[0001] The present application claims the benefit of priority to U.S. Provisional Patent Application No. 63/364,939 filed on May 18, 2022, which application is incorporated in its entirety as if fully set forth herein.

BACKGROUND

[0002] Remyelination is a process facilitated by the differentiation of oligodendrocyte progenitor cells (OPCs) into functional, myelin sheath-forming oligodendrocytes (OLs). In multiple sclerosis (MS), inflammatory lesions progressively fail to remyelinate, contributing to irreversible axonal loss and neurodegeneration that correlates with clinical disability. MS is a debilitating autoimmune disease that is characterized by episodes of focal inflammation leading to the primary demyelination of axons, neuronal dysfunction, and ultimately axonal loss ¹² ' ¹⁴ . Inflammatory and immune attacks in MS target oligodendrocytes (OLs), which are the cell type within the central nervous system (CNS) that produce and maintain the myelin sheath surrounding axons. Approved treatments for MS primarily consist of anti-inflammatory and immunomodulatory drugs. Despite the beneficial impact that these drugs have on disease severity and frequency of relapse, immune targeting alone ultimately fails and MS invariably progresses to a state of chronic demyelination, permanent disability, and reduced lifespan ¹⁸ .

[0003] As the most prevalent demyelinating disease of the CNS, MS affects approximately 2.5 million people worldwide ¹² . It is the most common neurological disease of young adults in North America, with typical onset around the third decade of life ^{15 16} . Hence, the socioeconomic burden associated with MS is significant due to the high cost of treatment and additional care associated with neurological disability ¹⁷ .

[0004] The development of regenerative therapeutics to address the observed impairment of remyelination that occurs during progressive phases of MS can be recognized as a major unmet medical need. Remyelination is a regenerative process that persists throughout adulthood in mammals and involves the differentiation of oligodendrocyte progenitor cells (OPCs) into myelinating oligodendrocytes (OLs). The ability of OPCs to facilitate remyelination diminishes with disease progression, age, and in the presence of inhibitory milieu.

[0005] Genetic fate mapping studies have shown that OL progenitor cells (OPCs), a highly abundant population of cells that persist throughout adulthood within the mammalian CNS, give rise to remyelinating OLs ^19,20 . OPCs, which are identified by distinct lineage markers including PDGFRa (platelet-derived growth factor receptor alpha) and NG2 (neural/glial antigen 2), are estimated to make up ~5% of total cells within the CNS ²¹ . In response to demyelination, OPCs become activated to drive remyelination, a regenerative process that is defined by the recruitment and subsequent differentiation of OPCs, resulting in the generation of mature, myelinating OLs. ²² ' ²⁷ . Based on the abundant numbers of OPCs that are often observed to be present in MS lesions ¹ , it is widely believed that inhibition of OPC differentiation plays a major role in disease progression ²⁸ ' ³² . OLs that survive demyelination can participate in myelin repair, but they do so in a limited capacity compared to newly -generated OLs ³³ ' ³⁵ .

SUMMARY

[0006] With an increasing rate of MS incidence throughout the world, along with a rise in disability within aging populations, there is a need for regenerative therapies to promote repair and functional recovery ^2,3 .

[0007] Accordingly, the present disclosure provides, in embodiments, a method for the treatment of a demyelinating disease in a subject suffering therefrom. The method comprises administering to the subject at least one muscarinic receptor antagonist and at least one vitamin D receptor (VDR) agonist.

[0008] In additional embodiments, the present disclosure provides a method for remyelination of demyelinated axons in a subject. The method comprises administering to the subject at least one muscarinic receptor antagonist and at least one vitamin D receptor (VDR) agonist. [0009] A muscarinic 1 receptor (MIR) antagonist, clemastine, was recently evaluated in a placebo-controlled Phase II clinical trial as a remyelinating therapy for MS (ReBUILD, UCSF) ¹⁰ . Using a maximally tolerated 5.36 mg twice per day dose of clemastine, the study met its primary efficacy endpoint based on impact on full field visual evoked potentials, an accepted biomarker of remyelination in MS patients, with an observed reduction in latency delay by 1.7 ms/eye (95% CI 0.5-2.9; p=O.OO48) ¹⁰ . While this clinical result provides evidence for the ability of MIR antagonists to promote functional remyelination in MS patients, , the magnitude of the effect was modest at the evaluated dose. It is evident that dose-limiting toxicity is a significant obstacle to the ability of clemastine to achieve optimal remyelination efficiency and there is a need for drugs that can improve the efficacy of such compounds and abrogate the toxic effects of, e.g., high dosage.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG. 1. Structures and comparative myelin basic protein (MBP)+ activity of MIR antagonists clemastine, orphenadrine, doxepin, or escitalopram.

[0011] FIG. 2. Structures and MBP+ activity of vitamin D3 analogues.

[0012] FIG. 3. Impact of supplementation with vitamin D3 analogue calcipotriol on MIR antagonist-induced OL maturation / MBP+ activity.

[0013] FIGS. 4A - 41. Impact of VDR agonist added to additional MIR antagonists. A. Double gradient heat map showing doxepin +/- calcipotriol activity as average (n=3) % of T3-induced MBP+ OLs (blue = 0%; yellow = optimal % for doxepin; red = largest value). B. Comparative impact on % T3- induced MBP+ OLs by doxepin at range of doses vs. doxepin + calcipotriol (100 nM). C. Comparative impact on % T3- induced MBP+ OLs by doxepin (7 nM) vs. with calcipotriol (100 nM) added. D. Double gradient heat map showing orphenadrine +/- calcipotriol activity as average (n=3) % of T3 -induced MBP+ OLs (blue = 0%; yellow = optimal % for orphenadrine; red = largest value). E. Comparative impact on % T3 -induced MBP+ OLs by orphenadrine at range of doses vs. orphenadrine + calcipotriol (100 nM). F. Comparative impact on % T3-induced MBP+ OLs by orphenadrine (7 nM) vs. with calcipotriol (100 nM) added. G. Double gradient heat map showing escitalopram +/- calcipotriol activity as average (n=3) % of T3-induced MBP+ OLs (blue = 0%; yellow = optimal % for escitalopram; red = largest value). H. Comparative impact on % T3 -induced MBP+ OLs by escitalopram at range of doses vs. escitalopram + calcipotriol (100 nM). I. Comparative impact on % T3-induced MBP+ OLs by escitalopram (60 or 185 nM) vs. with calcipotriol (100 nM) added. and “***” represent p < 0.01 and p < 0.001; respectively.

[0014] FIG. 5A and FIG. 5B. Impact of VDR modulators on clemastine or clemastine + calcipotriol-induced differentiation. A. Comparative impact on % T3-induced MBP+ OLs by clemastine (100 nM) +/- calcipotriol (100 nM) or VDR antagonist ZK 159222 (200 nM). B. Comparative impact on % T3-induced MBP+ OLs by low-dose clemastine (60 nM) vs. with calcipotriol (100 nM) or calcifediol (25-hydroxycholecalciferol; 100 nM) added. “****” represents p < 0.0001.

[0015] FIG. 6. Image-based quantification of MBP+ OLs following 4 d treatment with calcipotriol (100 nM), clemastine (60 nM) or a combination using OPCs isolated from young rats. Treatment was initiated following 48 h postisolation recovery. Representative images show impact on 3 m/o rat whole brain- derived MBP+ OLs following 4 d treatment with T3 (1 pM), clemastine (60 nM or 200 nM), calcipotriol (100 nM), or calcipotriol + clemastine (60 nM). “****” represents p < 0.0001.

[0016] FIG. 7 The Ml muscarinic receptor (MIR) agonist carbachol blocks the oligodendrocyte progenitor cell (OPC) differentiation-inducing activities of doxepine, orphenadrine, and escitalopram as determined based on Western blot analysis of induced myelin basic protein (MBP) expression in treated OPCs.

The results demonstrate that MIR antagonism is responsible for OPC inducingactivity.

DETAILED DESCRIPTION [0017] The present disclosure is premised in part upon the surprising and significant enhancement of OL maturation above levels achieved by the monotherapy in a combination of muscarinic receptor antagonists, such as MIR antagonists, and Vitamin D Receptor (VDR) agonists. More specifically, the present disclosure is premised in part upon a combination of agents (i.e., differentiation-inducing and maturation-inducing) that surprisingly induces overall OPC differentiation and maturation. The present disclosure also relates to the identification of pharmacological agents, or combinations thereof, that can directly stimulate the process of remyelination by inducing the differentiation of OPCs into mature OLs. Agents that promote functional OL maturation translate to disease-modifying treatments for demyelinating diseases. Furthermore, coadministration of agents that modulate multiple, relevant remyelination targets, such as the muscarinic or vitamin D receptors, result in superior treatments for such diseases.

[0018] Well-tolerated non-MIR differentiation-inducing agents were based upon screening of the drug repurposing ReFRAME (Repurposing, Focused Rescue, and Accelerated Medchem) collection. The ReFRAME collection is a set of 12,000 compounds that have been clinically evaluated or have undergone extensive preclinical profiling ¹⁰⁹ . One of the major advantages of screening this collection is that the drugs, approximately 38% of which are FDA-approved, have demonstrated safety profiles, thereby significantly reducing the time required for preclinical optimization (e.g., pharmacokinetics and safety) following discovery.

[0019] Definitions

[0020] As used herein, and unless otherwise clear from context or specified to the contrary, the term “compound” is inclusive in that it encompasses a compound or a pharmaceutically acceptable salt thereof.

[0021] In this disclosure, a “pharmaceutically acceptable salt” is a pharmaceutically acceptable, organic or inorganic acid or base salt of a compound described herein. Representative pharmaceutically acceptable salts include, e.g., alkali metal salts, alkali earth salts, ammonium salts, water-soluble and water-insoluble salts, such as the acetate, amsonate (4,4-diaminostilbene- 2,2-disulfonate), benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium, calcium edetate, camsylate, carbonate, chloride, citrate, clavulariate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexafluorophosphate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methyl sulfate, mucate, napsylate, nitrate, N-methylglucamine ammonium salt, 3 -hydroxy -2-naphthoate, oleate, oxalate, palmitate, pamoate (l,l-methene-bis-2- hydroxy-3 -naphthoate, einbonate), pantothenate, phosphate/diphosphate, picrate, polygalacturonate, propionate, p-toluenesulfonate, salicylate, stearate, subacetate, succinate, sulfate, sulfosaliculate, suramate, tannate, tartrate, teoclate, tosylate, triethiodide, and valerate salts. A pharmaceutically acceptable salt can have more than one charged atom in its structure. In this instance the pharmaceutically acceptable salt can have multiple counterions. Thus, a pharmaceutically acceptable salt can have one or more charged atoms and/or one or more counterions.

[0022] As used herein, the term “oligodendrocyte precursor cell” or “OPC” refers to an undifferentiated progenitor cell with the capacity to self-renew and differentiate into a myelinating oligodendrocyte. A “mature myelinating cell fate” refers to a cell that is capable of forming myelin, e.g., a myelinating oligodendrocyte. “Differentiation” refers to the process by which a specialized cell type is formed from a less specialized cell type, for example, a myelinating oligodendrocyte from an OPC. In some embodiments, an OPC is identified by morphology and/or by the presence of a biomarker, e.g., PDGFR-a or NG2. In some embodiments, a myelinating oligodendrocyte is identified by morphology and/or by the presence of a marker, e.g., myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), 2'3'-cyclic-nucleotide 3' phosphodiesterase (CNP), galactocebroside (GalC), 01 antigen (01), or 04 antigen (04). [0023] As used herein, the term “remyelination” refers to inducing an increased amount of myelin surrounding an axon, e.g., by administering an agent that induces the differentiation of oligodendrocyte precursor cells to a mature myelinating cell fate, as compared to the amount of myelin surrounding the axon in the absence of the agent being administered. In some embodiments, an agent stimulates “increased” myelination when the amount of myelin surrounding the axon in a sample (e.g., a brain tissue sample from a subject having a demyelinating disease) subsequent to administration of an agent that induces the differentiation of OPCs to a mature myelinating cell fate is at least about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more as compared to the amount of myelin surrounding the axon in the sample prior to administration of the agent. The amount of myelin surrounding an axon can be measured by any method known in the art, e.g., using magnetic resonance imaging (MRI). In some embodiments, an agent stimulates increased myelination when one or more characteristics of a demyelinating disease (e.g., multiple sclerosis) improves subsequent to administration of an agent that induces differentiation of OPCs to a mature myelinating cell fate as compared to the characteristic of the diseases prior to administration of the agent. As a nonlimiting example, an agent is said to stimulate increased myelination in a subject having multiple sclerosis when the frequency and/or severity of inflammatory attacks decreases subsequent to administration of an agent as compared to the frequency and/or severity of inflammatory attacks prior to administration of the agent.

[0024] As used herein, the term “demyelinating disease” refers to a disease or condition of the nervous system characterized by damage to or loss of the myelin sheath of neurons. A demyelinating disease can be a disease affecting the central nervous system or a disease affecting the peripheral nervous system. Examples of demyelinating diseases include, but are not limited to, multiple sclerosis, idiopathic inflammatory demyelinating disease, transverse myelitis, Devic's disease, progressive multifocal leukoencephalopathy, optic neuritis, leukodystrophy, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, autoimmune peripheral neuropathy, Charcot-Marie-Tooth disease, acute disseminated encephalomyelitis, adrenoleukodystrophy, adrenomyeloneuropathy, Leber's hereditary optic neuropathy, or HTLV- associated myelopathy. In some embodiments, the demyelinating disease is multiple sclerosis.

[0025] The terms “treat”, “treating” and “treatment” refer to the amelioration or eradication of a disease or symptoms associated with a disease. In various embodiments, the terms refer to minimizing the spread or worsening of the disease resulting from the administration of one or more prophylactic or therapeutic compounds described herein to a patient with such a disease.

[0026] The terms “prevent,” “preventing,” and “prevention” refer to the prevention of the onset, recurrence, or spread of the disease in a patient resulting from the administration of a compound described herein.

[0027] The term “effective amount” refers to an amount of a compound as described herein or other active ingredient sufficient to provide a therapeutic or prophylactic benefit in the treatment or prevention of a disease or to delay or minimize symptoms associated with a disease. Further, a therapeutically effective amount with respect to a compound as described herein means that amount of therapeutic agent alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or prevention of a disease. Used in connection with a compound as described herein, the term can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of disease, or enhances the therapeutic efficacy of or is synergistic with another therapeutic agent.

[0028] A “patient” or subject” includes an animal, such as a human, cow, horse, sheep, lamb, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit or guinea pig. In accordance with some embodiments, the animal is a mammal such as a non-primate and a primate (e.g., monkey and human). In one embodiment, a patient is a human, such as a human infant, child, adolescent or adult. In the present disclosure, the terms “patient” and “subject” are used interchangeably.

[0029] METHODS OF USE

[0030] Thus, in various embodiments, the present disclosure provides method for the treatment of a demyelinating disease in a subject suffering therefrom. The method comprises administering to the subject at least one muscarinic receptor antagonist and at least one vitamin D receptor (VDR) agonist.

[0031] In some embodiments, the muscarinic receptor antagonist is a muscarinic Ml receptor antagonist. Illustrative examples of Ml receptor antagonists suitable for this purpose include clemastine, orphenadrine, doxepin, escitalopram, pirenzepine, telenzepine, scopolamine, atropine, biperiden, V-[3- Oxo-3-[4-(4-pyridinyl)-l-piperazinyl]propyl]-2,l,3-benzothia diazole-4- sulfonamide (VU 0255035), dicyclomine, trihexyphenidyl, and pharmaceutically acceptable salts thereof. In an embodiment, the MR receptor antagonist is clemastine.

[0032] In various embodiments, the VDR agonist is selected from dihydrotachysterol, calcipotriol, eldecalcitol, elocalcitol, calcitriol, tacalcitol, seocalcitol, and pharmaceutically acceptable salts thereof. In an exemplary embodiment, the VDR agonist is calcipotriol.

[0033] Various combinations of the muscarinic receptor antagonist and VDR agonist are contemplated. For example, in an embodiment, the muscarinic receptor antagonist is clemastine and the VDR agonist is calcipotriol.

[0034] In some embodiments, the demyelinating disease is a demyelinating disease of the central nervous system (CNS). Demyelinating diseases of the CNS include, in various embodiments, multiple sclerosis, neuromyelitis optica (Devic's disease), an idiopathic inflammatory demyelinating disease, a leukodystrophic disease, acute disseminated encephalomyelitis, optic neuritis, transverse myelitis, adrenoleukodystrophy, adrenomyeloneuropathy, central pontine myelinolysis, and a leukoencephalopathy. An illustrative demyelinating disease of the CNS is multiple sclerosis. [0035] In an embodiment, the muscarinic receptor antagonist is clemastine, the VDR agonist is calcipotriol, and the demyelinating disease is multiple sclerosis.

[0036] In other embodiments, the demyelinating disease is a demyelinating disease of the peripheral nervous system. Examples of such a disease includes Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, anti-MAG peripheral neuropathy, Charcot-Marie-Tooth disease, hereditary neuropathy with liability to pressure palsy, a copper deficiency-associated condition, and progressive inflammatory neuropathy. In various embodiment, the copper deficiency-associated condition is selected from peripheral neuropathy, myelopathy, and optic neuropathy.

[0037] Treatment of the disease comprises, in one embodiment, retarding the rate of disease progression. Thus, therapeutic intervention as contemplated herein acknowledges that some demyelinating diseases may not be fully eradicated from the subject who is treated, and that acceptable therapy resides an extension of quality of life, motor skills, and the like, that would otherwise not occur but for the treatment as described herein. In other embodiments, treatment can result in the arrest of disease progression as determined, for example, by clinical assessment of symptoms and imaging techniques for direct observation of nervous system integrity. In still other embodiments, the methods described herein can comprise a reversal of disease progression, thereby restoring or improving at least some functionality lost to the subject through disease. In additional embodiments, the treatment reduces the frequency and/or severity of disease symptoms, such as those suffered by a subject in multiple sclerosis relapse or flare-up, including new and old symptoms, e.g., fatigue, dizziness, balance and coordination difficulty, vision trouble, incontinence, numbing or tingling sensations, memory difficulty, and trouble with mental concentration.

[0038] In additional embodiments, optionally in combination with any other embodiment described herein, the present disclosure provides a method for remyelination of demyelinated axons in a subject. As used herein, “remyelination” can refer to partial or complete remyelination. The method comprises administering to the subject at least one muscarinic receptor antagonist and at least one vitamin D receptor (VDR) agonist as described herein.

[0039] In some embodiments, the axons are partially demyelinated. In other embodiments, the axons are completely demyelinated. In still additional embodiments, the axons are in the central nervous system of the subject.

[0040] PHARMACEUTICAL COMPOSITION

[0041] The disclosure also provides, optionally for use in combination with the methods described herein, a pharmaceutical composition comprising a therapeutically effective amount of one or more compounds or a pharmaceutically acceptable salt described herein, in admixture with a pharmaceutically acceptable carrier. In some embodiments, the composition further contains, in accordance with accepted practices of pharmaceutical compounding, one or more additional therapeutic agents, pharmaceutically acceptable excipients, diluents, adjuvants, stabilizers, emulsifiers, preservatives, colorants, buffers, and/or flavor imparting agents.

[0042] In one embodiment, the pharmaceutical composition comprises a muscarinic receptor antagonist as described herein, a vitamin D receptor (VDR) agonist as described herein, and a pharmaceutically acceptable carrier.

[0043] The pharmaceutical composition of the present disclosure is formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular subject being treated, the clinical condition of the subject, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.

[0044] Amounts

[0045] The “therapeutically effective amount” of a compound or a pharmaceutically acceptable salt thereof that is administered is governed by considerations known to those in the art, and is the minimum amount necessary to induce oligodendrocyte progenitor cell (OPC) differentiation, to induce maturation of myelin sheath-forming oligodendrocytes (OLs), to remyelinate demyelinated axons, and combinations thereof. Such an amount may be below the amount that is toxic to normal cells, or the subject as a whole. Generally, the initial therapeutically effective amount of each muscarinic receptor antagonist and vitamin D receptor agonist that is administered is in the range of about 0.01 to about 200 mg/kg or about 0.1 to about 20 mg/kg of patient body weight per day, with the typical initial range being about 0.3 to about 15 mg/kg/day. Oral unit dosage forms, such as tablets and capsules, may contain from about 0.1 mg to about 1000 mg of a compound of the present disclosure. In another embodiment, such dosage forms contain from about 50 mg to about 500 mg of a compound of the present disclosure. In yet another embodiment, such dosage forms contain from about 25 mg to about 200 mg of a compound of the present disclosure. In still another embodiment, such dosage forms contain from about 10 mg to about 100 mg of a compound of the present disclosure. In a further embodiment, such dosage forms contain from about 5 mg to about 50 mg of a compound of the present disclosure. In any of the foregoing embodiments the dosage form can be administered multiple times per day, for example, once a day or twice per day.

[0046] Surprisingly, the combination of a muscarinic receptor (MR) antagonist and a vitamin D receptor (VDR) agonist achieves an additive or synergistic effect on remyelination, relative to the effect achieved by either agent alone. In some embodiments, including those in which the effect is additive, an advantage resides in the ability to use suboptimal concentrations of either single agent, thereby reducing or eliminating toxicity or off-target concerns attributable to use of optimal concentrations of a single agent. In embodiments, the ratio of MR antagonist to VDR agonist is about 200: 1 to about 0.05: 1, about 50: 1 to about 0.2: 1, about 10: 1 to about 0.3: 1, about 5: 1 to about 0.4: 1, or about 3: 1 to about 0.5: 1. Exemplary ratios include about 0.05: 1, 0.08: 1, 0.1 : 1, 0.2: 1, 0.3: 1. 0.4: 1. 0.5: 1, 0.6: 1, 0.7: 1, 0.8: 1, 0.9: 1, 1 : 1, 1.5: 1, and 2: 1. The amounts of MR antagonist and VDR agonist are chosen, in part, upon various factors known to the skilled clinician, including disease to be treated and health of the subject.

[0047] Routes of Administration and Formulations

[0048] The compositions of the present disclosure can be administered orally, topically, parenterally, by inhalation, spray, or rectally; in dosage unit formulations. The term parenteral as used herein includes, for example, subcutaneous injections, intravenous, intramuscular, intrasternal injection, or infusion techniques.

[0049] Suitable oral compositions as described herein include without limitation tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, syrups, or elixirs.

[0050] In an embodiment, also encompassed are pharmaceutical compositions suitable for single unit dosages that comprise a compound of the disclosure or its pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier.

[0051] The compositions of the present disclosure that are suitable for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions. For example, liquid formulations of the compounds of the present disclosure contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically palatable preparations of a compound of the present disclosure.

[0052] For tablet compositions, a compound of the present disclosure in admixture with non-toxic pharmaceutically acceptable excipients is used for the manufacture of tablets. Examples of such excipients include without limitation inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, com starch, or alginic acid; binding agents, for example, starch, gelatin or acacia; and lubricating agents, for example, magnesium stearate, stearic acid, or talc. The tablets may be uncoated or they may be coated by known coating techniques to delay disintegration and absorption in the gastrointestinal tract and thereby to provide a sustained therapeutic action over a desired time period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed.

[0053] Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin, or olive oil.

[0054] For aqueous suspensions, a compound of the present disclosure is admixed with excipients suitable for maintaining a stable suspension. Examples of such excipients include without limitation are sodium carboxymethylcellulose, methylcellulose, hydroxpropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum acacia.

[0055] Oral suspensions can also contain dispersing or wetting agents, such as naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example, heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example, polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example, ethyl, or n-propyl p- hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

[0056] Oily suspensions may be formulated by suspending a compound of the present disclosure in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol.

[0057] Sweetening agents such as those set forth above, and flavoring agents may be added to provide palatable oral preparations. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.

[0058] Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide a compound of the present disclosure in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present.

[0059] Pharmaceutical compositions of the present disclosure may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally-occuring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monoleate, and condensation reaction products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monoleate. The emulsions may also contain sweetening and flavoring agents.

[0060] Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, and flavoring and coloring agents.

[0061] The pharmaceutical composition may be in the form of a sterile injectable, an aqueous suspension or an oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3 -butanediol. Among the vehicles and solvents that may be employed are water, Ringer’s solution and isotonic sodium chloride solution. In addition, sterile, fixed oils can be conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

[0062] The compound as described herein may also be administered in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials are cocoa butter and polyethylene glycols.

[0063] Compositions for parenteral administrations are administered in a sterile medium. Depending on the vehicle used and concentration the concentration of the drug in the formulation, the parenteral formulation can either be a suspension or a solution containing dissolved drug. Adjuvants such as local anesthetics, preservatives and buffering agents can also be added to parenteral compositions.

[0064] The following examples provide further illustrative embodiments of the present disclosure.

[0065] EXAMPLES

[0066] General Methods

[0067] Screening of ReFRAME compound library. Using the high-content imaging assay described in B. A. Beyer el al., Nature Chemical Biology 14 (2018) 22 - 28, ReFRAME compounds were screened at 5 uM, confirmed in triplicate, and confirmed hit compounds (>15% T3 -induced activity) were tested in a 12-point dose response format at 10 pM with 3-fold dilutions down. A list of these compounds, their optimal activity, and reported toxicity, if any, is shown in Table 1.

[0068] Table 1

[0069] Pairwise combination screening. For combination screening, representatives were selected from 11 hit classes for evaluation at 8-12 concentrations pairwise with clemastine, the primary anchor compound, to identify additive, synergistic and/or dose-sparing activity. These were tested in the described combinations at 9-12 doses, with concentrations starting in the low nM range and increasing 3 -fold, and transferred onto plates in triplicate. Immunofluorescence staining was quantified based on % MBP ⁺ OLs. Data are represented in double-gradient heat maps that were generated in Prism (graphpad.com).

[0070] Isolation of rat oligodendrocyte progenitor cells (OPCs). A 3-month- old rat was euthanized using isoflurane and decapitated as soon as the heartbeat and breathing stopped. The brain was then quickly removed and placed into a 50ml conical tube containing 15 to 20ml of ice-cold Hibernate A (cat. HACAMG, Transnetyx). The brain was placed onto a 60 mm x 15 mm petri dish containing 2 ml of HBSS without Mg ²⁺ and Ca ²⁺ (cat. #141750095, GIBCO) and divided along the midsagittal plane. Meninges, the olfactory bulb, and white matter were mechanically removed using sterilized forceps. The brain was then cut into approximately 1mm ³ pieces using a scalpel and rinsed with 15 ml of HBSS without Mg ²⁺ and Ca ²⁺ and transferred into a 15 ml conical tube. Tissue was collected by centrifugation at lOOxg for 1 minute at room temperature. The supernatant was discarded and the tissue was gently resuspended in 10 ml dissociation solution containing 34 U/ml papain (cat. #LS003126, Worthington) and 40 pg/ml Type IV DNase (cat. #D5025, Sigma-Aldrich) in Hibernate A. The brain tissue was dissociated on an orbital shaker at 50 rpm for 40 minutes at 35°C. Nine ml of ice cold HBSS without Mg ²⁺ and Ca ²⁺ was added to conical tubes to stop digestion and centrifuged at 200xg for 3 minutes for tissue collection. To obtain a single cell suspension, supernatant was replaced with 5 ml of trituration solution containing 2 mM pyruvate (cat. #11360070, ThermoFisher Scientific) and 2% B27 without vitamin A (cat. #12587010, ThermoFisher Scientific) in Hibernate A. Using a 5 ml serological pipette, tissue was triturated very slowly for 10 times and the supernatant was transferred through a 70 pm cell strainer (cat. #542070, Greiner Bio-One) to a 50 ml conical tube filled with 12 ml of 90% Percoll® solution (cat. #17-0891-01, GE Healthcare). The tissue was triturated three more times using fire polished Pasteur pipettes with 2 ml of fresh trituration solution each time. Twenty -four ml of DMEMF12 with HEPES (cat. #11039-021, Gib co) was added to make the final Percoll concentration of 22.5%. Tubes were inverted to mix the solution and centrifuged at 800xg for 20 minutes at room temperature with no brake. Then the supernatant was aspirated from 1~2 ml of the solution and the pellet resuspended in 10ml of HBSS without Mg ²⁺ and Ca ²⁺ then centrifuged at 300xg for 5 minutes. After removing the supernatant, pellet was resuspended in 1 ml of red blood cell lysis buffer (cat. #R7757, Sigma-Aldrich) for 90 s. Nine ml of HBSS was added and centrifuged at 300 g for 5 min. The cell pellet was resuspended in 500 pl of wash buffer containing lx PBS, 2 mM pyruvate, 0.5% BSA, and 2 mM EDTA with 2.5 pg of A2B5 primary antibody (cat. #MAB312, Millipore) for every 10 million cells. The suspension was mixed and incubated at 4°C for 15 min. Seven ml of cold wash buffer was added to the tube and centrifuged at 300 g for 5 min. The pellet was resuspended in 80 pl of wash buffer and 20 pl of rat-anti-mouse-IgM antibody (cat. #130-047-302, Miltenyi) and incubated at 4°C for 15 min. Seven ml of cold wash buffer was then added to the tube and centrifuged at 300 g for 5 min. The pellet was resuspended in 500 pl of wash buffer and the OPCs were then selected using an MS column (cat. #130- 042-201, Miltenyi) on MiniMACS™ Separator (cat. #130-042-102, Miltenyi). MS column was pre-wet with 500 pl of wash buffer before adding cell suspension. After the cell suspension was passed through, the column was washed three times, each with 500 pl of wash buffer. A2B5 selected OPCs were plunged out of the column with ImL of OPC culture medium composed of 1 mM pyruvate, 10 pg/ml insulin (cat. #12585014, ThermoFisher Scientific), and 4% Brightcell SOS Neuronal Supplement (cat. #SCM147, Sigma-Aldrich) in BrainPhys Neuronal Medium (cat. #05790, Stemcell Technologies) supplemented with PDGF-AA at 50 ng/ml.

[0071] OPC differentiation assay using whole brain-derived rat OPCs.

OPCs were isolated as described above, and were seeded at 8K cells per well onto poly-d-lysine-coated black clear-bottom 96 well plates. They were given a 48 h recovery period in medium containing 1 mM pyruvate, 10 pg/ml insulin, and 4% Brightcell™ SOS Neuronal Supplement in BrainPhys™ Neuronal Medium supplemented with PDGF-AA at 50 ng/ml. At 48 h, the recovery medium was removed and replaced with OPC differentiation supplemented with B27-without vitamin A, lx non-essential amino acids, lx Glutamax, lx Antibiotic- Antimycotic, P-mercaptoethanol, and PDGF-AA at 2 ng/ml. Cells were treated with clemastine (60 nM, 200 nM), calcipotriol (100 nM), clemastine (60 nM) + calcipotriol (100 nM), or T3 (1 pM). The plates were incubated at 37 °C with 5% CO2 for 6 d. On day 6, cells were fixed, stained for MBP, and analyzed.

[0072] To evaluate clinically viable small molecules with reported muscarinic receptor antagonism, a cell-based phenotypic assay which quantifies the percentage of mature, myelin basic protein (MBP)-positive OLs was used. There were identified select compounds with lower ECso values and higher achievable human exposure compared to clemastine, a compound that was previously evaluated in the clinic (Figure 1). Activity was quantified by immunofluorescence analysis of percentage MBP ⁺ OLs after 6 days of compound treatment, with vehicle / DMSO, clemastine, and triiodothyronine (T3) controls on each plate using rat optic nerve derived OPCs. Following this initial set of profiling, additional Ml antagonists were evaluated for OPC differentiation-inducing activity. The majority of these small molecules were structurally orthogonal compounds or stereoisomers of doxepin, orphenadrine, and escitalopram, the prioritized candidates that demonstrated superior cellbased potency in the initial screen. A cluster of VDR agonists demonstrated MBP+ activity that reached -40-70% of that induced by T3 (Figure 2).

[0073] Combination screening proceeded with clemastine, doxepin, orphenadrine and escitalopram as exemplary muscarinic receptor antagonists. The impact of calcipotriol, a vitamin D3 analogue identified in initial singleagent screening, was evaluated for its activity on MIR antagonists and there was observed additive and dose-sparing activity with clemastine (Figure 3) and all muscarinic receptor antagonists that were profiled. The results show that a simultaneous Ml antagonism and VDR activation surprisingly enhances functional OL maturation. Calcipotriol supplementation with clemastine improves the ratio of Ml potency (IC50 = 35 nM) to the effective concentration required for OPC differentiation, which is as low as 60 nM with calcipotriol versus 0.25 pM alone. These data indicate that the use of a VDR agonist in combination with an MIR antagonist can reduce the concentration of an MR antagonist that is necessary for inducing OPC differentiation.

[0074] Additional MIR antagonists and VDR. VDR agonism induced a similar effect as observed for all MIR antagonists, including escitalopram, which provides more evidence that this SSRI induces differentiation via the same MIR mechanism as clemastine, doxepin and orphenadrine (FIG. 4A-4I).

[0075] Target Activity. The VDR antagonist ZK 159222 was used to probe on- target activity of calcipotriol alone and in combination with an MIR antagonist. ZK 159222 significantly inhibited calcipotriol -induced differentiation and reduced the combination effect with calcipotriol and clemastine (FIG. 5A). ZK 159222 is described as a partial VDR antagonist in that it binds VDR with similar affinity as vitamin D, likely explaining the incomplete inhibition of calcipotriol- or calcipotriol + clemastine-induced differentiation. 25- hydroxycholecalciferol (calcifediol) is hydroxylated to form calcitriol, the active form of vitamin D. 25-hydroxycholecalciferol demonstrated an activity profile consistent with calcipotriol in the differentiation assay and enhanced suboptimal clemastine-induced OL maturation in a similar manner (FIG. 5B). This result indicates that OPCs can convert 25-hydroxycholecalciferol to the active form of vitamin D.

[0076] VDR/M1R combination effect on OPCs. To ensure that the observed additive VDR/M1R combination effect was not exclusive to OPCs derived from the rat optic nerve, the combination was evaluated using whole brain-derived OPCs from 3-month-old rats isolated using an A2B5-MACS bead-based method. The differentiation assay was optimized in using this cell population: the most robust difference in vehicle vs. T3-induced MBP ⁺ OLs occurred between 4-5 days following a 48h recovery time. A significant increase in OPC differentiation was observed following treatment with calcipotriol or co-treatment with calcipotriol supplemented with suboptimal clemastine (FIG. 6). These findings provide evidence that the VDR/M1R combination effect is effective in both 5 day postnatal (P5) rat optic nerve-derived OPCs as well as whole brain OPCs isolated from 3 month old rats.

[0077] Useful references, including numbered references appearing throughout the present disclosure, are incorporated herein in their entireties, and they are as follows:

1 Chang, A., Tourtellotte, W. W., Rudick, R. & Trapp, B. D. Premyelinating oligodendrocytes in chronic lesions of multiple sclerosis. The New England journal of medicine 346, 165-173, doi: 10.1056/NEJMoaO 10994 (2002).

2 Feinstein, A., Freeman, J. & Lo, A. C. Treatment of progressive multiple sclerosis: what works, what does not, and what is needed. The Lancet. Neurology 14, 194- 207, doi: 10.1016/sl474- 4422(14)70231-5 (2015).

3 Franklin, R. J. & Gallo, V. The translational biology of remyelination: past, present, and future. Glia 62, 1905-1915, doi:10.1002/glia.22622 (2014). 4 Johnson, C. H., Ivanisevic, J. & Siuzdak, G. Metabolomics: beyond biomarkers and towards mechanisms. Nat Rev Mol Cell Biol 17, 451-459, doi: 10.1038/nrm.2016.25 (2016).

5 Shyh-Chang, N. & Ng, H.-H. The metabolic programming of stem cells. Genes & development 31, 336-346, doi:10.1101/gad.293167.116 (2017).

6 Beyer, B. A. et al. Metabolomics-based discovery of a metabolite that enhances oligodendrocyte maturation. Nature chemical biology 14, 22-28, doi: 10.1038/nchembio.2517 (2018).

7 Mei, F. et al. Micropillar arrays as a high-throughput screening platform for therapeutics in multiple sclerosis. Nature medicine 20, 954-960, doi: 10.1038/nm.3618 (2014).

8 Deshmukh, V. A. et al. A regenerative approach to the treatment of multiple sclerosis. Nature 502, 327-332, doi: 10.1038/naturel2647 (2013).

9 Huang, J. K. et al. Retinoid X receptor gamma signaling accelerates CNS remyelination. Nat Neurosci 14, 45-53, doi : 10.1038/nn.2702 (2011).

10 Green, A. J. et al. Clemastine fumarate as a remyelinating therapy for multiple sclerosis (ReBUILD): a randomised, controlled, double-blind, crossover trial. Lancet (London, England) 390, 2481- 2489, doi:10.1016/s0140-6736(17)32346-2 (2017).

11 Brown, J. W. L. et al. Safety and efficacy of bexarotene in patients with relapsing- remitting multiple sclerosis (CCMR One): a randomised, double-blind, placebo- controlled, parallel-group, phase 2a study. The Lancet. Neurology 20, 709-720, doi : 10.1016/S 1474-4422(21 )00179-4 (2021 ).

12 Pouly, S. & Antel, J. P. Multiple sclerosis and central nervous system demyelination. Journal of autoimmunity 13, 297-306, doi: 10.1006/jaut.1999.0321 (1999). 13 Reich, D. S., Lucchinetti, C. F. & Calabresi, P. A. Multiple Sclerosis. The New England journal of medicine 378, 169-180, doi:10.1056/NEJMral401483 (2018).

14 Loma, I. & Heyman, R. Multiple sclerosis: pathogenesis and treatment. Current neuropharmacology 9, 409-416, doi:10.2174/157015911796557911 (2011).

15 Rolak, L. A. Multiple sclerosis: it's not the disease you thought it was. Clin Med Res 1, 57-60 (2003).

16 Wallin, M. T. etal. The prevalence of MS in the United States: A population-based estimate using health claims data. Neurology 92, el029-el040, doi: 10.1212/WNL.0000000000007035 (2019).

17 Paz-Zulueta, M., Paras-Bravo, P., Cantarero-Prieto, D., Blazquez- Femandez, C. & Oterino-Duran, A. A literature review of cost-of- illness studies on the economic burden of multiple sclerosis. Mult Scler Relat Disord 43, 102162, doi: 10.1016/j.msard.2020.102162 (2020).

18 Gholamzad, M. et al. A comprehensive review on the treatment approaches of multiple sclerosis: currently and in the future. Inflamm Res 68, 25-38, doi: 10.1007/s00011-018-1185-0 (2019).

19 Zawadzka, M. et al. CNS-resident glial progenitor/ stem cells produce Schwann cells as well as oligodendrocytes during repair of CNS demyelination. Cell Stem Cell 6, 578-590, doi:10.1016/j. stem.2010.04.002 (2010). 0 Crawford, A. H., Tripathi, R. B., Richardson, W. D. & Franklin, R. J. M. Developmental Origin of Oligodendrocyte Lineage Cells Determines Response to Demyelination and Susceptibility to Age- Associated Functional Decline. Cell Rep 15, 761-773, doi:10.1016/j.celrep.2016.03.069 (2016). 1 Dawson, M. R., Polito, A., Levine, J. M. & Reynolds, R. NG2- expressing glial progenitor cells: an abundant and widespread population of cycling cells in the adult rat CNS. Mol Cell Neurosci 24, 476-488, doi: 10.1016/sl044-7431(03)00210- 0 (2003). Franklin, R. J. & Ffrench-Constant, C. Remyelination in the CNS: from biology to therapy. Nature reviews. Neuroscience 9, 839-855, doi:10.1038/nm2480 (2008). Franklin, R. J. M. & Ffrench-Constant, C. Regenerating CNS myelin - from mechanisms to experimental medicines. Nature reviews. Neuroscience 18, 753- 769, doi: 10.1038/nrn.2017.136 (2017). Kremer, D., Aktas, O., Hartung, H. P. & Kiiry, P. The complex world of oligodendroglial differentiation inhibitors. Ann Neurol 69, 602-618, doi: 10.1002/ana.22415 (2011). Patel, J. R. & Klein, R. S. Mediators of oligodendrocyte differentiation during remyelination. FEBS Lett 585, 3730-3737, doi: 10.1016/j.febslet.2011.04.037 (2011). Chari, D. M., Huang, W. L. & Blakemore, W. F. Dysfunctional oligodendrocyte progenitor cell (OPC) populations may inhibit repopulation of OPC depleted tissue. Journal of neuroscience research 73, 787-793, doi:10.1002/jnr, 10700 (2003). Wolswijk, G. Chronic stage multiple sclerosis lesions contain a relatively quiescent population of oligodendrocyte precursor cells.

The Journal of neuroscience : the official journal of the Society for Neuroscience 18, 601-609 (1998). Hart, I. K., Richardson, W. D., Bolsover, S. R. & Raff, M. C.

PDGF and intracellular signaling in the timing of oligodendrocyte differentiation. The Journal of cell biology 109, 3411-3417 (1989). Billon, N., Tokumoto, Y., Forrest, D. & Raff, M. Role of thyroid hormone receptors in timing oligodendrocyte differentiation. Dev Biol 235, 110-120, doi : 10.1006/dbio.2001.0293 (2001). Kuhlmann, T. et al. Differentiation block of oligodendroglial progenitor cells as a cause for remyelination failure in chronic multiple sclerosis. Brain : a journal of neurology 131, 1749-1758, doi:10.1093/brain/awn096 (2008). 31 Tokumoto, Y. M., Tang, D. G. & Raff, M. C. Two molecularly distinct intracellular pathways to oligodendrocyte differentiation: role of a p53 family protein. EMBO J 20, 5261-5268, doi:10.1093/emboj/20.18.5261 (2001).

32 Fernandez, M. et al. Thyroid hormone administration enhances remyelination in chronic demyelinating inflammatory disease. Proceedings of the National Academy of Sciences of the United States of America 101, 16363-16368, doi : 10.1073/pnas.0407262101 (2004).

33 Neely, S. A. et al. New oligodendrocytes exhibit more abundant and accurate myelin regeneration than those that survive demyelination. Nat Nenrosci. doi: 10.1038/s41593-021-01009-x (2022).

34 Orthmann-Murphy, J. et al. Remyelination alters the pattern of myelin in the cerebral cortex. eLife 9, doi: 10.7554/eLife.56621 (2020).

35 Bacmeister, C. M. et al. Motor learning promotes remyelination via new and surviving oligodendrocytes. Nat Neurosci 23, 819-831, doi:10.1038/s41593-020- 0637-3 (2020).

36 Najm, F. J. et al. Drug-based modulation of endogenous stem cells promotes functional remyelination in vivo. Nature 522, 216-220, doi:10.1038/naturel4335 (2015).

37 Jakel, S. et al. Altered human oligodendrocyte heterogeneity in multiple sclerosis. Nature 566, 543-547, doi:10.1038/s41586-019- 0903-2 (2019).

38 Marisca, R. et al. Functionally distinct subgroups of oligodendrocyte precursor cells integrate neural activity and execute myelin formation. Nat Neurosci 23, 363- 374, doi : 10.1038/s41593-019-0581 -2 (2020).

39 Neumann, B. et al. Metformin Restores CNS Remyelination Capacity by Rejuvenating Aged Stem Cells. Cell Stem Cell 25, 473-485. e478, doi: 10.1016/j.stem.2019.08.015 (2019). 40 Hampton, D. W. etal. Focal immune-mediated white matter demyelination reveals an age-associated increase in axonal vulnerability and decreased remyelination efficiency. Am J Pathol 180, 1897-1905, doi: 10.1016/j.ajpath.2012.01.018 (2012). 109 Janes, J. et al. The ReFRAME library as a comprehensive drug repurposing library and its application to the treatment of cryptosporidiosis. Proceedings of theNational Academy of Sciences of the United States of America 115, 10750-10755, doi:10.1073/pnas.1810137115 (2018).

U.S. Patent No. 9,592,288.