WO2024107448A1 - Apilimod combination therapy - Google Patents

Apilimod combination therapy Download PDF

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Publication number
WO2024107448A1
WO2024107448A1 PCT/US2023/037275 US2023037275W WO2024107448A1 WO 2024107448 A1 WO2024107448 A1 WO 2024107448A1 US 2023037275 W US2023037275 W US 2023037275W WO 2024107448 A1 WO2024107448 A1 WO 2024107448A1
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subject
methylcobalamin
administered
apilimod
pharmaceutically acceptable
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PCT/US2023/037275
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French (fr)
Inventor
Peter Ronald Young
Carlo SPIRLI
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Orphai Therapeutics Inc.
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Publication of WO2024107448A1 publication Critical patent/WO2024107448A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7135—Compounds containing heavy metals
    • A61K31/714—Cobalamins, e.g. cyanocobalamin, i.e. vitamin B12
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • the invention relates to compositions and methods comprising Apilimod 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 and was originally identified in a screen of PI3K class IA inhibitors and 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 with other therapeutic agents, e.g., methylcobalamin.
  • a PIKfyve inhibitor e.g., any PIKfyve inhibitors known or described herein, including but not limited to apilimod, APY0201, and YM201636
  • methylcobalamin 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) and/or antioxidants (e.g., edaravone).
  • neurological diseases e.g., ALS
  • glutamatergic agents e.g., riluzole
  • antioxidants e.g., edaravone
  • 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 apilimod or a pharmaceutically acceptable salt thereof and methylcobalamin.
  • apilimod or a pharmaceutically acceptable salt thereof and methylcobalamin are in one composition.
  • apilimod or a pharmaceutically acceptable salt thereof and methylcobalamin 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 methylcobalamin, wherein the subject is receiving or has received treatment with 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 apilimod or a pharmaceutically acceptable salt thereof, wherein the subject is receiving or has received treatment with methylcobalamin.
  • the subject is administered 500-750 pg of methylcobalamin.
  • methylcobalamin is administered to the subject twice daily.
  • methylcobalamin is administered to the subject three times daily.
  • the composition is administered orally.
  • the subject is administered 25 mg - 50 mg of methylcobalamin. In some embodiments, the subject is administered 50 mg of methylcobalamin. In some embodiments, methylcobalamin is administered to the subject twice weekly. In some embodiments, methylcobalamin is administered intramuscularly.
  • apilimod is apilimod dimesylate. In some embodiments, apilimod is administered orally. In some embodiments, the subject is administered 30-300 mg of apilimod or a pharmaceutically acceptable salt thereof. In some embodiments, or a pharmaceutically acceptable salt thereof is administered daily. In some embodiments, apilimod or a pharmaceutically acceptable salt thereof is administered twice daily.
  • 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 of has received treatment with a glutamatergic agent.
  • the glutamatergic agent is riluzole.
  • the method further comprises administering to the subject an antioxidant.
  • the antioxidant is edaravone.
  • edaravone is administer 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.
  • the antioxidant is administered at a daily dose of 105 mg.
  • the antioxidant is administered orally.
  • the subject is not receiving or has not received treatment with an antioxidant.
  • the antioxidant is edaravone.
  • the method further comprises administering to the subject a glutamatergic agent and an antioxidant.
  • the subject is not receiving or has not received treatment with a glutamatergic agent or an antioxidant.
  • the glutamatergic agent is riluzole and the antioxidant is edaravone.
  • 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.
  • ALS amyotrophic lateral sclerosis
  • 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) obsessive compulsive disorder
  • Parkinson's disease Rett syndrome
  • 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.
  • the neurological disease or disorder is amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD).
  • the subject in need of treatment is one having repeat expansions of the C9ORF72 gene.
  • the subject in need of treatment is one having a mutation in the SOD1 gene.
  • the subject in need of treatment is one having a mutation in the TDP-43 gene.
  • the subject in need of treatment is one having accumulation of TDP-34 aggregates.
  • the subject is human.
  • PIKFyve inhibitor e.g., any PIKfyve inhibitors known or described herein, including but not limited to apilimod, APY 0201 , and YM201636 or a pharmaceutically acceptable salt thereof
  • methylcobalamin e.g., any PIKfyve inhibitors known or described herein, including but not limited to apilimod, APY 0201 , and YM201636 or a pharmaceutically acceptable salt thereof
  • PIKFyve inhibitor e.g., any PIKfyve inhibitors known or described herein, including but not limited to apilimod, APY 0201 , and YM201636 or a pharmaceutically acceptable salt thereof
  • a PIKFyve inhibitor e.g., any PIKfyve inhibitors known or described herein, including but not limited to apilimod, APY 0201 , and YM201636 or a pharmaceutically acceptable salt thereof
  • methylcobalamin 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 (e.g., any PIKfyve inhibitors known or described herein, including but not limited to apilimod, APY0201, and YM201636 or a pharmaceutically acceptable salt thereof).
  • a PIKFyve inhibitor e.g., any PIKfyve inhibitors known or described herein, including but not limited to apilimod, APY0201, and YM201636 or a pharmaceutically acceptable salt 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 methylcobalamin (1 nM) and apilimod (6.25 nM) in combination to increase cell viability synergistically after glutamate exposure in iPSC-derived motoneurons from a C9orf72-ALS patient.
  • the combination provides an increase in viability of 34.65% with respect to glutamate while Apilimod and methylcobalamin provide an increase of 7.72% and 2.63%, respectively (see also, Table 4).
  • FIGURE 2 is a graph demonstrating the ability of methylcobalamin (1 nM) 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 ⁇ 0.001; The combination provides an increase in viability of 34.65% with respect to glutamate while Apilimod and methylcobalamin provide an increase of 7.72% and 2.63%, respectively (see also, Table 5).
  • FIGURE 3 is a graph demonstrating the ability of methylcobalamin (1 nM) and apilimod (6.25 nM) in combination to increase cell viability after growth factor deprivation in iPSC-derived motoneurons from a TDP-43-ALS patient. ** denotes significance of p ⁇ 0.001; * denotes significance of p ⁇ 0.05.
  • the combination provides an increase in viability of 26.67% with respect to cells growth-factors deprived (-GF), while Apilimod and methylcobalamin provide an increase of 5.32% and 3.63%, respectively (see also, Table 6).
  • FIGURE 4 is a graph demonstrating the ability of methylcobalamin (1 nM) 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 ⁇ 0.0001; ** denotes significance of p ⁇ 0.001. The combination provides an increase in viability of 46.43% with respect to glutamate, while Apilimod and methylcobalamin provide an increase of 14.95% and 4.48%, respectively (see also, Table
  • FIGURE 5 is a graph demonstrating the ability of methylcobalamin (1 nM) 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 of p ⁇ 0.05.
  • the combination provides an increase in viability of 35.35% with respect to glutamate while YM201636 and methylcobalamin provide an increase of 6.96% and 9.66%, respectively (see also, Table
  • FIGURE 6 is a graph demonstrating the ability of methylcobalamin (1 nM) and YM201636 (12.5 nM) in combination to increase cell viability synergistically after glutamate exposure in iPSC-derived motoneurons from a SOD1-ALS patient.
  • the combination provides an increase in viability of 35.75% with respect to glutamate while YM201636 and methylcobalamin provide an increase of 13.81% and 12.72%, respectively (see also, Table
  • FIGURE 7 is a graph demonstrating the ability of methylcobalamin (1 nM) and YM201636 (12.5 nM) in combination to increase cell viability synergistically after growth factor deprivation in iPSC-derived motoneurons from a TDP-43-ALS patient.
  • the combination provides an increase in viability of 33% with respect to cells growth-factors deprived (-GF), while YM201636 and methylcobalamin provide an increase of 6.18% and 8.91%, respectively (see also, Table 10).
  • FIGURE 8 is a graph demonstrating the ability of methylcobalamin (1 nM) and YM201636 (12.5 nM) in combination to increase cell viability synergistically after glutamate exposure in iPSC-derived motoneurons from a Sporadic-ALS patient.
  • the combination provides an increase in viability of 41.21% with respect to glutamate, while YM201636 and methylcobalamin provide an increase of 10.26% and 9.28%, respectively (see also, Table 11).
  • FIGURE 9 is a graph demonstrating the ability of methylcobalamin (1 nM) and APY0201 (6.25 nM) in combination to synergistically increase cell viability after glutamate exposure in iPSC-derived motoneurons from a C9orf72-ALS patient. ** denotes significance of p ⁇ 0.001; * denotes significance of p ⁇ 0.05. The combination provides an increase in viability of 37.38% with respect to glutamate while APY0201 and methylcobalamin provide an increase of 9.74% and 12%, respectively (see also, Table 12).
  • FIGURE 10 is a graph demonstrating the ability of methylcobalamin (1 nM) and APY0201 (6.25 nM) in combination to synergistically increase cell viability after glutamate exposure in iPSC-derived motoneurons from a SOD-l-ALS patient. ** denotes significance of p ⁇ 0.001; * denotes significance of p ⁇ 0.05. The combination provides an increase in viability of 38.45% with respect to glutamate while APY0201 and methylcobalamin provide an increase of 11.11% and 10.02%, respectively (see also, Table 13).
  • FIGURE 11 is a graph demonstrating the ability of methylcobalamin (1 nM) and APY0201 (6.25 nM) in combination to synergistically increase cell viability after growth factor deprivation in iPSC-derived motoneurons from a TDP-43-ALS patient. ** denotes significance of p ⁇ 0.001; * denotes significance of p ⁇ 0.05.
  • the combination provides an increase in viability of 42.9% with respect to cells growth- factors deprived (- GF), while APY0201and methylcobalamin provide an increase of 7.6% and 6.61%, respectively (see also, Table 14).
  • FIGURE 12 is a graph demonstrating the ability of methylcobalamin (1 nM) and APY0201 (6.25 nM) in combination to synergistically increase cell viability after glutamate exposure in iPSC-derived motoneurons from a Sporadic-ALS patient. *** denotes significance of p ⁇ 0.0001.
  • the combination provides an increase in viability of 42.12% with respect to glutamate while APY0201 and methylcobalamin provide an increase of 4.56% and 6.59%, respectively (see also, Table 15).
  • 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., apilimod, YM-201636, APY0201).
  • 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), mal onate, 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,l
  • the salt of apilimod comprises methanesulfonate.
  • pharmaceutically acceptable salt may also refer to a salt prepared from a compound described herein (e.g., apilimod, YM-201636, APY0201), 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-hydroxyethyl)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-
  • pharmaceutically acceptable salt may also refer to a salt prepared from apilimod (e.g., 2- [2-Pyridin-2-yl)-ethoxy]-4-N’-(3-methyl-benzilidene)-hydrazino]-6-(morpholin-4-yl)- pyrimidine), having a basic functional group, such as an amino functional group, and a pharmaceutically acceptable inorganic or organic acid.
  • apilimod e.g., 2- [2-Pyridin-2-yl)-ethoxy]-4-N’-(3-methyl-benzilidene)-hydrazino]-6-(morpholin-4-yl)- pyrimidine
  • 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 (e.g., 2-[2-Pyridin-2-yl)-ethoxy]-4-N’-(3-methyl-benzilidene)-hydrazino]-6- (morpholin-4-yl)-pyrimidine) by conventional chemical methods.
  • parent compound e.g., 2-[2-Pyridin-2-yl)-ethoxy]-4-N’-(3-methyl-benzilidene)-hydrazino]-6- (morpholin-4-yl)-pyrimidine
  • such salts can be prepared by reacting the parent compound (e.g., 2-[2-Pyridin-2-yl)-ethoxy]-4-N’-(3- methyl-benzilidene)-hydrazino]-6-(morpholin-4-yl)-pyrimidine) with the appropriate acid in water or in an organic solvent, or in a mixture of the two.
  • parent compound e.g., 2-[2-Pyridin-2-yl)-ethoxy]-4-N’-(3- methyl-benzilidene)-hydrazino]-6-(morpholin-4-yl)-pyrimidine
  • 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., apilimod, YM-201636, APY0201) 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., apilimod, YM-201636, APY0201) 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., apilimod, YM-201636, APY0201) 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., apilimod, YM-201636, APY0201
  • 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., apilimod, YM-201636, APY0201) 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., apilimod, YM-201636, APY0201) 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., apilimod, YM-201636, APY0201).
  • 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 apilimod and methylcobalamin in (iPSC)-derived motoneurons from ALS patients has synergistic results in cell viability.
  • the combination therapy of methylcobalamin and apilimod provides unexpected benefits on neuronal survival that are greater than the application of either therapy individually.
  • compositions and methods related to the use of a PIKFyve inhibitor e.g., any PIKfyve inhibitors known or described herein
  • 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 methylcobalamin.
  • a method described herein comprises administering to a subject a composition (e.g., in a solid oral dosing form such as a tablet) comprising a PIKfyve inhibitor (e.g., any PIKfyve inhibitors known or described herein) or a pharmaceutically acceptable salt thereof, and methylcobalamin.
  • a composition e.g., in a solid oral dosing form such as a tablet
  • a PIKfyve inhibitor e.g., any PIKfyve inhibitors known or described herein
  • 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 methylcobalamin.
  • a method described herein comprises administering methylcobalamin 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.
  • 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
  • a PIKfyve inhibitor used in accordance with the present disclosure is a selective PIKfyve inhibitor (i.e., inhibitor that selectively inhibits PIKfyve versus other kinases).
  • the selective PIKfyve inhibitor is apilimod, or an analog or salt thereof.
  • 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
  • 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[ 1 ,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:
  • YM201636 is a PIKFyve inhibitor with a morpholino-pyrimidine (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 , 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).
  • 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., 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 1H- 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; 15(10): 1694-1718, incorporated herein by reference).
  • 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) 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.
  • WWL MLS000703078
  • benzaldehyde [2,6-di(4- morpholinyl)-4-pyrimidinyl]hydrazone represented by Formula (XI):
  • 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 (MLS001167897), refers to N-(4-ethylphenyl)- 4,6- dimorpholino-l,3,5-triazin-2-amine hydrochloride, represented by Formula (XIII):
  • XBA is described in Sharma G, et al. Autophagy. 2019; 15(10): 1694-1718, incorporated herein by reference.
  • the PIKfyve inhibitor is apilimod or a pharmaceutically acceptable salt thereof.
  • 30-300 e.g., 30-300, 30-200, 30-100, 30-80, 30-60, 30-50, 30-40, 40-300, 40-200, 40-100, 40-80, 40-60, 40-50, 50-300, 50-200, 50- 100, 50-80, 50-60, 60-300, 60-200, 60-100, 60-80, 80-300, 80-200, 80-100, 100-300, 1000- 200, 200-300
  • milligrams per day of apilimod or a pharmaceutically acceptable salt thereof may be administered to a subject.
  • any one of the methods described herein in any one of the methods described herein, about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, or 300 milligrams per day of apilimod or a pharmaceutically acceptable salt thereof, may be administered to a subject. In some embodiments, in any one of the methods described herein, about 100-300 milligrams/day of apilimod or a pharmaceutically acceptable salt thereof, is administered to the subject. In some embodiments, in any one of the methods described herein, about 200 or 250 milligrams/day of apilimod or a pharmaceutically acceptable salt thereof, is administered to the subject.
  • a subject in any one of the methods described herein, is administered apilimod twice a day, and each administration provides to the subject 75-150 (e.g., 75-150, 75-125, 75-100, 100-150, 100-125, or 125-150 mg) milligrams of apilimod.
  • a subject in any one of the methods described herein, is administered apilimod twice a day, and each administration provides to the subject 75 milligrams, 100 milligrams, 125 milligrams, or 150 milligrams of apilimod.
  • apilimod or a pharmaceutically acceptable salt thereof 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, apilimod or a pharmaceutically acceptable salt thereof, is administered to the subject 1, 2, or 3 times daily. In some embodiments, in any one of the methods described herein, apilimod or a pharmaceutically acceptable salt thereof, is administered to the subject daily. In some embodiments, in any one of the methods described herein, apilimod or a pharmaceutically acceptable salt thereof, is administered to the subject twice daily.
  • apilimod or a pharmaceutically acceptable salt thereof is administered to the subject over a period of time.
  • the period of time may be 1-60 (e.g., 1-60, 1-52, 1-50, 1-40, 1-30, 1-20, 1-16, 1-10, 1-8, 1-4, 4-60, 4-52, 4-50, 4-40, 4-30, 4-20, 4-16, 4-10, 4-8, 8-60, 8-52, 8-50, 8-40, 8-30, 8-20, 8-16, 8-10, 10-60, 10- 52, 10-50, 10-40, 10-30, 10-20, 10-16, 16-60, 16-52, 16-50, 16-40, 16-30, 16-20, 20-60, 20-52, 20-50, 20-40, 20-30, 30-60, 30-52, 30-50, 30-40, 40-60, 40-52, 40-50, 50-60, SO- 52, 52-60) weeks.
  • the period of time is about 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 52, 60 weeks. In some embodiments, in any one of the methods described herein, the period of time is about 4-16 weeks. In some embodiments, in any one of the methods described herein, the period of time is about 8 weeks. In some embodiments, in any one of the methods described herein, the period of time is about 52 weeks. In some embodiments, in any one of the methods described herein, the period time is the remainder of the subject’s lifetime.
  • apilimod or a pharmaceutically acceptable salt thereof is administered to the subject according to a dosage regimen of about 30-300 mg per day for at least 1 week. In some embodiments, in any one of the methods described herein, apilimod or a pharmaceutically acceptable salt thereof, is administered to the subject according to a dosage regimen of about 100-300 mg per day for about 4 or 16 weeks. In some embodiments, in any one of the methods described herein, apilimod or a pharmaceutically acceptable salt thereof, is administered to the subject according to a dosage regimen of about 100 mg per day, twice a day for about 8 weeks, or optionally for about 52 weeks or longer.
  • apilimod or a pharmaceutically acceptable salt thereof is administered to the subject according to a dosage regimen of about 100-300 mg per day, twice a day for the remainder of the subject’s lifetime. In some embodiments, in any one of the methods described herein, apilimod or a pharmaceutically acceptable salt thereof, is administered to the subject according to a dosage regimen of about 100-300 mg per day, twice a day until the subject is no longer suitable for receiving treatment with apilimod.
  • a PIKfyve inhibitor e.g., apilimod, YM- 201636, APY0201
  • a pharmaceutically acceptable salt thereof is used in combination with methylcobalamin for treating neurological diseases and disorders.
  • 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- lH-corrin-24-id-3-yl]propanoylamino]propan-2-yl hydrogen phosphate.
  • the molecular formula of methylcobalamin is C63H92CON13O14P, with
  • methylcobalamin is a form of vitamin B12, known as cobalamin.
  • Methylcobalamin contains metal-alkyl bonds and an octahedral cobalt(III) center and can be obtained as a bright red crystal.
  • methylcobalamin functions as a cofactor to methionine synthase.
  • Methionine synthase catalyzes the remethylation of homocysteine to methionine.
  • methylcobalamin has an important role in the transfer of one carbon (e.g., as described in Grober U., et al, Neuroenhancement with Vitamin B12 — Underestimated Neurological Significance.
  • vitamin B12 deficiency has been linked to depression, irritability, and psychosis. Long-term deficiency can lead to cardiovascular disease and elevated levels of homocysteine in the blood. Lesions in the central nervous system, including in the spinal cord have been linked to vitamin B12 deficiency, indicating an important role for the vitamin B12 in the nervous system. Methylcobalamin is a standard treatment in patients with vitamin B12 deficiency, has been used in treat peripheral neuropathy in Japan, and recently entered clinical trials for the use in ALS.
  • Methylcobalamin has been found to inhibit neuronal degradation through the decrease of homocysteine and the induction of neurite outgrowth, prolonging neuronal survival. Oral treatment with methylcobalamin results in improved peripheral neuropathy. In clinical studies intramuscular (IM) injection of ultra-high doses of methylcobalamin slowed the functional decline in early-stage ALS patients.
  • IM intramuscular
  • methylcobalamin is administered to the subject over a period of time.
  • the period of time may be 1 -60 (e.g., 1-60, 1-52, 1-50, 1-40, 1-30, 1-20, 1-16, 1-10, 1-8, 1-4, 4-60, 4-52, 4-50, 4-40, 4-30, 4-20, 4-16, 4-10, 4-8, 8-60, 8-52, 8-50, 8-40, 8-30, 8-20, 8-16, 8-10, 10-60, 10-52, 10-50, 10-40, 10-30, 10-20, 10-16, 16-60, 16-52, 16-50, 16-40, 16-30, 16-20, 20-60, 20-52, 20- 50, 20-40, 20-30, 30-60, 30-52, 30-50, 30-40, 40-60, 40-52, 40-50, 50-60, 50-52, 52-60) weeks.
  • the period of time is about 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 52, 60 weeks. In some embodiments, in any one of the methods described herein, the period of time is about 4-16 weeks. In some embodiments, in any one of the methods described herein, the period of time is about 8 weeks. In some embodiments, in any one of the methods described herein, the period of time is about 52 weeks. In some embodiments, in any one of the methods described herein, the period time is the remainder of the subject’s lifetime.
  • 1000-1500 e.g., 1000-1500, 1000-1100, 1100-1200, 1200-1300, 1300-1400, 1400-1500, 1000-1200, 1000-1300, 1000-1400, 1100-1500, 1200-1500, 1300-1500, 1100-1300, 1100-1400, 1100- 1500, 1200-1400, 1200-1500, 1300-1500
  • micrograms per day of methylcobalamin may be administered (e.g., orally) to a subject.
  • about 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500 micrograms per day of methylcobalamin may be administered (e.g., orally) to a subject.
  • about 1000-1500 micrograms/day of methylcobalamin is administered (e.g., orally) to the subject.
  • about 1000 or 1500 micrograms/day of methylcobalamin is administered (e.g., orally) to the subject.
  • a subject in any one of the methods described herein, is administered (e.g., orally) methylcobalamin twice a day, and each administration provides to the subject SOO- SO (e.g., 500-550, 550-600, 600-650, 650-700, or 700-750 pg) micrograms of methylcobalamin.
  • SOO- SO e.g., 500-550, 550-600, 600-650, 650-700, or 700-750 pg
  • micrograms of methylcobalamin in any one of the methods described herein, a subject is administered (e.g., orally) methylcobalamin twice a day, and each administration provides to the subject 500 micrograms, 550 micrograms, 600 micrograms, 650 micrograms, 700 micrograms or 750 micrograms of methylcobalamin.
  • a subject in any one of the methods described herein, is administered (e.g., orally) methylcobalamin three times a day, and each administration provides to the subject 350-500 (e.g., 350-400, 400-450, or 450-500 pg) micrograms of methylcobalamin.
  • a subject in any one of the methods described herein, is administered (e.g., orally) methylcobalamin three times a day, and each administration provides to the subject 350 micrograms, 400 micrograms, 450 micrograms, 500 micrograms, or 750 micrograms of methylcobalamin.
  • methylcobalamin 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, methylcobalamin is administered to the subject 1, 2, or 3 times daily. In some embodiments, in any one of the methods described herein, methylcobalamin is administered to the subject daily. In some embodiments, in any one of the methods described herein, methylcobalamin is administered to the subject twice daily.
  • methylcobalamin is administered (e.g., orally) to the subject according to a dosage regimen of about 1000-1500 pg per day for at least 1 week. In some embodiments, in any one of the methods described herein, methylcobalamin is administered (e.g., orally) to the subject according to a dosage regimen of about 1000-1500 pg per day for about 4 or 16 weeks. In some embodiments, in any one of the methods described herein, methylcobalamin is administered (e.g., orally) to the subject according to a dosage regimen of about 500-750 pg twice a day for about 8 weeks, or optionally for about 52 weeks or longer.
  • methylcobalamin is administered (e.g., orally) to the subject according to a dosage regimen of about 1000-1500 pg twice a day for the remainder of the subject’s lifetime. In some embodiments, in any one of the methods described herein, methylcobalamin is administered (e.g., orally) to the subject according to a dosage regimen of about 500-750 jag twice a day until the subject is no longer suitable for receiving treatment with methylcobalamin.
  • methylcobalamin is administered (e.g., orally) to the subject according to a dosage regimen of about 350-500 jag three times a day for about 8 weeks, or optionally for about 52 weeks or longer. In some embodiments, in any one of the methods described herein, methylcobalamin is administered (e.g., orally) to the subject according to a dosage regimen of about 350-500 pg three times a day for the remainder of the subject’s lifetime.
  • methylcobalamin is administered (e.g., orally) to the subject according to a dosage regimen of about 350-500 pg three times a day until the subject is no longer suitable for receiving treatment with methylcobalamin.
  • 50-100 e.g., 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, 95-100, 60-100, 70-100, 80-100, 90-100, 50-70, 50-80, 50-90, 60-80, 60-90, 70-90
  • milligrams per week of methylcobalamin may be administered (e.g., intramuscularly) to a subject.
  • about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 milligrams per week of methylcobalamin may be administered (e.g., intramuscularly) to a subject.
  • about 50-100 milligrams/week of methylcobalamin is administered (e.g., intramuscularly) to the subject.
  • about 50 or 100 milligrams/week of methylcobalamin is administered (e.g., intramuscularly) to the subject.
  • a subject is administered (e.g., intramuscularly) methylcobalamin twice a week, and each administration provides to the subject 25-50 (e.g., 25-30, 30-35, 35-40, 40-45, or 45- 50 mg) milligrams of methylcobalamin.
  • a subject is administered (e.g., intramuscularly) methylcobalamin twice a week, and each administration provides to the subject 25 milligrams, 30 milligrams, 35 milligrams, 40 milligrams, 45 milligrams, or 50 milligrams of methylcobalamin.
  • a subject is administered (e.g., intramuscularly) methylcobalamin twice a week, and each administration provides to the subject 50 milligrams.
  • methylcobalamin is administered (e.g., intramuscularly) to the subject 1-2 times weekly. In some embodiments, in any one of the methods described herein, methylcobalamin is administered (e.g., intramuscularly) to the subject 1 or 2 times weekly. In some embodiments, in any one of the methods described herein, methylcobalamin is administered (e.g., intramuscularly) to the subject weekly. In some embodiments, in any one of the methods described herein, methylcobalamin is administered (e.g., intramuscularly) to the subject twice a week.
  • methylcobalamin is administered (e.g., intramuscularly) to the subject according to a dosage regimen of about 50-100 mg per week for at least 1 week. In some embodiments, in any one of the methods described herein, methylcobalamin administered (e.g., intramuscularly) to the subj ect according to a dosage regimen of about 50-100 mg per week for about 4 or 16 weeks. In some embodiments, in any one of the methods described herein, methylcobalamin is administered (e.g., intramuscularly) to the subject according to a dosage regimen of about 50-100 mg twice a week, for about 8 weeks, or optionally for about 52 weeks or longer.
  • methylcobalamin is administered (e.g., intramuscularly) to the subject according to a dosage regimen of about 50-100 mg twice a week for the remainder of the subject’s lifetime. In some embodiments, in any one of the methods described herein, methylcobalamin is administered (e.g., intramuscularly) to the subject according to a dosage regimen 25-50 mg twice a week, until the subject is no longer suitable for receiving treatment with methylcobalamin. In some embodiments methylcobalamin is administered intramuscularly to the subject. [0076] In some embodiments, 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.
  • 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)-2- carboxypiperazin-4
  • 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 (e.g., apilimod, YM-201636, APY0201) or a pharmaceutically acceptable salt thereof, and methylcobalamin, 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 (e.g., apilimod, YM-201636, APY0201), or a pharmaceutically acceptable salt thereof, and methylcobalamin, wherein the subject is receiving or has received treatment with riluzole.
  • a method of treating a neurological disorder comprises administering a PIKfyve inhibitor (e.g., apilimod, YM-201636, APY0201), or a pharmaceutically acceptable salt thereof, and methylcobalamin, wherein the subject is not receiving or has not received treatment with a glutamatergic agent (e.g., riluzole).
  • a PIKfyve inhibitor e.g., apilimod, YM-201636, APY0201
  • methylcobalamin e.g., a pharmaceutically acceptable salt thereof, and methylcobalamin
  • 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.
  • any one of the methods described herein further comprises administering to the subject an antioxidant.
  • 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.
  • a method of treating a neurological disorder comprises administering a PIKfyve inhibitor (e.g., apilimod, YM-201636, APY0201), or a pharmaceutically acceptable salt thereof, and methylcobalamin, wherein the subject is receiving or has received treatment with an antioxidant.
  • a method of treating a neurological disorder comprises administering a PIKfyve inhibitor (e.g., apilimod, YM-201636, APY0201), or a pharmaceutically acceptable salt thereof, and methylcobalamin, wherein the subject is receiving or has received treatment with edaravone.
  • a method of treating a neurological disorder comprises administering a PIKfyve inhibitor (e.g., apilimod, YM-201636, APY0201), or a pharmaceutically acceptable salt thereof, and methylcobalamin, wherein the subject is not receiving or has not received treatment with an antioxidant (e.g., edaravone).
  • a PIKfyve inhibitor e.g., apilimod, YM-201636, APY0201
  • an antioxidant e.g., edaravone
  • a method of treating a neurological disorder comprises administering a PIKfyve inhibitor (e.g., apilimod, YM-201636, APY0201), or a pharmaceutically acceptable salt thereof, and methylcobalamin, wherein the subject is receiving or has received treatment with an antioxidant and/or (e.g., and) a glutamatergic agent.
  • a PIKfyve inhibitor e.g., apilimod, YM-201636, APY0201
  • a method of treating a neurological disorder comprises administering a PIKfyve inhibitor (e.g., apilimod, YM-201636, APY0201), or a pharmaceutically acceptable salt thereof, and methylcobalamin, wherein the subject is receiving or has received treatment with edaravone and/or (e.g., and) riluzole.
  • a PIKfyve inhibitor e.g., apilimod, YM-201636, APY0201
  • a method of treating a neurological disorder comprises administering a PIKfyve inhibitor (e.g., apilimod, YM-201636, APY0201), or a pharmaceutically acceptable salt thereof, and methylcobalamin, wherein the subject is not receiving or has not received treatment with an antioxidant (e.g., edaravone) or a glutamatergic agent (e.g., riluzole).
  • a PIKfyve inhibitor e.g., apilimod, YM-201636, APY0201
  • an antioxidant e.g., edaravone
  • glutamatergic agent e.g., riluzole
  • 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.
  • edaravone 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. [0087] 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.
  • 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
  • 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.
  • 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.
  • 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 methylcobalamin, and/or a PIKfyve inhibitor e.g., apilimod, YM-201636, APY0201
  • the glutamatergic agent e.g., riluzole
  • the administration route of the antioxidant e.g., edaravone
  • the administration route of the methylcobalamin may be intramuscularly or orally.
  • 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
  • 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.
  • Various forms of 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. 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.
  • 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 bi
  • ⁇ 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).
  • SMA1 infantile spinal muscular atrophy
  • SMA3 juvenile spinal muscular atrophy
  • 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.
  • adjuviate 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 prevent 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.
  • 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.
  • administration of a PIKfyve inhibitor may be simultaneous with or sequential to the administration of one or more additional therapeutic agents (e.g., methylcobalamin, and/or the glutamatergic agent, and/or the antioxidant).
  • additional therapeutic agents e.g., methylcobalamin, and/or the glutamatergic agent, and/or the antioxidant.
  • 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 (e.g., apilimod, YM-201636, APY0201), or a pharmaceutically acceptable salt thereof.
  • a PIKfyve inhibitor e.g., apilimod, YM-201636, APY0201
  • 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 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 e.g., apilimod, YM- 201636, APY0201
  • the a PIKfyve inhibitor e.g., apilimod, YM- 201636, APY0201
  • methylcobalamin and/or the glutamatergic agent and/or the antioxidant are administered in a single dosage form, or in separate dosage forms.
  • 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.
  • 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).
  • ALS a progressive 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.
  • a bulbar onset of the disease presenting with dysarthria, dysphagia, dysphonia, or more rarefy with masseter weakness.
  • 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.
  • FTD frontotemporal dementia
  • Apilimod dimesylate capsule is a first-in-class inhibitor of phosphatidylinositol-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 phosphatidylinositol-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 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.
  • TLR toll-like receptor
  • Methylcobalamin (Vitamin B12) is one of the two forms of biologically active vitamin B12 and is an essential cofactor in the enzyme methionine synthase, which functions to transfer methyl groups for the regeneration of methionine from homocysteine.
  • Methylcobalamin features an octahedral cobalt (III) center and can be obtained as bright red crystals.
  • vitamin B12 is an essential factor for regulating the mammalian nervous system functions by supporting neurite outgrowth, protecting neurons from glutamate toxicity, and by playing a role in recovery from nerve injury. (Ito, 2017).
  • the mechanisms of action of methylcobalamin are still not completely known.
  • the elimination of homocysteine which is neurotoxic, may be protective for the nervous tissue.
  • the antioxidant and anti-inflammatory properties of cobalamin may directly lead to beneficial results. (Oki, 2022).
  • 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 day 32 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.
  • 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).
  • 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.
  • iPSC-derived motoneurons from a TDP-43-ALS patient Apilimod and methylcobalamin provided an increase in viability of 26.67% with respect to cells growth-factors deprived (-GF), while Apilimod and methylcobalamin alone provide an increase of 5.32% and 3.63%, respectively (Table 6 and FIGURE 3).
  • Apilimod and methylcobalamin provided an increase in viability of 46.43% with respect to glutamate while Apilimod and methylcobalamin alone provide an increase of 14.95% and 4.48%, respectively (Table 7 and FIGURE 4).
  • the combination of Apilimod with methylcobalamin showed a synergistic effect (i.e. more than the sum of the improved viability with each compound alone) with respect to each single compound in any line tested.
  • iPSC-derived motoneurons from a C9orf72-ALS patient YM201636 and methylcobalamin provided an increase viability of 35.35% with respect to glutamate while YM201636 and methylcobalamin alone provide an increase of 6.95% and 9.66%, respectively (Table 8 and FIGURE 5).
  • iPSC-derived motoneurons from a SOD-1- ALS patient YM201636 and methylcobalamin provided an increase in viability of 35.75% with respect to glutamate while YM201636 and methylcobalamin alone provide an increase of 13.81% and 12.72%, respectively (Table 9 and FIGURE 6).
  • YM201636 and methylcobalamin provided an increase in viability of 33% with respect to cells growth-factors deprived (-GF), while YM201636 and methylcobalamin alone provide an increase of 6.18% and 8.91%, respectively (Table 10 and FIGURE 7).
  • YM201636 and methylcobalamin provided an increase in viability of 41.21 % with respect to glutamate while YM201636 and methylcobalamin alone provide an increase of 10.96% and 9.98%, respectively (Table 11 and FIGURE 8).
  • the combination of YM201636 with methylcobalamin showed a synergistic effect (i.e. more than the sum of the improved viability with each compound alone) with respect to each single compound in any line tested.
  • iPSC-derived motoneurons from a C9orf72-ALS patient APY0201 and methylcobalamin provided an increase viability of 37.38% with respect to glutamate while APY0201 and methylcobalamin alone provide an increase of 9.74% and 12%, respectively (Table 12 and FIGURE 9).
  • APY0201 and methylcobalamin provided an increase in viability of 38.45% with respect to glutamate while APY0201 and methylcobalamin alone provide an increase of 11.11% and 10.02%, respectively (Table 13 and FIGURE 10).
  • APY0201 and methylcobalamin provided an increase in viability of 42.9% with respect to cells growth-factors deprived (-GF), while APY0201 and methylcobalamin alone provide an increase of 7.6% and 6.61%, respectively (Table 14 and FIGURE 11).
  • APY0201 and methylcobalamin provided an increase in viability of 42.1% with respect to glutamate while APY0201 and methylcobalamin alone provide an increase of 4.56% and 6.59%, respectively (Table 15 and FIGURE 12).
  • the combination of APY0201 with methylcobalamin showed a synergistic effect (i.e. more than the sum of the improved viability with each compound alone) with respect to each single compound in any line tested.
  • Table 4 Increase in Cell Viability with respect to Glutamate in iPSC-derived motoneurons from a C9orf72-ALS patient.
  • Table 5 Increase in Cell Viability with respect to Glutamate in iPSC-derived motoneurons from a SOD-l-ALS patient.
  • Table 6 Increase in Cell Viability with respect to cells growth-factors deprivation in iPSC-derived motoneurons from a TDP-43-ALS patient.
  • Table 7 Increase in Cell Viability with respect to Glutamate in iPSC-derived motoneurons from a Sporadic-ALS patient.
  • Table 8 Increase in Cell Viability with respect to Glutamate in iPSC-derived motoneurons from a C9orf72-ALS patient.
  • Table 9 Increase in Cell Viability with respect to Glutamate in iPSC-derived motoneurons from a SOD-l-ALS patient.
  • Table 10 Increase in Cell Viability with respect to cells growth-factors deprivation, in iPSC-derived motoneurons from a TDP -43-ALS patient.
  • Table 11 Increase in Cell Viability with respect to Glutamate in iPSC-derived motoneurons from a Sporadic-ALS patient.
  • Table 12 Increase in Cell Viability with respect to Glutamate in iPSC-derived motoneurons from a C9orf72-ALS patient.
  • Table 13 Increase in Cell Viability with respect to Glutamate in iPSC-derived motoneurons from a SOD-l-ALS patient.
  • Table 14 Increase in Cell Viability with respect cells growth- factors deprivation in iPSC-derived motoneurons from a TDP-43-ALS patient.

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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 apilimod, APY0201, and YM201636 or a pharmaceutically acceptable salt thereof), or a pharmaceutically acceptable salt thereof and methylcobalamin. In some embodiments, further therapeutic agents (e.g., glutamatergic agents and/or antioxidants) are administered to the subject.

Description

APILIMOD COMBINATION THEREAPY
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63/425,434 filed November 15, 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 Apilimod 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
Figure imgf000003_0001
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 and was originally identified in a screen of PI3K class IA inhibitors and 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, including but not limited to apilimod, APY0201, and YM201636) for treating neurological diseases and disorders, particularly in combination with other therapeutic agents, e.g., methylcobalamin. As demonstrated herein, the combination of a PIKfyve inhibitor (e.g., any PIKfyve inhibitors known or described herein, including but not limited to apilimod, APY0201, and YM201636) and methylcobalamin 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) and/or antioxidants (e.g., edaravone).
[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 apilimod or a pharmaceutically acceptable salt thereof and methylcobalamin. In some embodiments, apilimod or a pharmaceutically acceptable salt thereof and methylcobalamin are in one composition. In some embodiments, apilimod or a pharmaceutically acceptable salt thereof and methylcobalamin 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
Figure imgf000004_0001
administering to the subject methylcobalamin, wherein the subject is receiving or has received treatment with 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 apilimod or a pharmaceutically acceptable salt thereof, wherein the subject is receiving or has received treatment with methylcobalamin.
[0009] In some embodiments, the subject is administered 500-750 pg of methylcobalamin. In some embodiments, methylcobalamin is administered to the subject twice daily. In some embodiments, methylcobalamin is administered to the subject three times daily. In some embodiments, the composition is administered orally.
[0010] In some embodiments, the subject is administered 25 mg - 50 mg of methylcobalamin. In some embodiments, the subject is administered 50 mg of methylcobalamin. In some embodiments, methylcobalamin is administered to the subject twice weekly. In some embodiments, methylcobalamin is administered intramuscularly.
[0011] In some embodiments, apilimod is apilimod dimesylate. In some embodiments, apilimod is administered orally. In some embodiments, the subject is administered 30-300 mg of apilimod or a pharmaceutically acceptable salt thereof. In some embodiments, or a pharmaceutically acceptable salt thereof is administered daily. In some embodiments, apilimod or a pharmaceutically acceptable salt thereof is administered twice daily.
[0012] 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. In some embodiments, the subject is not receiving of has received treatment with a glutamatergic agent. In some embodiments, the glutamatergic agent is riluzole.
[0013] In some embodiments, the method further comprises administering to the subject an antioxidant. In some embodiments, the antioxidant is edaravone. In some embodiments, edaravone is administer 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-
Figure imgf000005_0001
day period, further followed by 14 days of no administration. In some embodiments, the antioxidant is administered intravenously. In some embodiments, the antioxidant is administered at a daily dose of 105 mg. In some embodiments, the antioxidant is administered orally. In some embodiments, the subject is not receiving or has not received treatment with an antioxidant. In some embodiments, the antioxidant is edaravone.
[0014] In some embodiments, the method further comprises administering to the subject a glutamatergic agent and an antioxidant. In some embodiments, the subject is not receiving or has not received treatment with a glutamatergic agent or an antioxidant. In some embodiments, the glutamatergic agent is riluzole and the antioxidant is edaravone.
[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, wherein 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). In some embodiments, the subject 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 a mutation in the TDP-43 gene. In some embodiments, the subject in need of treatment is one having accumulation of TDP-34 aggregates. In some embodiments, the subject is human.
[0016] Further provided herein are uses of a PIKFyve inhibitor (e.g., any PIKfyve inhibitors known or described herein, including but not limited to apilimod, APY 0201 , and
Figure imgf000006_0001
YM201636 or a pharmaceutically acceptable salt thereof) and methylcobalamin in a method of treating a neurological disease or disorder in a subject in need thereof
[0017] Further provided herein are uses of a PIKFyve inhibitor (e.g., any PIKfyve inhibitors known or described herein, including but not limited to apilimod, APY 0201 , and YM201636 or a pharmaceutically acceptable salt thereof) 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 methylcobalamin.
[0018] Further provided herein are uses of methylcobalamin 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 (e.g., any PIKfyve inhibitors known or described herein, including but not limited to apilimod, APY0201, and YM201636 or a pharmaceutically acceptable salt thereof).
BRIEF DESCRIPTION OF DRAWINGS
[0019] 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.
[0020] FIGURE 1 is a graph demonstrating the ability of methylcobalamin (1 nM) 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 significance of p<0.0001; ** denotes significance of p<0.001; * denotes significance of p<0.05. The combination provides an increase in viability of 34.65% with respect to glutamate while Apilimod and methylcobalamin provide an increase of 7.72% and 2.63%, respectively (see also, Table 4).
[0021] FIGURE 2 is a graph demonstrating the ability of methylcobalamin (1 nM) 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<0.001; The combination provides an increase in viability of 34.65% with respect to glutamate while Apilimod and methylcobalamin provide an increase of 7.72% and 2.63%, respectively (see also, Table 5).
Figure imgf000007_0001
[0022] FIGURE 3 is a graph demonstrating the ability of methylcobalamin (1 nM) and apilimod (6.25 nM) in combination to increase cell viability after growth factor deprivation in iPSC-derived motoneurons from a TDP-43-ALS patient. ** denotes significance of p<0.001; * denotes significance of p<0.05. The combination provides an increase in viability of 26.67% with respect to cells growth-factors deprived (-GF), while Apilimod and methylcobalamin provide an increase of 5.32% and 3.63%, respectively (see also, Table 6).
[0023] FIGURE 4 is a graph demonstrating the ability of methylcobalamin (1 nM) 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<0.0001; ** denotes significance of p<0.001. The combination provides an increase in viability of 46.43% with respect to glutamate, while Apilimod and methylcobalamin provide an increase of 14.95% and 4.48%, respectively (see also, Table
7).
[0024] FIGURE 5 is a graph demonstrating the ability of methylcobalamin (1 nM) 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 of p<0.05. The combination provides an increase in viability of 35.35% with respect to glutamate while YM201636 and methylcobalamin provide an increase of 6.96% and 9.66%, respectively (see also, Table
8).
[0025] FIGURE 6 is a graph demonstrating the ability of methylcobalamin (1 nM) 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 ofp<0.0001; * denotes significance of p<0.05; The combination provides an increase in viability of 35.75% with respect to glutamate while YM201636 and methylcobalamin provide an increase of 13.81% and 12.72%, respectively (see also, Table
9).
[0026] FIGURE 7 is a graph demonstrating the ability of methylcobalamin (1 nM) and YM201636 (12.5 nM) in combination to increase cell viability synergistically after growth
Figure imgf000008_0001
factor deprivation in iPSC-derived motoneurons from a TDP-43-ALS patient. *** denotes significance of p<0.0001; * denotes significance of p<0.05. The combination provides an increase in viability of 33% with respect to cells growth-factors deprived (-GF), while YM201636 and methylcobalamin provide an increase of 6.18% and 8.91%, respectively (see also, Table 10).
[0027] FIGURE 8 is a graph demonstrating the ability of methylcobalamin (1 nM) 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 of p<0.001; The combination provides an increase in viability of 41.21% with respect to glutamate, while YM201636 and methylcobalamin provide an increase of 10.26% and 9.28%, respectively (see also, Table 11).
[0028] FIGURE 9 is a graph demonstrating the ability of methylcobalamin (1 nM) and APY0201 (6.25 nM) in combination to synergistically increase cell viability after glutamate exposure in iPSC-derived motoneurons from a C9orf72-ALS patient. ** denotes significance of p<0.001; * denotes significance of p<0.05. The combination provides an increase in viability of 37.38% with respect to glutamate while APY0201 and methylcobalamin provide an increase of 9.74% and 12%, respectively (see also, Table 12). [0029] FIGURE 10 is a graph demonstrating the ability of methylcobalamin (1 nM) and APY0201 (6.25 nM) in combination to synergistically increase cell viability after glutamate exposure in iPSC-derived motoneurons from a SOD-l-ALS patient. ** denotes significance of p<0.001; * denotes significance of p<0.05. The combination provides an increase in viability of 38.45% with respect to glutamate while APY0201 and methylcobalamin provide an increase of 11.11% and 10.02%, respectively (see also, Table 13).
[0030] FIGURE 11 is a graph demonstrating the ability of methylcobalamin (1 nM) and APY0201 (6.25 nM) in combination to synergistically increase cell viability after growth factor deprivation in iPSC-derived motoneurons from a TDP-43-ALS patient. ** denotes significance of p<0.001; * denotes significance of p<0.05. The combination provides an increase in viability of 42.9% with respect to cells growth- factors deprived (-
Figure imgf000009_0001
GF), while APY0201and methylcobalamin provide an increase of 7.6% and 6.61%, respectively (see also, Table 14).
[0031] FIGURE 12 is a graph demonstrating the ability of methylcobalamin (1 nM) and APY0201 (6.25 nM) in combination to synergistically increase cell viability after glutamate exposure in iPSC-derived motoneurons from a Sporadic-ALS patient. *** denotes significance of p<0.0001. The combination provides an increase in viability of 42.12% with respect to glutamate while APY0201 and methylcobalamin provide an increase of 4.56% and 6.59%, respectively (see also, Table 15).
DEFINITIONS
[0032] 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., apilimod, YM-201636, APY0201). 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), mal onate, 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, l-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., apilimod, YM-201636, APY0201), 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.
[0033] 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
Figure imgf000010_0001
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-hydroxyethyl)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 apilimod (e.g., 2- [2-Pyridin-2-yl)-ethoxy]-4-N’-(3-methyl-benzilidene)-hydrazino]-6-(morpholin-4-yl)- pyrimidine), 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.
[0034] The salts of a compound (e.g., apilimod) may be synthesized from the parent compound (e.g., 2-[2-Pyridin-2-yl)-ethoxy]-4-N’-(3-methyl-benzilidene)-hydrazino]-6- (morpholin-4-yl)-pyrimidine) by conventional chemical methods. Generally, such salts can be prepared by reacting the parent compound (e.g., 2-[2-Pyridin-2-yl)-ethoxy]-4-N’-(3- methyl-benzilidene)-hydrazino]-6-(morpholin-4-yl)-pyrimidine) with the appropriate acid in water or in an organic solvent, or in a mixture of the two.
[0035] One salt form of a compound described herein (e.g., apilimod, YM-201636, APY 0201) 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
Figure imgf000011_0001
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.
[0036] As described herein, the term “polymorph” may refer to solid crystalline forms of a compound of the present disclosure (e.g., apilimod, YM-201636, APY0201) 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.
[0037] As described herein, the term “hydrate” may describe a compound of the present disclosure (e.g., apilimod, YM-201636, APY0201) or a salt thereof, which further includes a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces.
[0038] As described herein, the term “clathrate” may refer to a compound of the present disclosure (e.g., apilimod, YM-201636, APY0201) 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.
[0039] As described herein, the term “prodrug” may refer to a derivative of a compound described herein (e.g., apilimod, YM-201636, APY0201) 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
Figure imgf000012_0001
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.
[0040] Some of the compounds suitable for use in the methods described in this disclosure (e.g., apilimod, YM-201636, APY0201) 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., apilimod, YM-201636, APY0201) 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., apilimod, YM-201636, APY0201) are expressly included in the present disclosure.
[0041] 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., apilimod, YM-201636, APY0201). The term solvate may include hydrates (e.g., hemi-hydrate, mono-hydrate, dihydrate, trihydrate, tetrahydrate, and the like).
[0042] 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
Figure imgf000013_0001
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
[0043] 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. Methylcobalamin (Vitamin B 12) is currently undergoing clinical trials for the treatment of ALS. The present disclosure, in some aspects, demonstrates that administration of apilimod and methylcobalamin in (iPSC)-derived motoneurons from ALS patients has synergistic results in cell viability. The combination therapy of methylcobalamin and apilimod provides unexpected benefits on neuronal survival that are greater than the application of either therapy individually.
[0044] 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 methylcobalamin 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 methylcobalamin. In some embodiments, a method described herein comprises administering to a subject a composition (e.g., in a solid oral dosing form such as a tablet) comprising a PIKfyve inhibitor (e.g., any PIKfyve inhibitors known or described herein) or a pharmaceutically acceptable salt thereof, and methylcobalamin.
[0045] 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 methylcobalamin. In some embodiments a method described herein comprises administering methylcobalamin to a subject in need thereof, wherein the subject is receiving or has received treatment with a PIKfyve inhibitor (e.g.,
Figure imgf000014_0001
any PIKfyve inhibitors known or described herein), or a pharmaceutically acceptable salt thereof.
[0046] 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
Figure imgf000015_0001
Genomics; prodrug of VRG50648), VRG50648 (by Verge Genomics; active form of VRG50635) and XBA (MLS001167909).
[0047] 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, WO 19046316, 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.
[0048] In some embodiments, a PIKfyve inhibitor used in accordance with the present disclosure is a selective PIKfyve inhibitor (i.e., inhibitor that selectively inhibits PIKfyve versus other kinases). In some embodiments, the selective PIKfyve inhibitor is apilimod, or an analog or salt thereof. 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.
[0049] 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 imgf000016_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.
[0050] The term “APY0201” refers to 2-[7-(4-morpholinyl)-2-(4- pyridinyl)pyrazolo[ 1 ,5-a]pyrimidin-5-yl]hydrazone, 3-methyl-benzaldehyde, represented by Formula II:
Figure imgf000017_0001
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).
[0051] The term “YM201636” refers to a compound with CAS number of 371942-69- 7 and a structure represented by Formula III:
Figure imgf000017_0002
Figure imgf000018_0001
or any pharmaceutically acceptable salt, solvate, clathrate, hydrate, polymorph, metabolite, prodrug, analog, or derivative thereof. YM201636 is a PIKFyve inhibitor with a morpholino-pyrimidine (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).
[0052] The term “MOMIPP” refers to a compound with CAS number of 1363421-46- 8 and a structure represented by Formula IV:
Figure imgf000018_0002
or any pharmaceutically acceptable salt, solvate, clathrate, hydrate, polymorph, metabolite, prodrug, analog, or derivative thereof.
[0053] The term “MF4” refers to a compound with a structure represented by Formula (V):
Figure imgf000018_0003
Figure imgf000019_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.
[0054] The term “AS2677131” refers to a compound with CAS number of 2171502- 44-4 and a structure represented by Formula (VI):
Figure imgf000019_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 , 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).
[0055] 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 imgf000019_0003
Figure imgf000020_0001
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).
[0056] The term “Vacuolin-1” refers to a compound with CAS number of 351986-85- 1 and a structure represented by Formula (VIII):
Figure imgf000020_0002
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).
[0057] The term “WX8” (MLS000543798; PubChem CID 135510930) refers to 1H- indole-3-carbaldehyde [4-anilino-6-(4-morpholinyl)-l,3,5-triazin-2-yl]hydrazine, a compound represented by Formula (IX):
Figure imgf000020_0003
Figure imgf000021_0001
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).
[0058] The term “NDF” (MLS000699212; PubChem CID 9629709) refers to 3- methylbenzaldehyde (2,6-dimorpholin-4-ylpyrimidin-4-yl)hydrazine, represented by Formula (X):
Figure imgf000021_0002
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.
[0059] The term “WWL” (MLS000703078) refers to benzaldehyde [2,6-di(4- morpholinyl)-4-pyrimidinyl]hydrazone, represented by Formula (XI):
Figure imgf000021_0003
Figure imgf000022_0001
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.
[0060] 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 imgf000022_0002
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.
[0061] The term “XB6” (MLS001167897), refers to N-(4-ethylphenyl)- 4,6- dimorpholino-l,3,5-triazin-2-amine hydrochloride, represented by Formula (XIII):
Figure imgf000022_0003
Figure imgf000023_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.
[0062] In some embodiments, in any one of the methods described herein, the PIKfyve inhibitor is apilimod or a pharmaceutically acceptable salt thereof. In some embodiments, in any one of the methods described herein, 30-300 (e.g., 30-300, 30-200, 30-100, 30-80, 30-60, 30-50, 30-40, 40-300, 40-200, 40-100, 40-80, 40-60, 40-50, 50-300, 50-200, 50- 100, 50-80, 50-60, 60-300, 60-200, 60-100, 60-80, 80-300, 80-200, 80-100, 100-300, 1000- 200, 200-300) milligrams per day of apilimod or a pharmaceutically acceptable salt thereof, may be administered to a subject. In some embodiments, in any one of the methods described herein, about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, or 300 milligrams per day of apilimod or a pharmaceutically acceptable salt thereof, may be administered to a subject. In some embodiments, in any one of the methods described herein, about 100-300 milligrams/day of apilimod or a pharmaceutically acceptable salt thereof, is administered to the subject. In some embodiments, in any one of the methods described herein, about 200 or 250 milligrams/day of apilimod or a pharmaceutically acceptable salt thereof, is administered to the subject. In some embodiments, in any one of the methods described herein, a subject is administered apilimod twice a day, and each administration provides to the subject 75-150 (e.g., 75-150, 75-125, 75-100, 100-150, 100-125, or 125-150 mg) milligrams of apilimod. In some embodiments, in any one of the methods described herein, a subject is administered apilimod twice a day, and each administration provides to the subject 75 milligrams, 100 milligrams, 125 milligrams, or 150 milligrams of apilimod.
Figure imgf000023_0002
[0063] In some embodiments, in any one of the methods described herein, apilimod or a pharmaceutically acceptable salt thereof, 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, apilimod or a pharmaceutically acceptable salt thereof, is administered to the subject 1, 2, or 3 times daily. In some embodiments, in any one of the methods described herein, apilimod or a pharmaceutically acceptable salt thereof, is administered to the subject daily. In some embodiments, in any one of the methods described herein, apilimod or a pharmaceutically acceptable salt thereof, is administered to the subject twice daily.
[0064] In some embodiments, in any one of the methods described herein, apilimod or a pharmaceutically acceptable salt thereof, is administered to the subject over a period of time. In some embodiments, in any one of the methods described herein, the period of time may be 1-60 (e.g., 1-60, 1-52, 1-50, 1-40, 1-30, 1-20, 1-16, 1-10, 1-8, 1-4, 4-60, 4-52, 4-50, 4-40, 4-30, 4-20, 4-16, 4-10, 4-8, 8-60, 8-52, 8-50, 8-40, 8-30, 8-20, 8-16, 8-10, 10-60, 10- 52, 10-50, 10-40, 10-30, 10-20, 10-16, 16-60, 16-52, 16-50, 16-40, 16-30, 16-20, 20-60, 20-52, 20-50, 20-40, 20-30, 30-60, 30-52, 30-50, 30-40, 40-60, 40-52, 40-50, 50-60, SO- 52, 52-60) 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, 14, 16, 18, 20, 30, 40, 50, 52, 60 weeks. In some embodiments, in any one of the methods described herein, the period of time is about 4-16 weeks. In some embodiments, in any one of the methods described herein, the period of time is about 8 weeks. In some embodiments, in any one of the methods described herein, the period of time is about 52 weeks. In some embodiments, in any one of the methods described herein, the period time is the remainder of the subject’s lifetime.
[0065] In some embodiments, in any one of the methods described herein, apilimod or a pharmaceutically acceptable salt thereof, is administered to the subject according to a dosage regimen of about 30-300 mg per day for at least 1 week. In some embodiments, in any one of the methods described herein, apilimod or a pharmaceutically acceptable salt thereof, is administered to the subject according to a dosage regimen of about 100-300 mg per day for about 4 or 16 weeks. In some embodiments, in any one of the methods described herein, apilimod or a pharmaceutically acceptable salt thereof, is administered to the subject according to a dosage regimen of about 100 mg per day, twice a day for about 8 weeks, or
Figure imgf000024_0001
optionally for about 52 weeks or longer. In some embodiments, in any one of the methods described herein, apilimod or a pharmaceutically acceptable salt thereof, is administered to the subject according to a dosage regimen of about 100-300 mg per day, twice a day for the remainder of the subject’s lifetime. In some embodiments, in any one of the methods described herein, apilimod or a pharmaceutically acceptable salt thereof, is administered to the subject according to a dosage regimen of about 100-300 mg per day, twice a day until the subject is no longer suitable for receiving treatment with apilimod.
[0066] According to the present disclosure, a PIKfyve inhibitor (e.g., apilimod, YM- 201636, APY0201) or a pharmaceutically acceptable salt thereof is used in combination with methylcobalamin for treating neurological diseases and disorders.
[0067] 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- lH-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.
[0068] As described herein, methylcobalamin is a form of vitamin B12, known as cobalamin. Methylcobalamin contains metal-alkyl bonds and an octahedral cobalt(III) center and can be obtained as a bright red crystal. Within the cytosol of the cell methylcobalamin functions as a cofactor to methionine synthase. Methionine synthase catalyzes the remethylation of homocysteine to methionine. In methyl group transfer methylcobalamin has an important role in the transfer of one carbon (e.g., as described in Grober U., et al, Neuroenhancement with Vitamin B12 — Underestimated Neurological Significance. Nutrients. 2013 Dec; 5(12): 5031-5045., incorporated herein by reference). Commonly vitamin B12 deficiency has been linked to depression, irritability, and psychosis. Long-term deficiency can lead to cardiovascular disease and elevated levels of homocysteine in the blood. Lesions in the central nervous system, including in the spinal cord have been linked to vitamin B12 deficiency, indicating an important role for the vitamin B12 in the nervous system. Methylcobalamin is a standard treatment in patients with vitamin B12 deficiency, has been used in treat peripheral neuropathy in Japan, and
Figure imgf000025_0001
recently entered clinical trials for the use in ALS. Methylcobalamin has been found to inhibit neuronal degradation through the decrease of homocysteine and the induction of neurite outgrowth, prolonging neuronal survival. Oral treatment with methylcobalamin results in improved peripheral neuropathy. In clinical studies intramuscular (IM) injection of ultra-high doses of methylcobalamin slowed the functional decline in early-stage ALS patients.
[0069] In some embodiments, in any one of the methods described herein, methylcobalamin is administered to the subject over a period of time. In some embodiments, in any one of the methods described herein, the period of time may be 1 -60 (e.g., 1-60, 1-52, 1-50, 1-40, 1-30, 1-20, 1-16, 1-10, 1-8, 1-4, 4-60, 4-52, 4-50, 4-40, 4-30, 4-20, 4-16, 4-10, 4-8, 8-60, 8-52, 8-50, 8-40, 8-30, 8-20, 8-16, 8-10, 10-60, 10-52, 10-50, 10-40, 10-30, 10-20, 10-16, 16-60, 16-52, 16-50, 16-40, 16-30, 16-20, 20-60, 20-52, 20- 50, 20-40, 20-30, 30-60, 30-52, 30-50, 30-40, 40-60, 40-52, 40-50, 50-60, 50-52, 52-60) 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, 14, 16, 18, 20, 30, 40, 50, 52, 60 weeks. In some embodiments, in any one of the methods described herein, the period of time is about 4-16 weeks. In some embodiments, in any one of the methods described herein, the period of time is about 8 weeks. In some embodiments, in any one of the methods described herein, the period of time is about 52 weeks. In some embodiments, in any one of the methods described herein, the period time is the remainder of the subject’s lifetime.
[0070] In some embodiments, in any one of the methods described herein, 1000-1500 (e.g., 1000-1500, 1000-1100, 1100-1200, 1200-1300, 1300-1400, 1400-1500, 1000-1200, 1000-1300, 1000-1400, 1100-1500, 1200-1500, 1300-1500, 1100-1300, 1100-1400, 1100- 1500, 1200-1400, 1200-1500, 1300-1500) micrograms per day of methylcobalamin may be administered (e.g., orally) to a subject. In some embodiments, in any one of the methods described herein, about 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500 micrograms per day of methylcobalamin, may be administered (e.g., orally) to a subject. In some embodiments, in any one of the methods described herein, about 1000-1500 micrograms/day of methylcobalamin is administered (e.g., orally) to the subject. In some embodiments, in any one of the methods described herein, about 1000 or 1500
Figure imgf000026_0001
micrograms/day of methylcobalamin, is administered (e.g., orally) to the subject. In some embodiments, in any one of the methods described herein, a subject is administered (e.g., orally) methylcobalamin twice a day, and each administration provides to the subject SOO- SO (e.g., 500-550, 550-600, 600-650, 650-700, or 700-750 pg) micrograms of methylcobalamin. In some embodiments, in any one of the methods described herein, a subject is administered (e.g., orally) methylcobalamin twice a day, and each administration provides to the subject 500 micrograms, 550 micrograms, 600 micrograms, 650 micrograms, 700 micrograms or 750 micrograms of methylcobalamin. In some embodiments, in any one of the methods described herein, a subject is administered (e.g., orally) methylcobalamin three times a day, and each administration provides to the subject 350-500 (e.g., 350-400, 400-450, or 450-500 pg) micrograms of methylcobalamin. In some embodiments, in any one of the methods described herein, a subject is administered (e.g., orally) methylcobalamin three times a day, and each administration provides to the subject 350 micrograms, 400 micrograms, 450 micrograms, 500 micrograms, or 750 micrograms of methylcobalamin.
[0071] In some embodiments, in any one of the methods described herein, methylcobalamin, 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, methylcobalamin is administered to the subject 1, 2, or 3 times daily. In some embodiments, in any one of the methods described herein, methylcobalamin is administered to the subject daily. In some embodiments, in any one of the methods described herein, methylcobalamin is administered to the subject twice daily.
[0072] In some embodiments, in any one of the methods described herein, methylcobalamin is administered (e.g., orally) to the subject according to a dosage regimen of about 1000-1500 pg per day for at least 1 week. In some embodiments, in any one of the methods described herein, methylcobalamin is administered (e.g., orally) to the subject according to a dosage regimen of about 1000-1500 pg per day for about 4 or 16 weeks. In some embodiments, in any one of the methods described herein, methylcobalamin is administered (e.g., orally) to the subject according to a dosage regimen of about 500-750 pg twice a day for about 8 weeks, or optionally for about 52 weeks or longer. In some
Figure imgf000027_0001
embodiments, in any one of the methods described herein, methylcobalamin is administered (e.g., orally) to the subject according to a dosage regimen of about 1000-1500 pg twice a day for the remainder of the subject’s lifetime. In some embodiments, in any one of the methods described herein, methylcobalamin is administered (e.g., orally) to the subject according to a dosage regimen of about 500-750 jag twice a day until the subject is no longer suitable for receiving treatment with methylcobalamin. In some embodiments, in any one of the methods described herein, methylcobalamin is administered (e.g., orally) to the subject according to a dosage regimen of about 350-500 jag three times a day for about 8 weeks, or optionally for about 52 weeks or longer. In some embodiments, in any one of the methods described herein, methylcobalamin is administered (e.g., orally) to the subject according to a dosage regimen of about 350-500 pg three times a day for the remainder of the subject’s lifetime. In some embodiments, in any one of the methods described herein, methylcobalamin is administered (e.g., orally) to the subject according to a dosage regimen of about 350-500 pg three times a day until the subject is no longer suitable for receiving treatment with methylcobalamin.
[0073] In some embodiments, in any one of the methods described herein, 50-100 (e.g., 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, 95-100, 60-100, 70-100, 80-100, 90-100, 50-70, 50-80, 50-90, 60-80, 60-90, 70-90) milligrams per week of methylcobalamin, may be administered (e.g., intramuscularly) to a subject. In some embodiments, in any one of the methods described herein, about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 milligrams per week of methylcobalamin, may be administered (e.g., intramuscularly) to a subject. In some embodiments, in any one of the methods described herein, about 50-100 milligrams/week of methylcobalamin is administered (e.g., intramuscularly) to the subject. In some embodiments, in any one of the methods described herein, about 50 or 100 milligrams/week of methylcobalamin, is administered (e.g., intramuscularly) to the subject. In some embodiments, in any one of the methods described herein, a subject is administered (e.g., intramuscularly) methylcobalamin twice a week, and each administration provides to the subject 25-50 (e.g., 25-30, 30-35, 35-40, 40-45, or 45- 50 mg) milligrams of methylcobalamin. In some embodiments, in any one of the methods described herein, a subject is administered (e.g., intramuscularly) methylcobalamin twice
Figure imgf000028_0001
a week, and each administration provides to the subject 25 milligrams, 30 milligrams, 35 milligrams, 40 milligrams, 45 milligrams, or 50 milligrams of methylcobalamin. In some embodiments in any one of the methods described herein, a subject is administered (e.g., intramuscularly) methylcobalamin twice a week, and each administration provides to the subject 50 milligrams.
[0074] In some embodiments, in any one of the methods described herein, methylcobalamin, is administered (e.g., intramuscularly) to the subject 1-2 times weekly. In some embodiments, in any one of the methods described herein, methylcobalamin is administered (e.g., intramuscularly) to the subject 1 or 2 times weekly. In some embodiments, in any one of the methods described herein, methylcobalamin is administered (e.g., intramuscularly) to the subject weekly. In some embodiments, in any one of the methods described herein, methylcobalamin is administered (e.g., intramuscularly) to the subject twice a week.
[0075] In some embodiments, in any one of the methods described herein, methylcobalamin is administered (e.g., intramuscularly) to the subject according to a dosage regimen of about 50-100 mg per week for at least 1 week. In some embodiments, in any one of the methods described herein, methylcobalamin administered (e.g., intramuscularly) to the subj ect according to a dosage regimen of about 50-100 mg per week for about 4 or 16 weeks. In some embodiments, in any one of the methods described herein, methylcobalamin is administered (e.g., intramuscularly) to the subject according to a dosage regimen of about 50-100 mg twice a week, for about 8 weeks, or optionally for about 52 weeks or longer. In some embodiments, in any one of the methods described herein, methylcobalamin is administered (e.g., intramuscularly) to the subject according to a dosage regimen of about 50-100 mg twice a week for the remainder of the subject’s lifetime. In some embodiments, in any one of the methods described herein, methylcobalamin is administered (e.g., intramuscularly) to the subject according to a dosage regimen 25-50 mg twice a week, until the subject is no longer suitable for receiving treatment with methylcobalamin. In some embodiments methylcobalamin is administered intramuscularly to the subject.
Figure imgf000029_0001
[0076] 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.
[0077] 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).
[0078] 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.
[0079] 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.
[0080] In some embodiments, a method of treating a neurological disorder comprises administering a PIKfyve inhibitor (e.g., apilimod, YM-201636, APY0201) or a pharmaceutically acceptable salt thereof, and methylcobalamin, 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 (e.g., apilimod, YM-201636, APY0201), or a pharmaceutically acceptable salt thereof, and methylcobalamin, wherein the subject is receiving or has received treatment with riluzole.
Figure imgf000030_0001
In some embodiments, a method of treating a neurological disorder comprises administering a PIKfyve inhibitor (e.g., apilimod, YM-201636, APY0201), or a pharmaceutically acceptable salt thereof, and methylcobalamin, wherein the subject is not receiving or has not received treatment with a glutamatergic agent (e.g., riluzole).
[0081] 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. [0082] 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.
[0083] In some embodiments, any one of the methods described herein further comprises administering to the subject an antioxidant. 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.
[0084] In some embodiments, a method of treating a neurological disorder comprises administering a PIKfyve inhibitor (e.g., apilimod, YM-201636, APY0201), or a pharmaceutically acceptable salt thereof, and methylcobalamin, wherein the subject is
Figure imgf000031_0001
receiving or has received treatment with an antioxidant. In some embodiments, a method of treating a neurological disorder comprises administering a PIKfyve inhibitor (e.g., apilimod, YM-201636, APY0201), or a pharmaceutically acceptable salt thereof, and methylcobalamin, wherein the subject is receiving or has received treatment with edaravone. In some embodiments, a method of treating a neurological disorder comprises administering a PIKfyve inhibitor (e.g., apilimod, YM-201636, APY0201), or a pharmaceutically acceptable salt thereof, and methylcobalamin, wherein the subject is not receiving or has not received treatment with an antioxidant (e.g., edaravone).
[0085] In some embodiments, a method of treating a neurological disorder comprises administering a PIKfyve inhibitor (e.g., apilimod, YM-201636, APY0201), or a pharmaceutically acceptable salt thereof, and methylcobalamin, wherein the subject is receiving or has received treatment with an antioxidant and/or (e.g., and) a glutamatergic agent. In some embodiments, a method of treating a neurological disorder comprises administering a PIKfyve inhibitor (e.g., apilimod, YM-201636, APY0201), or a pharmaceutically acceptable salt thereof, and methylcobalamin, wherein the subject is receiving or has received treatment with edaravone and/or (e.g., and) riluzole. In some embodiments, a method of treating a neurological disorder comprises administering a PIKfyve inhibitor (e.g., apilimod, YM-201636, APY0201), or a pharmaceutically acceptable salt thereof, and methylcobalamin, wherein the subject is not receiving or has not received treatment with an antioxidant (e.g., edaravone) or a glutamatergic agent (e.g., riluzole).
[0086] 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.
Figure imgf000032_0001
[0087] 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.
[0088] 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.
[0089] 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.
[0090] 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
Figure imgf000033_0001
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 methylcobalamin, and/or a PIKfyve inhibitor (e.g., apilimod, YM-201636, APY0201), 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 the administration route of the methylcobalamin may be intramuscularly or orally.
[0091] 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.
[0092] 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
Figure imgf000034_0001
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).
[0093] 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 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. 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.
Figure imgf000035_0001
[0094] 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.
[0095] 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.
[0096] \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).
[0097] Neurodevelopmental disorders that may be treated according to the methods described herein may include but is not limit to Rett syndrome.
[0098] 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
Figure imgf000036_0001
subject’s response to therapy as not clinically adequate to relieve one or more symptoms associated with the neurological disease” or disorder.
[0099] 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.
[0100] 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.
[0101] 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
Figure imgf000037_0001
as part of separate monotherapy regimens that incidentally and arbitrarily result in the combinations of the present disclosure.
[0102] In the context of combination therapy, administration of a PIKfyve inhibitor (e.g., apilimod, YM-201636, APY0201), or a pharmaceutically acceptable salt thereof, may be simultaneous with or sequential to the administration of one or more additional therapeutic agents (e.g., methylcobalamin, and/or the glutamatergic agent, and/or the antioxidant). 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 (e.g., apilimod, YM-201636, APY0201), or a pharmaceutically acceptable salt thereof.
[0103] 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.
[0104] 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
Figure imgf000038_0001
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.
[0105] 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.
[0106] 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.
Figure imgf000039_0001
[0107] 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.
[0108] 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. [0109] 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
Figure imgf000040_0001
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™.
[0110] 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.
[0111] 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.
[0112] 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.
[0113] 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
Figure imgf000041_0001
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.
[0114] 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.
[0115] 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
Figure imgf000042_0001
and their amphiphilic derivatives, amino acids and their salts, and organic acids and their esters and anhydrides.
[0116] 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 (e.g., apilimod, YM- 201636, APY0201), may be administered at the same time or at a different time, the context of the combination therapy with methylcobalamin, and/or the glutamatergic agent, and/or the antioxidant. In some embodiments, the a PIKfyve inhibitor (e.g., apilimod, YM- 201636, APY0201), and/or methylcobalamin and/or the glutamatergic agent and/or the antioxidant are administered in a single dosage form, or in separate dosage forms.
[0117] 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.
[0118] 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.
[0119] 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
Figure imgf000043_0001
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
[0120] 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).
[0121] 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).
[0122] 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 rarefy 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
Figure imgf000044_0001
be two ends of a spectrum due to the overlap in molecular mechanisms underlying both neurodegenerative disorders (Goutman 2022).
[0123] 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-binding 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.
[0124] To date, there are a handful of FDA approved drugs for use in the treatment of ALS, e.g., Qalsody, Relyviro, Radicava, Rilutek and Tiglutik.
[0125] There are several other drugs currently in clinical trials including ultra-high dose methylcobalamin which was efficacious in slowing functional decline in patients with early-stage ALS in a randomized clinical trial in Japan (Oki, 2022).
[0126] Apilimod dimesylate capsule (Apilimod) is a first-in-class inhibitor of phosphatidylinositol-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.
Figure imgf000045_0001
[0127] 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.
[0128] Methylcobalamin (Vitamin B12) is one of the two forms of biologically active vitamin B12 and is an essential cofactor in the enzyme methionine synthase, which functions to transfer methyl groups for the regeneration of methionine from homocysteine. Methylcobalamin features an octahedral cobalt (III) center and can be obtained as bright red crystals. Beside other functions, vitamin B12 is an essential factor for regulating the mammalian nervous system functions by supporting neurite outgrowth, protecting neurons from glutamate toxicity, and by playing a role in recovery from nerve injury. (Ito, 2017). The mechanisms of action of methylcobalamin are still not completely known. On one hand the elimination of homocysteine, which is neurotoxic, may be protective for the nervous tissue. On the other hand, the antioxidant and anti-inflammatory properties of cobalamin may directly lead to beneficial results. (Oki, 2022).
[0129] Results
[0130] 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
Figure imgf000046_0001
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 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). 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 day 32 of differentiation.
Figure imgf000047_0001
[0131] 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).
[0132] 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.
[0133] For the iPSC-derived motoneurons from a C9orf72-ALS patient Apilimod and methylcobalamin provided an increase viability of 34.65% with respect to glutamate while Apilimod and methylcobalamin alone provide an increase of 7.72% and 2.63%, respectively (Table 4 and FIGURE 1). For the iPSC-derived motoneurons from a SOD-1- ALS patient Apilimod and methylcobalamin provided an increase in viability of 34.65% with respect to glutamate while Apilimod and methylcobalamin alone provide an increase of 7.72% and 2.63%, respectively (Table 5 and FIGURE 2). For the iPSC-derived motoneurons from a TDP-43-ALS patient Apilimod and methylcobalamin provided an increase in viability of 26.67% with respect to cells growth-factors deprived (-GF), while Apilimod and methylcobalamin alone provide an increase of 5.32% and 3.63%, respectively (Table 6 and FIGURE 3). For the iPSC-derived motoneurons from a Sporadic- ALS patient Apilimod and methylcobalamin provided an increase in viability of 46.43% with respect to glutamate while Apilimod and methylcobalamin alone provide an increase
Figure imgf000048_0001
of 14.95% and 4.48%, respectively (Table 7 and FIGURE 4). In summary, the combination of Apilimod with methylcobalamin showed a synergistic effect (i.e. more than the sum of the improved viability with each compound alone) with respect to each single compound in any line tested.
[0134] For the iPSC-derived motoneurons from a C9orf72-ALS patient YM201636 and methylcobalamin provided an increase viability of 35.35% with respect to glutamate while YM201636 and methylcobalamin alone provide an increase of 6.95% and 9.66%, respectively (Table 8 and FIGURE 5). For the iPSC-derived motoneurons from a SOD-1- ALS patient YM201636 and methylcobalamin provided an increase in viability of 35.75% with respect to glutamate while YM201636 and methylcobalamin alone provide an increase of 13.81% and 12.72%, respectively (Table 9 and FIGURE 6). For the iPSC-derived motoneurons from a TDP-43-ALS patient YM201636 and methylcobalamin provided an increase in viability of 33% with respect to cells growth-factors deprived (-GF), while YM201636 and methylcobalamin alone provide an increase of 6.18% and 8.91%, respectively (Table 10 and FIGURE 7). For the iPSC-derived motoneurons from a Sporadic-ALS patient YM201636 and methylcobalamin provided an increase in viability of 41.21 % with respect to glutamate while YM201636 and methylcobalamin alone provide an increase of 10.96% and 9.98%, respectively (Table 11 and FIGURE 8). In summary, the combination of YM201636 with methylcobalamin showed a synergistic effect (i.e. more than the sum of the improved viability with each compound alone) with respect to each single compound in any line tested.
[0135] For the iPSC-derived motoneurons from a C9orf72-ALS patient APY0201 and methylcobalamin provided an increase viability of 37.38% with respect to glutamate while APY0201 and methylcobalamin alone provide an increase of 9.74% and 12%, respectively (Table 12 and FIGURE 9). For the iPSC-derived motoneurons from a SOD-l-ALS patient APY0201 and methylcobalamin provided an increase in viability of 38.45% with respect to glutamate while APY0201 and methylcobalamin alone provide an increase of 11.11% and 10.02%, respectively (Table 13 and FIGURE 10). For the iPSC-derived motoneurons from a TDP-43-ALS patient APY0201 and methylcobalamin provided an increase in viability of 42.9% with respect to cells growth-factors deprived (-GF), while APY0201 and
Figure imgf000049_0001
methylcobalamin alone provide an increase of 7.6% and 6.61%, respectively (Table 14 and FIGURE 11). For the iPSC-derived motoneurons from a Sporadic-ALS patient APY0201 and methylcobalamin provided an increase in viability of 42.1% with respect to glutamate while APY0201 and methylcobalamin alone provide an increase of 4.56% and 6.59%, respectively (Table 15 and FIGURE 12). In summary, the combination of APY0201 with methylcobalamin showed a synergistic effect (i.e. more than the sum of the improved viability with each compound alone) with respect to each single compound in any line tested.
[0136] Table 1. Cell viability (Apilimod+ methylcobalamin) with 10 pM Glutamate or growth factor depletion (TDP-43) treatment
Figure imgf000050_0002
Figure imgf000050_0001
[0137] Table 2. Cell viability (YM201636 + methylcobalamin) with 10 pM Glutamate or growth factor depletion (TDP-43) treatment
Figure imgf000051_0002
[0138] Table 3. Cell viability (APY0201 + methylcobalamin) with 10 pM Glutamate or growth factor depletion (TDP-43) treatment
Figure imgf000051_0003
Figure imgf000051_0001
Figure imgf000052_0002
[0139] Table 4. Increase in Cell Viability with respect to Glutamate in iPSC-derived motoneurons from a C9orf72-ALS patient.
Figure imgf000052_0003
[0140] Table 5. Increase in Cell Viability with respect to Glutamate in iPSC-derived motoneurons from a SOD-l-ALS patient.
Figure imgf000052_0004
Figure imgf000052_0001
[0141] Table 6. Increase in Cell Viability with respect to cells growth-factors deprivation in iPSC-derived motoneurons from a TDP-43-ALS patient.
Figure imgf000053_0002
[0142] Table 7. Increase in Cell Viability with respect to Glutamate in iPSC-derived motoneurons from a Sporadic-ALS patient.
Figure imgf000053_0003
[0143] Table 8. Increase in Cell Viability with respect to Glutamate in iPSC-derived motoneurons from a C9orf72-ALS patient.
Figure imgf000053_0004
[0144] Table 9. Increase in Cell Viability with respect to Glutamate in iPSC-derived motoneurons from a SOD-l-ALS patient.
Figure imgf000053_0005
Figure imgf000053_0001
Figure imgf000054_0002
[0145] Table 10. Increase in Cell Viability with respect to cells growth-factors deprivation, in iPSC-derived motoneurons from a TDP -43-ALS patient.
Figure imgf000054_0003
[0146] Table 11. Increase in Cell Viability with respect to Glutamate in iPSC-derived motoneurons from a Sporadic-ALS patient.
Figure imgf000054_0004
[0147] Table 12. Increase in Cell Viability with respect to Glutamate in iPSC-derived motoneurons from a C9orf72-ALS patient.
Figure imgf000054_0005
[0148] Table 13. Increase in Cell Viability with respect to Glutamate in iPSC-derived motoneurons from a SOD-l-ALS patient.
Figure imgf000054_0001
Figure imgf000055_0002
[0149] Table 14. Increase in Cell Viability with respect cells growth- factors deprivation in iPSC-derived motoneurons from a TDP-43-ALS patient.
Figure imgf000055_0003
[0150] Table 15. Increase in Cell Viability with respect to Glutamate in iPSC-derived motoneurons from a Sporadic-ALS patient.
Figure imgf000055_0004
Figure imgf000055_0001
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Figure imgf000058_0001
[0152] 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.
Figure imgf000059_0001

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 methylcobalamin.
2. The method of claim 1, wherein the PIKfyve inhibitor or a pharmaceutically acceptable salt thereof and methylcobalamin are in one composition.
3. The method of claim 1, wherein the PIKfyve inhibitor or a pharmaceutically acceptable salt thereof and methylcobalamin 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 methylcobalamin, wherein the subject is receiving or has received treatment with a PIKfyve inhibitor 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 methylcobalamin.
6. The method of any one of claims 1 -5 , wherein the PIKfyve inhibitor is apilimod, 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 apilimod, YM-201636, APY0201, or a pharmaceutically acceptable salt thereof.
8. The method of any one of claims 1-7, wherein the subject is administered 500-
Figure imgf000060_0001
9. The methods of any one of claims 1-8, wherein methylcobalamin is administered to the subject twice daily.
10. The methods of any one of claims 1-8, wherein methylcobalamin is administered to the subject three times daily.
11. The method of any one of claims 8-10, wherein the composition is administered orally.
12. The methods any one of claims 1-7, wherein the subject is administered 25 mg - 50 mg of methylcobalamin.
13. The methods of any one claim 1-7, wherein the subject is administered 50 mg of methylcobalamin.
14. The methods of claim 12 or 13, wherein methylcobalamin is administered to the subject twice weekly.
15. The method of any one of claims 12-14, wherein methylcobalamin is administered intramuscularly.
16. The method of any one of claims 7-15, wherein apilimod is apilimod dimesylate.
17. The method of any one of claims 7-16, wherein apilimod is administered orally.
18. The method claims 7-17, wherein the subject is administered 30-300 mg of apilimod or a pharmaceutically acceptable salt thereof.
19. The method of claims 7-18, wherein apilimod or a pharmaceutically acceptable salt thereof is administered daily.
20. The method of claims 7-18, wherein apilimod or a pharmaceutically acceptable salt thereof is administered twice daily.
Figure imgf000061_0001
21. The method of any one of claims 1-20, further comprising administering to the subject a glutamatergic agent.
22. The method of claim 21, wherein the glutamatergic agent is riluzole.
23. The method of claim 22, wherein riluzole is administered to the subject twice daily and 50 mg of riluzole is administered in each administration.
24. The method of any one of claims 21-23, wherein the glutamatergic agent is administered orally.
25. The method of any one of claims 1-20, wherein the subject is not receiving of has received treatment with a glutamatergic agent.
26. The method of claim 25, wherein the glutamatergic agent is riluzole.
27. The method of any claims 1-26, further comprising administering to the subject an antioxidant.
28. The method of claim 27, wherein the antioxidant is edaravone.
29. The method of claim 28, 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.
30. The method of claims 27-29, wherein the antioxidant is administered intravenously.
31. The method of claim 28, wherein edaravone is administered at a daily dose of 105 mg.
32. The method of claim 31 , wherein edaravone is administered orally.
Figure imgf000062_0001
33. The method of any one of claims 1-26, wherein the subject is not receiving or has not received treatment with an antioxidant.
34. The method of claim 29, wherein the antioxidant is edaravone.
35. The method of any one of claims 1-20, further comprising administering to the subject a glutamatergic agent and an antioxidant.
36. The method of any one of claims 1-20, wherein the subject is not receiving or has not received treatment with a glutamatergic agent or an antioxidant.
37. The method of claim 35 or claim 36, wherein the glutamatergic agent is riluzole and the antioxidant is edaravone.
38. The method of any one of claims 1-37, 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.
39. The method of claim 38, wherein the neurological disease or disorder is dementia.
40. The method of claim 39, 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.
41. The method of claim 40, wherein the neurological disease or disorder is amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD).
Figure imgf000063_0001
42. The method of claim 41, wherein the subject is in need of treatment is one having repeat expansions of the C9ORF72 gene.
43. The method of claim 41 , wherein the subject in need of treatment is one having a mutation in the SOD 1 gene.
44. The method of claim 41 , wherein the subject in need of treatment is one having accumulation of TDP-34 aggregates.
45. The method of any one of claims 1 -44, wherein the subject is human.
46. Use of a PIKfyve inhibitor and methylcobalamin in a method of treating a neurological disease or disorder in a subject in need thereof.
47. 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 methylcobalamin.
48. Use of methylcobalamin 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.
49. The use of any one of claims 46-48, wherein the PIKfyve inhibitor is apilimod, 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.
50. The use of claim 49, wherein the PIKfyve inhibitor is apilimod, YM-201636, APY 0201, or a pharmaceutically acceptable salt thereof.
Figure imgf000064_0001
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