WO2025179205A1 - Methods for the treatment of neurological disorders - Google Patents

Abstract

Provided are combination therapies for the treatment of neurological conditions comprising administering to a subject 4-((2-(2,2,2- trifluoroethy 1)-1H-imidazol-4-yl)methyl)pyridine and NAD+ or a derivative thereof.

Description

METHODS FOR THE TREATMENT OF NEUROLOGICAL DISORDERS

CROSS-REFERENCE

[0001] This application claims benefit of U.S. Provisional Patent Application No. 63/557,412, filed on February 23, 2024, which is incorporated herein by reference in its entirety.

BACKGROUND

[0002] Aging constitutes the main risk factor for the development of neurodegenerative diseases. Axonal degeneration is an important pathological event in many neurodegenerative and neurological disorders, including peripheral neuropathy and traumatic brain injury (Gerdts, J. et al., Neuron, 2016, 89, 449-60). Axonal degeneration has also been implicated in, for example, Alzheimer’s disease, Parkinson’s disease, and Amyotrophic Lateral Sclerosis, where degeneration precedes symptom onset and widespread neuronal loss (Kurowska, Z. et al., J. Parkinson’s Dis., 2016, 6, 703-07). While these neurological conditions have unique underlying etiologies, inhibition of axonal degeneration in the conditions’ early stages may slow or prevent their progression by preventing the loss of functional synapses and maintaining neuronal connectivity (Essuman, K. et al., Neuron, 2017 Mar. 22, 93(6), 1334-43).

[0003] Axonal degeneration after injury occurs both toward the proximal cell body (termed retrograde degeneration) and toward the distal axon terminal (termed Wallerian or orthograde degeneration) (Kanamori A. et al., Am. J. Pathol. 2012 Jul; 18 l(l):62-73). Wallerian degeneration, which occurs in that section of the axon that is distal to the site of injury, occurs after axonal injury in both the peripheral nervous system (PNS) and the central nervous system (CNS). Wallerian degeneration usually begins within 24-36 hours of a lesion. Prior to degeneration, the distal section of the axon tends to remain electrically excitable, while after injury, the axonal skeleton disintegrates, and the axonal membrane breaks apart.

[0004] The processes of death of the cell body and degeneration of the axon are independent events. As alluded to above, evidence exists indicating that the degeneration of axons precedes clinical symptoms in neurodegenerative diseases and occurs before cell body loss. Thus, axonal degeneration constitutes an early event in pathological processes and provides a potential therapeutic target to treat neurodegeneration prior to neuronal cell death (Salvadores, N. et al., Front. Neurosci., 2017, 11, 451).

[0005] In view of the above, new modalities are needed for the treatment of neurological disorders such as neurodegenerative disease by the prevention of axonal degeneration. SUMMARY

[0006] In certain aspects, described herein is a method of treating a neurological condition in a subject, comprising administering to the subject a therapeutically effective amount of Compound 1 and a therapeutically effective amount of nicotinamide adenine dinucleotide (NAD+) or a derivative thereof. In some embodiments, the method comprises comprising adminsitering to the subject a therapeutically effective amount of Compound 1 and a therapeutically effective amount of NAD+. In some embodiments, the NAD+ derivative is selected from the list consisting of nicotinamide riboside (NR), nicotinic acid (NA), nicotinic acid riboside (NaR), nicotinamide (NAM), nicotinamide mononucleotide (NMN), nicotinic acid mononucleotide (NaMN), tryptophan, vitamin B3, nicotinic acid adenine dinucleotide (NAAD), quinolinic acid, 3-hydroxyanthranilate, 3 -hydroxy kynurenine, kynurenine, and N- formylkynurenine. In some embodiments, Compound 1 and the NAD+ or derivative thereof are administered substantially simultaneously. In some embodiments, Compound 1 and the NAD+ or derivative thereof are administered separately. In some embodiments, Compound 1 and the NAD+ or derivative thereof are administered sequentially. In some embodiments, Compound 1 and the NAD+ or derivative thereof are co-formulated for administration by a single formulation. In some embodiments, Compound 1 and the NAD+ or derivative thereof are administered in separate compositions. In some embodiments, Compound 1 and the NAD+ or derivative thereof are admixed prior to administration. In some embodiments, the neurological condition is depression or a disorder related to depression. In some embodiments, the depression is major depressive disorder, persistent depressive disorder, bipolar disorder, treatment resistant depression (TRD), postpartum depression, premenstrual dysphoric disorder, or seasonal affective disorder. In some embodiments, the disease or disorder related to depression is anxiety. In some embodiments, Compound 1 and the NAD+ or derivative thereof act synergistically.

[0007] In certain aspects, described herein is a method of reducing axon degeneration, the method comprising comprising administering to the subject a therapeutically effective amount of Compound 1 and a therapeutically effective amount of nicotinamide adenine dinucleotide (NAD+) or a derivative thereof. In some embodiments, the method comprises comprising adminsitering to the subject a therapeutically effective amount of Compound 1 and a therapeutically effective amount of NAD+. In some embodiments, the NAD+ derivative is selected from the list consisting of nicotinamide riboside (NR), nicotinic acid (NA), nicotinic acid riboside (NaR), nicotinamide (NAM), nicotinamide mononucleotide (NMN), nicotinic acid mononucleotide (NaMN), tryptophan, vitamin B3, nicotinic acid adenine dinucleotide (NAAD), quinolinic acid, 3-hydroxyanthranilate, 3 -hydroxy kynurenine, kynurenine, and N- formylkynurenine. In some embodiments, Compound 1 and the NAD+ or derivative thereof are administered substantially simultaneously. In some embodiments, Compound 1 and the NAD+ or derivative thereof are administered separately. In some embodiments, Compound 1 and the NAD+ or derivative thereof are administered sequentially. In some embodiments, Compound 1 and the NAD+ or derivative thereof are co-formulated for administration by a single formulation. In some embodiments, Compound 1 and the NAD+ or derivative thereof are administered in separate compositions. In some embodiments, Compound 1 and the NAD+ or derivative thereof are admixed prior to administration. In some embodiments, Compound 1 and the NAD+ or derivative thereof act synergistically.

INCORPORATION BY REFERENCE

[0008] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0010] Fig. 1A depicts the dose response of NAD in sensory neuron axotomy assay. Fig. IB depicts the dose response of NAM in sensory neuron axotomy assay. Fig. 1C depicts the dose response of NR in sensory neuron axotomy assay.

[0011] Fig. 2 depicts the dose response of Compound 1 with adjuvant vehicle or NAD in DRG axotomy assay.

[0012] Fig. 3 depicts the dose response of Compound 1 with adjuvant vehicle or NAM in DRG axotomy assay.

[0013] Fig. 4 depicts the dose response of Compound 1 with adjuvant vehicle or NR in DRG axotomy assay.

[0014] Fig. 5 depicts neuroprotective efficacy post-spared nerve injury (SNI) co-dosing of NR and Compound 1 compared to either treatment in isolation.

[0015] Fig. 6 depicts plasma concentrations of Compound 1 measured in SNI animals treated with Compound 1 or a combination of Compound 1 and NR. DETAILED DESCRIPTION

[0016] Similar to programmed cell death pathways (e.g., apoptosis), axonal degeneration in response to injury or disease stimulates a local signaling cascade that causes destruction of the injured axon segment (Summers D. W., et al., PNAS USA, 2016 Oct 11, 113 (41 ):E6271— E6280). Following injury, the axonal skeleton disintegrates, and the axonal membrane breaks apart. Subsequent to axonal degeneration, the myelin sheath degrades and infiltration by macrophages follows; the macrophages, along with Schwann cells, clear the cellular debris resulting from the degeneration (Coleman M.P., et al., PNAS USA, 1998 Aug, 95( 17): 9985-90). [0017] SARM1 (sterile alpha and TIR motif-containing 1) protein (NP 055892) is a 724 amino acid protein involved in axon degeneration. It has also been implicated in infectious and inflammatory disorders. The SARM1 protein, also known as FLJ36296, KIAA0524, MyD88-5, SAM domain-containing protein 2, and SAMD2, comprises four domains, i) a mitochondrial localization signal, ii) an auto-inhibitory N-terminus region consisting of armadillo/HEAT motifs, iii) two sterile alpha motifs responsible for multimerization, and iv) a C-terminus Toll/Interleukin-1 receptor that possesses enzymatic activity (Essuman K., et al., Neuron 2017 Mar., 93(6): 1334-43.e5).

[0018] SARM1 protein plays a critical role in the Wallerian degeneration pathway. Activation of SARM1 triggers a rapid collapse of NAD+ levels in the distal section of the injured axon, which then undergoes degeneration (Gerdts J. et al., Science 2015 Apr. 348(6233):453 -57). Promoting dimerization of the Toll/interleukin receptor (TIR) domain of SARM1 has been shown to be sufficient to promote NAD+ loss and axon degeneration.

[0019] SARMl’s activity is responsible for, at least in part, the protective nature of the survival factor NMNAT2, as NMKNAT enzymes have been found to prevent S ARM 1 -mediated depletion of NAD+. Other pro-degeneration signaling pathways, including the MAP kinase pathway, have been linked to SARM1 activation. MAPK signaling has been shown to promote the loss of NMNAT2, which promotes SARM1 activation (See, e.g., Yang J. et al., Cell 2015 Jan 160(1-2): 161-76).

[0020] SARM1 is involved in the innate immune response. It promotes neuronal cell death in response to stress and other stimuli. SARM1 acts as a negative regulator of TICAMl/TRIF- dependent Toll-like receptor signaling by inhibiting induction of TLR3- and TLR4-dependent genes, which play a pivotal role in activating axonal degeneration following injury. In addition, SARM1 specifically blocks TICAMl/TRIF-dependent transcription factor activation and gene induction, without affecting the MYD88- dependent pathway or non-TLR signaling. It is also a negative regulator of NF- kappa-B and IRF activation. (See, e.g., Summers, D.W. et al., J Neurosci., 2014 Jul 9, 34(28):9338-50). [0021] In some embodiments, described herein are inhibitors of SARM1. SARM1 activation can cause a rapid reduction in NAD+ levels in injured axons, which then undergo degeneration. In particular embodiments, the compounds inhibit axonal degeneration, including axonal degeneration that results from reduction or depletion of NAD+ (e.g., inhibition of SARM1 NADase).

[0022] Further described herein are active-site SARM1 NAD hydrolase inhibitors. In some aspects, the inhibitors described herein act in the catalytic pocket but do not directly compete with substrate binding. In some aspects, the inhibitors are uncompetitive, pro-inhibitors that function by opportunistically intercepting the NAD hydrolysis reaction and undergoing covalent conjugation with the reaction product adenosine diphosphate ribose (ADPR). In some embodiments, the resulting small molecule-ADPR adducts confer knock-out like axon protection in vivo - reducing levels of the translatable biomarker neurofilament light and conferring functional protection. In some embodiments, described herein is a mode of pharmacologic inhibition that has implications not just for SARM1 but for a broader panel of related NAD hydrolases linked to age-related decline and disease.

[0023] Described herein is 4-((2-(2,2,2-trifluoroethyl)-lH-imidazol-4-yl)methyl)pyridine (Compound 1), a SARM1 inhibitor. Also described herein is nicotinamide adenine dinucleotide (NAD+) and derivatives thereof. The combination of Compound 1 and NAD+ or a derivative thereof may be effective in the treatment of neurodegenerative diseases as described herein.

METHODS

[0024] In certain aspects, described herein are methods of treating a neurological condition in a subject, the methods comprising administering to the subject a therapeutically effective amount of Compound 1 and a therapeutically effective amount of nicotinamide adenine dinucleotide (NAD+) or a derivative thereof. In certain embodiments, the neurological condition is depression. In certain embodiments, the depression is major depressive disorder, persistent depressive disorder, bipolar disorder, treatment resistant depression (TRD), postpartum depression, premenstrual dysphoric disorder, or seasonal affective disorder. In certain embodiments, the depression is anxiety.

Compound 1

[0025] In one embodiment is 4-((2-(2,2,2-trifluoroethyl)-lH-imidazol-4-yl)methyl)pyridine. “Compound 1” or “4-((2-(2,2,2-trifluoroethyl)-lH-imidazol-4-yl)methyl)pyridine” refers to the compound with the following structure: [0026] A variety of pharmaceutically acceptable salts are formed from Compound 1 and include:

- acid addition salts formed by reacting Compound 1 with an organic acid, which includes aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyl alkanoic acids, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, amino acids, etc. and include, for example, acetic acid, adipic acid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like;

- acid addition salts formed by reacting Compound 1 with an inorganic acid, which includes hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, hydroiodic acid, hydrofluoric acid, phosphorous acid, and the like.

[0027] The term “pharmaceutically acceptable salts” in reference to Compound 1 refers to a salt of Compound 1, which does not cause significant irritation to a mammal to which it is administered and does not substantially abrogate the biological activity and properties of the compound.

[0028] It should be understood that a reference to a pharmaceutically acceptable salt includes the solvent addition forms (solvates). Solvates contain either stoichiometric or non- stoichiometric amounts of a solvent, and are formed during the process of product formation or isolation with pharmaceutically acceptable solvents such as water, ethanol, methanol, methyl tert-butyl ether (MTBE), diisopropyl ether (DIPE), ethyl acetate, isopropyl acetate, isopropyl alcohol, methyl isobutyl ketone (MIBK), methyl ethyl ketone (MEK), acetone, nitromethane, tetrahydrofuran (THF), dichloromethane (DCM), dioxane, heptanes, toluene, anisole, acetonitrile, and the like. In one aspect, solvates are formed using, but not limited to, Class 3 solvent(s). Categories of solvents are defined in, for example, the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH), “Impurities: Guidelines for Residual Solvents, Q3C(R3), (November 2005). Hydrates are formed when the solvent is water, or alcoholates are formed when the solvent is alcohol. In some embodiments, solvates of Compound 1, or pharmaceutically acceptable salts thereof, are conveniently prepared or formed during the processes described herein. In some embodiments, solvates of Compound 1 are anhydrous. In some embodiments, Compound 1, or pharmaceutically acceptable salts thereof, exist in unsolvated form. In some embodiments, Compound 1, or pharmaceutically acceptable salts thereof, exist in unsolvated form and are anhydrous. NAD+ and derivatives thereof

[0029] The methods described herein comprise administering a therapy comprising NAD+ or a combination thereof. In some embodiments, the one or more additional pharmaceutically active agent can be NAD+ or an NAD+ precursor. In some embodiments, the NAD+ derivative is selected from the list consisting of nicotinamide riboside (NR), nicotinic acid (NA), nicotinic acid riboside (NaR), nicotinamide (NAM), nicotinamide mononucleotide (NMN), nicotinic acid mononucleotide (NaMN), tryptophan, vitamin B3, nicotinic acid adenine dinucleotide (NAAD), quinolinic acid, 3-hydroxyanthranilate, 3 -hydroxy kynurenine, kynurenine, and N- formylkynurenine. In some embodiments, the NAD+ derivative is nicotinamide riboside (NR). In some embodiments, the NAD+ derivative is nicotinic acid (NA). In some embodiments, the NAD+ derivative is nicotinic acid riboside (NaR). In some embodiments, the NAD+ derivative is nicotinamide (NAM). In some embodiments, the NAD+ derivative is nicotinamide mononucleotide (NMN). In some embodiments, the NAD+ derivative is nicotinic acid mononucleotide (NaMN). In some embodiments, the NAD+ derivative is tryptophan. In some embodiments, the NAD+ derivative is vitamin B3. In some embodiments, the NAD+ derivative is nicotinic acid adenine dinucleotide (NAAD). In some embodiments, the NAD+ derivative is quinolinic acid. In some embodiments, the NAD+ derivative is 3-hydroxyanthranilate. In some embodiments, the NAD+ derivative is 3 -hydroxykynurenine. In some embodiments, the NAD+ derivative is kynurenine. In some embodiments, the NAD+ derivative is N-formylkynurenine.

Neurological conditions

[0030] The compounds of the invention are useful in the treatment and prevention of various diseases associated with abnormal expression or activity of SARM1. For example, the compounds of the invention are useful in the treatment and prevention of neurological disorders. The term "neurological disorder" generally refers to a disorder affecting the nervous system, including the central nervous system or the peripheral nervous system. The term “neurological disorder” also includes ocular indications having a nexus to the nervous system.

[0031] In some embodiments, the neurological disorder treatable or preventable by administration of a compound of the invention includes neurodegenerative diseases. Neurodegenerative diseases are characterized by damage to the central nervous system and can be identified by progressive dysfunction, degeneration and death of specific populations of neurons which are often synaptically interconnected. Examples of neurodegenerative diseases include Parkinson’s disease (PD), Alzheimer’s disease (AD), Huntington’s disease (HD), prion disease, motor neuron diseases (MND), spinocerebellar ataxia (SCA), spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), and epilepsy. [0032] Examples of neurological disorders treatable or preventable according to the methods of the invention include spinal muscular atrophy (SMA), Chemotherapy Induced Peripheral Neuropathy (representative chemotherapeutic agents include vinca-alkaloids, taxols and platins), multiple sclerosis (MS), traumatic brain injury (TBI), spinal cord injury, stroke, Parkinson’ disease, glaucoma, Huntington’s disease, Alzheimer’s disease, Char cot-Marie-Tooth disease (CMT), retinitis pigmentosa (RP), age-related macular degeneration (AMD), small fiber neuropathies, peripheral neuropathy (e.g., viral neuropathy), spinocerebellar ataxias, cystic fibrosis, familial amyloidotic polyneuropathy, spongiform encephalopathies, spinal and bulbar muscular atrophy, hereditary dentatorubral-pallidoluysian atrophy, adrenoleukodystrophy, adrenomyeloneuropathy, Alexander’s disease, amyotrophic lateral sclerosis (ALS), Bassen- Kornzweig syndrome, Bell’s palsy, progressive supra nuclear palsy (PSP), central pontine myelolysis, cluster headache, congenital hypomyelination, corticobasal degeneration, Creutzfeldt- Jakob disease, epilepsy, dementia (e.g., frontotemporal dementia and Lewy body dementia), demyelination disorders (e.g., ischemic demyelination), encephalomyelitis, Friedrich’s ataxia, Gaucher’s disease, hereditary sensory and autonomic neuropathy (HSAN), Hurler syndrome, Krabbe’s disease, metachromatic leukodystrophy, migraine and tension headaches, mild cognitive impairment, motor spinoneuron disease, neuromyelitis optica, Niemann-Pick disease, optic neuritis, Pelizaeus Merzbacher disease, peripheral neuropathy, periventricular leukomalacia, post-herpetic neuralgia, prion disease, progressive supranuclear palsy, progressive multifocal leukoencephalopathy, Tay-Sacks disease, thoracic disc herniation, traverse myelitis, trigeminal neuralgia, Wallerian degeneration, cerebellar degeneration, chiari malformation, dystonia, encephalitis (e.g., pediatric viral encephalitis and La Crosse virus encephalitis), hyperekplexia, multifocal motor neuropathy, muscular dystrophy, myasthenia gravis, myopathy, neurofibromatosis, neuronal ceroid lipofuscinosis, neuropathies (e.g., peripheral neuropathy), pseudobulbar affect, restless legs syndrome, spina bifida, syringomyelia, thoracic outlet syndrome, and transverse myelitis.

[0033] In other embodiments, the neurological disorder treatable or preventable by administration of a compound of the invention is a neuropathy. As used herein, the term “neuropathy” refers broadly to diseased conditions of the nervous system, including polyneuropathy; neuropathy, ataxia, and retinosa pigmentosa (NARP); familial amyloid neuropathies; diabetic neuropathy (peripheral neuropathy due to diabetes mellitus); peripheral neuropathy (e.g., chemotherapy-induced peripheral neuropathy (CIPN), including CIPN caused by vinca alkaloids, bortezomib, Ixabepilone, thalidomide and its analogs, taxanes, and platinumbased agents); and cranial neuropathy (e.g., auditory neuropathy and optic neuropathy). The term also includes other neuropathies associated with genetic disorders (e.g., NMNAT2 genetic mutation disorders).

[0034] In still other embodiments, the neurological disorder treatable or preventable by administration of a compound of the invention is an ocular neuropathy (e.g., optic neuropathy). The term “optic neuropathy” refers to damage to the optic nerve from a number of causes. Types of optic neuropathy include ischemic optic neuropathy (e.g., anterior and posterior ischemic optic neuropathy); optic neuritis (e.g., chronic relapsing inflammatory optic neuropathy (CRION), single isolated optic neuritis (SION), and relapsing isolated optic neuritis); compressive optic neuropathy; infiltrative optic neuropathy; traumatic optic neuropathy; mitochondrial optic neuropathies; and hereditary optic neuropathies (e.g., Leber’s hereditary optic neuropathy (LHON), hereditary neuropathy with liability to pressure palsy (HNPP), and dominant optic atrophy).

[0035] In still other embodiments, the neurological disorder treatable or preventable by administration of a compound of the invention is multiple sclerosis (MS), chemotherapy- induced peripheral neuropathy (CIPN), amyotrophic lateral sclerosis (ALS), glaucoma, traumatic brain injury (TBI), or stroke.

[0036] In certain embodiments, the disease or disorder is depression. In certain embodiments, the depression is major depressive disorder, persistent depressive disorder, bipolar disorder, treatment resistant depression (TRD), postpartum depression, premenstrual dysphoric disorder, or seasonal affective disorder. In certain embodiments, the depression is anxiety.

Methods of administration

[0037] In some embodiments, Compound 1 and the NAD+ or derivative thereof are administered at the same time. In some embodiments, Compound 1 and the NAD+ or derivative thereof are administered simultaneously. In some embodiments, Compound 1 and the NAD+ or derivative thereof are administered substantially simultaneously. In some embodiments, Compound 1 and the NAD+ or derivative thereof are administered sequentially. In some embodiments, Compound 1 and the NAD+ or derivative thereof are administered separately. [0038] In some embodiments, Compound 1 and the NAD+ or derivative thereof are administered in a combined synergistic amount. In some embodiments, Compound 1 and the NAD+ or derivative thereof are co-formulated for administration by a single formulation. In some embodiments, Compound 1 and the NAD+ or derivative thereof are admixed prior to administration. In some embodiments, Compound 1 and the NAD+ or derivative thereof are administered in separate compositions. [0039] Compound 1 and the NAD+ or derivative thereof described herein may be administered through a variety of routes, including, without limitation, subcutaneous, intraperitoneal, intravenous, intramuscular, intracerebral, or orally.

[0040] In certain aspects, described herein is a method of reducing axon degeneration in a subject, the method comprising administering to the subject a therapeutically effective amount of Compound 1 and a therapeutically effective amount of NAD+ or a derivative thereof. In some embodiments, axon degeneration is reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In some embodiments, axon degeneration is reduced compared to a subject who was not administered Compound 1 or a NAD+ derivative. In some embodiments, axon degeneration is reduced compared to a subject who was administered Compound 1 alone. In some embodiments, Compound 1 and the NAD+ derivative reduce axon degeneration in a synergistic manner. In some embodiments, Compound 1 and the NAD+ derivative reduce axon degeneration in an additive manner.

Additional therapeutics

[0041] One or more additional pharmaceutically active agents or treatment methods can be used in combination with Compound 1 as described herein. The agents can be combined with Compound 1 in a single dosage form, or the agents can be administered simultaneously or sequentially as separate dosage forms. Examples of additional agents include acamprosate, agomelatine, almotriptan, amantadine, amisulpride, amitriptyline, apomorphine, aripiprazole, asenapine, atomoxetine, baclofen, botulinum toxin type A, bromocriptine, buccal midazolam, buprenorphine, buspirone, cabergoline, carbamazepine, chlordiazepoxide, chlorpromazine, citalopram, clobazam, clomethiazole, clomipramine, clonazepam, clozapine, denzapine, co- beneldopa, co-careldopa, dantrolene, dexamfetamine, diazepam, divalproex sodium, donepezil, doxepin, duloxetine, eletriptan, entacapone, epinephrine, escitalopram, eslicarbazepine, ethosuximide, fmgolimod, fluoxetine, flupentixol, flupentixol, fluphenazine long-acting injection (modecate), fluvoxamine (Faverin), frovatriptan, gabapentin, galantamine, haloperidol, imipramine, lacosamide, lamotrigine, levetiracetam, levomepromazine, lisdexamfetamine, lithium, lofepramine, loprazolam, lorazepam, lormetazepam, lurasidone, melatonin, memantine, methylphenidate, mianserin, mirtazapine, moclobemide, modafinil, naratriptan, neostigmine, nitrazepam, nortriptyline, olanzapine, orlistat, orphenadrine, oxazepam, oxcarbazepine, paliperidone, paliperidone, paroxetine, perampanel, pergolide, pericyazine, phenobarbital, phenytoin, piracetam, pizotifen, pramipexole, pregabalin, primidone, prochlorperazine, procyclidine, pyridostigmine, quetiapine, rasagiline, reboxetine, risperidone, rivastigmine, rizatriptan, ropinirole, rotigotine, rufmamide, selegiline, sertraline, sodium oxybate, sodium valproate, sulpiride, sumatriptan, temazepam, tetrabenazine, tiagabine, tizanidine, tolcapone, topiramate, trazodone, trihexyphenidyl, trimipramine, valproate semisodium, venlafaxine, vigabatrin, vortioxetine, zolmitriptan, zolpidem, zonisamide, zopiclone, and zuclopenthixol. [0042] In some embodiments, the one or more additional pharmaceutically active agent can include a neuroprotective agent. In some embodiments, the neuroprotective agent is a dual leucine-zipper kinase (DLK) inhibitor. In some embodiments, the neuroprotective agent is a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor.

PHARMACEUTICAL FORMULATIONS AND DOSAGE FORMS

[0043] A pharmaceutical composition refers to a combination of a compound described herein, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier.

[0044] These compositions can be prepared in a manner well known in the pharmaceutical art, and can be administered by a variety of routes, depending upon whether local or systemic treatment is desired and upon the area to be treated. Pharmaceutical compositions may be formulated in a conventional manner using one or more physiologically acceptable carriers including excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. Additional details about suitable excipients for pharmaceutical compositions described herein may be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999), herein incorporated by reference for such disclosure. [0045] Administration may be oral, topical (including ophthalmic and to mucous membranes including intranasal, vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), ocular (e.g., eye drops or intravitreal, subconjunctival, subtenon, or retrobulbar injection), or parenteral.

[0046] In some embodiments provided herein are pharmaceutical compositions which contain, as the active ingredient, Compound 1 described herein in combination with one or more pharmaceutically acceptable carriers. In making the compositions of the invention, the active ingredient is typically mixed with an excipient, diluted by an excipient or enclosed within such a carrier in the form of, for example, a capsule, sachet, paper, or other container. When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, carrier or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.

[0047] The compositions can be formulated in a unit dosage form. The term "unit dosage form" refers to a physically discrete unit suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient. [0048] The active compound can be effective over a wide dosage range and is generally administered in a pharmaceutically effective amount. It will be understood, however, that the amount of the compound actually administered will usually be determined by a physician, according to the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.

[0049] For preparing solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical excipient to form a solid pre-formulation composition containing a homogeneous mixture of Compound 1 described herein. When referring to these preformulation compositions as homogeneous, the active ingredient is typically dispersed evenly throughout the composition so that the composition can be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid pre-formulation is then subdivided into unit dosage forms of the type described above.

[0050] The tablets or pills described herein can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.

[0051] The amount of compound or composition administered to a patient will vary depending upon what is being administered, the purpose of the administration, such as prophylaxis or therapy, the state of the patient, the manner of administration, and the like. In therapeutic applications, compositions can be administered to a patient already suffering from a disease in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications. Effective doses will depend on the disease condition being treated as well as by the judgment of the attending clinician depending upon factors such as the severity of the disease, the age, weight and general condition of the patient, and the like.

[0052] The compositions administered to a patient can be in the form of pharmaceutical compositions described above. These compositions can be sterilized by conventional sterilization techniques, or may be sterile filtered.

[0053] The therapeutic dosage of Compound 1 can vary according to, for example, the particular use for which the treatment is made, the manner of administration of the compound, the health and condition of the patient, and the judgment of the prescribing physician. The proportion or concentration of a compound of the invention in a pharmaceutical composition can vary depending upon a number of factors including dosage, chemical characteristics (e.g., hydrophobicity), and the route of administration. The dosage is likely to depend on such variables as the type and extent of progression of the disease or disorder, the overall health status of the particular patient, the relative biological efficacy of the compound selected, formulation of the excipient, and its route of administration. Effective doses can be extrapolated from doseresponse curves derived from in vitro or animal model test systems.

[0054] The following examples are offered for illustrative purposes, and are not intended to limit the invention in any manner. Those of skill in the art will readily recognize a variety of non-critical parameters which can be changed or modified to yield essentially the same results.

DEFINITIONS

[0055] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the claimed subject matter belongs. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any subject matter claimed. In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. In this application, the use of “or” means “and/or” unless stated otherwise. Furthermore, use of the term “including” as well as other forms, such as “include”, “includes,” and “included,” is not limiting. The term "comprising" (and related terms such as "comprise" or "comprises" or "having" or "including") is not intended to exclude that in other certain embodiments, for example, an embodiment of any composition of matter, composition, method, or process, or the like, described herein, may "consist of' or "consist essentially of' the described features. The term "about" when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and thus the number or numerical range may vary between 1% and 15% of the stated number or numerical range.

[0056] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in the application including, but not limited to, patents, patent applications, articles, books, manuals, and treatises are hereby expressly incorporated by reference in their entirety.

[0057] The term “acceptable” or “pharmaceutically acceptable”, with respect to a formulation, composition or ingredient, as used herein, means having no persistent detrimental effect on the general health of the subject being treated or does not abrogate the biological activity or properties of the compound, and is relatively nontoxic.

[0058] As used herein, “amelioration” of the symptoms of a particular disease, disorder, or condition by administration of a particular compound or pharmaceutical composition refers to any lessening of severity, delay in onset, slowing of progression, or shortening of duration, whether permanent or temporary, lasting or transient that can be attributed to or associated with administration of the compound or composition.

[0059] “Bioavailability” refers to the percentage of Compound 1 dosed that is delivered into the general circulation of the animal or human being studied. The total exposure (AUC(o-co)) of a drug when administered intravenously is usually defined as 100% bioavailable (F%). “Oral bioavailability” refers to the extent to which Compound 1 is absorbed into the general circulation when the pharmaceutical composition is taken orally as compared to intravenous injection.

[0060] “Blood plasma concentration” refers to the concentration of Compound 1 in the plasma component of blood of a subject. It is understood that the plasma concentration of Compound 1 may vary significantly between subjects, due to variability with respect to metabolism and/or possible interactions with other therapeutic agents. In accordance with one embodiment disclosed herein, the blood plasma concentration of Compound 1 may vary from subject to subject. Likewise, values such as maximum plasma concentration (Cmax) or time to reach maximum plasma concentration (T_max), or total area under the plasma concentration time curve (AUC(o-co)) may vary from subject to subject. Due to this variability, the amount necessary to constitute “a therapeutically effective amount” of Compound 1 may vary from subject to subject.

[0061] The terms “co-administration” or the like, as used herein, are meant to encompass administration of the selected therapeutic agents to a single patient, and are intended to include treatment regimens in which the agents are administered by the same or different route of administration or at the same or different time.

[0062] The terms “effective amount” or “therapeutically effective amount,” as used herein, refer to a sufficient amount of an agent or a compound being administered which will relieve to some extent one or more of the symptoms of the disease or condition being treated. The result can be reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. For example, an “effective amount” for therapeutic uses is the amount of the composition including a compound as disclosed herein required to provide a clinically significant decrease in disease symptoms without undue adverse side effects. An appropriate “effective amount” in any individual case may be determined using techniques, such as a dose escalation study. The term “therapeutically effective amount” includes, for example, a prophylactically effective amount. An “effective amount” of a compound disclosed herein is an amount effective to achieve a desired pharmacologic effect or therapeutic improvement without undue adverse side effects. It is understood that “an effect amount” or “a therapeutically effective amount” can vary from subject to subject, due to variation in metabolism of Compound 1, age, weight, general condition of the subject, the condition being treated, the severity of the condition being treated, and the judgment of the prescribing physician. By way of example only, therapeutically effective amounts may be determined by a dose escalation clinical trial.

[0063] The terms “enhance” or “enhancing” means to increase or prolong either in potency or duration a desired effect. By way of example, “enhancing” the effect of therapeutic agents refers to the ability to increase or prolong, either in potency or duration, the effect of therapeutic agents on during treatment of a disease, disorder, or condition. An “enhancing-effective amount,” as used herein, refers to an amount adequate to enhance the effect of a therapeutic agent in the treatment of a disease, disorder, or condition. When used in a patient, amounts effective for this use will depend on the severity and course of the disease, disorder, or condition, previous therapy, the patient's health status and response to the drugs, and the judgment of the treating physician.

[0064] The term “prophylactically effective amount,” as used herein, refers that amount of a composition applied to a patient which will relieve to some extent one or more of the symptoms of a disease, condition or disorder being treated. In such prophylactic applications, such amounts may depend on the patient's state of health, weight, and the like. As an example, one can determine such prophylactically effective amounts by a dose escalation clinical trial. [0065] The term “subject” as used herein, refers to an animal which is the object of treatment, observation or experiment. By way of example only, a subject may be, but is not limited to, a mammal including, but not limited to, a human.

[0066] As used herein, the term “target activity” refers to a biological activity capable of being modulated by a selective modulator. Certain exemplary target activities include, but are not limited to, binding affinity, signal transduction, enzymatic activity, tumor growth, inflammation or inflammation-related processes, and amelioration of one or more symptoms associated with a disease or condition.

[0067] A "tautomer" refers to a molecule wherein a proton shift from one atom of a molecule to another atom of the same molecule is possible. In certain embodiments, Compound 1 described herein exists as a tautomer. In circumstances where tautomerization is possible, a chemical equilibrium of the tautomers will exist. The exact ratio of the tautomers depends on several factors, including physical state, temperature, solvent, and pH.

[0068] The terms “treat,” “treating” or “treatment”, as used herein, include alleviating, abating or ameliorating a disease or condition symptoms, preventing additional symptoms, ameliorating or preventing the underlying metabolic causes of symptoms, inhibiting the disease or condition, e.g., arresting the development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, relieving a condition caused by the disease or condition, or stopping the symptoms of the disease or condition. The terms “treat,” “treating” or “treatment”, include, but are not limited to, prophylactic and/or therapeutic treatments.

[0069] As used herein, IC50 refers to a dosage, concentration or amount of a particular test compound that elicits a dose-dependent response at 50% of maximal expression of a particular response that is induced, provoked or potentiated by the particular test compound.

[0070] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

EXAMPLES

[0071] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

List of abbreviations

[0072] As used throughout the description of the invention, the following abbreviations, unless otherwise indicated, shall be understood to have the following meanings:

ACN or MeCN acetonitrile Bn benzyl

BOC or Boc ter -butyl carbamate

Z-Bu tert-butyl

Cy cyclohexyl

DCE di chloroethane (CICH2CH2CI)

DCM d ichloromethane (CH2CI2)

DIPEA or DIEA diisopropylethylamine

DMAP 4-(A,A-dimethylamino)pyridine

DMF dimethylformamide

DMA /f-di methyl acetamide

DMSO dimethylsulfoxide eq or equiv e quivalent(s) Et ethyl

Et2O diethyl ether

EtOH ethanol

EtOAc ethyl acetate

HPLC high performance liquid chromatography

IPA isopropanol

Me methyl

MeOH methanol

MS mass spectroscopy

GC gas chromatography h hour(s)

KF Karl Fischer min minutes

MsOH methanesulfonic acid

NMR nuclear magnetic resonance

RP-HPLC reverse phase-high performance liquid chromatography r.t. room temperature

TFA trifluoroacetic acid

THF tetrahydrofuran

TLC thin layer chromatography

V volumes Example 1: Synthesis of Compound 1

[0073] Step 1: Ethyl 3-(((l-amino-3,3,3-trifluoropropylidene)amino)oxy)acrylate. To a flask containing sodium metal (843 mg, 36.7 mmol) in MeOH (10 mL), a suspension of NH2OH HCI (2.55 g, 36.7 mmol) in MeOH (10 mL) was added slowly using an addition funnel. The resulting mixture was stirred for 15 min. The suspension was filtered, the filtrate was cooled to 0 °C and 3,3,3-trifluoropropionitrile (4 g, 36.7 mmol) was added. The reaction mixture was stirred for 1 h. The solvent was evaporated under reduced pressure and the resulting oil was dissolved in MeCN (150 mL) and TEA (3.72 g, 36.7 mmol) was added to the solution. The resulting mixture was heated to 80 °C and a solution of ethyl propiolate (3.6 g, 36.7 mmol) in MeCN (20 mL) was added. The resulting reaction was stirred at 80 °C for 18 h. The reaction was then cooled to room temperature, the solvent was evaporated under reduced pressure and the crude product was purified by silica gel column chromatography to yield ethyl 3-(((l-amino- 3,3,3-trifluoropropylidene)amino)oxy)acrylate (7.1 g, 81%) as a yellow oil. LCMS ESI-MS m/z 241 [M+H]⁺.

[0074] Step 2: Ethyl 2-(2.2.2-trifluoroethyl)-l//-imidazole-5-carboxylate. Ethyl 3-(((l- amino-3,3,3-trifluoropropylidene)amino)oxy)acrylate (5 g, 20.8 mmol) and PI12O (60 mL) was heated to 180 °C and stirred for 30 min. The reaction was cooled and the crude mixture was purified via silica gel column chromatography to yield the ethyl 2-(2,2,2-trifluoroethyl)-UT- imidazole-5-carboxylate (1.8 g) as a yellow oil. This compound was used in the next step without further purification. LCMS ESI-MS m/z 1 [M+H]⁺.

[0075] Step 3: (2-(2,2,2-Trifluoroethyl)-lH-imidazol-5-yl)methanol. To a solution of ethyl 2-(2,2,2-trifluoroethyl)-U/-imidazole-5-carboxylate (1 g, 4.5 mmol) in anhydrous THF (50 mL) was added 1 M LAH in THF (11.2 mL, 11.2 mmol) at 0 °C. The resulting mixture was warmed to room temperature and stirred for 3 h. The reaction was quenched by the addition of water and filtered. The filtrate was dried over Na2SO4, filtered, and concentrated under reduced pressure to yield (2-(2,2,2-trifluoroethyl)- lrt-imidazol-5-yl)methanol (560 mg) as a yellow solid. This compound was used in the next step without further purification. LCMS ESI-MS m/z 181 [M+H]⁺.

[0076] Step 4: 2-(2,2,2-Trifluoroethyl)-lH-imidazole-4-carbaldehyde. To a solution of (2-(2,2,2-trifluoroethyl)-lJ/-imidazol-5-yl)methanol (560 mg, 3.11 mmol) in DCM (50 mL) and MeCN (50 mL) was added MnCL (2.64 g, 31.1 mmol), and the resulting mixture was stirred at room temperature for 3 h. The reaction mixture was filtered and concentrated under reduced pressure to yield the crude 2-(2,2,2-trifluoroethyl)-U/-imidazole-4-carbaldehyde (498 mg) as a yellow solid. This compound was used in the next step without further purification. LCMS ESIMS m/z 179 [M+H]⁺.

[0077] Step 5: tert-Butyl 4-formyl-2-(2,2,2-trifluoroethyl)-lZ/-imidazole-l -carboxylate.

A solution of 2-(2,2,2-trifluoroethyl)-lrt-imidazole-4-carbaldehyde (1 g, 5.61 mmol), TEA (1.13 g, 11.2 mmol) and (Boc)2O (2.45 g, 11.2 mmol) in DCM (40 mL) was stirred at room temperature for 16 h. The mixture was quenched by the addition of H2O (20 mL) and the organics were extracted into EtOAc (3 x 100 mL). The organic layers were combined, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified via silica gel column chromatography to yield tert-butyl 4-formyl-2-(2,2,2-trifluoroethyl)-lJT- imidazole-1 -carboxylate (716 mg, 46%) as a brown oil. LCMS ESI-MS m/z 179 [M+H-Boc]⁺. [0078] Step 6: tert-Butyl 4-(hydroxy(pyridin-4-yl)methyl)-2-(2,2,2-trifluoroethyl)-lH- imidazole-l-carboxylate. A solution of 4-iodopyridine (839 mg, 4.09 mmol) in THF (30 mL) was cooled to 0 °C, and EtMgBr (4.10 mL, 4.09 mmol, 1.0 M in THF) was added to it dropwise. The mixture was stirred at 0 °C for 1 h, and tert-butyl 4-formyl-2-(2,2,2-trifluoroethyl)-lJT- imidazole-1 -carboxylate (716 mg, 2.57 mmol) was added at 0 °C. After the addition, the resulting mixture was warmed to room temperature and stirred for 16 h. The mixture was quenched with the addition of H2O (20 mL) and the organics were extracted into EtOAc (3 x 80 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to yield the crude tert-butyl 4-(hydroxy(pyridin-4- yl)methyl)-2-(2,2,2-trifluoroethyl)-U/-imidazole-l-carboxylate (2.87 g) as a brown oil. This compound was used in the next step without further purification. LCMS ESI-MS m/z 358 [M+H]⁺.

[0079] Step 7: 4-((2-(2,2,2-Trifluoroethyl)-lH-imidazol-4-yl)methyl)pyridine. A mixture of crude tert-butyl 4-(hydroxy(pyridin-4-yl)methyl)-2-(2,2,2-trifluoroethyl)-lJ/-imidazole-l- carboxylate (2.87 g, 8.03 mmol) and Zn (5.22 g, 80.3 mmol) in AcOH (50 mL) was heated to 130 °C for 16 h. The resulting mixture was filtered, and the precipitate rinsed with MeOH (50 mL). The filtrate was concentrated under reduced pressure. To this residue was added saturated NaHCCh (50 mL) and the organics were extracted into DCM/MeOH (10: 1, 3 x 50 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by prep-HPLC to yield 4-((2-(2,2,2-trifluoroethyl)- l//-imidazol-4-yl)methyl)pyridine (Compound 1) (308 mg, 31%) as a white solid. 'H NMR (400 MHz, DMSO-t/6, ppm): 5 12.01 (s, 1 H), 8.44 (d, J= 6.0 Hz, 2 H), 7.23 (d, J= 6.0 Hz, 2 H), 6.84 (s, 1 H), 3.84 (s, 2 H), 3.65 (q, J= 10.8 Hz, 2 H). LCMS ESI-MS m/z 242 [M+H]⁺.

Example 2: Axon protection using Compound 1 and adjuvant NAD, NAM or NR

[0080] The following data demonstrate robust enhancement of axon protection using a combination treatment of a SARM1 inhibitor primary therapy with adjuvant nicotinamide adenine dinucleotide (NAD), nicotinamide (NAM), or NR (nicotinamide riboside). The dorsal root ganglion (DRG) sensory neuron axotomy assay was used to assess axon protection. In brief, primary sensory neurons were plated in a spot in the center of the well. Axons grow outward from concentrated cell bodies and injury was applied by microscalpel to the axons. Within 24 hours post-axotomy, axon distal to the injury site were completely fragmented and degenerated. Lack of SARM1 via genetic knockdown or knockout or SARM1 inhibition effectively halted axon degeneration in this assay.

Methods

[0081] Primary neuron culture and axotomy assay: DRGs were dissected from C57/BL6 mouse embryos on embryonic day 13.5 and mechanically dissociated following incubation with 0.05% trypsin-EDTA at 37C. Cell suspension (luL) was spotted on poly-D-lysine/laminin coated 24-well plates and cultured in Neurobasal media supplemented with 2% B27, 0.45% D- glucose, 1% glutamine, 100 U/mL penicillin, and 100 U/mL streptomycin. 5pM 5-fluoro-2’- deoxyuridine and 5pM uridine were added to the culture media to inhibit non-neuronal cell proliferation at DIV 1. Media was exchanged every 3-5 days. Experiments were performed on DIV 10-15. Treatments were mixed in DMSO (dimethyl sulfoxide) or Neurobasal media and added 1 before axotomy. Axotomy was performed with a microscalpel. At 24 hours post- axotomy neurons were fixed in 4% paraformaldehyde, washed in PBS with 0.1% Triton X-100, blocked in wash buffer plus 5% goat serum, stained overnight with anti-TubulinP3 (Biolegend), washed, stained with anti-mouse Alexa Fluor 488, and mounted in Fluoromount-G with DAPI (Invitrogen). Axons were imaged on a Zeiss CD7 microscope using Plan-Apochromat 5x/0.35 objective plus Optovar 2x tubulens and degeneration was quantified by identifying intact versus fragmented axons using a custom thresholding program in Zeiss Zen analysis software. The degree of degeneration is presented as a degeneration index (fragmented axon area / total axonal area) where 1.0 represents complete axon fragmentation and 0.0 represents completely intact axons within the analyzed field of view. Data was plotted in GraphPad Prism 9.5. Curves were fit with nonlinear regression ([Inhibitor] vs. response: variable slope (four parameters)) and used to calculate IC50 values.

[0082] To assess additive effects of NAD, NAM, and NR it was first determined whether these molecules could prevent axon degeneration without SARM1 inhibition. Figs. 1A-C demonstrate that NAD, NR, and NAM protected axons at 24 hours post-injury when treated at high concentrations with IC50 values of 1.9 mM, 0.5 mM, and 0.2 mM, respectively.

[0083] To assess the potential to enhance the potency of SARM1 inhibitor, two ‘subprotecting’ doses of NAD, NAM, and NR were chosen to test in combination with a dose response from 1.2 to 0.017 pM of the SARM1 inhibitor, Compound 1. These concentrations did not confer axonal protection and are indicated by the dashed lines on Fig. 1A at 410 pM & 140 pM of NAD, on Fig. IB at 45 pM & 15 pM of NAM, and on Fig. 1C at 45 pM & 15 pM of NR. All treatments were run in parallel from the same batch of DRGs on the same day and fixed at 24 hours post-axotomy. In Fig. 2, NAD supplementation at 410 pM or 140 pM significantly enhanced the potency of axon protection compared to Compound 1 only (Table 1, column 5). Without Compound 1, vehicle, NAD at 410 pM, or 140 pM failed to confer any axon protection (hollow squares) and has no effect in uninjured axons (solid squares). In Fig. 3, NAM supplementation at 45 pM or 15 pM left significantly enhanced the potency of axon protection compared to Compound 1 only (Table 1, column 5). Without Compound 1, vehicle, NAM at 45 pM, or 15 pM pM failed to confer any axon protection (hollow squares) and has no effect in uninjured axons (solid squares). In Fig. 4, NR supplementation at 45 pM or 15 pM significantly enhanced the potency of axon protection compared to Compound 1 only (Table 1, column 5). Without Compound 1, vehicle, NR at 45 pM, or 15 pM pM failed to confer any axon protection (hollow squares) and has no effect in uninjured axons (solid squares).

[0084] Calculated IC50 from best fit curves are presented in Table 1. Adjuvant enhancement fold changes were calculated by dividing IC50 from Compound 1 treatment alone by the IC50 of Compound 1 with NAD, NAM, or NR treatments. P values were calculated using one-way ANOVA.

Table 1

[0085] In summary, the axon protective potency Compound 1 was enhanced by 2-fold or greater with the addition of NAD, NAM, or NR in primary neurons subjected to axotomy.

Example 3: Co-dosing Compound 1 and Nicotinamide Riboside in Spared Nerve Injury (SNI) Rat Model

[0086] SARM1 knockout or inhibition effectively prevents neurodegeneration in peripheral nerve injury models, such as spared nerve injury (SNI). Oral supplements that raise tissue nicotinamide adenine dinucleotide (NAD) levels such as nicotinamide riboside (NR) may be neuroprotective.

[0087] To determine whether NR and uncompetitive SARM1 inhibitors act synergistically to enhance SAR 1 inhibition in vivo, NR and a SAR 1 inhibitor were tested in a rat model of SNI individually or in combination. In isolation, oral NR (400 mg/kg QD) failed to prevent elevation of a plasma biomarker of neurodegeneration, neurofilament light chain (NfL), on days 2 and 3 post-SNI compared to vehicle treated animals (Fig. 5). A suboptimal oral dose of a SARM1 inhibitor (Compound 1, 7.5 mg/kg BID) in isolation was able to partially suppress plasma NfL on day 2 post-SNI, but not on day 3. The combination treatment of Compound 1 and NR at the same dose levels effectively suppressed plasma NfL elevation on day 2 and day 3 post-SNI compared to vehicle and to a greater extent than either treatment in isolation (Fig. 5). For Fig. 5, plasma NfL was measured in SNI animals treated with vehicle, vehicle; Compound 1 at 7.5 mg/kg PO BID, NR at 400 mg/kg PO QD; vehicle, or NR at 400 mg/kg PO QD;

Compound 1 at 7.5 mg/kg PO BID at day 2, and 3 post-SNI. Data represent mean ± SD. 2-way ANOVA with Holm-Sidak's multiple comparisons test was applied to each time point. Significance notation: ns > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0088] NR had no effect on plasma exposure of Compound 1 at 2h post-dose on day 2 post- SNI (Fig. 6). For Fig. 6, plasma concentrations of Compound 1 were measured in SNI animals treated with vehicle; Compound 1 at 7.5 mg/kg PO BID or NR at 400 mg/kg PO QD;

Compound 1 at 7.5 mg/kg PO BID at day 2 post-SNI. Data represent mean ± SD. Unpaired t-test was applied to Day 2. Significance notation: ns > 0.05. Materials and Methods

[0089] Formulation and dosing via oral gavage:

Compound 1 formulation: 1.875 mg/mL in 1% Methylcellulose, 0.5% Tween 80, in Normal saline.

Nicotinamide riboside formulation: 100 mg/mL in water Oral gavage: Formulations were brought to room temperature and stirred until a homogenous suspension was obtained. Rats were dosed at 4 mL/kg using feeding needles attached to syringes.

[0090] Animal information: Sprague Dawley rats were obtained from the Charles River Laboratories. Rats were maintained in a 12 h light/ dark cycle with ad libitum access to food and water while housed in the Innovive rack system. All animal experiments were conducted in accordance with the NIH Guide for the Care and Use of Laboratory animals. Age-matched and weight-matched adult rats were used in all in vivo experiments.

[0091] Spared nerve injury model: Rats were anesthetized via isoflurane and the surgical site on the left thigh was shaved and washed via alternating ethanol and betadine scrubs. A small incision was made on the posterior face of the thigh to uncover the muscle, and forceps were used to create a gap in the biceps femoris and expose the sciatic nerve. Next, the sciatic nerve was tracked distal to the branch point of the sural, peroneal, and tibial nerves and the tibial + peroneal nerves were severed with scissors, while the sural nerve was left intact. A drop of bupivacaine (diluted to 0.125% in saline) was applied directly to the surgical site. The wound was closed using wound clips. Following surgery, buprenorphine (diluted to 0.03 mg/mL in saline) was dosed subcutaneously at 0.1 mg/kg and rats were recovered in a warming cage. The surgical site was monitored continuously for wound opening or infection. Sham surgery replicated all steps described above excluding nerve injury.

[0092] Blood collection and plasma preparation (tail vein collection): Rats were anesthetized via vaporized isoflurane (~3%) and blood was collected from either the left or right tail veins. Briefly, the collection area was cleaned by with ethanol wipes. While the tail was held straight, the needle was inserted into the vein and blood was collected by applying negative pressure to the syringe by withdrawing the plunger. To collect plasma, the blood was centrifuged at 14,000 rpm for 5 mins at 4°C and the supernatant (plasma) was aspirated into a new 1.5ml tube or 96 well plate. Plasma was stored at -80°C until analysis.

Bioanalytical Assay for SARM1 Inhibitors

[0093] Sample preparation procedure: An aliquot of 20 pL of plasma sample was extracted with 100 pL of acetonitrile containing Bucetin (internal standard). The mixture was shaken on a shaker for 15 minutes and subsequently centrifuged at 4000 rpm for 15 minutes. An aliquot of 70 pL of the supernatant was mixed with 70 pL of water for the injection to the LC/MS/MS. Calibration standards and quality control samples were prepared by spiking the test compound into corresponding blank plasma and the resulting plasma samples were processed with unknown samples in the same batch.

[0094] LC/MS/MS conditions for Compound 1: LC/MS/MS measurement was run on Shimadzu LC30AD System using the following conditions: Column: Waters Acquity UPLC BEH C18, 50 x 2.1 mm, 1.7p; Mobile phase: (A) 5mM ammonium acetate in water, (B) 0.1% formic acid in acetonitrile; Elution program: Gradient from 10% B to 95% B over 1.3 minutes, 95% B for 0.5 minutes, return to 10% B over 0.05 minutes and hold for 0.65 minutes; Flow Rate: 0.5 ml/min; Temperature: 50 °C; Detection: Sciex API 5000, multiple reaction monitoring, turbo ion spray for Compound 1 (QI m/z: 242.051; Q3 m/z: 91.90); positive ionization for Bucetin internal standard (QI m/z: 224.200; Q3 m/z: 136).

Plasma Neurofilament Light (pNfL) measurement (MSD)

[0095] Plasma was diluted in assay kit dilution buffer and NfL was measured using the MESO QuickPlex SQ 120 per manufacturer’s instructions.

[0096] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS What is claimed is:

1. A method of treating a neurological condition in a subject, comprising administering to the subject a therapeutically effective amount of 4-((2-(2,2,2-trifluoroethyl)-lH- imidazol-4-yl)methyl)pyridine (Compound 1) and a therapeutically effective amount of nicotinamide adenine dinucleotide (NAD+) or a derivative thereof.

2. The method of claim 1, comprising administering to the subject a therapeutically effective amount of Compound 1 and a therapeutically effective amount of NAD+.

3. The method of claim 1, wherein the NAD+ derivative is selected from the list consisting of nicotinamide riboside (NR), nicotinic acid (NA), nicotinic acid riboside (NaR), nicotinamide (NAM), nicotinamide mononucleotide (NMN), nicotinic acid mononucleotide (NaMN), tryptophan, vitamin B3, nicotinic acid adenine dinucleotide (NAAD), quinolinic acid, 3-hydroxyanthranilate, 3 -hydroxy kynurenine, kynurenine, and N -formyl kynurenine .

4. The method of any of the preceding claims, wherein Compound 1 and the NAD+ or derivative thereof are administered substantially simultaneously.

5. The method of any of the preceding claims, wherein Compound 1 and the NAD+ or derivative thereof are administered separately.

6. The method of any of the preceding claims, wherein Compound 1 and the NAD+ or derivative thereof are administered sequentially.

7. The method of any of the preceding claims, wherein Compound 1 and the NAD+ or derivative thereof are co-formulated for administration by a single formulation.

8. The method of any of the preceding claims, wherein Compound 1 and the NAD+ or derivative thereof are administered in separate compositions.

9. The method of any of the preceding claims, wherein Compound 1 and the NAD+ or derivative thereof are admixed prior to administration.

10. The method of any one of the preceding claims, wherein the neurological condition is depression or a disorder related to depression.

11. The method of claim 10, wherein the depression is major depressive disorder, persistent depressive disorder, bipolar disorder, treatment resistant depression (TRD), postpartum depression, premenstrual dysphoric disorder, or seasonal affective disorder.

12. The method of claim 10, wherein the disease or disorder related to depression is anxiety.

13. The method of any one of the preceding claims, wherein Compound 1 and the NAD+ or derivative thereof act synergistically.

14. A method of reducing axon degeneration, the method comprising comprising administering to a subject a therapeutically effective amount of 4-((2-(2,2,2- trifluoroethyl)-lH-imidazol-4-yl)methyl)pyridine (Compound 1) and a therapeutically effective amount of nicotinamide adenine dinucleotide (NAD+) or a derivative thereof.

15. The method of claim 14, comprising administering to the subject a therapeutically effective amount of Compound 1 and a therapeutically effective amount of NAD+.

16. The method of claim 14, wherein the NAD+ derivative is selected from the list consisting of nicotinamide riboside (NR), nicotinic acid (NA), nicotinic acid riboside (NaR), nicotinamide (NAM), nicotinamide mononucleotide (NMN), nicotinic acid mononucleotide (NaMN), tryptophan, vitamin B3, nicotinic acid adenine dinucleotide (NAAD), quinolinic acid, 3-hydroxyanthranilate, 3 -hydroxy kynurenine, kynurenine, and N -formyl kynurenine .

17. The method of any one of claims 14-16, wherein Compound 1 and the NAD+ or derivative thereof are administered substantially simultaneously.

18. The method of any one of claims 14-16, wherein Compound 1 and the NAD+ or derivative thereof are administered separately.

19. The method of any one of claims 14-16, wherein Compound 1 and the NAD+ or derivative thereof are administered sequentially.

20. The method of any one of claims 14-16, wherein Compound 1 and the NAD+ or derivative thereof are co-formulated for administration by a single formulation.

21. The method of any one of claims 14-16, wherein Compound 1 and the NAD+ or derivative thereof are administered in separate compositions.

22. The method of any one of claims 14-16, wherein Compound 1 and the NAD+ or derivative thereof are admixed prior to administration.

23. The method of any one of claims 14-22, wherein Compound 1 and the NAD+ or derivative thereof act synergistically.