WO2019126759A1 - Compounds and methods of promoting myelination - Google Patents

Abstract

The present invention relates to a method of promoting myelination of central nervous system neurons in a subject suffering from a myelin-related disorder, the method comprising administering to the subject a therapeutically effective amount of a compound represented by structural Formula (I) or a pharmaceutically acceptable salt thereof.

Description

COMPOUNDS AND METHODS OF PROMOTING MYELINATION

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.

62/609,908, filed December 22, 2017, which is hereby incorporated by reference in its entirety.

BACKGROUND

Myelin-related disorders are disorders that result in abnormalities of the myelin sheath (e.g„ dysmyelination,demyelination and hypomyelination) in a subject’s neural cells, e.g., CNS neurons including their axons. Degradation of the myelin sheath in such disorders, produces a slowing or cessation of nerve cell conduction. The resulting myelin related disorders are characterized by deficits in sensation, motor function, cognition, or other physiological functions. Myelin related disorders include, but are not limited to, multiple sclerosis (MS), neuromyelitis optica (NMO), optic neuritis, pediatric leukodystrophies, neonatal white matter injury, age- related dementia, schizophrenia, progressive multifocal leukoencephalopathy (PML), encephalomyelitis (EPL), central pontine myelolysis (CPM), adrenoleukodystrophy, Alexander's disease, Pelizaeus Merzbacher disease (PMD), Vanishing White Matter Disease, Wallerian Degeneration, transverse myelitis, amylotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease, Parkinson's disease, spinal cord injury, traumatic brain injury, post radiation injury, neurologic complications of chemotherapy, stroke, acute ischemic optic neuropathy, vitamin E deficiency, isolated vitamin E deficiency syndrome, Bassen-Komzweig syndrome, Marchiafava-Bignami syndrome, metachromatic leukodystrophy, trigeminal neuralgia, acute disseminated encephalitis, Guillian-Barre syndrome, Charcot-Marie-Tooth disease, Bell's palsy and radiation-induced demyelination.

MS is the most common myelin-related disorder affecting several million people globally and is estimated to result in about 18,000 deaths per year. MS is a complex neurological disease characterized by deterioration of central nervous system (CNS) myelin. Myelin, composed in its majority by lipids (70% lipids, 30% protein), protects axons and makes saltatory conduction possible, which speeds axonal electric impulse. Demyelination of axons in chronic MS can result in axon degeneration and neuronal cell death. This damage disrupts the ability of parts of the nervous system to communicate, resulting in a range of signs and symptoms, including physical, mental, and sometimes psychiatric problems. Specific symptoms can include double vision, blindness in one eye, muscle weakness, trouble with sensation, or trouble with coordination.

The three main characteristics of multiple sclerosis are the formation of lesions in the central nervous system (also called plaques), inflammation, and the destruction of myelin sheaths of neurons. Multiple sclerosis also involves the loss of oligodendrocytes, the cells responsible for creating and maintaining a fatty layer— known as the myelin sheath— which helps the neurons carry electrical signals (action potentials). This results in a thinning or complete loss of myelin and, as the disease advances, the breakdown of the axons of neurons. When the myelin is lost, a neuron can no longer effectively conduct electrical signals. A repair process, called remyelination, takes place in early phases of the disease, but the oligodendrocytes are unable to completely rebuild the cell's myelin sheath. Repeated attacks lead to successively less effective remyelinations, until a scar-like plaque is built up around the damaged axons. These scars are the origin of the symptoms.

Several phenotypes (commonly termed types), or patterns of progression, have been described. Phenotypes use the past course of the disease in an attempt to predict the future course. They are important not only for prognosis but also for treatment decisions. Currently, the United States National Multiple Sclerosis Society and the Multiple Sclerosis International Federation, describes four types of MS (revised in 2013):

1. Clinically isolated syndrome (CIS)

2. Relapsing-remitting MS (RRMS)

3. Primary progressive MS (PPMS)

4. Secondary progressive MS (SPMS)

Relapsing-remitting multiple sclerosis is characterized by unpredictable relapses followed by periods of months to years of relative quiet (remission) with no new signs of disease activity. Deficits that occur during attacks may either resolve or leave problems, the latter in about 40% of attacks and being more common the longer a person has had the disease. This describes the initial course of 80% of individuals with multiple sclerosis. The relapsing-remitting subtype usually begins with a clinically isolated syndrome (CIS). In CIS, a person has an attack suggestive of demyelination, but does not fulfill the criteria for multiple sclerosis. 30 to 70% of persons experiencing CIS later develop multiple sclerosis.

Primary progressive multiple sclerosis occurs in approximately 10-20% of individuals, with no remission after the initial symptoms. It is characterized by progression of disability from onset, with no, or only occasional and minor, remissions and improvements. The usual age of onset for the primary progressive subtype is later than of the relapsing-remitting subtype. It is similar to the age that secondary progressive usually begins in relapsing-remitting multiple sclerosis, around 40 years of age.

Secondary progressive multiple sclerosis occurs in around 65% of those with initial relapsing-remitting multiple sclerosis, who eventually have progressive neurologic decline between acute attacks without any definite periods of remission. Occasional relapses and minor remissions may appear. The most common length of time between disease onset and conversion from relapsing-remitting to secondary progressive multiple sclerosis is 19 years.

At present, there is no cure for myelin-related disorders such as multiple sclerosis. While there are a number of treatments available for multiple sclerosis, these treatments are generally effective mostly for the relapsing-remitting multiple sclerosis and none are able to promote remyelination. Because demyelination is prominent in primary progressive multiple sclerosis and secondary progressive multiple sclerosis, the available treatments for these types of multiple sclerosis are inadequate. There is the potential to develop effective treatments of these stages of multiple sclerosis by identifying compounds which promote the differentiation, maturation and proliferation of oligodendrocyte progenitors, which can stimulate and enhance the generation of new oligodendrocytes and intrinsic myelination and/or remyelination. Therefore, there is a need for compounds and therapeutic methods capable of enhancing the generation of new oligodendrocytes.

SUMMARY OF THE INVENTION

In one embodiment, the present invention relates to a method of promoting myelination of central nervous system neurons in a subject suffering from a myelin- related disorder, the method comprising administering to the subject a therapeutically effective amount of a compound represented by structural Formula (I) ([(Z)-3-(4- adamantan-2-yl-3,5-dichlorophenyl)propen-2-yl]cyclohexylethylamine):

or a pharmaceutically acceptable salt thereof.

In another embodiment, the present invention relates to the compound of Formula I or a pharmaceutically acceptable salt thereof for promoting myelination of central nervous system neurons in a subject suffering from a myelin-related disorder.

In another embodiment, the present invention relates to the use of a compound of Formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for promoting myelination of central nervous system neurons in a subject suffering from a myelin-related disorder.

In yet another embodiment, the present invention relates to a method of inducing endogenous oligodendrocyte precursor cell (OPC) differentiation in a subject suffering from a myelin-related disorder, the method comprising

administering to the subject a therapeutically effective amount of a compound represented by structural Formula (I).

In another embodiment, the present invention relates to the compound of Formula I or a pharmaceutically acceptable salt thereof for inducing endogenous oligodendrocyte precursor cell (OPC) differentiation in a subject suffering from a myelin-related disorder.

In another embodiment, the present invention relates to the use of a compound of Formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for inducing endogenous oligodendrocyte precursor cell (OPC) differentiation in a subject suffering from a myelin-related disorder.

In yet another embodiment, the present invention relates to a method of treating secondary progressive multiple sclerosis in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a compound of Formula (I) or a salt thereof.

In another embodiment, the present invention relates to a compound of Formula (I) or a pharmaceutically acceptable salt thereof for treating secondary progressive multiple sclerosis in a subject in need thereof.

In another embodiment, the present invention relates to the use of a compound of Formula (I) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating secondary progressive multiple sclerosis in a subject in need thereof.

In yet another embodiment, the present invention relates to a method of treating primary progressive multiple sclerosis in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a compound of Formula (I) or a salt thereof.

In another embodiment, the present invention relates to a compound of Formula (I) or a pharmaceutically acceptable salt thereof for treating primary progressive multiple sclerosis in a subject in need thereof.

In another embodiment, the present invention relates to the use of a compound of Formula (I) or a pharmaceutically acceptable salt thereof for the manufacture of a medicmant for treating primary progressive multiple sclerosis in a subject in need thereof.

In a particular embodiment, the compound of Formula (I) is an HC1 salt.

In certain embodiments, the subject is suffering from a myelin-related disorder selected from one or more of the following: secondary progressive multiple sclerosis (SPMS), primary progressive multiple sclerosis (SPMS), neuromyelitis optica (NMO), optic neuritis, pediatric leukodystrophies, neonatal white matter injury, age- related dementia, schizophrenia, progressive multifocal leukoencephalopathy (PML), encephalomyelitis (EPL), central pontine myelolysis (CPM), adrenoleukodystrophy, Alexander's disease, Pelizaeus Merzbacher disease (PMD), Vanishing White Matter Disease, Wallerian Degeneration, transverse myelitis, amylotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease, Parkinson's disease, spinal cord injury, traumatic brain injury, post radiation injury, neurologic complications of chemotherapy, stroke, acute ischemic optic neuropathy, vitamin E deficiency, isolated vitamin E deficiency syndrome, Bassen-Komzweig syndrome, Marchiafava-Bignami syndrome, metachromatic leukodystrophy, trigeminal neuralgia, acute disseminated encephalitis, Guillian-Barre syndrome, Charcot-Marie-Tooth disease, Bell's palsy and radiation-induced demyelination.

In a particular embodiment, the subject is suffering from a disorder selected from: neuromyelitis optica (NMO), optic neuritis, pediatric leukodystrophies, neonatal white matter injury, age-related dementia and schizophrenia.

In a further embodiment, the subject being treated is a human, for example a female.

In a particular embodiment, the compound of Formula (I) is administered parenterally or enterally. For example, the compound of Formula (I) can be administered intravenously, intrathecally, subcutaneously, intramuscularly, intranasally or orally.

In yet another embodiment, the compound of Formula (I) or a

pharmaceutically acceptable salt thereof is in a pharmaceutical composition with a pharmaceutically acceptable carrier.

In another embodiment, the subject is suffering from secondary progressive or primary progressive multiple sclerosis and the method further comprises

administration of a therapeutically effect amount of an MS therapeutic agent. An MS therapeutic agent can be an agent that is primarily an immunomodulatory agent. In one aspect, the MS therapeutic agent is selected from: glatiramer acetate, Ocrevus

(ocrelizumab), Campath (Lemtrada or alemtuzumab), Gilenya, Ampyra

(dalfampridine), Tysabri (natalizumab), Aubagio (teriflunomide), Rebif , Avonex,

Betaseron, Plegridy, Interferon Beta-la, dimethyl fumarate, fingolimod, rituximab,

Zinbryta, Ofatumymab, Nerventra (laquinimod), Masitinib, Siponimod, Ozanimod,

Ponesimod, ibudilast, vatelizumab, minocycline, ibrutinib, PRN2246, Cladripine, GNBAC1, daclizumab, and MD1003 (biotin). . In another aspect, the MS therapeutic agent is administered simultaneously with the compound represented by Formula (I) or a pharmaceutically acceptable salt thereof. In yet another aspect, the MS therapeutic agent is administered prior to administration of the compound represented by Formula (I) or a pharmaceutically acceptable salt thereof. In a further aspect, the MS therapeutic agent is administered following the administration of the compound represented by Formula (I) or a pharmaceutically acceptable salt thereof.

In another embodiment, the subject is administered the compound represented by Formula I for an on-drug cycle of at least three months. For example, the subject is administered the compound represented by Formula I for an on-drug cycle of at least six months.

In a specific embodiment, the subject is administered the compound represented by Formula (I) using the following dosing regimen:

a. on-drug cycle for at least six months;

b. off-drug cycle for at least three months;

wherein the on-drug and off-drug cycles are optionally repeated.

In a further embodiments, the method of promoting myelination of central nervous system neurons in a subject suffering from a myelin-related disorder, or the method of inducing endogenous oligodendrocyte precursor cell (OPC) differentiation in a subject in need thereof or the method of treating secondary progressive multiple sclerorsis in a subject in need thereof or the method of treating primary progressive multiple sclerosis in a subject in need thereof, the compound represented by Formula (I) inhibits enzyme mediated synthesis of one or more sterol intermediates in the cholesterol biosynthesis pathway. In one aspect, the compound represented by Formula (I) promotes accumulation of A8,9-unsaturated sterol intermediates in the cholesterol biosynthesis pathway. For example, the compound of Formula (I) inhibits one or more of CYP51, sterol- l4-reductase, SC4MOL, NSDHL, TM7SF2 or EBP enzyme mediated synthesis of sterol intermediates in the cholesterol biosynthesis pathway. In a particular embodiment, when the compound of Formula (I) is administered, EBP (Emopamil Binding Protein) in inhibited and the levels of zymostenol in the brain increases.

In an additional embodiment, administration of the compound of Formula (I) induces, promotes, and/or modulates oligodendrocyte precursor cell (OPC) differentiation, proliferation and/or maturation. In one aspect, the induction of OPC differentiation is characterized by an increase in myelin basic protein (MBP) expression.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the cholesterol biosynthesis cascade. A narrow window of the cholesterol biosynthesis cascade (i.e., the enzymes CYP51, TM7SF2, and EBP) can be targeted to enhance oligodendrocyte generation. Inhibiting each of these enzymes leads to an increase in the specific cholesterol intermediate directly upstream of the target of the inhibitor. The cholesterol intermediate can be measured by laboratory tests and provides a mechanistic biomarker of drug activity.

FIG. 2 shows an exemplary synthetic procedure for producing the compound of Formula (I).

FIG. 3 shows the ¹ FI NMR of the compound of Formula (I).

FIG. 4 is a chart that shows the percent of cells that stained positive for MBP following treatment with test compounds. After 72 hours of exposure to test compounds, cells were fixed and stained using anti-MBP antibody and DAPI. Plates were imaged using a PerkinElmer Operetta microscope. Six fields per well were assayed, and the percent of cells that displayed peri-nuclear MBP staining was assessed. Results are presented as the mean and standard deviation of three biological replicates. Individual biological replicates are represented as dots.

FIG. 5 is a pair graph that show that the compound of formula (I) inhibits EBP and increases zymostenol in mouse OPCs in a dose-dependent manner.

DETAILED DESCRIPTION OF THE INVENTION

DEFINITIONS

For convenience, certain terms employed in the specification, examples, and appended claims are collected here. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The articles“a” and“an” are used herein to refer to one or to more than one (/._<?., to at least one) of the grammatical object of the article. By way of example,“an element” means one element or more than one element.

The terms“comprise,”“comprising,”“include,”“including,”“have,” and “having” are used in the inclusive, open sense, meaning that additional elements may be included. The terms“such as”,“e.g”, as used herein are non-limiting and are for illustrative purposes only.“Including” and“including but not limited to” are used interchangeably.

As used herein, the term“and/or” includes any and all combinations of one or more of the associated listed items.

The term“or” as used herein should be understood to mean“and/or”, unless the context clearly indicates otherwise.

As used herein, the term“about” or“approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In one embodiment, the term“about” or“approximately” refers a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length ± 15%, ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2%, or ± 1% about a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

The phrases“parenteral administration” and“administered parenterally” are art-recognized terms, and include modes of administration other than enteral and topical administration, such as injections, and include, without limitation, intravenous, intramuscular, intranasal, intrapleural, intravascular, intrapericardial, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal and intrastemal injection and infusion.

The term“treating” is art-recognized and includes inhibiting a disease, disorder or condition in a subject, e.g., impeding its progress; and relieving the disease, disorder or condition, e.g., causing regression of the disease, disorder and/or condition. Treating the disease or condition includes ameliorating at least one symptom of the particular disease or condition, even if the underlying

pathophysiology is not affected.

The term“preventing” is art-recognized and includes stopping a disease, disorder or condition from occurring in a subject, which may be predisposed to the disease, disorder and/or condition but has not yet been diagnosed as having it.

Preventing a condition related to a disease includes stopping the condition from occurring after the disease has been diagnosed but before the condition has been diagnosed.

The term“pharmaceutical composition” refers to a formulation containing the disclosed agents, in a form suitable for administration to a subject. In a preferred embodiment, the pharmaceutical composition is in bulk or in unit dosage form. The unit dosage form is any of a variety of forms, including, for example, a capsule, an IV bag, a tablet, a single pump on an aerosol inhaler, or a vial. The quantity of active ingredient (e.g., the compound of Formula (I) or salts thereof) in a unit dose of composition is an effective amount and is varied according to the particular treatment involved. One skilled in the art will appreciate that it is sometimes necessary to make routine variations to the dosage depending on the age and condition of the patient.

The dosage will also depend on the route of administration· A variety of routes are contemplated, including oral, pulmonary, rectal, parenteral, transdermal,

subcutaneous, intravenous, intramuscular, intraperitoneal, intranasal, inhalational, and the like. Dosage forms for the topical or transdermal administration of a compound described herein includes powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, nebulized compounds, and inhalants. In a preferred embodiment, the active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that are required.

The phrase“pharmaceutically acceptable” is art-recognized. In certain embodiments, the term includes compositions, polymers and other materials and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. The phrase“pharmaceutically acceptable carrier” is art-recognized, and includes, for example, pharmaceutically acceptable materials, compositions or vehicles, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any subject composition from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be“acceptable” in the sense of being compatible with the other ingredients of a subject composition and not injurious to the patient. In certain embodiments, a pharmaceutically acceptable carrier is non-pyrogenic. Some examples of materials which may serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer’s solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

The compound of Formula (I) is capable of forming salts. All of these forms are also contemplated herein. A“pharmaceutically acceptable salt” of the compound of Formula (I) means a salt that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound. For example, the salt can be an acid addition salt. One embodiment of an acid addition salt is a hydrochloride salt. Other acceptable acid addition salts include hydrobromide, sulphate, hydrogen sulphate, dihydrogen phosphate, maleate, fumarate, 2naphthalenesulphonate or para- toluenesulphonate. The structural representation of compound of Formula (I) includes both the Z-E stereoisomers.

Additionally, the salts of the compound of Formula (I) described herein, can exist in either hydrated or unhydrated (the anhydrous) form or as solvates with other solvent molecules. Nonlimiting examples of hydrates include monohydrates, dihydrates, etc. Nonlimiting examples of solvates include ethanol solvates, acetone solvates, etc.

The term“oligodendrocyte precursor cells” or“OPCs” as used herein refers to a neural progenitor cell capable to generate new oligodendrocyte cells.

Oligodendrocyte precursor cells can be identified by the expression of a number of surface antigens. For example, the surface antigens known as platelet-derived growth factor-alpha receptor subunit (PDGFRa), NG2 chondroitin sulfate proteoglycan, and ganglioside GD3, are commonly used to identify oligodendrocyte precursor cells.

Immature oligodendrocyte precursors are generated in ventral areas of the developing brain from a common glial progenitor. The immature cells actively migrate, proliferate, and populate the CNS to finally differentiate to premyelinating oligodendrocytes (04+). Oligodendrocyte precursor differentiation and maturation is characterized by an extension of multiple processes, increase in cell body size and formation of myelin.

Central nervous system neurons are nerve impulse-conducting cells found in the central nervous system. A central nervous system neuron comprises nucleated soma and processes (axons and dendrites).

A“patient,”“subject,” or“host” to be treated by the subject method may mean either a human or non-human animal, such as a mammal, a fish, a bird, a reptile, or an amphibian. Thus, the subject of the herein disclosed methods can be a human, non human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent.

The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. In one aspect, the subject is a mammal (e.g., a male of female human). A patient refers to a subject afflicted with a disease or disorder.

With respect to the compound of Formula (I), the present application is intended to include all isotopes of atoms occurring in the present compounds.

Isotopes include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon include C-13 and C-14.

Throughout the description, where compositions are described as having, including, or comprising, specific components, it is contemplated that compositions also consist essentially of, or consist of, the recited components. Similarly, where methods or processes are described as having, including, or comprising specific process steps, the processes also consist essentially of, or consist of, the recited processing steps. Further, it should be understood that the order of steps or order for performing certain actions is immaterial so long as the compositions and methods described herein remains operable. Moreover, two or more steps or actions can be conducted simultaneously.

All percentages and ratios used herein, unless otherwise indicated, are by weight.

COMPOUNDS OF FORMULA (I) AND SALTS THEREOF

The enhancement and/or inducement of the accumulation of A8,9-unsaturated sterol intermediates of the cholesterol biosynthesis pathway in oligodendrocyte progenitor cells (OPCs) can induce Oligodendrocyte generation. Enhancement and/or inducement of the accumulation of A8,9-unsaturated sterol intermediates can be provided by modulating and/or inhibiting enzymes within the cholesterol biosynthesis pathway in OPCs that inhibit A8,9-unsaturated sterol intermediate accumulation and/or for which the A8,9-unsaturated sterol intermediates are substrates as well as directly and/or indirectly administering A8,9-unsaturated sterol intermediates to the OPCs. Enhancement and/or inducement of the accumulation of A8,9-unsaturated sterol intermediates can promote OPC differentiation, survival, proliferation and/or maturation and treat disease and/or disorders in subjects where myelination is beneficial to the subject.

As such, an agent, such as the compound of Formula (I), that can enhance and/or induce accumulation of A8,9-unsaturated sterol intermediates of the cholesterol biosynthesis pathway in the OPCs can be administered to a subject and/or the OPCs at an amount effective to promote and/or induce OPC differentiation, proliferation and/or maturation as well as oligodendrocyte generation. In some such embodiments, the agent, for example the compound of Formula (I), is a compound that inhibits enzyme mediated synthesis of one or more sterol intermediates in the cholesterol biosynthesis pathway of the OPCs and/or promotes accumulation of A8,9-unsaturated sterol intermediates.

In some embodiments, the compound used in the methods described herein

(i.e., the compound of Formula (I)) can modulate and/or inhibit one or more enzyme mediated conversion steps of the cholesterol biosynthesis pathway from lanosterol to cholesterol, for example, between lanosterol and/or lathosterol, of OPCs to promote and/or induce oligodendrocyte generation. For example, the compound of Formula (I) can inhibit CYP51, sterol l4-reductase, SC4MOL, NSDHL, TM7SF2 and/or EBP enzyme mediated synthesis of sterol intermediates in the cholesterol biosynthesis pathway. In a specific embodiment, the compound of Formula (I) can inhibit CYP51, sterol l4-reductase and/or EBP. In an even more specific embodiment, the compound of Formula (I) can inhibit EBP.

For example, in some embodiments, the compound of Formula (I) used in the methods described herein can inhibit enzyme mediated conversion of zymostenol to lathosterol through the inhibition of emopamil binding protein (EBP) isomerase enzyme activity. Alternatively, a compound used in the methods described herein can inhibit sterol C14 reductase enzyme activity or CYP51 enzyme activity in the cholesterol biosynthesis pathway.

Emopamil Binding Protein (EBP) is an enzyme responsible for one of the final steps in the production of cholesterol. Specifically, EBP converts zymostenol to lathosterol, where other enzymes then modify lathosterol to produce cholesterol. EBP is also referred to as A8-A7-sterol isomerase, 3 -beta-hydroxy steroid- Delta(8),Delta(7)-isomerase, CDPX2, CH02, CPX, or CPXD). The compound of Formula (I) has been shown to have nanomolar affinity for EBP (See B. Bourrie et al., European Journal of Pharmacology 456 (2002) 123-131).

Without being bound by a particular theory, it is believed that the compound of Formula (I) can inhibit EBP mediated conversion of zymostenol to lathosterol in the cholesterol biosynthesis pathway of OPCs resulting in enhancement and/or inducement of the accumulation of A8,9-unsaturated sterol intermediates.

Enhancement and/or inducement of the accumulation of A8,9-unsaturated sterol intermediates can promote OPC differentiation, survival, proliferation and/or maturation and treat disease and/or disorders in subjects where myelination or remyelination is beneficial to the subject (e.g., secondary progressive multiple sclerosis or primary progressive multiple sclerosis). This mechanism of promoting myelination is distinct from the primary action of immunomodulatory agents that are often used to treat myelin-related disorders. Synthetic preparation of the compounds of the invention are known in the art. Specifically, detailed synthetic protocols for preparing the compound of Formula (I) can be found in U.S. Patent No. 6,482,986, incorporated herein by reference in its entirety. In addition, an alternative synthetic scheme for prepareing of the compound of Formula (I) is found in FIG. 2.

The salts of the compounds according to the invention are prepared according to techniques that are well known to those skilled in the art.

The functional groups that may be present in the compound of Formula (I) and in the reaction intermediates can be protected, either in permanent form or in temporary form, with protecting groups which ensure an unequivocal synthesis of the expected compounds. The protection and deprotection reactions are carried out according to techniques that are well known to those skilled in the art. The expression “temporary protecting group for amines, alcohols, phenolthiols or carboxylic acids” means protecting groups such as those described in Protective Groups in Organic Synthesis, Greene T. W. and Wuts P. G. M., ed. John Wiley and Sons, 1991 and in Protecting Groups, Kocienski P. J., 1994, Georg Thieme Verlag. A person skilled in the art will be capable of selecting the appropriate protecting groups. The compound of Formula (I) can comprise precursor groups for other functions which are generated subsequently in one or more steps.

PHARMACEUTICAL COMPOSITIONS AND METHODS OF ADMINISTRATION

Pharmaceutical compositions for use in the methods of the present invention preferably have a therapeutically effective amount of the compound or salts thereof in a dosage in the range of .01 to 1,000 mg/kg of body weight of the subject, and more preferably in the range of from about 10 to 100 mg/kg of body weight of the patient.

The overall dosage will be a therapeutically effective amount depending on several factors including the overall health of a subject, the subject’s disease state, severity of the condition, the observation of improvements and the formulation and route of administration of the selected agent(s). Determination of a therapeutically effective amount is within the capability of those skilled in the art. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the subject’s condition. The terms“prophylactic” or“therapeutic” treatment is art-recognized and includes administration to the subject of the compound of Formula (I) either alone or in combination with a second agent.. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal such as, but not limited to, myelination disturbances, myelin deficiencies, myelin loss and ineffective myelin repair) then the treatment is prophylactic, i.e., it protects the host against developing the unwanted condition, whereas if it is administered after manifestation of the unwanted condition, the treatment is therapeutic (i.e., it is intended to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereof).

The phrase“therapeutically effective amount” is an art-recognized term. In certain embodiments, the term refers to an amount of a therapeutic agent that produces some desired effect at a reasonable benefit/risk ratio applicable to any medical treatment. In certain embodiments, the term refers to that amount necessary or sufficient to eliminate, reduce or maintain a target of a particular therapeutic regimen. The effective amount may vary depending on such factors as the disease or condition being treated, the particular targeted constructs being administered, the size of the subject or the severity of the disease or condition. One of ordinary skill in the art may empirically determine the effective amount of a particular compound without necessitating undue experimentation.

The present invention provides a method of treating myelin-related disorders in a subject by promoting myelination of central nervous system neurons. In one embodiment, a therapeutically effective amount is the amount necessary to promote myelination of central nervous system neurons in a subject suffering from a myelin- related disorder. It is believed that myelination is promoted due to the differentiation and/or proliferation of oligodendrocyte precursors in a subject. The method includes administering to the subject in need thereof a therapeutically effective amount of a pharmaceutical compound in accordance with the present invention. As described above, one or more of the compounds can be administered in association with one or more non-toxic, pharmaceutically acceptable carriers and/or diluents and/or adjuvants and if desired other active ingredients.

In certain embodiments, the compound of Formula (I) can be administered in an amount effective to promote myelination of CNS neurons in a subject thereby increasing in the amount of myelin proteins (e.g., MBP) of at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, or 1000% as compared to the level of myelin proteins of untreated CNS neurons or subject.

In other embodiments, the compound of Formula (I) can be administered in an amount effective to promote survival of CNS neurons in a subject by an increase in the number of surviving neurons of at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, or 1000% as compared to the number of surviving neurons in untreated CNS neurons or subject.

In some embodiments, the compound of Formula (I) can be administered in an amount effective to enhance generation of OPCs in the subject’s central nervous system by an increase in the amount of OPC generation of at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, or 1000% as compared to the amount of OPC generation in untreated OPCs or subject.

In some embodiments, the compound of Formula (I) can be administered in an amount effective to induce endogenous oligodendrocyte precursor cell (OPC) differentiation in the subject’s central nervous system by an increase in the amount of OPC differentiation of at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%,

75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, or 1000% as compared to the amount of OPC differentiation in untreated OPCs or subject.

In some embodiments, the compound of Formula (I) can be administered in an amount effective to modulate the cholesterol biosynthesis pathway in a OPC cells in a subject by a decrease in the amount of cholesterol and/or increase in one or more cholesterol precursors in OPCs of at least 5%, 10%, 20%, 25%, 30%, 40%, 50%,

60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% as compared to the amount of cholesterol and/or one or more sterol intermediates synthesis in untreated OPCs or subject.

The pharmaceutical compositions of the present invention can be administered to a subject by any means that achieve their intended purpose. For example, administration can be by parenteral, subcutaneous, intravenous, intraarticular, intrathecal, intramuscular, intraperitoneal, or intradermal injections, or by transdermal, buccal, oromucosal, ocular routes or via inhalation. Alternatively, or concurrently, administration can be by the oral route.

Formulation of the pharmaceutical compounds for use in the modes of administration noted above (and others) are known in the art and are described, for example, in Remington’s Pharmaceutical Sciences (18th edition), ed. A. Gennaro, 1990, Mack Publishing Company, Easton, Pa. (also see, e.g., M. J. Rathbone, ed.,

Oral Mucosal Drug Delivery, Drugs and the Pharmaceutical Sciences Series, Marcel Dekker, Inc., N.Y., U.S.A., 1996; M. J. Rathbone et ak, eds., Modified-Release Drug Delivery Technology, Drugs and the Pharmaceutical Sciences Series, Marcel Dekker, Inc., N.Y., U.S.A., 2003; Ghosh et ak, eds., Drug Delivery to the Oral Cavity, Drugs and the Pharmaceutical Sciences Series, Marcel Dekker, Inc., N.Y., U.S.A., 2005; and Mathiowitz et ak, eds., Bioadhesive Drug Delivery Systems, Drugs and the

Pharmaceutical Sciences Series, Marcel Dekker, Inc., N.Y., U.S.A., 1999.

Compounds of the invention can be formulated into pharmaceutical compositions containing pharmaceutically acceptable non-toxic excipients and carriers. The excipients are all components present in the pharmaceutical formulation other than the active ingredient or ingredients. Suitable excipients and carriers useful in the present invention are composed of materials that are considered safe and effective and may be administered to an individual without causing undesirable biological side effects, or unwanted interactions with other medications. Suitable excipients and carriers are those, which are composed of materials that will not affect the bioavailability and performance of the agent. As generally used herein“excipient” includes, but is not limited to surfactants, emulsifiers, emulsion stabilizers, emollients, buffers, solvents, dyes, flavors, binders, fillers, lubricants, and preservatives. Suitable excipients include those generally known in the art such as the“Handbook of Pharmaceutical Excipients”, 4th Ed., Pharmaceutical Press, 2003. METHODS OF TREATING MYELIN-RELATED DISORDERS

The compound of Formula (I) can be administered alone or in combination with another agent to a subject suffering from a myelin-related disorder to promote myelination of neurons (e.g., neuronal axons). A myelin-related disorder can include any disease, condition (e.g., those occurring from traumatic spinal cord injury and cerebral infarction), or disorder resulting in abnormalities of the myelin sheath.

Abnormalities can be caused by loss of myelin referred to as demyelination, dysfunctional myelin referred to as dysmyelination or failure to form enough myelin referred to as hypomyelination. A myelin related disorder as used herein can arise from a genetic disorder or from a variety of neurotoxic insults.

“Demyelination” as used herein, refers to the act of demyelinating, or the loss of the myelin sheath insulating the nerves, and is the hallmark of myelin-related disorders.

Myelin related disorders include, but are not limited to, secondary progressive multiple sclerosis and primary progressive multiple sclerosis (MS), neuromyelitis optica (NMO), optic neuritis, pediatric leukodystrophies, neonatal white matter injury, age-related dementia, schizophrenia, progressive multifocal leukoencephalopathy (PML), encephalomyelitis (EPL), central pontine myelolysis (CPM),

adrenoleukodystrophy, Alexander's disease, Pelizaeus Merzbacher disease (PMD), Vanishing White Matter Disease, Wallerian Degeneration, transverse myelitis, amylotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease, Parkinson's disease, spinal cord injury, traumatic brain injury, post radiation injury, neurologic complications of chemotherapy, stroke, acute ischemic optic neuropathy, vitamin E deficiency, isolated vitamin E deficiency syndrome, Bassen-Kornzweig syndrome, Marchiafava-Bignami syndrome, metachromatic leukodystrophy, trigeminal neuralgia, acute disseminated encephalitis, Guillian-Barre syndrome, Charcot-Marie-Tooth disease, Bell's palsy and radiation-induced demyelination.

Both acquired and inherited myelin disorders share a poor prognosis leading to major disability. Thus, some embodiments of the present invention can include methods for the treatment of myelin-related disorders in a subject.

Multiple sclerosis (MS) is one of the most common neurological disorders frequently leading to permanent disability in young adults. The clinical course is unpredictable and highly variable. The most common disease course for MS is relapsing-remitting MS (RRMS) characterized by episodes of acute exacerbations, followed by partial or complete recovery of the deficits. About 85% of people diagnosed with MS are initially diagnosed with RRMS. RRMS typically begins in the second or third decade of life and after a medium time to conversion of around 19 years approximately 70% of the patients subsequently develop secondary progressive MS (SPMS). Secondary progressive multiple sclerosis is a form of MS that typically follows relapsing-remitting multiple sclerosis. The rate of conversion to SPMS is approximately 2-3% per year. Secondary progression is usually defined as a period of clinical worsening and steady accumulation of disability, which is independent of relapses and sustained for at least six months. When an attack does occur, recovery is usually slow and, in many cases, incomplete. Existing symptoms can get worse and physical mobility becomes increasingly difficult. The time of conversion is sometimes difficult to pinpoint as it slowly builds up and remains unnoticed by the patient and the clinician for some time. Another challenge is to distinguish the chronic progression from residual symptoms that remain after patients have experienced acute relapses. Another disease course for MS is primary progressive MS (PPMS) characterized by worsening neurologic function (accumulation of disability) from the onset of symptoms, without early relapses or remissions. Approximately 15% of people with MS are diagnosed with PPMS. Primary progressive multiple sclerosis is identified by steadily worsening neurologic functions from the onset of symptoms without distinct relapses (attacks or exacerbations) or remission. The rate of progression may vary with occasional plateaus and temporary minor improvements, but declining neurologic progression is continuous.

Demyelination of axons in chronic MS can result in axon degeneration and neuronal cell death, but more specifically, MS destroys oligodendrocytes, the highly specialized CNS cells that generate and maintain myelin.

Neuromyelitis Optica (NMO), is also referred to as Devic’s disease. NMO is a disorder of the central nervous system (CNS) that predominantly affects the optic nerve and spinal cord of patients.

Leukodystrophies are a group of progressive, metabolic, genetic diseases that affect the brain, spinal cord and often the peripheral nerves. Each type of leukodystrophy is caused by a specific gene abnormality that leads to abnormal development or destruction of the myelin sheath of the brain. Each type of leukodystrophy affects a different part of the myelin sheath, leading to a range of neurological problems. Exemplary leukodystrophies which may be treated or ameliorated by the methods of the present invention include, but are not limited to, adult-onset autosomal dominant leukodystrophy (ADLD), Aicardi-Goutieres syndrome, Alexander disease, CADASIL, Canavan disease, CARASIL,

cerebrotendionous xanthomatosis, childhood ataxia and cerebral hypomyelination (CACH)/ vanishing white matter disease (VWMD), Fabry disease, fucosidosis, GM1 gangliosidosis, Krabbe disease, L-2-hydroxyglutaric aciduria, megalencephalic leukoencephalopathy with subcortical cysts, metachromatic leukodystrophy, multiple sulfatase deficiency, Pelizaeus-Merzbacher disease (PMD), Pol Ill-related leukodystrophies, Refsum disease, salla disease (free sialic acid storage disease), Sjogren- Larsson syndrome, X- linked adrenoleukodystrophy, and Zellweger syndrome spectrum disorders.

Myelin-related disorders which can be treated or ameliorated by the methods of the present invention include a disorder characterized by a myelin deficiency. Insufficient myelination in the central nervous system has been implicated in a wide array of neurological disorders. Among these are forms of cerebral palsy wherein a congenital deficit in forebrain myelination in children with periventricular leukomalacia, contributes to neurological morbidity (Goldman et ak, 2008) Goldman, S. A., Schanz, S., and Windrem, M. S. (2008). Stem cell-based strategies for treating pediatric disorders of myelin. Hum Mol Genet. 17, R76-83. At the other end of the age spectrum, myelin loss and ineffective repair may contribute to the decline in cognitive function associated with senescence (Kohama et ak, 2011) Kohama, S. G., Rosene, D. L., and Sherman, L. S. (2011) Age (Dordr). Age-related changes in human and non-human primate white matter: from myelination disturbances to cognitive decline. Therefore, it is contemplated that effective compounds and methods of enhancing myelination and/or remyelination may have substantial therapeutic benefits in halting disease progression and restoring function in MS and in a wide array of neurological disorders.

Myelination of neurons requires oligodendrocytes. The term“myelination”, as used herein, refers to the generation of the nerve’s myelin sheath by replacing myelin producing cells or restoring their function. “Promoting Myelination” as used herein refers to increasing the rate of myelin production rather than a mere net increase in the amount of myelin as compared to a baseline level of myelin production rate in a subject. An increase in the rate of myelin production can be determined using imaging techniques or functional measurements.

A“baseline level of myelin production rate” as used herein, refers to the rate of myelin production in subject being treated before the onset of treatment.

“MS therapeutic agents” as used herein, refers to therapeutic agents known to be used in treating MS. Such therapeutic agents include, but are not limited to, Copaxone (glatiramer acetate), Ocrevus (ocrelizumab), Campath (Lemtrada or alemtuzumab), Gilenya, Ampyra (dalfampridine), Tysabri (natalizumab), Aubagio (teriflunomide), Rebif , Avonex, Betaseron, Plegridy, Interferon Beta- la, dimethyl fumarate, fingolimod, rituximab, Zinbryta, Ofatumymab, Nerventra (laquinimod), Masitinib, Siponimod, Ozanimod, Ponesimod, ibudilast, vatelizumab, minocycline, ibrutinib, PRN2246, Cladripine, GNBAC1, daclizumab, and MD1003 (biotin).

In certain embodiments, the compound of Formula (I) or a pharmaceutically acceptable salts thereof can be administered in combination with cognitive enhancing (nootropic) agents. Exemplary agents include any drugs, supplements, or other substances that improve cognitive function, particularly executive functions, memory, creativity, or motivation, in healthy individuals. Non-limiting examples include race tarns (e.g., piracetam, oxiracetam, and aniracetam), nutraceuticals (e.g., bacopa monnieri, panax ginseng, ginko biloba, and GABA), stimulants (e.g., amphetamine pharmaceuticals, methylphenidate, eugeroics, xanthines, and nicotine), L-Theanine, Tolcapone, Levodopa, Atomoxetine, and Desipramine

A further embodiment for treating a subject suffering from a myelin-related disorder is to administer a therapeutically effective amount of a compound described herein along with a therapeutically effective amount of additional oligodendrocyte differentiation and/or proliferation inducing agent(s) and/or anti-neurodegenerative disease agent. Examples of anti-neurodegenerative disease agents include L-dopa, cholinesterase inhibitors, anticholinergics, dopamine agonists, steroids,

immunomodulators including interferons, monoclonal antibodies, and glatiramer acetate and modulators (e.g., inhibitors) of SARM1 a new class of NADase enzyme

(See, Essuman, Neuron, Vol. 93, Issue 6, pa 1334, March 22, 2017). Therefore, in a further aspect of the invention, the oligodendrocyte precursor differentiation and/or proliferation inducing compound of Formula (I) described herein can be administered as part of a combination therapy with adjunctive therapies for treating neurodegenerative and myelin related disorders.

The phrase“combination therapy” embraces the administration of the compound of Formula (I) and an additional therapeutic agent as part of a specific treatment regimen intended to provide a beneficial effect from the co-action of each. When administered as a combination, the oligodendrocyte precursor differentiation inducing compound (the compound of Formula (I) and an additional therapeutic agent can be formulated as separate compositions. Administration of these therapeutic agents in combination typically is carried out over a defined time period (usually minutes, hours, days or weeks depending upon the combination selected).

“Combination therapy” is intended to embrace administration of these therapeutic agent (the compound of Formula (I) and an additional therapeutic agent) in a sequential manner, that is, wherein each therapeutic agent is administered at a different time, as well as administration of these therapeutic agents, or at least two of the therapeutic agents, in a substantially simultaneous manner. Substantially simultaneous administration can be accomplished, for example, by administering to the subject a single capsule having a fixed ratio of each therapeutic agent or in multiple, single capsules for each of the therapeutic agents. Sequential or substantially simultaneous administration of each therapeutic agent can be effected by any appropriate route including, but not limited to, oral routes, intravenous routes, intramuscular routes, and direct absorption through mucous membrane tissues. The therapeutic agents can be administered by the same route or by different routes. For example, a first therapeutic agent of the combination selected may be administered by intravenous injection while the other therapeutic agents of the combination may be administered orally. Alternatively, for example, all therapeutic agents may be administered orally or all therapeutic agents may be administered by intravenous injection. The sequence wherein the therapeutic agents are administered is not narrowly critical.“Combination therapy” also can embrace the administration of the therapeutic agents as described above in further combination with other biologically active ingredients (such as, but not limited to, a second and different therapeutic agent) and non-drug therapies (e.g., surgery). In another aspect of the invention, the therapeutic agents administered in a combination therapy with the oligodendrocyte differentiation and/or proliferation inducing compound described herein (the compound of Formula (I) or a salt thereof) can include at least one anti-neurodegenerative agent such as but not limited to, an immunotherapeutic agent.

Administration of the Compound of Formula (I) and a Second Agent for Treating MS

For treatment of Secondary Progressive and Primary Progresive Multiple Sclerosis, the compound of Formula (I) can be administered in conjunction with one or more MS therapeutic agents.“MS therapeutic agents” as used herein, refers to therapeutic agents known to be used in treating MS by targeting the immune component of the disorder and/or the acute inflammatory response evidenced during an acute attack in remitting-relapsing multiple sclerosis. Such therapeutic agents include, but are not limited to, Copaxone (glatiramer acetate), Ocrevus (ocrelizumab), Campath (Lemtrada or alemtuzumab), Gilenya, Ampyra (dalfampridine), Tysabri (natalizumab), Aubagio (teriflunomide), Rebif , Avonex, Betaseron, Plegridy, Interferon Beta- la, dimethyl fumarate, fingolimod, rituximab, Zinbryta,

Ofatumymab, Nerventra (laquinimod), Masitinib, Siponimod, Ozanimod, Ponesimod, ibudilast, vatelizumab, minocycline, ibrutinib, PRN2246, Cladripine, GNBAC1, daclizumab, and MD1003 (biotin). Administration may be simultaneous (for example, administration of a mixture of the compound of Formula (I) and the one or more MS therapeutic agents) or sequential in any order. For example, in one embodiment the compound of Formula (I) and the one or more MS therapeutic agent are administered at the same time either in separate dosage units or in one dosage unit. In an alternative embodiment, the one or more MS therapeutic agent is administered first followed by the compound of Formula (I). In another embodiment, the compound of Formula (I) is administered first and the one or more MS therapeutic agent is administered second.

In one embodiment of the method described herein (promoting myelination of central nervous system neurons in a subject suffering from a myelin-related disorder) the subject is administered the compound represented by Formula I for an on-drug cycle of at least three months. For example, the subject is administered the compound represented by Formula (I) for an on-drug cycle of at least six months. In a particular embodiment of the method described herein (promoting myelination of central nervous system neurons (e.g., the axons of the neurons) in a subject suffering from a myelin-related disorder), the subject is administered the compound represented by Formula I using the following dosing regimen: on-drug cycle for at least six months; off-drug cycle for at least three months; wherein the on- drug and off-drug cycles are optionally repeated.

An“on-drug cycle” is the period of time (e.g., number of days or weeks) deemed appropriate by a skilled medical professional that the drug is being administered to the subject, and will vary depending on the nature of the disease, the dose of the drug being administered, the health of the patient, the intended result, and the like. An“off-drug cycle” is the period of time between on-drug cycles. By way of example, a cycle of treatment regimen for treating multiple sclerosis can be on- drug cycle for at least six months, followed by an off drug cycle for at least three months, wherein the on-drug and off-drug cycles are optionally repeated. As will be appreciated by those of skill in the art, a cycle having any combination of the number of“on” and“off’ drug days can be designed as deemed appropriate by a skilled medical professional.

In one embodiment, the subject suffering from multiple sclerosis has an EDSS score from about 1 to about 9.5. In a particular aspect, the subject suffering from multiple sclerosis has an EDSS score from about 2 to about 8.5. In another aspect, the subject suffering from multiple sclerosis has an EDSS score from about 2.5 to about 8.0. In a further aspect, the subject suffering from multiple sclerosis has an EDSS score from about 3.0 to about 7.5, such as from about 3.0 to about 7, from about 3.0 to about 6.5, from about3.5 to about 6.5 or from about 4.0 to about 6.5.

In another embodiment, the subject suffering from multiple sclerosis has an EDSS score of at least 1.5. In one aspect, the subject suffering from multiple sclerosis has an EDSS score of at least 2.0. In another aspect, the subject suffering from multiple sclerosis has an EDSS score of at least 2.5. In yet another aspect, the subject suffering from multiple sclerosis has an EDSS score of at least 3.0. In another aspect, the subject suffering from multiple sclerosis has an EDSS score of at least 3.5. In a further aspect, the subject suffering from multiple sclerosis has an EDSS score of at least 4.0. In another aspect, the subject suffering from multiple sclerosis has an EDSS score of at least 4.5. In another aspect, the subject suffering from multiple sclerosis has an EDSS score of at least 5.0. In another aspect, the subject suffering from multiple sclerosis has an EDSS score of at least 5.5. In another aspect, the subject suffering from multiple sclerosis has an EDSS score of at least 6.0. In another aspect, the subject suffering from multiple sclerosis has an EDSS score of at least 6.5. In another aspect, the subject suffering from multiple sclerosis has an EDSS score of at least 7.0. In another aspect, the subject suffering from multiple sclerosis has an EDSS score of at least 7.5. In another aspect, the subject suffering from multiple sclerosis has an EDSS score of at least 8.0. In another aspect, the subject suffering from multiple sclerosis has an EDSS score of at least 8.5. In another aspect, the subject suffering from multiple sclerosis has an EDSS score of at least 9.0.

In another embodiment the subject suffering from multiples sclerosis has an EDSS score of 1.0, 1.5, 2.0, 2.5, 3.0. 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0 or 9.5.

As used herein, the Expanded Disability Status Scale (EDSS) is a method of quantifying disability in multiple sclerosis and monitoring changes in the level of disability over time. It is widely used in clinical trials and in the assessment of people with MS. (See: Kurtzke JF. Rating neurologic impairment in multiple sclerosis: an expanded disability status scale (EDSS). Neurology. 1983 Nov; 33(11): 1444-52 and Haber A, LaRocca NG. eds. Minimal Record of Disability for multiple sclerosis.

New York: National Multiple Sclerosis Society; 1985.)

The EDSS scale ranges from 0 to 10 in 0.5 unit increments that represent higher levels of disability. Scoring is based on an examination by a medical professional, usually a neurologist.

EDSS steps 1.0 to 4.5 refer to people with MS who are able to walk without any aid and is based on measures of impairment in eight functional systems (FS): pyramidal - weakness or difficulty moving limbs; cerebellar - ataxia, loss of coordination or tremor; brainstem - problems with speech, swallowing and nystagmus; sensory -numbness or loss of sensation; bowel and bladder function; visual function; cerebral (or mental) functions and other. Each functional system is scored on a scale of 0 (no disability) to 5 or 6 (more severe disability). EDSS steps 5.0 to 9.5 are defined by the impairment to walking. Although the scale takes account of the disability associated with advanced MS, most people will never reach these scores.

Expanded Disability Status Scale (EDSS)

*Excludes cerebral function grade 1

The invention is further illustrated by the following examples, which are not intended to limit the scope of the claims.

BIOLOGICAL TESTING METHODS AND SYNTHETIC PROCEDURES SCREENING OF OPCs

The compound of Formula (I) can be subjected to a primary screen where the compound is added to OPC cells seeded and incubated on a 96- or 384- well plate.

The cells can then be visually screened for oligodendrocyte precursor morphology changes. In a secondary screen, differentiation and proliferation induced by the compound of Formula (I) can be further validated by fluorescence microscopy.

Further oligodendrocyte precursor proliferation and maturation in response to the compound of Formula (I) can then be assessed by induction of myelin protein expression as determined by, for example, immunocytochemistry and western blot. Examples of assays that can be used in the primary and secondary screening are described in Najm et al. Nat Methods. 2011 Sep 25;8(l l):957-62; Bai et al. Neurosci Bull. 2013 Apr;29(2):239-50; Yang et al. Dev Biol. 2011 Feb l;350(l): l27-38;Cho et al. Curr Neuropharmacol. 2007 March; 5(1): 19-33; and Najm et al. Nature 522, 216- 220, 2015.

More specifically, OPCs can be treated with vehicle alone (e.g, 0.05%

(v/v)DMSO) as a negative control, a known OPC differentiation inducer as a positive control, or drug dissolved in DMSO at a pre-determined concentration (e.g., 5 mM). After 72 h, cells can be fixed and labelled with antibodies to myelin basic protein (MBP) and the length and intensity of MBP labelled oligodendrocyte processes can be measured. These features are known to be reliable indicators of alteration in cellular phenotype. The experimental data for the tested drug can then be normalized against ketoconazole (set value of 100) on a per plate basis. On the basis of this analysis, enhanced oligodendrocyte formation can be determined. In an even more specific example of the phenotypic screening of OPCs, EpiSC-derived OPCs (see, Najm et al. Nature 522, 216-220, 2015) can be seeded onto poly-D-lysine 96-well Viewplate or CellCarrier plates (PerkinElmer) coated with laminin (Sigma, L2020; 10 mgml2l) using electronic multichannel pipetors. For the primary screen, 30,000 cells can be seeded per well in screening medium

(DMEM/F12 supplemented with N2 (R&D Systems), B-27 (Life Technologies), neurotrophin 3 (R&D Systems; 10 ng ml2l), cAMP (Sigma; 50 mM), IGF-l (R&D Systems; 100 ng ml2l), noggin (R&D Systems; 100 ngml2l)) and can be allowed to attach for 2 h before addition of drug. Drugs can be added to assay plates with 0.1 ml pin replicators (Molecular Devices, Genetix; X5051), resulting in a final primary screening concentration of 5 mM. Positive control and DMSO vehicle controls can be included in each assay plate. Cells can be incubated under standard conditions (37 °C, 5% CO2) for 3 days and can be fixed with 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS). Fixed plates can be permeabilized with 0.1% Triton X-100 and blocked with 10% donkey serum (v/v) in PBS for 2 h. Cells can be labelled with MBP antibodies (Abeam, ab7349; 1:100) for 1 h at room temperature detection can be assessed with Alexa Fluorconjugated secondary antibodies (1:500) for 45 min. Nuclei can be visualized by DAPI staining (Sigma; 1 mgml2l). All plates for the primary screen can be processed and analysed simultaneously to eliminate variability

The compound of Formula (I) can be further assessed using a GCMS assay to monitor levels of cholesterol and intermediates en route to cholesterol, including squalene, squalene epoxide, lanosterol, FF-MAS, T-MAS, other meiosis activating sterols, zymosterol, zymostenol, lathosterol, dehydrolathosterol dehydrodesmosterol, desmosterol, 7-DHC, 8-DHC, and others. Such assays are described in Korade et al.

J. Med. Chem., 2016 59 (3), 1102-1115, among others. Briefly, cells can be treated with the compound of Formula (I) and then lysed using an organic solvent, such as methanol or chloroform to extract lipophilic metabolites. Following silylation using BTMSA or an equivalent silylating reagent, samples are injected onto the GC-MS and sterol abundance is determined by comparison of integrated peak intensities to genuine sterol reference standards.

While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

EXEMPLIFICATION

Example 1: Synthesis of the compound of formula (I).

The compound of formula (I) was manufactured according to the Scheme in

FIG. 2. The ¹ H NMR of the compound of formula (I) is shown in FIG. 3.

Alternatively, the compound of formula (I) and its pharmaceutically acceptable salts:

can be achieved as follows.

Step 1: Preparation of r3-(4-Adamantan-2-yl-3,5-dichlorophenyl)prop-2- vnyllcvclohexylethylamine Hydrochloride

8.6 ml of 36% formaldehyde are added to 11.2 ml of cyclohexylethylamine in 100 ml of 1 ,2-dimethoxyethane and stirring is continued at room temperature for 2 hours. This solution is added to a mixture of 16 g of 2-(4-ethynyl-3,5- dichlorophenyl)adamantane and 0.58 g of copper II chloride dihydrate in 400 ml of l,2-dimethoxy ethane. The reaction mixture is refluxed for 2 hours and the solvents are then evaporated off under reduced pressure. The compound obtained is taken up in diethyl ether, hydrogen chloride is bubbled through and the precipitate obtained is filtered off and dried.

Step 2: Preparation of r(Z)-3-(4-adamantan-2-yl-3,5-dichlorophenyl)propen-2- yll cyclohexylethylamine hydrochloride

3 g of the compound of Step 1 in 50 ml of petroleum ether are hydrogenated, under an inert atmosphere and at atmospheric pressure, in the presence of 3 ml of cyclohexene and 0.3 g of palladium on calcium carbonate poisoned with 3.5% lead (Lindlar catalyst). The reaction mixture is filtered through Celite, the solvents are evaporated off and the residue obtained is purified by chromatography on a column of silica gel, eluting with a 95/5 (v/v) dichloromethane/ethanol mixture. The oily residue obtained is taken up in diethyl ether and hydrogen chloride is bubbled through. The precipitate is filtered off and dried under reduced pressure.

Example 2: Preparation of Inhibitor Doses

Control compounds and maximum doses of the compound of formula (I) were laid out in 96- well format. Test compounds were serially diluted 1 : 1 with vehicle to set up smaller doses. Compounds were transferred to freshly plated OPCs using a manual pinning tool at 1:1000. The following final concentrations were used:

Drugs were prepared with DMSO as the vehicle.

Example 3: Differentiation of OPCs with the compound of formula (I)

OPCs were generated from the EpiSC5 cell line. EpiSC-derived OPCs were obtained from the EpiSC5 cells using in vitro differentiation protocols and culture conditions described previously (see, Najm et al. Nature 522, 216-220, 2015 and Najm et al, 2012, Nature Methods). To ensure uniformity throughout all in vitro screening experiments, EpiSC-derived OPCs were sorted to purity by fluorescence activated cell sorting at passage five with conjugated CD l40a-APC (eBioscience, 17- 1401; 1:80) and NG2-AF488 (Millipore, AB5320A4; 1: 100) antibodies In vitro phenotypic screening of OPCs: EpiSC-derived OPCs were grown and expanded in poly-L-omithine (PO) and laminin -coated flasks in N2B27 media (DMEM/F12 (Gibco), N2-MAX (R&D Systems), B-27 (ThermoFisher), and

GlutaMax (Gibco)) supplemented with FGF2 (10 mg/mL, R&D systems, 233-FB- 025) and PDGF-AA (10 mg/mL, R&D systems, 233-AA-050) before harvesting for experiments. The cells were seeded onto poly-L-omithine or poly-D-lysine coated 96- well CellCarrier Ultra plates (PerkinElmer) coated with laminin (Sigma, L2020) using a multi-channel pipet. Fifty-thousand cells were seeded per well in media containing N2 and B27 without growth factors, and allowed to attach for 30 min before addition of drug. For dose-response testing, a lOOOx compound stock in dimethyl sulphoxide (DMSO) was added to assay plates with 0.1 mL solid pin multi-blot replicators (V &

P Scientific; VP 409), resulting in a final primary screening dose curve of 12 doses between 4000nM and 2nM . A positive control (ketoconazole) and DMSO vehicle control were included in each assay plate. Cells were incubated under standard conditions (37 °C, 5% C02) for 3 days and fixed with 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) for 20 min. Fixed plates were washed with PBS (200 mL per well) twice, permeabilized with 0.1% Triton X-100 and blocked with 10% donkey serum (v/v) in PBS for 40 min. Then, cells were labelled with MBP antibodies (Abeam, ab7349; 1:200) for 1 h at room temperature followed by detection with Alexa Fluor conjugated secondary antibodies (1:500) for 45 min. Nuclei were visualized by DAPI staining (Sigma; 1 pg/ml). During washing steps, PBS was added using a multi-channel pipet and aspiration was performed using Biotek EL406 washer dispenser (Biotek) equipped with a 96-well aspiration manifold.

High-content imaging and analysis: Plates were imaged on the Operetta High

Content Imaging and Analysis system (PerkinElmer) and a set of 6 fields captured from each well. Analysis (PerkinElmer Harmony and Columbus software) began by identifying intact nuclei stained by DAPI; that is, those traced nuclei that were larger than 300 mm2 in surface area. Each traced nucleus region was then expanded by 50% and cross-referenced with the mature myelin protein (MBP) stain to identify oligodendrocyte nuclei, and from this the percentage of oligodendrocytes was calculated. EC50 values were calculated using Collaborative Drug Discovery, using

The Levenberg-Marquardt algorithm to fit a Hill equation to dose-response data

(2nM to 4000nM). FIG. 4 is a plot showing the increase in the number of differentiated MBP positive oligodendrocytes following exposure of the EpiSC- derived OPCs to varying concentrations of the compound of Formula (I). The EC50 value is between 100 and 200nM.

Example 4: The compound of formula (I) increases zymostenol in mouse OPCs.

GCMS-based sterol profiling: Sterols were monitored using a modified Folch wash protocol (Hubler et al, 2018, Nature). EpiSC-derived OPCs were plated at one million cells per well in PO- and laminin-coated six-well plates in N2B27 media without growth factors. After 24 hours, cells were dissociated with Accutase, rinsed with saline, and cell pellets were frozen. Cells were lysed in methanol (Sigma- Aldrich) with agitation for 30 minutes and cell debris removed by centrifugation at 10,000 rpm for 15 min. Cholesterol-d7 standard (Cambridge Isotope Laboratories) was added before drying under nitrogen stream and derivatization with 55 ml of bis(trimethylsilyl) trifluoroacetamide. After derivatization, 1 ml was analyzed by gas chromatography / mass spectrometry using an Agilent 5973 Network Mass Selective Detector equipped with a 6890 gas chromatograph system and a HP-5MS capillary column (60m x 0.25mm x 0.25mm). Samples were analyzed in full scan mode using electron impact ionization; ion fragment peaks were integrated to calculate sterol abundance, and quantitation was relative to cholesterol-d7. The following ion fragments were used to quantitate each metabolite: cholesterol-d7 (465), FF-Mas (482), cholesterol (368), zymostenol (458), zymosterol (456), Desmosterol (456, 343), 7-dehydrocholesterol (456, 325), lanosterol (393), lathosterol (458), 14- dehydrozymostenol (456, 351). All standards were obtained from Avanti Polar Lipids unless otherwise indicated. Calibration curves were generated by injecting varying concentrations of sterol standards and maintaining a fixed amount of cholesterol-D7.

The results of testing in FIG. 5 show that the compound of Formula (I) inhibits EBP on mouse OPCs resulting in an increase in measured levels of zymostenol.

Claims

What is claimed is: 1. A method of treating secondary progressive multiple sclerosis in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound represented by structural Formula (I):

or a pharmaceutically acceptable salt thereof.

2. A method of treating primary progressive multiple sclerosis in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound represented by structural Formula (I):

or a pharmaceutically acceptable salt thereof.

3. The method of claim 1 or claim 2, wherein the subject is a human.

4. The method of claim 3, wherein the human is a female.

5. The method of any one of Claims 1-4, wherein the compound of Formula (I) is administered intravenously, intrathecally, subcutaneously, intramuscularly, intranasally or orally.

6. The method of any one of claims 1-5, wherein the compound of Formula (I) or a pharmaceutically acceptable salt thereof is in a pharmaceutical composition with a pharmaceutically acceptable carrier.

The method of any one of Claims 1-6, wherein the compound of Formula (I) is an HC1 salt.

8. The method of any one of Claims 1-7 further comprising administering a therapeutically effect amount of an MS therapeutic agent.

9. The method of Claim 8, wherein the MS therapeutic agent is selected from: glatiramer acetate, Ocrevus (ocrelizumab), Campath (Lemtrada or

alemtuzumab), Gilenya, Ampyra (dalfampridine), Tysabri (natalizumab), Aubagio (teriflunomide), Rebif , Avonex, Betaseron, Plegridy, Interferon Beta- la, dimethyl fumarate, fingolimod, rituximab, Zinbryta, Ofatumymab, Nerventra (laquinimod), Masitinib, Siponimod, Ozanimod, Ponesimod, ibudilast, vatelizumab, minocycline, ibrutinib, PRN2246, Cladripine, GNBAC1, daclizumab, and MD1003 (biotin).

10. The method of claim 8 or claim 9, wherein the MS therapeutic agent is

administered simultaneously with the compound represented by Formula (I) or a pharmaceutically acceptable salt thereof.

11. The method of claim 8 or Claim 9, wherein the MS therapeutic agent is

administered prior to administration the compound represented by Formula (I) or a pharmaceutically acceptable salt thereof.

12. The method of claim 8 or claim 9 wherein the MS therapeutic agent is administered following the administration of the compound represented by Formula (I) or a pharmaceutically acceptable salt thereof.

13. The method of any one of Claims 1-12, wherein the subject is administered the compound represented by Formula I for an on-drug cycle of at least three months.

14. The method of Claim 13, wherein the subject is administered the compound represented by Formula I for an on-drug cycle of at least six months.

15. The method of any one of Claims 1-12, wherein the subject is administered the compound represented by Formula I using the following dosing regimen: a. on-drug cycle for at least six months;

b. off-drug cycle for at least three months;

wherein the on-drug and off-drug cycles are optionally repeated.

16. A method of promoting myelination of central nervous system neurons in a subject suffering from a myelin-related disorder, the method comprising administering to the subject a therapeutically effective amount of a compound represented by structural Formula (I):

or a pharmaceutically acceptable salt thereof.

17. The method of Claim 16, wherein the myelin-related disorder is selected from; secondary progressive multiple sclerosis (MS), primary progressive multiple sclerosis, neuromyelitis optica (NMO), optic neuritis, pediatric

leukodystrophies, neonatal white matter injury, age-related dementia, schizophrenia, progressive multifocal leukoencephalopathy (PML), encephalomyelitis (EPL), central pontine myelolysis (CPM),

adrenoleukodystrophy, Alexander's disease, Pelizaeus Merzbacher disease (PMD), Vanishing White Matter Disease, Wallerian Degeneration, transverse myelitis, amylotrophic lateral sclerosis (ALS), Huntington's disease,

Alzheimer's disease, Parkinson's disease, spinal cord injury, traumatic brain injury, post radiation injury, neurologic complications of chemotherapy, stroke, acute ischemic optic neuropathy, vitamin E deficiency, isolated vitamin E deficiency syndrome, Bassen-Kornzweig syndrome, Marchiafava-Bignami syndrome, metachromatic leukodystrophy, trigeminal neuralgia, acute disseminated encephalitis, Guillian-Barre syndrome, Charcot-Marie-Tooth disease, Bell's palsy and radiation-induced demyelination.

18. The method of Claim 17, wherein the disorder is secondary progressive MS (SPMS).

19. The method of claim 17, wherein the disorder is primary progressive MS (PPMS).

20. The method of claim 18 or claim 19, wherein the subject is a human.

21. The method of claim 20, wherein the human is a female.

22. The method of any one of claims 16-21, wherein the compound of Formula (I) is administered intravenously, intrathecally, subcutaneously, intramuscularly, intranasally or orally.

23. The method of any one of claims 16-22, wherein the compound of Formula (I) or a pharmaceutically acceptable salt thereof is in a pharmaceutical composition with a pharmaceutically acceptable carrier.

24. The method of any one of claims 16-23, wherein the compound of Formula (I) is an HC1 salt.

25. The method of any one of claims 18-24, further comprising administering a therapeutically effect amount of an MS therapeutic agent.

26. The method of claim 25, wherein the MS therapeutic agent is selected from: glatiramer acetate, Ocrevus (ocrelizumab), Campath (Lemtrada or

alemtuzumab), Gilenya, Ampyra (dalfampridine), Tysabri (natalizumab), Aubagio (teriflunomide), Rebif , Avonex, Betaseron, Plegridy, Interferon Beta- la, dimethyl fumarate, fingolimod, rituximab, Zinbryta, Ofatumymab, Nerventra (laquinimod), Masitinib, Siponimod, Ozanimod, Ponesimod, ibudilast, vatelizumab, minocycline, ibrutinib, PRN2246, Cladripine, GNBAC1, daclizumab, and MD1003 (biotin).

27. The method of claim 25 or claim 26, wherein the MS therapeutic agent is administered simultaneously with the compound represented by Formula (I) or a pharmaceutically acceptable salt thereof.

28. The method of claim 25 or claim 26, wherein the MS therapeutic agent is administered prior to administration the compound represented by Formula (I) or a pharmaceutically acceptable salt thereof.

29. The method of Claim 25 or claim 26 wherein the MS therapeutic agent is administered following the administration of the compound represented by Formula (I) or a pharmaceutically acceptable salt thereof.

30. The method of any one of claims 16-29, wherein the subject is administered the compound represented by Formula I for an on-drug cycle of at least three months.

31. The method of claim 30, wherein the subject is administered the compound represented by Formula I for an on-drug cycle of at least six months.

32. The method of any one of claims 16-29, wherein the subject is administered the compound represented by Formula I using the following dosing regimen: a. on-drug cycle for at least six months;

b. off-drug cycle for at least three months;

wherein the on-drug and off-drug cycles are optionally repeated.

33. The method of any one of claims 16-32, wherein the compound of Formula (I) inhibits EBP enzyme mediated synthesis of sterol intermediates in the cholesterol biosynthesis pathway.

34. The method of any one of claims 16-33, wherein the compound of Formula (I) induces, promotes, and/or modulates oligodendrocyte precursor cell (OPC) differentiation, proliferation and/or maturation.

35. The method of claim 34, wherein the induction of OPC differentiation is characterized by an increase in myelin basic protein (MBP) expression.

36. A method of inducing endogenous oligodendrocyte precursor cell (OPC) differentiation in a subject in need thereof, the method comprising

administering to the subject a therapeutically effective amount of a compound represented by structural Formula (I):

or a pharmaceutically acceptable salt thereof.

37. The method of claim 36, wherein the subject suffers from a myelin-related disorder.

38. The method of claim 37, wherein the subject is suffering from multiple sclerosis.

39. The method of any one of claims 36-38, wherein the subject is human.

40. The method of any one of claims 36-38, further comprising administering a therapeutically effect amount of an MS therapeutic agent.