF = Growth factor deprived)
[0126] Table 4. Increase in Cell Viability with respect to Glutamate or growth factor deprivation (Apilimod+Edaravone)
Figure imgf000038_0003
Figure imgf000039_0001
[0127] Table 5. Increase in Cell Viability with respect to Glutamate or growth factor deprivation (YM201636+Edaravone)
Figure imgf000039_0002
[0128] Table 6. Increase in Cell Viability with respect to Glutamate or growth factor deprivation (AP Y 0201 +E daravone)
Figure imgf000039_0003
[0129] REFERENCES
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18) Hayakawa N, Noguchi M, Takeshita S, Eviryanti A, Seki Y, Nishio H, Yokoyama R, Noguchi M, Shuto M, Shima Y, Kuribayashi K, Kageyama S, Eda H, Suzuki M, Hatta T, lemura S, Natsume T, Tanabe I, Nakagawa R, Shiozaki M, Sakurai K, Shoji M, Andou A, Yamamoto T. Structure-activity relationship study, target identification, and pharmacological characterization of a small molecular IL-12/23 inhibitor, APY0201. Bioorg Med Chem. 2014;22(l l):3021-3029.
19) David H. Drewry, Frances M. Potjewyd, Armin Bayati, Jeffery L. Smith, Rebekah J. Dickmander, Stefanie Howell, Sharon Taft-Benz, Sophia M. Min, Mohammad Anwar Hossain, Mark Heise, Peter S. McPherson, Nathaniel J. Moorman, and Alison D. Axtman. Identification and Utilization of a Chemical Probe to Interrogate the Roles of PIKfyve in the Lifecycle of 0- Coronaviruses. Journal of Medicinal Chemistry 2022 65 (19), 12860-12882. [0130] Although the invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.

Claims

CLAIMS What is claimed is:
1. A method of treating a neurological disease or disorder in a subject in need thereof, the method comprising administering to the subject a PIKfyve inhibitor and an antioxidant, wherein the PIKfyve inhibitor is not apilimod or a pharmaceutically acceptable salt thereof.
2. The method of claim 1 , wherein the PIKfyve inhibitor or a pharmaceutically acceptable salt thereof and the antioxidant are in one composition.
3. The method of claim 1 , wherein the PIKfyve inhibitor or a pharmaceutically acceptable salt thereof and the antioxidant are in different compositions.
4. A method of treating a neurological disease or disorder in a subject in need thereof, the method comprising administering to the subject the antioxidant, wherein the subject is receiving or has received treatment with a PIKfyve inhibitor or a pharmaceutically acceptable salt thereof, wherein the PIKfyve inhibitor is not apilimod or a pharmaceutically acceptable salt thereof.
5. A method of treating a neurological disease or disorder in a subject in need thereof, the method comprising administering to the subject a PIKfyve inhibitor or a pharmaceutically acceptable salt thereof, wherein the subject is receiving or has received treatment with an antioxidant, wherein the PIKfyve inhibitor is not apilimod or a pharmaceutically acceptable salt thereof.
6. The method of any one of claims 1-5, wherein the PIKfyve inhibitor is YM-201636, MOMIPP, MF4, APY0201, AS2677131, AS2795440, VRG101, Vacuolin-1, WX8, NDF, WWL, XB6, AS-201, AS-202, VRG50635, VRG50648, XBA, or analogs or salts thereof.
7. The method of any one of claims 1-5, wherein the PIKfyve inhibitor is YM-201636, APY0201, or a pharmaceutically acceptable salt thereof.
8. The method of any one of claims 1-5, wherein the antioxidant is ascorbic acid, cysteine, cysteamine, edaravone, glutathione and bilirubin, amifostine (WR-2721), vitamin A, vitamin C, vitamin E, and flavonoids such as Indian holy basil (Ocimum sanctum), orientin or vicenin.
9. The method of claim 8, wherein the antioxidant is administered orally.
10. The method of claim 8, wherein the antioxidant is edaravone.
11. The method of claim 9, wherein edaravone is administered at a daily dose of 60 mg for
14 days, followed by 14 days of no administration, further followed by a daily dose of 60 mg for 10 days in a 14-day period, further followed by 14 days of no administration.
12. The method of claim 10 or claim 11, wherein the antioxidant is administered intravenously.
13. The method of claim 10, wherein edaravone is administered at a daily dose of 105 mg.
14. The method of claim 12, wherein edaravone is administered orally
15. The method of any one of claims 1-14, wherein the PIKfyve inhibitor is administered orally.
16. The method of claim 2, wherein the composition is administered orally.
17. The method of any one of claims 1-16, further comprising administering to the subject a glutamatergic agent.
18. The method of claim 17, wherein the glutamatergic agent is riluzole.
19. The method of claim 18, wherein riluzole is administered to the subject twice daily and
50 mg of riluzole is administered in each administration.
20. The method of any one of claims 17-19, wherein the glutamatergic agent is administered orally.
21. The method of any one of claims 1-16, wherein the subject is not receiving or has not received treatment with a glutamatergic agent.
22. The method of claim 21, wherein the glutamatergic agent is riluzole.
23. The method of any one of claims 1-22, wherein the neurological disease of disorder is selected from Alzheimer's disease, amyotrophic lateral sclerosis (ALS), attention deficit hyperactivity disorder, autism, cerebellar ataxia, Charcot-Marie-Tooth disease, Creutzfeldt- Jakob disease, dementia, epilepsy, Friedreich's ataxia, Huntington's disease, multiple sclerosis, obsessive compulsive disorder (OCD), Parkinson's disease, Rett syndrome, senile chorea, spinal ataxia, spinal cord injury, supranuclear palsy, traumatic brain injury.
24. The method of claim 23, wherein the neurological disease or disorder is dementia.
25. The method of claim 24, wherein the dementia is selected from AIDS dementia complex (ADC), dementia associated with Alzheimer’s disease (AD), dementia pugilistica, diffuse Lewy body disease, frontotemporal dementia, mixed dementia, senile dementia of Lewy body type, and vascular dementia.
26. The method of claim 25, wherein the neurological disease or disorder is amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD).
27. The method of claim 26, wherein the subject is in need of treatment is one having repeat expansions of the C9ORF72 gene.
28. The method of claim 26, wherein the subject in need of treatment is one having a mutation in the SOD 1 gene.
29. The method of claim 26, wherein the subject in need of treatment is one having accumulation of TDP-34 aggregates.
30. The method of claim 26, wherein the subject in need of treatment is one having a mutation in the TDP-43 gene.
31. The method of any one of claims 1-30, wherein the subject is human.
32. The method of any one of claims 1-31, wherein the subject is not receiving or has not received treatment with AMX0035, PBA, TUDCA, methylcobalamin, or any combinations thereof.
33. Use of a PIKfyve inhibitor and an antioxidant in a method of treating a neurological disease or disorder in a subject in need thereof, wherein the PIKfyve inhibitor is not apilimod or a pharmaceutically acceptable salt thereof.
34. Use of a PIKfyve inhibitor in a method of treating a neurological disease or disorder in a subject in need thereof, wherein the subject is receiving or has received treatment with an antioxidant, wherein the PIKfyve inhibitor is not apilimod or a pharmaceutically acceptable salt thereof.
35. Use of an antioxidant in a method of treating a neurological disease or disorder in a subject in need thereof, wherein the subject is receiving or has received treatment with a PIKfyve inhibitor, and wherein the PIKfyve inhibitor is not apilimod or a pharmaceutically acceptable salt thereof.
36. The use of any one of claims 33-35, wherein the PIKfyve inhibitor is YM-201636, MOMIPP, MF4, APY0201, AS2677131, AS2795440, VRG101, Vacuolin-1, WX8, NDF, WWL, XB6, AS-201, AS-202, VRG50635 , VRG50648, XBA, or analogs or salts thereof.
37. The use of claim 36, wherein the PIKfyve inhibitor is YM-201636, APY0201 or a pharmaceutically acceptable salt thereof.
38. The use of any one of claims 33-37, wherein the antioxidant is ascorbic acid, cysteine, cysteamine, edaravone, glutathione and bilirubin, amifostine (WR-2721), vitamin A, vitamin C, vitamin E, and flavonoids such as Indian holy basil (Ocimum sanctum), orientin or vicenin
39. The use of claim 38, wherein the antioxidant is edaravone.
40. The use of any one of claims 33-39, wherein the subject is not receiving or has not received treatment with AMX0035, PBA, TUDCA, methylcobalamin, or any combinations thereof.
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