Compounds and methods for promoting myelination

Abstract

To provide a method of enhancing the generation of oligodendrocytes.SOLUTION: The method includes administering to an oligodendrocyte precursor cell (OPC) an agent that enhances and / or induces accumulation of Δ8,9-unsaturated sterol intermediates of the cholesterol biosynthesis pathway in the OPC.SELECTED DRAWING: Figure 1-1

Description

[Technical Field]

[0001] Related Applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 367,416, filed July 27, 2016, and U.S. Provisional Patent Application No. 62 / 452,204, filed January 30, 2017, the subject matter of which is incorporated herein by reference in its entirety.

[0002] government financial support

[0002] This invention was made with government support under Grant No. R01NS095280 awarded by the National Institutes of Health. The United States Government has certain rights in this invention. [Background technology]

[0003]

[0003] Multiple sclerosis (MS) is a complex neurological disease characterized by the deterioration of central nervous system (CNS) myelin. This insulating substance, composed mostly of lipids (70% lipid, 30% protein), protects axons and allows for saltatory conduction, which accelerates axonal electrical impulses. Demyelination of axons in chronic MS can lead to axonal degeneration and neuronal death, but more specifically, MS destroys oligodendrocytes, the highly specialized CNS cells that generate and maintain myelin.

[0004]

[0004] Immature oligodendrocytes, oligodendrocyte precursors (PDGFRα+, NG2-proteoglycan+), are generated from a common glial progenitor in the ventral region of the developing brain, actively migrate and proliferate, populate the CNS, and ultimately differentiate into premyelinating oligodendrocytes (O4+). At this mature stage, oligodendrocytes either target and extend myelin sheaths along axons or disappear. However, the hypothesis that myelination and / or remyelination can be enhanced by either endogenous oligodendrocyte precursors or transplanted cells has been less explored. Summary of the Invention [Problem to be solved by the invention]

[0005]

[0005] Inducing differentiation and / or promoting survival during maturation of endogenous oligodendrocyte progenitors can stimulate and enhance the generation of new oligodendrocytes and endogenous myelination and / or remyelination. Thus, there is a need for compounds and therapeutic methods that can enhance the generation of new oligodendrocytes. [Means for solving the problem]

[0006]

[0006] The embodiments described herein generally relate to agents, compounds, and methods for enhancing oligodendrocyte production by inducing, promoting, and / or regulating the differentiation, proliferation, and / or maturation of oligodendrocyte precursor cells, as well as methods for treating diseases or disorders in subjects where myelination or remyelination is beneficial to the subject.

[0007] It has been discovered that enhancing and / or inducing the accumulation of Δ8,9-unsaturated sterol intermediates of the cholesterol biosynthetic pathway in oligodendrocyte progenitor cells (OPCs) can induce oligodendrogenesis. Enhancement and / or induction of the accumulation of Δ8,9-unsaturated sterol intermediates can be achieved by inhibiting the accumulation of Δ8,9-unsaturated sterol intermediates and / or modulating and / or inhibiting enzymes in the cholesterol biosynthetic pathway in OPCs for which Δ8,9-unsaturated sterol intermediates are substrates, as well as by inhibiting the accumulation of Δ8,9-unsaturated sterol intermediates. Sterol intermediates can be obtained by directly and / or indirectly administering them to OPCs. Enhancing and / or inducing the accumulation of Δ8,9-unsaturated sterol intermediates can promote the differentiation, survival, proliferation, and / or maturation of OPCs and can treat diseases and / or disorders in a subject where myelination or remyelination is beneficial to the subject.

[0008] In some embodiments, an agent that enhances and / or induces the accumulation of Δ8,9-unsaturated sterol intermediates of the cholesterol biosynthetic pathway in OPCs can be administered to a subject and / or OPCs in an amount effective to promote and / or induce differentiation, proliferation, and / or maturation of OPCs and oligodendrocyte production. In one example, the agent can include at least one compound that inhibits enzyme-mediated synthesis of one or more sterol intermediates in the cholesterol biosynthetic pathway in OPCs and / or promotes the accumulation of Δ8,9-unsaturated sterol intermediates.

[0009] In some embodiments, the compounds can modulate and / or inhibit one or more enzyme-mediated conversion steps of lanosterol to cholesterol, e.g., between lanosterol and / or lathosterol, in OPCs, promoting and / or inducing oligodendrogenesis. For example, the compounds can inhibit CYP51, sterol 14-reductase, and / or EBP enzyme-mediated synthesis of sterol intermediates in the cholesterol biosynthetic pathway. In yet another embodiment, the compounds can modulate and / or inhibit the enzyme-mediated conversion of lanosterol to 4,4-dimethylcholesta-8(9),14,24-trien-3β-ol, 4,4-dimethylcholesta-8(9),14,24-trien-3β-ol to zymostenol, and / or zymostenol to lathosterol, enhancing oligodendrogenesis.

[0010]

[0010] In some embodiments, the compound is capable of modulating and / or inhibiting one or more enzyme-mediated conversion steps of the cholesterol biosynthetic pathway from lanosterol to dehydrolanthosterol or lathosterol.

[0011] In some embodiments, the compounds used in the methods described herein can inhibit the enzyme-mediated conversion of zymostenol to lathosterol through inhibition of emopamil-binding protein (EBP) isomerase enzyme activity. Alternatively, the compounds used in the methods described herein can inhibit sterol C14 reductase enzyme activity or CYP51 enzyme activity in the cholesterol biosynthetic pathway.

[0012]

[0012] In some embodiments, the compound is selected from the group consisting of bifonazole, clotrimazole, miconazole, butoconazole, ketoconazole, fulvestrant, amorolfine, ifenprodil, ziprasidone, Tasin-1, tamoxifen, benztropine, clemastine, trans-U50488, EPZ005687, raloxifene, hydroxyzine, vesamicol, L-745,870, TMB-8, cis-N-cyclohexyl-N-ethyl-3-(3-chloro-4-cyclohexylphenyl)prop-2-enylamine, p-fluorohexahydro-silazifenidol, pramoxine, toremifene, and combinations thereof.

[0013] In another embodiment, the compound can be an interfering RNA, such as an siRNA, or a CRISPR / Cas nuclease that inhibits expression of an enzyme that synthesizes one or more sterol intermediates in the cholesterol biosynthesis pathway in OPCs. For example, an interfering RNA CRISPR / Cas nuclease can inhibit expression of CYP51, sterol 14-reductase, and / or EBP enzymes.

[0014] In another embodiment, the agent can comprise a Δ8,9-unsaturated sterol intermediate or derivative thereof of at least one of the cholesterol biosynthetic pathway in OPCs, wherein the Δ8,9-unsaturated sterol intermediate or derivative thereof enhances oligodendrocyte production from OPCs. The Δ8,9-unsaturated sterol intermediate can include at least one of lanosterol, 14-dehydrozymostenol, FF-MAS, MAS-412, zymosterol, zymostenol, and derivatives thereof.

[0015] In some embodiments, the compounds described herein can be used to treat neurodegenerative diseases and disorders in a subject in need thereof. In some embodiments, the neurodegenerative disease or disorder is a myelin-associated disorder. Myelin-associated diseases or disorders include diseases, disorders, or injuries associated with myelin dysplasia or loss of myelin in nerve cells, e.g., CNS neurons, of a subject. Examples of myelin-associated diseases and disorders include multiple sclerosis (MS), neuromyelitis optica (NMO), progressive multifocal leukoencephalopathy (PML), encephalomyelitis (EPL), central pontine myelinolysis (CPM), adrenoleukodystrophy, Alexander disease, Pelizaeus-Merzbacher disease (PMD), vanishing white matter disease, Wallerian degeneration, optic neuritis, transverse myelitis, amyotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease, Parkinson's disease, and Parkinson's disease. These include: Kinson's disease, spinal cord injury, traumatic brain injury, post-radiation injury, neurological complications of chemotherapy, stroke, acute ischemic optic neuropathy, vitamin E deficiency, isolated vitamin E deficiency syndrome, Bassen-Korn-Zweig syndrome, Marchia-Farba-Bignami syndrome, metachromatic leukodystrophy, trigeminal neuralgia, acute disseminated encephalitis, Guillain-Barré syndrome, Charcot-Marie-Tooth disease, Bell's palsy, and mental health disorders such as schizophrenia. [Brief explanation of the drawings]

[0016] [Figure 1-1]Figure 1 (A-J) illustrates structures, plots, graphs, and images showing that CYP51 is a functional target whereby imidazole antifungal agents enhance oligodendrogenesis. A) Diagram of azole molecules with varying degrees of potency for mammalian CYP51 inhibition. [Figure 1-2] Figure 1 (A-J) illustrates structures, plots, graphs, and images showing that CYP51 is a functional target whereby imidazole antifungals enhance oligodendrogenesis. B) Rat CYP51 enzyme activity as measured by LC / MS-based quantification of the CYP51 product FF-MAS (4,4-dimethyl-5a-cholesta-8,14,24-trien-3β-ol) after treatment with azoles. n=2 independent enzyme assays. [Figure 1-3] Figure 1 (A-J) illustrates structures, plots, graphs, and images demonstrating that CYP51 is a functional target by which imidazole antifungals enhance oligodendrogenesis. C) Percentage of MBP+ oligodendrocytes generated from OPCs 72 hours after treatment with azoles. n=4 replicates per condition, >1,000 cells analyzed per replicate. [Figure 1-4] Figure 1(A-J) illustrates structures, plots, graphs, and images showing that CYP51 is a functional target whereby imidazole antifungals enhance oligodendrogenesis. D) Representative images of OPCs treated with the indicated imidazole antifungals for 72 hours. [Figure 1-5] Figure 1 (A-J) illustrates structures, plots, graphs, and images showing that CYP51 is a functional target by which imidazole antifungals enhance oligodendrogenesis. E) GC / MS-based quantification of lanosterol levels in OPCs treated with 2.5 μM of the indicated azoles for 24 h. n=2 replicates per condition. [Figure 1-6]Figure 1 (A-J) illustrates structures, plots, graphs, and images showing that CYP51 is a functional target whereby imidazole antifungals enhance oligodendrogenesis. F) GC / MS-based quantification of lanosterol levels in OPCs treated for 24 h with the indicated doses of ketoconazole. n=2 replicates per condition. [Figure 1-7] Figure 1 (A-J) illustrates structures, plots, graphs, and images demonstrating that CYP51 is a functional target, thereby enhancing oligodendrocyte formation, with imidazole antifungals. G) CYP51 mRNA levels measured by RT-qPCR after 4 days of treatment with non-targeting or CYP51-targeting pools of cell-permeable siRNA. n=2 replicates, with four qPCR measurements per replicate. H) GC / MS-based quantification of lanosterol levels in OPCs treated with the indicated reagents for 96 hours. n=2 replicates per condition, ketoconazole, 2.5 μM. I) Percentage of MBP+ oligodendrocytes generated from OPCs at 72 hours after treatment with the indicated reagents. n=2 independent experiments, four replicates per condition, >1,000 cells analyzed per replicate. Two-tailed t-test, **P<0.01 for siRNA group compared to its respective non-targeting control treatment group. J) Percentage of MBP+ oligodendrocytes generated from OPCs 72 hours after treatment with exogenous lanosterol, n=4 replicates per condition, and >1,000 cells analyzed per replicate. [Figure 2-1]

[0017] Figure 2 (A-D) illustrates schematics and graphs showing the effect of small molecule inhibition of enzymes in the cholesterol biosynthetic pathway on enhancing oligodendrogenesis. A) A concise diagram of the cholesterol biosynthetic pathway, including intermediate metabolites and labeled selective inhibitors. [Figure 2-2]Figure 2 (A-D) illustrates a schematic and graphs showing the effect of small molecule inhibition of enzymes in the cholesterol biosynthesis pathway on enhancing oligodendrocyte formation. B) Percentage of MBP+ oligodendrocytes generated from OPCs at 72 hours after treatment with the indicated pathway inhibitors. n=4 replicates per condition, >1,000 cells analyzed per replicate. [Figure 2-3] Figure 2 (A-D) illustrates a schematic and graphs showing the effect of small molecule inhibition of enzymes in the cholesterol biosynthetic pathway on enhancing oligodendrogenesis. C) GC / MS-based quantification of 14-dehydrozymostenol levels in OPCs treated for 24 hours with the indicated doses of amorolfine or TASIN-1. n=2 replicates per condition. D) Percentage of MBP+ oligodendrocytes generated from OPCs at 72 hours after treatment with the indicated purified sterol intermediates. n=4 replicates per condition, and >1,000 cells analyzed per replicate. [Figure 3-1]

[0018] Figure 3 (A–F) illustrates graphs showing that an inhibitory step between CYP51 and EBP is a unifying mechanism for many small enhancers of oligodendrogenesis identified by high-throughput screening. A) Percentage of MBP+ oligodendrocytes generated from OPCs at 72 h after treatment with a uniform dose of 5 μM ketoconazole, nine molecules identified by bioactivity screening, and nine randomly selected library members. n = 4 replicates per condition, and >1,000 cells analyzed per replicate. B) GC / MS-based quantification of zymosterol, zymostenol, and 14-dehydrozymostenol levels in OPCs treated with 2 μM of the indicated screening hits and randomly selected library members for 24 h. One replicate per condition was performed, and results were confirmed with a second OPC induction (Figure 9B). Molecules are categorized by the enzyme targeted (top labels). [Figure 3-2]Figure 3 (A-F) illustrates graphs demonstrating that the inhibitory step between CYP51 and EBP is a unifying mechanism for many small enhancers of oligodendrogenesis identified by high-throughput screening. C) GC / MS-based quantification of zymostenol levels in OPCs treated for 24 hours with the indicated previously reported oligodendrogenesis enhancers. Unless otherwise noted, the following concentrations were used: benztropine, 2 μM; clemastine, 1 μM; tamoxifen, 100 nM; U50488, 5 μM; bexarotene, 1 μM; and liothyronine, 3 μM. D) GC / MS-based quantification of cholesterol levels in OPCs treated for 24 hours with the indicated previously reported oligodendrogenesis enhancers. [Figure 3-3] Figure 3 (A-F) illustrates graphs showing that an inhibitory step between CYP51 and EBP is a unifying mechanism for many small enhancers of oligodendrogenesis identified by high-throughput screening. E) GC / MS-based quantification of EBP enzyme activity in a biochemical assay after treatment with small molecules (10 μM) that inhibit or do not inhibit EBP in OPCs. n=3. F) Percentage of MBP+ oligodendrocytes generated from OPCs at 72 hours after treatment with the indicated combinations of oligodendrogenesis enhancers. n=4 replicates per treatment condition, and >1,000 cells analyzed per replicate. Keto = ketoconazole, 2.5 μM. [Figure 4-1]

[0019] Figure 4 (A-D) illustrates graphs and images showing the effect of small molecules on sterol intermediate accumulation and enhancing remyelination in vivo. A) GC / MS-based quantification of sterol levels in mouse brain after 3 days of daily dosing with miconazole (10 mg per kg), ifenprodil (10 mg per kg), and tamoxifen (2 mg per kg). n = 4 animals per group. B) Quantification of remyelinated axons within toluidine blue-stained sections of LPC-lesioned spinal cord from mice treated for 8 days with the molecules at the doses indicated in panel a. n = 6 animals per group, except for vehicle (n = 4). Data are shown as mean + / - SEM. [Figure 4-2] Figure 4 (A-D) illustrates graphs and images showing the effect of small molecules on sterol intermediate accumulation and enhancing remyelination in vivo. C) Representative images within toluidine blue-stained sections of LPC-lesioned dorsal spinal cord from mice treated for 8 days with the molecules at the doses indicated in panel a. Scale bar, 20 µm. [Figure 4-3] Figure 4 (A-D) illustrates graphs and images showing the effect of small molecules on sterol intermediate accumulation and enhancing remyelination in vivo. D) Representative electron microscopy images of sections of LPC-lesioned dorsal spinal cord from mice treated for 8 days with the molecules at the doses indicated in panel a. Scale bar, 5 μm. Mann-Whitney, *U<0.05 and **U<0.01 for drug-treated groups compared to their respective vehicle-treated groups. [Figure 5-1]

[0020] Figure 5 (A-N) illustrates graphs and images showing that CYP51 is a functional target whereby imidazole antifungals enhance oligodendrogenesis. A) GC / MS-based quantification of cholesterol levels in OPCs (OPC-5) treated with 2.5 μM of the indicated azoles for 24 hours. n=2 replicates per condition. B) Percentage of MBP+ oligodendrocytes from a second independent induction of OPCs (OPC-1) at 72 hours after treatment with the indicated concentrations of azoles. n=4 replicates per condition, and >1,000 cells analyzed per replicate. [Figure 5-2] Figure 5 (A-N) illustrates graphs and images showing that CYP51 is a functional target whereby imidazole antifungals enhance oligodendrogenesis. C) GC / MS-based quantification of lanosterol levels in second induction OPCs (OPC-1) treated with 2.5 μM of the indicated azoles for 24 hours. n=2 replicates per condition. D) GC / MS-based quantification of cholesterol levels in OPCs (OPC-1) treated with 2.5 μM of the indicated azoles for 24 hours. n=2 replicates per condition. [Figure 5-3] Figure 5 (A-N) illustrates graphs and images demonstrating that CYP51 is a functional target by which imidazole antifungals enhance oligodendrogenesis. E) Percentage of MBP+ oligodendrocytes generated from mouse primary OPCs 72 hours after treatment with 3 μM of the indicated imidazole antifungal agent. n=4 replicates per condition, and >1,000 cells analyzed per replicate. F) GC / MS-based quantification of lanosterol levels in mouse primary OPCs treated with 3 μM of the indicated imidazole antifungal agent for 24 hours. n=2 replicates per condition. G) Assessment of oligodendrogenesis using an alternative image quantification measure, the fold increase in total neurite length. Panels are reanalysis of data shown in Figure 1C. n=4 replicates per condition, and >1,000 cells analyzed per replicate. [Figure 5-4]Figure 5 (A-N) illustrates graphs and images demonstrating that CYP51 is a functional target, thereby enhancing oligodendrocyte formation, through imidazole antifungals. H) Percentage of oligodendrocytes generated from OPCs 72 hours after treatment with the indicated concentrations of azoles, as measured by PLP1 immunostaining. Left, OPC-5; right, OPC-1. n=4 replicates per condition, and >1,000 cells analyzed per replicate. I) GC / MS-based quantification of lanosterol levels in a second, independent batch of OPCs treated with the indicated doses of ketoconazole for 24 hours. n=2 replicates per condition. Concentrations shown in panel i reflect those shown in panel b. J) Representative images of OPC-5 cells treated with the indicated siRNA reagents for 72 hours. Nuclei are labeled with DAPI (blue), and oligodendrocytes are indicated by immunostaining for myelin basic protein (green). Scale bar, 100 μm. [Figure 5-5] Figure 5 (A-N) illustrates graphs and images demonstrating that CYP51 is a functional target, whereby imidazole antifungals enhance oligodendrogenesis. K) GC / MS-based quantification of lanosterol levels in a second independent batch of OPCs (OPC-1) treated with the indicated pooled siRNA reagents for 96 hours. n=2 replicates per condition. L) Percentage of MBP+ oligodendrocytes from a second independent batch of OPCs (OPC-1) at 72 hours after treatment with the indicated reagents. n=4 replicates per condition, and >1,000 cells analyzed per replicate. Two-tailed t-test, **P<0.01 for siRNA groups compared with their respective non-targeting control treatment groups. M) Representative images of OPC-5 OPCs treated with lanosterol (47 μM) for 72 hours. Nuclei were labeled with DAPI, and oligodendrocytes were visualized by immunostaining for myelin basic protein. [Figure 6]

[0021] Figure 6 illustrates a schematic diagram showing an expanded cholesterol synthesis pathway diagram. A cascade of squalene epoxide cyclizations is catalyzed by lanosterol synthase (LSS) to provide the first sterol, lanosterol. Processing of lanosterol to cholesterol can proceed via either the Kandutsch-Russell or Bloch pathway. These pathways use the same enzymes and process substrates and differ only in the presence or absence of a C24 double bond. The blue intermediate was confirmed in our GC / MS-based sterol profiling assay using authentic standards. Sterol 14-reductase activity in mice is shared by two genes, TM7SF2 and LBR. [Figure 7-1]

[0022] Figure 7 (AK) illustrates graphs and images showing the effect of small molecule targeting of enzymes in the cholesterol biosynthesis pathway on enhancing oligodendrogenesis. A) GC / MS-based quantification of basal sterol levels in OPCs treated for 24 hours with the indicated cholesterol biosynthesis inhibitors. Left: cholesterol; right: desmosterol. n=2 replicates per condition. Inhibitors were used at the following doses unless otherwise noted: mevastatin, ketoconazole, MGI-39, 2.5 μM; YM53601, 2 μM; Ro48-8071, amorolfine, TASIN-1, 100 nM; AY9944, 200 nM. B) GC / MS-based quantification of basal sterol levels in a second batch of OPCs (OPC-1). Left: cholesterol; right: desmosterol. n=2 replicates per condition. [Figure 7-2]Figure 7 (AK) illustrates graphs and images showing the effect of small molecule targeting of enzymes in the cholesterol biosynthesis pathway on enhancing oligodendrogenesis. C) GC / MS-based quantification of sterol intermediates predicted to accumulate after 24 hours of treatment of OPCs with the indicated cholesterol biosynthesis inhibitors. n=2 replicates per condition. D) GC / MS-based quantification of sterol intermediates predicted to accumulate after 24 hours of treatment of a second induction of OPCs (OPC-1) with the indicated cholesterol biosynthesis inhibitors. n=2 replicates per condition. In C and D, accumulation of other sterol intermediates, indicative of off-target effects within the cholesterol pathway, was not observed. [Figure 7-3] Figure 7 (AK) illustrates graphs and images showing the effect of small molecule targeting of enzymes in the cholesterol biosynthesis pathway on enhancing oligodendrogenesis. E) Representative images of OPC-5 cells treated with the indicated small molecules for 72 hours. All treatments are at the highest concentrations shown in Figure 2. Scale bar, 100 μm. [Figure 7-4] Figure 7 (AK) illustrates graphs and images showing the effect of small molecule targeting of enzymes in the cholesterol biosynthesis pathway on enhancing oligodendrocyte formation. F) Percentage of MBP+ oligodendrocytes from a second batch of OPCs (OPC-1) at 72 hours after treatment with the indicated cholesterol pathway inhibitors. n=4 replicates per condition, and >1,000 cells analyzed per replicate. [Figure 7-5]Figure 7 (AK) illustrates graphs and images showing the effect of small molecule targeting of enzymes in the cholesterol biosynthesis pathway on enhancing oligodendrocyte formation. G) Percentage of MBP+ oligodendrocytes generated from mouse primary OPCs at 72 hours after treatment with 300 nM of the indicated cholesterol pathway inhibitors. n=4 replicates per condition, and >1,000 cells analyzed per replicate. H) GC / MS-based quantification of sterol intermediate levels in mouse primary OPCs treated with 300 nM of the indicated cholesterol biosynthesis inhibitors for 24 hours. Left, 14-dehydrozymostenol levels after treatment with amorolfine; right, zymostenol levels after treatment with TASIN-1. n=2 replicates per condition. [Figure 7-6] Figure 7 (AK) illustrates graphs and images showing the effect of small molecule targeting of enzymes in the cholesterol biosynthesis pathway on enhancing oligodendrogenesis. I) GC / MS-based quantification of sterol intermediate levels in OPC-1 OPCs treated for 24 hours with the indicated doses of cholesterol biosynthesis inhibitors. Left, 14-dehydrozymostenol levels after treatment with amorolfine; right, zymostenol levels after treatment with TASIN-1. n=2 replicates per condition. Concentrations shown in panel i reflect those shown in panel f. [Figure 7-7] Figure 7 (AK) illustrates graphs and images showing the effect of small molecule targeting of enzymes in the cholesterol biosynthetic pathway on enhancing oligodendrocyte formation. J) Percentage of MBP+ oligodendrocytes generated from OPC-1 OPCs at 72 hours after treatment with the indicated purified sterol intermediates. n=4 replicates per condition and >1,000 cells analyzed per replicate. Green boxes highlight metabolites that accumulate after treatment that enhances oligodendrocyte formation in panel e. K) Percentage of MBP+ oligodendrocytes generated from OPC-5 and OPC-1 OPCs at 72 hours after treatment with the indicated concentrations of cholesterol. n=4 replicates per condition and >1,000 cells analyzed per replicate. [Figure 8-1]

[0023] Figure 8 (A-I) illustrates plots, graphs, and images showing the effects of EPZ005687 and related EZH2 inhibitors on cellular EBP function and oligodendrocyte formation. A) Percentage of MBP+ oligodendrocytes (relative to DMSO control replicates) obtained from OPCs (OPC-1 induced) at 72 hours following treatment with a library of 3,000 bioactive small molecules at 2 μM each. B) Four structurally analogous EZH2 inhibitors included in the bioactive library screened in panel a. [Figure 8-2] Figure 8 (A-I) illustrates plots, graphs, and images showing the effects of EPZ005687 and related EZH2 inhibitors on cellular EBP function and oligodendrocyte formation. C) Percentage of MBP+ oligodendrocytes generated from OPCs 72 hours after treatment with the indicated structural analog EZH2 inhibitors. n=4 replicates per condition, and >1,000 cells analyzed per replicate. D) Percentage of MBP+ oligodendrocytes from a second batch of OPCs 72 hours after treatment with the indicated structural analog EZH2 inhibitors. n=4 replicates per condition, and >1,000 cells analyzed per replicate. E) Percentage of MBP+ oligodendrocytes generated from mouse primary OPCs 72 hours after treatment with EPZ005687. n=4 replicates per condition, and >1,000 cells analyzed per replicate. [Figure 8-3] Figure 8 (A-I) illustrates plots, graphs, and images showing the effects of EPZ005687 and related EZH2 inhibitors on cellular EBP function and oligodendrogenesis. F) Representative images of OPCs treated with the indicated EZH2 inhibitors for 72 hours. All treatments are at 2 μM. Scale bar, 100 μm. [Figure 8-4]Figure 8 (A-I) illustrates plots, graphs, and images showing the effects of EPZ005687 and related EZH2 inhibitors on cellular EBP function and oligodendrocyte formation. G) GC / MS-based quantification of two sterol intermediates after treating OPCs with 1 μM of the indicated EZH2 inhibitor for 24 hours. Left, zymostenol; right, zymosterol. n=2 replicates per condition. H) GC / MS-based quantification of two sterol intermediates after treating a second induction of OPCs with 1 μM of the indicated EZH2 inhibitor for 24 hours. Left, zymostenol; right, zymosterol. n=2 replicates per condition. [Figure 8-5] Figure 8 (A-I) illustrates plots, graphs, and images showing the effects of EPZ005687 and related EZH2 inhibitors on cellular EBP function and oligodendrogenesis. I) GC / MS-based quantification of two sterol intermediates after 24 h treatment of mouse primary OPCs with 2 μM EPZ005687. Left, zymostenol; right, zymosterol. n=2 replicates per condition. [Figure 9-1]

[0024] Figure 9 (A-H) illustrates that an inhibitory step between CYP51 and EBP is a unifying mechanism for many small enhancers of oligodendrogenesis identified by high-throughput screening. A) Percentage of MBP+ oligodendrocytes from the second induction of OPCs at 72 hours after treatment with a uniform dose of 5 μM ketoconazole, nine molecules identified by bioactivity screening, and nine randomly selected library members. n=4 replicates per condition, and >1,000 cells analyzed per replicate. [Figure 9-2]Figure 9 (A-H) illustrates that an inhibitory step between CYP51 and EBP is a unifying mechanism for many small enhancers of oligodendrogenesis identified by high-throughput screening. B) GC / MS-based quantification of zymosterol, zymostenol, and 14-dehydrozymostenol levels in a second batch of OPCs treated with 2 μM of the indicated screening hit compounds and randomly selected library members for 24 hours. n=2 replicates per condition. Molecules are sorted by the enzyme targeted (top labeled). C) Percentage of MBP+ oligodendrocytes generated from OPCs at 72 hours after treatment with the indicated doses of fulvestrant. n=4 replicates per condition, and >1,000 cells analyzed per replicate. [Figure 9-3] Figure 9 (A-H) illustrates that an inhibitory step between CYP51 and EBP is a unifying mechanism for many small enhancers of oligodendrogenesis identified by high-throughput screening. D) GC / MS-based quantification of lanosterol levels in OPCs treated with 2 μM fulvestrant for 24 h. n=2 replicates per condition. E) Percentage of MBP+ oligodendrocytes generated from OPCs at 72 h after treatment with the indicated previously reported oligodendrogenesis enhancers. n=4 replicates per condition, and >1,000 cells analyzed per replicate. [Figure 9-4] Figure 9 (A-H) illustrates that an inhibitory step between CYP51 and EBP is a unifying mechanism for many small enhancers of oligodendrogenesis identified by high-throughput screening. F) Percentage of MBP+ oligodendrocytes from secondary induction of OPCs at 72 hours after treatment with the indicated previously reported oligodendrogenesis enhancers. n=4 replicates per condition, and >1,000 cells analyzed per replicate. G) Representative images of OPCs treated for 72 hours with the indicated small molecules. All treatments in G are at the highest concentration shown in panel E. Scale bar, 100 μm. [Figure 9-5] Figure 9 (A-H) illustrates that an inhibitory step between CYP51 and EBP is a unifying mechanism for many small enhancers of oligodendrogenesis identified by high-throughput screening. H) GC / MS-based quantification of two metabolite levels in the second induction of OPCs treated for 24 h with the following doses of the indicated previously reported oligodendrogenesis enhancers: benztropine, 2 μM; clemastine, 1 μM; tamoxifen, 100 nM; U50488, 5 μM; bexarotene, 1 μM; and liothyronine, 3 μM. Left, zymostenol; right, cholesterol. [Figure 10-1]

[0025] Figure 10 (A-M) illustrates graphs and images showing the effects of muscarinic receptor antagonists and selective estrogen receptor modulators on cellular EBP function and oligodendrogenesis. A) Diagram of the structure of the muscarinic receptor antagonists used in this study. B) Diagram of the percentage of MBP+ oligodendrocytes generated from OPCs 72 hours after treatment with 2 μM ketoconazole or the indicated muscarinic receptor modulators, the concentration used during screening. n=4 replicates per condition, and >1,000 cells analyzed per replicate. [Figure 10-2] Figure 10 (A-M) illustrates graphs and images showing the effects of muscarinic receptor antagonists and selective estrogen receptor modulators on cellular EBP function and oligodendrogenesis. C) Percentage of MBP+ oligodendrocytes from a second independent batch of OPCs 72 hours after treatment with 2 μM ketoconazole, the concentration used during screening, or the indicated muscarinic receptor modulators. n=4 replicates per condition, and >1,000 cells analyzed per replicate. D) Heatmap showing inhibition of muscarinic receptor isoforms M1, M3, and M5 by the indicated small molecules (2 μM) assayed using gene BLAzerNFAT-blaCHO-K1 cells. n=2 replicates per condition. [Figure 10-3]Figure 10 (A-M) illustrates graphs and images showing the effects of muscarinic receptor antagonists and selective estrogen receptor modulators on cellular EBP function and oligodendrogenesis. E) GC / MS-based quantification of three metabolite levels in OPC-5 OPCs treated with U50488 (5 μM) or the indicated muscarinic receptor modulators (2 μM) for 24 hours. Left, zymostenol; center, cholesterol; right, desmosterol. F) GC / MS-based quantification of three metabolite levels in OPC-1 OPCs treated with clemastine (1 μM) or 2 μM of the indicated muscarinic receptor modulators for 24 hours. Left, zymostenol; center, zymosterol; right, cholesterol. [Figure 10-4] Figure 10 (A-M) illustrates graphs and images showing the effects of muscarinic receptor antagonists and selective estrogen receptor modulators on cellular EBP function and oligodendrogenesis. G) Diagram of the structures of the two selective estrogen receptor modulators used in this study. H) Diagram of the percentage of MBP+ oligodendrocytes generated from OPCs 72 hours after treatment with ospemifene and toremifene. n=4 replicates per condition, and >1,000 cells analyzed per replicate. [Figure 10-5] Figure 10 (A-M) illustrates graphs and images showing the effects of muscarinic receptor antagonists and selective estrogen receptor modulators on cellular EBP function and oligodendrogenesis. I) Percentage of MBP+ oligodendrocytes from secondary induction of OPCs 72 hours after treatment with ospemifene and toremifene. n=4 replicates per condition, and >1,000 cells analyzed per replicate. J) Representative images of OPCs treated with the indicated small molecules for 72 hours. All molecules were treated at 300 nM. Scale bar, 100 μm. [Figure 10-6]Figure 10 (A-M) illustrates graphs and images showing the effects of muscarinic receptor antagonists and selective estrogen receptor modulators on cellular EBP function and oligodendrogenesis. K) GC / MS-based quantification of two metabolite levels in OPCs treated with 300 nM ospemifene and toremifene for 24 hours. Left, zymostenol; right, cholesterol. L) GC / MS-based quantification of two metabolite levels in a second induction of OPCs treated with 300 nM ospemifene and toremifene for 24 hours. Left, zymostenol; right, cholesterol. [Figure 10-7]

[0025] Figure 10 (A-M) illustrates graphs and images showing the effects of muscarinic receptor antagonists and selective estrogen receptor modulators on cellular EBP function and oligodendrogenesis. M) Effect of ospemifene and toremifene on estrogen-dependent growth of T47D cells. n=3 replicates per condition. All graphs show mean + / - standard deviation. [Figure 11-1]

[0026] Figure 11 (A-D) provides graphs and images illustrating the effect of small molecule treatment combinations on oligodendrogenesis. A) Percentage of MBP+ oligodendrocytes generated from OPCs 72 hours after treatment with the indicated combinations of liothyronine and oligodendrogenesis enhancers. Unless noted, the following concentrations were used: ketoconazole, 2.5 μM; benztropine, 2 μM; clemastine, 2 μM; tamoxifen, 200 nM; liothyronine, 3 μM. n=4 replicates per treatment condition, and >1,000 cells analyzed per replicate. Lio=liothyronine. [Figure 11-2]Figure 11 (A-D) provides graphs and images illustrating the effect of small molecule treatment combinations on oligodendrogenesis. B) Percentage of MBP+ oligodendrocytes obtained from a second batch of OPCs 72 hours after treatment with the indicated combinations of liothyronine and oligodendrogenesis enhancers. n=4 replicates per treatment condition, and >1,000 cells analyzed per replicate. Lio = liothyronine. C) Percentage of MBP+ oligodendrocytes obtained from a second independent induction of OPCs 72 hours after treatment with the indicated combinations of ketoconazole and oligodendrogenesis enhancers. n=4 replicates per treatment condition, and >1,000 cells analyzed per replicate. Keto = ketoconazole. [Figure 11-3] Figure 11 (A-D) provides graphs and images illustrating the effect of small molecule treatment combinations on oligodendrocyte formation. D) Representative images of OPCs treated with the indicated small molecules for 72 hours. Small molecule concentrations remain the same as in panel a. Scale bar, 100 μm. [Figure 12-1]

[0027] Figure 12 (A-D) illustrates graphs and images showing the effect of sterol-modulating small molecules on the ability of oligodendrocytes to track along and wrap around electrospun microfibers. A) Fold increase in MBP+ oligodendrocytes after plating OPCs onto microfibers and treating with the indicated pathway-modulating drugs for 14 days. n=2. Scale bar, 500 μm. B) Low-magnification image representing the majority of the microfiber area for the treatment in panel a. Green, MBP; blue, DAPI. [Figure 12-2] Figure 12 (A-D) illustrates graphs and images showing the effect of sterol-modulating small molecules on the ability of oligodendrocytes to track along and wrap around electrospun microfibers. C) High-resolution image of MBP+ oligodendrocytes tracking along a microfiber. Green, MBP; blue, DAPI. Ketoconazole, 2.5 μM. Scale bar, 50 μm. D) Confocal images of OPCs seeded on aligned microfibers and treated with ketoconazole (2.5 μM) for 14 days. [Figure 13-1]

[0028] Figure 13 (A-B) illustrates graphs showing the effect of oligodendrocyte-enhancing small molecules on sterol levels in human cells and human cortical spheroids. A) GC / MS-based quantification of three metabolite levels in human glioma cells (GBM528) treated for 24 hours with the following concentrations of the indicated small molecules: tamoxifen 100 nM, clemastine 2 μM, ifenprodil 2 μM, ketoconazole 2.5 μM, and amorolfine 100 nM. Left, lanosterol; center, zymostenol; right, 14-dehydrozymostenol. n=2 replicates per condition. [Figure 13-2] 13(A-B) illustrate graphs showing the effect of oligodendrocyte-enhancing small molecules on sterol levels in human cells and human cortical spheroids. B) GC / MS-based quantification of three metabolite levels in two independent batches of human cortical spheroids treated with 2 μM of the indicated small molecules for 24 hours. Left, lanosterol; center, zymostenol; right, zymosterol. n=3 replicates per condition. [Figure 14]

[0029] Illustrated are structures of small molecules shown to enhance oligodendrocyte formation and regulate sterol levels in OPCs. Molecules are categorized by the enzyme inhibited: CYP51, top; sterol 14-reductase, middle; EBP, bottom. [Figure 15]

[0030] We demonstrate that hit compounds from bioactivity screening, validated by GCMS assay, inhibit at least three steps of cholesterol synthesis and lead to OPC differentiation. [Figure 16]

[0031] 1 illustrates graphs showing the effect of the benzene derivative cis-N-cyclohexyl-N-ethyl-3-(3-chloro-4-cyclohexylphenyl)prop-2-enylamine on oligodendrocyte formation and EBP inhibition. DETAILED DESCRIPTION OF THE INVENTION

[0017]

[0032] For convenience, certain terms employed in the specification, examples, and appended claims are collected here. Unless otherwise defined, 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.

[0018]

[0033] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., 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.

[0019]

[0034] The terms "comprise," "comprising," "include," "including," "have," and "having" are used in an inclusive, open sense, meaning that additional elements may be included. As used herein, the terms "such as," "eg," and "e.g." are non-limiting and for illustrative purposes only. "Including" and "including but not limited to" are used interchangeably.

[0020]

[0035] As used herein, the term "or" should be understood to mean "and / or" unless the context clearly indicates otherwise.

[0036] As used herein, the term "about" or "approximately" refers to an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that differs by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% relative to the referenced amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term "about" or "approximately" refers to a range of ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length referenced.

[0021]

[0037] It should be noted that the structures of some of the compounds of this application contain asymmetric (chiral) carbon or sulfur atoms. Accordingly, isomers arising from such asymmetry are to be understood as included herein unless otherwise indicated. Such isomers can be prepared by classical separation. It can be obtained in substantially pure form by techniques and by stereochemically controlled synthesis. The compounds of the present application may exist in stereoisomeric forms and can therefore be produced as individual stereoisomers or as mixtures.

[0022]

[0038] The term "isomerism" refers to compounds that have identical molecular formulae but differ in the nature or sequence of bonding of their atoms or in the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are called "stereoisomers." Stereoisomers that are not mirror images of one another are called "diastereoisomers," and stereoisomers that are non-superimposable mirror images are called "enantiomers" or sometimes optical isomers. A carbon atom bonded to four non-identical substituents is called a "chiral center," while a sulfur bonded to three or four different substituents, such as a sulfoxide or sulfinimide, is also called a "chiral center."

[0023]

[0039] The term "chiral isomer" refers to a compound having at least one chiral center. It has two enantiomeric forms of opposite chirality and may exist either as individual enantiomers or as a mixture of enantiomers. A mixture containing equal amounts of individual enantiomeric forms of opposite chirality is called a "racemic mixture." A compound with more than one chiral center has 2n-1 enantiomeric pairs, where n is the number of chiral centers. A compound with more than one chiral center may exist either as individual diastereomers or as a mixture of diastereomers, called a "diastereomeric mixture." When one chiral center is present, a stereoisomer may be characterized by the absolute configuration (R or S) of that chiral center. Alternatively, when one or more chiral centers are present, a stereoisomer may be characterized as (+) or (-). Absolute configuration refers to the arrangement in space of the substituents attached to the chiral center. The substituents attached to the chiral center under consideration are ranked according to the sequencing rules of Cahn, Ingold and Prelog (Cahn et al., Angew. Chem. Inter. Edit. 1966, 5, 385; Correction, p. 511; Cahn et al., Angew. Chem. 1966, 78, 413; Cahn and Ingold, J. Chem. Soc. 1951 (London), 612; Cahn et al., Experientia 1956, 12, 81; Cahn, J., Chem. Educ. 1964, 41, 116).

[0024]

[0040] The term "geometric isomer" refers to diastereomers whose existence results from hindered rotation about a double bond. These configurations are distinguished in their names by the prefixes cis and trans, or Z and E, which indicate that the groups are on the same or opposite sides of the double bond of the molecule, according to the Cahn-Ingold-Prelog rules. Additionally, structures and other compounds discussed in this application include all atropisomers thereof.

[0025]

[0041] The term "atropisomer" refers to a type of stereoisomer in which the atoms of two isomers are arranged differently in space. Atropisomers owe their existence to restricted rotation, caused by the hindrance of rotation of large groups around a central bond. Such atropisomers typically exist as mixtures, but recent advances in chromatographic techniques have made it possible to selectively separate two atropisomer mixtures.

[0026]

[0042] The term "crystalline polymorph" or "polymorph" or "crystalline form" refers to a crystalline structure in which a compound (or a salt or solvate thereof) can crystallize into different crystal packing arrangements, all of which have the same elemental composition. Different crystalline forms typically have different X-ray diffraction patterns, infrared spectra, melting points, density, hardness, crystal shape, optical and electrical properties, stability, and Solubility. Recrystallization solvent, crystallization rate, storage temperature, and other factors may cause one crystalline form to dominate. Polymorphs of a compound can be prepared by crystallization under different conditions.

[0027]

[0043] The term "derivatives" refers to compounds that have a common core structure and are substituted with various groups as described herein.

[0044] The term "bioisostere" refers to a compound resulting from the exchange of an atom or group of atoms with another broadly similar atom or group of atoms. The purpose of the bioisosteric substitution is to generate a novel compound with biological properties similar to those of the parent compound. The bioisosteric substitution may be based on physicochemical or topology. Examples of carboxylic acid bioisosteres include acylsulfonimides, tetrazoles, sulfonates, and phosphonates. See, e.g., Patani and LaVoie, Chem. Rev. 96, pp. 3147-3176 (1996).

[0028]

[0045] The phrases "parenteral administration" and "administering parenterally" are art-recognized terms and include enteral administration and topical administration, such as modes of administration other than injection, including, but not limited to, intravenous, intramuscular, intrapleural, intravascular, intrapericardial, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intrathecal, and intrastemal injection and infusion.

[0029]

[0046] The term "treating" is art-recognized and includes inhibiting a disease, disorder, or condition in a subject, e.g., preventing its progression; and alleviating a disease, disorder, or condition, e.g., causing regression of a disease, disorder, and / or condition. Treating a disease or condition includes improving at least one symptom of a particular disease or condition, even if the underlying pathophysiology is unaffected.

[0030]

[0047] The term "prevent" is art-recognized and includes stopping a disease, disorder, or condition from occurring in a subject who may be predisposed to the disease, disorder, and / or condition but has not yet been diagnosed as exhibiting it. Preventing a condition associated with a disease includes stopping the condition from occurring after the disease has been diagnosed, but before the condition has been diagnosed.

[0031]

[0048] 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 present in bulk or in a unit dosage form. The unit dosage form may be in any of a variety of forms, for example, a capsule, an IV bag, a tablet, a single pump aerosol inhaler, or the like. The composition may contain a syringe, ...

[0032]

[0049] The term "flash dose" refers to a compound formulation that disperses rapidly.

[0050] The term "immediate release" is defined as the release of a compound from a dosage form in a relatively short period of time, generally up to about 60 minutes. The term "modified release" is defined to include delayed release, sustained release, and pulsed release. The term "pulse release" is defined as a series of releases of a drug from a dosage form. The term "extended release" or "sustained release" is defined as a continuous release of a compound from a dosage form over an extended period of time.

[0033]

[0051] The phrase "pharmaceutically acceptable" is art-recognized. In certain embodiments, the term includes compositions, polymers, and other materials and / or dosage forms that 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.

[0034]

[0052] The phrase "pharmaceutically acceptable carrier" is art-recognized and includes, for example, a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, that is involved in delivering or transporting any subject composition from one organ or body part to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the subject composition and not harmful to the patient. In certain embodiments, a pharmaceutically acceptable carrier is non-pyrogenic. Some examples of materials that can 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 carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository wax; (9) peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and (11) polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers 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; and (21) other non-toxic compatible substances used in pharmaceutical formulations.

[0035]

[0053] The compounds of the present application can further form salts, and all of these forms are also contemplated herein.

[0054] A "pharmaceutically acceptable salt" of a compound refers to a salt that is pharmaceutically acceptable and 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. Pharmaceutically acceptable salts can be synthesized from a parent compound containing a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or free base form of these compounds with a stoichiometric amount of an appropriate base or acid in water or an organic solvent, or a mixture of the two; generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. A list of salts can be found in Remington's Pharmaceutical Sciences, 18th Edition (Mack Publishing Company, 1990).

[0036]

[0055] The compounds described herein can also be prepared as esters, for example, pharmaceutically acceptable esters. For example, a carboxylic acid functional group in a compound can be converted to its corresponding ester, for example, a methyl, ethyl, or other ester. Also, an alcohol group in a compound can be converted to its corresponding ester, for example, an acetate, propionate, or other ester.

[0037]

[0056] The compounds described herein can also be prepared as prodrugs, e.g., pharmaceutically acceptable prodrugs. The terms "pro-drug" and "prodrug" are used interchangeably herein to refer to any compound that releases an active parent drug in vivo. Because prodrugs are known to enhance many desirable qualities of pharmaceuticals (e.g., solubility, bioavailability, manufacturing, etc.), compounds can be delivered in prodrug form. Thus, the compounds described herein are intended to include prodrugs of the compounds claimed herein, methods of delivering them, and compositions containing them. "Prodrug" is intended to include any covalently bonded carrier that releases the active parent drug in vivo when such prodrug is administered to a subject. Prodrugs are prepared by modifying functional groups present in the compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to yield the parent compound. Prodrugs include compounds in which a hydroxyl, amino, sulfhydryl, carboxy, or carbonyl group is bonded to any group that can be cleaved in vivo to form a free hydroxyl, free amino, free sulfhydryl, free carboxy, or free carbonyl group, respectively.

[0038]

[0057] Examples of prodrugs include, but are not limited to, esters (e.g., acetate, dialkylaminoacetate, formate, phosphate, sulfate, and benzoate derivatives) and carbamates (e.g., N,N-dimethylaminocarbonyl) of hydroxy functional groups in the compounds of Formula I; ester groups (e.g., ethyl ester, morpholinoethanol ester) of carboxyl functional groups; N-acyl derivatives (e.g., N-acetyl) of amino functional groups; N-Mannich bases, Schiff bases, and enaminones; and oxime, acetal, ketal, and enol esters of ketone and aldehyde functional groups. See Bundegaard, H., "Design of Prodrugs," pp. 1-92, Elesevier, New York-Oxford (1985).

[0039]

[0058] The term "protecting group" refers to a group of atoms that, when attached to a reactive group of a molecule, masks, reduces or prevents the reactivity. Examples of protecting groups can be found in Green and Wuts, Protective Groups in Organic Chemistry, (Wiley, 2nd Edition 1991); Harrison and Harrison et al., Compendium of Synthetic Organic Methods, Volumes 1-8 (John Wiley and Sons, 1971-1996); and Kocienski, Protecting Groups, (Verlag, 3rd Edition 2003).

[0040]

[0059] The term "amine protecting group" is intended to mean a functional group that converts an amine, amide, or other nitrogen-containing moiety into a different chemical group that is substantially inert to the conditions of a particular chemical reaction. Amine protecting groups are easily and selectively removed, preferably in good yield and under conditions that do not affect other functional groups of the molecule. Examples of amine protecting groups include, but are not limited to, formyl, acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, t-butyloxycarbonyl (Boc), p-methoxybenzyl, methoxymethyl, tosyl, trifluoroacetyl, trimethylsilyl (TMS), fluorenylmethyloxycarbonyl, 2-trimethylsilylethyoxycarbonyl, 1-methyl-1-(4-biphenylyl)ethoxycarbonyl, allyloxycarbonyl, benzyloxycarbonyl (CBZ), 2-trimethylsilylethanesulfonyl (SES), trityl and substituted trityl groups, 9-fluorenylmethyloxycarbonyl (FMOC), nitroveratryloxycarbonyl (NVOC), etc. Other suitable amine protecting groups can be identified by one of ordinary skill in the art.

[0041]

[0060] Representative hydroxy protecting groups include those in which the hydroxy group is either acylated or alkylated, such as benzyl, and trityl ethers, as well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers and allyl ethers.

[0042]

[0061] Furthermore, the salts of the compounds described herein can exist in either hydrated or non-hydrated (anhydrous) form, or as solvates with other solvent molecules.Non-limiting examples of hydrates include monohydrates, dihydrates, etc.Non-limiting examples of solvates include ethanol solvates, acetone solvates, etc.

[0043]

[0062] The term "solvate" refers to a solvent addition form containing either a stoichiometric or non-stoichiometric amount of solvent. Some compounds have a tendency to trap a certain molar ratio of solvent molecules in the crystalline solid state, thus forming a solvate. When the solvent is water, the solvate formed is a hydrate, and when the solvent is alcohol, the solvate formed is an alcoholate. A hydrate is formed by the combination of one or more water molecules with one of the substances, in which water retains its molecular state as HO, and such a combination can form one or more hydrates.

[0044]

[0063] The compounds, salts, and prodrugs described herein can exist in several tautomeric forms, including enol and imine forms, keto and enamine forms, and geometric isomers, as well as mixtures thereof. Tautomers exist as a mixture of tautomeric sets in solution. In solid form, one tautomer usually predominates. Although one tautomer may be described, the present application includes all tautomers of the compounds of the present invention. A tautomer is one of two or more structural isomers that exist in equilibrium and are readily converted from one isomeric form to another. This reaction involves the formal transfer of a hydrogen atom and the simultaneous rearrangement of adjacent conjugated double bonds. In solutions where tautomerization is possible, a chemical equilibrium of the tautomers must be achieved. The exact ratio of tautomers depends on several factors, including temperature, solvent, and pH. The concept of tautomers that are interchangeable through tautomerization is called tautomerism.

[0045]

[0064] Of the various types of tautomerism possible, two are commonly observed: keto-enol tautomerism, in which a simultaneous shift of an electron and a hydrogen atom occurs.

[0065] Tautomerization can be catalyzed by bases: 1. deprotonation; 2. formation of a delocalized anion (e.g., enolate); 3. protonation at a different position on the anion; and acids: 1. protonation; 2. formation of a delocalized cation; 3. deprotonation at a different position adjacent to the cation.

[0046]

[0066] The term "analog" refers to a chemical compound that is structurally similar to another chemical compound but differs slightly in composition (e.g., one atom is replaced by an atom of a different element, or in the presence of a particular functional group, or one functional group is replaced by another). Thus, an analog is a compound that is similar or equivalent to a reference compound in function and appearance, but not in structure or origin.

[0047]

[0067] As used herein, the term "oligodendrocyte precursor cell" or "OPC" refers to a neural progenitor cell that can generate new oligodendrocytes. Oligodendrocyte precursor cells can be identified by the expression of a number of surface antigens. For example, surface antigens known as platelet-derived growth factor alpha receptor subunit (PDGFRα), NG2 chondroitin sulfate proteoglycan, and ganglioside GD3 are commonly used to identify oligodendrocyte precursor cells.

[0048]

[0068] Immature oligodendrocyte precursors are generated from a common glial progenitor in the ventral region of the developing brain. These immature cells actively migrate, proliferate, and assemble in the CNS, where they terminally differentiate into premyelinating oligodendrocytes (O4+). Differentiation and maturation of oligodendrocyte precursors is characterized by the extension of multiple processes, an increase in cell body size, and the formation of myelin.

[0049]

[0069] "Patient," "subject," or "host" to be treated by the subject method can refer to any of humans or non-human animals, such as mammals, fish, birds, reptiles, or amphibians.Thus, the subject of the method disclosed herein can be humans, non-human primates, horses, pigs, rabbits, dogs, sheep, goats, cows, cats, guinea pigs, or rodents.The term does not indicate a specific age or sex.Thus, it is intended to include both male and female adult and newborn subjects and fetuses.In one embodiment, the subject is a mammal.Patient refers to a subject suffering from a disease or disorder.

[0050]

[0070] The terms "prophylactic" or "therapeutic" treatment are art-recognized and include administration of one or more of the subject compositions to a host. When administered prior to clinical sign of an undesirable condition (e.g., a disease or other undesirable condition in the host animal, such as, but not limited to, deranged myelination, myelin deficiency, myelin loss, and ineffective myelin repair), the treatment is prophylactic, i.e., protects the host against the onset of the undesirable condition, whereas when administered after sign of the undesirable condition, the treatment is therapeutic (i.e., intended to reduce, ameliorate, or stabilize an existing undesirable condition or side effect).

[0051]

[0071] The terms "therapeutic agent," "drug," "medication," and "bioactive substance" are art-recognized and include molecules and other agents that are biologically, physiologically, or pharmacologically active substances that act locally or systemically in a patient or subject to treat a disease or condition. The terms include, but are not limited to, pharmaceutically acceptable salts and prodrugs thereof. Such agents may be acidic, basic, or salts; they may be neutral molecules, polar molecules, or molecular complexes capable of hydrogen bonding; and they may be prodrugs in the form of ethers, esters, amides, etc., that become biologically activated upon administration to a patient or subject.

[0052]

[0072] The phrases "therapeutically effective amount" or "pharmaceutically effective amount" are art-recognized terms. 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 an amount necessary or sufficient to eliminate, reduce, or maintain the target of a particular therapeutic regimen. An effective amount may vary depending on factors such as the disease or condition being treated, the particular targeting construct 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. In certain embodiments, a therapeutically effective amount of a therapeutic agent for in vivo use will likely depend on numerous factors, including the release rate of the agent from the polymer matrix, which will depend in part on the chemical and physical properties of the polymer; the agent itself; the mode and method of administration; and any other materials incorporated into the polymer matrix in addition to the agent.

[0053]

[0073] The term "ED50" is art-recognized. In certain embodiments, ED50 means the dose of a drug which produces 50% of its maximum response or effect, or alternatively, the dose which produces a predetermined response in 50% of test subjects or formulations. The term "LD50" is art-recognized. In certain embodiments, LD50 refers to the dose of a drug which is lethal in 50% of test subjects. The term "therapeutic index" is art-recognized and refers to the therapeutic index of a drug, defined as LD50 / ED50.

[0054]

[0074] "I C 50 " or "50% maximal inhibitory concentration" is intended to refer to the concentration of a substance (e.g., a compound or drug) required to inhibit a biological process or a component of that process by 50%, including proteins, subunits, organelles, ribonucleoproteins, etc.

[0055]

[0075] With respect to any chemical compound, the present application is intended to include all isotopes of atoms occurring in the compounds of the present invention. Isotopes are 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.

[0056]

[0076] When a bond to a substituent is shown to cross a bond connecting two atoms in a ring, such substituent may be bonded to any atom in the ring. When substituents are listed without indicating the atom through which such substituent is bonded to the remainder of the compound of a given formula, such substituent may be bonded through any atom in such substituent. Combinations of substituents and / or variables are permissible, but only if such combinations result in stable compounds.

[0057]

[0077] Atoms or chemical moieties followed by a subscripted numerical range (e.g., C 1~6 ) means that the range includes each number in that range as well as all intermediate ranges. For example, "C 1~6 "Alkyl" is meant to include alkyl groups having 1, 2, 3, 4, 5, 6, 1-6, 1-5, 1-4, 1-3, 1-2, 2-6, 2-5, 2-4, 2-3, 3-6, 3-5, 3-4, 4-6, 4-5, and 5-6 carbons.

[0058]

[0078] The term "alkyl" is intended to include both branched (e.g., isopropyl, tert-butyl, isobutyl) and straight-chain (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl) groups, as well as cycloalkyl (e.g., alicyclic) groups (e.g., cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. Such aliphatic hydrocarbon groups have the specified number of carbon atoms. For example, C 1~6Alkyl is intended to include C1, C2, C3, C4, C5, and C6 alkyl groups. As used herein, "lower alkyl" refers to an alkyl group having from 1 to 6 carbon atoms in its carbon chain backbone. "Alkyl" further includes alkyl groups which have oxygen, nitrogen, sulfur or phosphorous atoms replacing one or more hydrocarbon backbone carbon atoms. In certain embodiments, a straight chain or branched chain alkyl has 6 or fewer, e.g., 4 or fewer, carbon atoms in its backbone (e.g., C1-C6 for straight chain, C3-C6 for branched chain). Likewise, certain cycloalkyls have 3-8 carbon atoms in their ring structure, such as 5 or 6 carbons in the ring structure.

[0059]

[0079] The term "alkenyl" refers to any straight or branched chain alkyl group containing at least one double bond and having from 2 to about 24 carbon atoms, such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl, eicosenyl, tetracosenyl, cyclopentenyl, cyclohexenyl, cyclooctenyl, and the like. or cyclic hydrocarbon groups. Overall, although again not required, alkenyl groups can contain from 2 to about 18 carbon atoms, more particularly from 2 to 12 carbon atoms. The term "lower alkenyl" refers to an alkenyl group of 2 to 6 carbon atoms, and the specific term "cycloalkenyl" contemplates a cyclic alkenyl group, preferably having from 5 to 8 carbon atoms. The term "substituted alkenyl" refers to an alkenyl substituted with one or more substituents, and the terms "heteroatom-containing alkenyl" and "heteroalkenyl" refer to an alkenyl or heterocycloalkenyl (e.g., heterocyclohexenyl) in which at least one carbon atom has been replaced with a heteroatom. Unless otherwise specified, the terms "alkenyl" and "lower alkenyl" include linear, branched, cyclic, unsubstituted, substituted, and / or heteroatom-containing alkenyl and lower alkenyl, respectively.

[0060]

[0080] The term "alkynyl" refers to a straight-chain or branched hydrocarbon group of 2 to 24 carbon atoms containing at least one triple bond, such as ethynyl, n-propynyl, etc. Overall, although again not required, an alkynyl group can contain from 2 to about 18 carbon atoms, more specifically from 2 to 12 carbon atoms. The term "lower alkynyl" contemplates an alkynyl group of 2 to 6 carbon atoms. The term "substituted alkynyl" refers to an alkynyl substituted with one or more substituents, and the terms "heteroatom-containing alkynyl" and "heteroalkynyl" refer to an alkynyl in which at least one carbon atom has been replaced with a heteroatom. Unless otherwise specified, the terms "alkynyl" and "lower alkynyl" include straight-chain, branched, unsubstituted, substituted, and / or heteroatom-containing alkynyl and lower alkynyl, respectively.

[0061]

[0081] The terms "alkyl," "alkenyl," and "alkynyl" are intended to include moieties that are diradicals, i.e., have two points of attachment. A non-limiting example of such an alkyl moiety, i.e., a diradical, is --CHCH--, i.e., a C alkyl group covalently bonded to the rest of the molecule through each terminal carbon atom.

[0062]

[0082] The term "alkoxy" refers to an alkyl group attached through a single, terminal ether linkage; i.e., an "alkoxy" group may be represented as --O-alkyl, where alkyl is as defined above. A "lower alkoxy" group intends an alkoxy group containing from 1 to 6 carbon atoms and includes, for example, methoxy, ethoxy, n-propoxy, isopropoxy, t-butyloxy, and the like. Preferred substituents identified herein as "C1-C6 alkoxy" or "lower alkoxy" contain 1 to 3 carbon atoms, and particularly preferred such substituents contain 1 or 2 carbon atoms (i.e., methoxy and ethoxy).

[0063]

[0083] The term "aryl" refers to an aromatic substituent containing a single aromatic ring or multiple aromatic rings fused together, directly bonded, or indirectly bonded (such that different aromatic rings are bonded to a common group, such as a methylene or ethylene moiety). Aryl groups can contain 5 to 20 carbon atoms, with particularly preferred aryl groups containing 5 to 14 carbon atoms. Examples of aryl groups include benzene, phenyl, pyrrole, furan, thiophene, thiazole, isothiazole, imidazole, triazole, tetrazole, pyrazole, oxazole, isoxazole, pyridine, pyrazine, pyridazine, and pyrimidine. Furthermore, the term "aryl" includes polycyclic aryl groups, e.g., tricyclic, bicyclic, e.g., naphthalene, benzoxazole, benzodioxazole, benzothiazole, benzimidazole, benzothiophene, methylenedioxyphenyl, quinoline, isoquinoline, naphthridine, indole, benzofuran, purine, benzofuran, deazapurine, or indolizine. Those aryl groups having heteroatoms in the ring structure are also referred to as "aryl heterocycles," "aryl heterocycles," The aromatic ring may also be referred to as a "heterocycle," "heteroaryl," or "heteroaromatic." The aromatic ring may contain, at one or more ring positions, for example, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminocarbonyl, aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate, phosphonato, phosphinato, cyano, amino(aralkyl), hydroxyl, ... The aryl group can be substituted with substituents such as those described above, such as alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or aromatic or heteroaromatic moieties. Aryl groups can also be fused or bridged with alicyclic or heterocyclic rings that are not aromatic, thereby forming polycyclic systems (e.g., tetralin, methylenedioxyphenyl). Unless otherwise indicated, the term "aryl" includes unsubstituted, substituted, and / or heteroatom-containing aromatic substituents.

[0064]

[0084] The term "alkaryl" refers to an aryl group having an alkyl substituent, and the term "aralkyl" refers to an alkyl group having an aryl substituent, where "aryl" and "alkyl" are defined above. Exemplary aralkyl groups contain 6 to 24 carbon atoms, with particularly preferred aralkyl groups containing 6 to 16 carbon atoms. Examples of aralkyl groups include, but are not limited to, benzyl, 2-phenyl-ethyl, 3-phenyl-propyl, 4-phenyl-butyl, 5-phenyl-pentyl, 4-phenylcyclohexyl, 4-benzylcyclohexyl, 4-phenylcyclohexylmethyl, 4-benzylcyclohexylmethyl, and the like. Examples of alkaryl groups include, for example, p-methylphenyl, 2,4-dimethylphenyl, p-cyclohexylphenyl, 2,7-dimethylnaphthyl, 7-cyclooctylnaphthyl, 3-ethyl-cyclopenta-1,4-diene, and the like.

[0065]

[0085] The terms "heterocyclyl" or "heterocyclic group" include closed ring structures, e.g., 3- to 10- or 4- to 7-membered ring structures, that contain one or more heteroatoms. A "heteroatom" includes an atom of any element other than carbon or hydrogen. Examples of heteroatoms include nitrogen, oxygen, sulfur, and phosphorus.

[0066]

[0086] Heterocyclyl groups can be saturated or unsaturated and include pyrrolidine, oxolane, thiolane, piperidine, piperazine, morpholine, lactones, lactams such as azetidinone and pyrrolidinone, sultams, and sultones. Heterocyclic groups such as pyrrole and furan can have aromatic properties. These include fused ring structures such as quinoline and isoquinoline. Other examples of heterocyclic groups include pyridine and purine. Heterocycles can be substituted at one or more positions with substituents such as those described above, such as halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, or an aromatic or heteroaromatic moiety. Heterocyclic groups can also be substituted at one or more constituent atoms with, for example, lower alkyl, lower alkenyl, lower alkoxy, lower alkylthio, lower alkylamino, lower alkylcarboxyl, nitro, hydroxyl, --CF3, or --CN.

[0067]

[0087] The term "halo" or "halogen" refers to fluoro, chloro, bromo, and iodo. "Counterion" is used to represent small, negatively charged species such as fluoride, chloride, bromide, iodide, hydroxide, acetate, and sulfate.

[0068]

[0088] The term "substituted," as in "substituted alkyl," "substituted aryl," etc., means that at least one hydrogen atom bonded to a carbon (or other) atom in an alkyl, aryl, or other moiety, as implied in some of the above definitions, is replaced with one or more non-hydrogen substituents. Examples of such substituents include, but are not limited to: halo, hydroxyl, silyl, sulfhydryl, C1-C6 24 Alkoxy, C2-C 24 Alkenyloxy, C2-C 24 Alkynyloxy, C5-C 20 Aryloxy, acyl (C2-C 24 Alkylcarbonyl (-CO-alkyl) and C6-C 20 Arylcarbonyl (including -CO-aryl), acyloxy (-O-acyl), C2-C 24 Alkoxycarbonyl (-(CO)-O-alkyl), C6-C 20 Aryloxycarbonyl (-(CO)-O-aryl), C2-C 24 Alkylcarbonate (-O-(CO)-O-alkyl), C6-C 20 Arylcarbonato (-O-(CO)-O-aryl), carboxy (-COOH), carboxylato (-COO-), carbamoyl (-(CO)-NH), mono-(C1-C 24 Alkyl) substituted carbamoyl (-(CO)-NH(C1-C 24 di-(C1-C4 alkyl)-substituted carbamoyl (-(CO)--N(C1-C 24 alkyl)2), monosubstituted arylcarbamoyl (-(CO)-NH-aryl), thiocarbamoyl (-(CS)-NH2), carbamido (-NH-(CO)-NH2), cyano (-CN), isocyano (-N + C - ), cyanate (-O--CN), isocyanate (-ON + C - ), isothiocyanato (-S-CN), azido (-N=N + =N -), formyl (-(CO)--H), thioformyl (-(CS)-H), amino (-NH), mono- and di-(C1-C 24 Alkyl) substituted amino, mono- and di-(C5-C 20 Aryl)substituted amino, C2-C 24 Alkylamide (-NH-(CO)-alkyl), C6-C 20 Arylamide (—NH—(CO)-aryl), imino (—CR═NH, where R=hydrogen, C1-C 24 Alkyl, C5-C 20 Aryl, C6-C 24 Alkaryl, C6-C 24 aralkyl, etc.), alkylimino (-CR=N(alkyl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), arylimino (-CR=N(aryl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (-NO2), nitroso (-NO), sulfo (-SO2-OH), sulfonato (-SO2-O - ), C1~C 24 Alkyl sulfanyl (-S-alkyl; also called "alkylthio"), aryl sulfanyl (-S-aryl; also called "arylthio"), C1-C 24 Alkylsulfinyl (--(SO)-alkyl), C5-C 20 Arylsulfinyl (-(SO)-aryl), C1-C 24 Alkylsulfonyl (-SO2-alkyl), C5-C 20 Arylsulfonyl (-SO2-aryl), phosphono (-P(O)(OH)2), phosphonato (-P(O)(O - )2), phosphinato (-P(O)(O - )), phospho (-PO2), and phosphino (-PH2); and hydrocarbyl moieties C1-C 24 Alkyl, C2-C 24 Alkenyl, C2-C 24 Alkynyl, C5-C 20 Aryl, C6-C 24 Alkaryl, and C6-C 24 There are functional groups such as aralkyl.

[0069]

[0089] Additionally, the aforementioned functional groups may be optionally combined with one or more additional functional groups or with one or more hydrocarbyl moieties such as those specifically listed above, where particular groups are permitted. Similarly, the hydrocarbyl moieties described above may be further substituted with one or more functional groups or additional hydrocarbyl moieties such as those specifically listed.

[0070]

[0090] When the term "substituted" appears before a list of possible substituents, it is intended that the term apply to all members of that group. For example, the phrase "substituted alkyls, alkenyls, and aryls" should be read as "substituted alkyls, substituted alkenyls, and substituted aryls." Similarly, when the term "heteroatom-containing" appears before a list of possible heteroatom-containing groups, it is intended that the term apply to all members of that group. For example, the phrase "heteroatom-containing alkyls, alkenyls, and aryls" should be read as "heteroatom-containing alkyls, substituted alkenyls, and substituted aryls."

[0071]

[0091] "Any" or "optionally" means that the subsequently stated condition may or may not occur, and thus the description includes cases where the condition occurs and cases where it does not occur. For example, the phrase "optionally substituted" means that a non-hydrogen substituent may or may not be present at a given atom, and thus the description includes structures where the non-hydrogen substituent is present and structures where the non-hydrogen substituent is not present.

[0072]

[0092] The terms "stable compound" and "stable structure" are meant to indicate a compound that is sufficiently robust to survive isolation, and if necessary purification from a reaction mixture, and formulation into an efficacious therapeutic agent.

[0073]

[0093] The term "free compound" is used herein to describe a compound in an unbound state.

[0094] Throughout the description, when a composition is described as having, including, or comprising certain components, it is contemplated that the composition also consists essentially of, or consists of, the recited components. Similarly, when a method or process is described as having, including, or comprising certain method steps, the process also consists essentially of, or consists of, the recited processing steps. Furthermore, 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 remain operable. Moreover, two or more steps or actions can be conducted simultaneously.

[0074]

[0095] A "zinc finger DNA-binding protein" (or binding domain) is a protein, or a domain within a larger protein, that binds to DNA in a sequence-specific manner through one or more zinc fingers, which are regions of amino acid sequence within the binding domain whose structure is stabilized through the coordination of a zinc ion. The term zinc finger DNA-binding protein is often abbreviated as zinc finger protein or ZFP.

[0075]

[0096] A "TALE DNA binding domain" or "TALE" is a polypeptide containing one or more TALE repeat domains / units. The repeat domains are responsible for binding of the TALE to its cognate target DNA sequence. A single "repeat unit" (also referred to as a "repeat") is typically 33-35 amino acids in length and exhibits at least some sequence homology with other TALE repeat sequences within naturally occurring TALE proteins.

[0076]

[0097] Zinc finger and TALE binding domains can be "genetically engineered," e.g., by substituting the gene for the recognition helix region of a naturally occurring zinc finger or TALE protein. They can bind to a predetermined nucleotide sequence through manipulation (changing one or more amino acids).Therefore, engineered DNA-binding proteins (zinc fingers or TALEs) are non-naturally occurring proteins.A non-limiting example of a method for engineering DNA-binding proteins is design and selection.Designed DNA-binding proteins are proteins that do not exist in nature, and their design / composition mainly comes from rational criteria.Rational criteria for design include the application of substitution rules and computerized algorithms to process information in databases that store information on existing ZFP and / or TALE design and binding data. See, e.g., U.S. Patent Nos. 6,140,081; 6,453,242; 6,534,261; and 8,585,526; also WO 98 / 53058; WO 98 / 53059; WO 98 / 53060; WO 02 / 016536; and WO 03 / 016496.

[0077]

[0098] A "selected" zinc finger protein or TALE is a protein not found in nature, whose production primarily results from experimental methods such as phage display, interaction trap, or hybrid selection. See, for example, U.S. Patent Nos. 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,200,759; 8,586,526; WO 95 / 19431; WO 96 / 06166; WO 98 / 53057; WO 98 / 54311; WO 00 / 27878; WO 01 / 60970; WO 01 / 88197; WO 02 / 099084.

[0078]

[0099] Generally, "CRISPR" (Clustered Regularly Interspaced Short Palindromic Repeats) Short Palindromic Repeats (SSRs), also known as SPIDRs (Spacer Interspersed Direct Repeats), refer to a family of DNA loci that are usually specific to particular bacterial species. CRISPR loci comprise a distinct class of interspersed short sequence repeats (SSRs) recognized in Escherichia coli (E. coli) (Ishino et al. (1987) J. Bacteriol., 169:5429-5433; and Nakata et al., J. Bacteriol. (1989) 171:3553-3556) and related genes. Similar interspersed SSRs have been identified in Haloferax mediterranei, Streptococcus pyogenes, Anabaena, and Mycobacterium tuberculosis (see Groenen et al. (1993) Mol. Microbiol. 10:1057-1065; Hoe et al. (1999) Emerg. Infect. Dis. 5:254-263; Masepohl et al. (1996) Biochim. Biophys. Acta 1307:26-30; and Mojica et al. (1995) Mol. Microbiol. 17:85-93). CRISPR loci typically differ from other SSRs in their repeat structure, which is referred to as short regularly interspaced repeats (SRSRs) (Janssen et al. (2002) OMICS J. Integ. Biol., 6:23-33; and Mojica et al. (2000) Mol. Microbiol., 36:244-246). Generally, repeats are short elements that occur in regularly spaced clusters with unique intervening sequences of substantially constant length (Mojica et al. (2000), supra). Repeat sequences are highly conserved between strains, but the number of interspersed repeats and the sequence of the spacer region typically vary between strains (van Embden et al., J. Bacteriol. (2002) 182:2393-2401). CRISPR loci have been identified in over 40 prokaryotes, including, but not limited to:Aeropyrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Halocarcula, Methanobacterium, Methanococcus, Methanosarcina, Methanopyrus, Pyrococcus, Picrophilus crophilus, Thermoplasma (Thernioplasnia), Corynebacterium, Mycobacterium, Streptomyces, Aquifrx, Porphyromonas, Chlorobium, Thermus, Bacillus, Listeria, Staphylococcus s), Clostridium, Thermoanaerobacter, Mycoplasma, Fusobacterium, Azarcus, Chromobacterium, Neisseria, Nitrosomonas, Desulfovibrio, Geobacter, Mylococcus rococcus), Campylobacter, Wolinella, Acinetobacter, Erwinia, Escherichia, Legionella, Methylococcus, Pasteurella, Photobacterium, Salmonella, Xanthomonas,Includes the genera Yersinia, Treponema, and Thermotoga.

[0079]

[0100] "CRISPR systems" refer collectively to CRISPR-associated ("Cas") genes. "CRISPR" refers to transcripts and other elements involved in the expression or direction of gene activity, including sequences encoding Cas genes, tracr (transactivating CRISPR) sequences (e.g., tracrRNA or active portion tracrRNA), tracr-mate sequences (including "direct repeats" and tracrRNA processing portion direct repeats in the context of endogenous CRISPR systems), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR systems), or other sequences and transcripts from a CRISPR locus. In some embodiments, one or more elements of a CRISPR system are derived from a Class 1 Type I or Type III CRISPR system. In some embodiments, one or more elements of a CRISPR system are derived from a Class 2 Type II or Type V CRISPR system. In some embodiments, one or more elements of a CRISPR system are derived from a particular organism that contains an endogenous CRISPR system, such as Streptococcus pyogenes. Generally, CRISPR systems are characterized by elements that promote the formation of a CRISPR complex at the location of the target sequence (also referred to as a protospacer in the context of endogenous CRISPR systems). In the context of CRISPR complex formation, a "target sequence" refers to a sequence that the guide sequence is designed to have complementarity with, where hybridization between the target sequence and the guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessary, provided there is sufficient complementarity to allow hybridization and promote the formation of a CRISPR complex. The target sequence may comprise any polynucleotide, such as a DNA or RNA polynucleotide. In some embodiments, the target sequence is located in the nucleus or cytoplasm of a cell. A sequence or template that may be used to transduce into a target locus containing a target sequence is referred to as an "editing template" or "editing polynucleotide" or "editing sequence." In an embodiment of the present invention, an exogenous template polynucleotide may be referred to as an editing template. In an embodiment of the present invention, the genetic recombination is homologous recombination.

[0080]

[0101] "NgAgo" is a prokaryotic protein thought to be involved in gene silencing. NgAgo is an Argonaute protein derived from the archaeon Natronobacterium gregoryi (see, e.g., Gao et al. (2016) Nature Biotechnology 34, 768-773). The "NgAgo system" refers to all of the components required for the NgAgo enzyme to cleave the system, including, for example, short-stranded guide DNA.

[0081]

[0102] The term "small molecule" is an art-recognized term. In certain embodiments, the term refers to molecules having a molecular weight of less than about 2000 amu, or less than about 1000 amu, or even less than about 500 amu.

[0082]

[0103] All percentages and ratios used herein are in percent unless otherwise indicated. By weight.

[0104] The embodiments described herein generally involve the differentiation, proliferation, and differentiation of oligodendrocyte precursor cells. The present invention relates to agents, compounds, and methods for enhancing oligodendrocyte production by inducing, promoting, and / or modulating proliferation and / or maturation, as well as methods for treating diseases or disorders in a subject where myelination or remyelination would be beneficial to the subject.

[0083]

[0105] Cholesterol biosynthesis in oligodendrocyte progenitor cells (OPCs) It has been discovered that enhancing and / or inducing the accumulation of Δ8,9-unsaturated sterol intermediates in the OPC pathway can induce oligodendrogenesis. Enhancement and / or induction of Δ8,9-unsaturated sterol intermediate accumulation can be achieved by inhibiting the accumulation of Δ8,9-unsaturated sterol intermediates and / or modulating and / or inhibiting enzymes in the cholesterol biosynthesis pathway in OPCs for which Δ8,9-unsaturated sterol intermediates are substrates, and by administering Δ8,9-unsaturated sterol intermediates directly and / or indirectly to OPCs. Enhancement and / or induction of Δ8,9-unsaturated sterol intermediate accumulation promotes differentiation, survival, proliferation, and / or maturation of OPCs and can treat diseases and / or disorders in a subject where myelination or remyelination is beneficial to the subject.

[0084]

[0106] In some embodiments, the Δ Agents that enhance and / or induce the accumulation of 8,9-unsaturated sterol intermediates can be administered to a subject and / or OPCs in an amount effective to promote and / or induce differentiation, proliferation, and / or maturation of OPCs and oligodendrocyte production. In one example, the agent can include at least one compound that inhibits enzyme-mediated synthesis of one or more sterol intermediates in the cholesterol biosynthetic pathway of OPCs and / or promotes the accumulation of Δ8,9-unsaturated sterol intermediates.

[0085]

[0107] In some embodiments, the compound inhibits the lanosterol-cholesterol conversion of OPCs. The compound can regulate and / or inhibit one or more enzyme-mediated conversion steps of the cholesterol biosynthesis pathway to sterols, for example, lanosterol and / or lanosterol, and promote and / or induce oligodendrogenesis.For example, the compound can inhibit CYP51, sterol 14-reductase, and / or EBP enzyme-mediated synthesis of sterol intermediates in the cholesterol biosynthesis pathway.In yet another embodiment, the compound can inhibit the 4,4-dimethylcholesterol- The enzyme-mediated conversion of 8(9),14,24-trien-3β-ol, 4,4-dimethylcholesta-8(9),14,24-trien-3β-ol to zymostenol, and / or zymostenol to lathosterol can be modulated and / or inhibited to enhance oligodendrogenesis.

[0086]

[0108] In some embodiments, the compound is prepared by dehydrogenating lanosterol from lanosterol. One or more enzyme-mediated conversion steps of the cholesterol biosynthetic pathway to sterol or lathosterol can be modulated and / or inhibited.

[0087]

[0109] In some embodiments, the compounds used in the methods described herein can inhibit the enzyme-mediated conversion of zymostenol to lathosterol through inhibition of emopamil-binding protein (EBP) isomerase enzyme activity. Alternatively, the compounds used in the methods described herein can inhibit sterol C14 reductase enzyme activity or CYP51 enzyme activity in the cholesterol biosynthetic pathway.

[0088]

[0110] Inducing OPC differentiation through modulation and / or inhibition of the cholesterol biosynthetic pathway Thus, compounds capable of enhancing oligodendrocyte production can be identified by measuring the levels of cholesterol and upstream metabolites in OPCs administered with the screened compound. In some embodiments, compounds capable of enhancing OPC differentiation via modulation and / or inhibition of the cholesterol biosynthetic pathway can be identified using high-throughput small molecule screening (HTS), which tends to select compounds that have both high potency and low toxicity in mammalian subjects and can promote the differentiation of oligodendrocyte precursors. As used herein, the term "small molecule" refers to a low-molecular-weight (e.g., <550 kDa) biologically active organic compound that can cross biological membranes and regulate intracellular processes.

[0089]

[0111] HTS involves adding small drug-like organic compounds (250-550 kDa) to cells. Primary screening can involve seeding cells in 96- or 384-well plates and incubating them in a 96- or 384-well plate. The cells can then be visually screened for morphological changes in oligodendrocyte precursors. In secondary screening, differentiation and proliferation induced by selected compounds can be further verified by fluorescence microscopy. Oligodendrocyte precursor proliferation and maturation in response to selected compounds can then be further assessed by inducing myelin protein expression, as determined by, for example, immunocytochemistry and Western blot. Examples of assays that can be used for primary and secondary screening are described in Najm et al., Nat Methods. 2011 Sep. 25;8(11):957-62; Bai et al., Neurosci Bull. 2013 Apr. 29(2):239-50; Yang et al., Dev Biol. 2011 Feb. 1;350(1):127-38; and Cho et al., Curr Neuropharmacol. 2007 Mar. 5(1):19-33.

[0090]

[0112] Compounds were further screened using a GCMS assay to determine cholesterol levels. Levels of cholesterol and intermediates on the way to cholesterol, including squalene, squalene epoxide, lanosterol, FF-MAS, T-MAS, other meiosis-activating sterols, zymostenol, lanosterol, dehydrolasosterol, dehydrodesmosterol, desmosterol, 7-DHC, 8-DHC, etc., can be monitored. Such assays are described, inter alia, in Korade et al., J. Med. Chem., 2016, 59(3), 1102-1115. Briefly, cells are treated with various molecules and then lysed using organic solvents such as methanol or chloroform, and lipophilic metabolites can be extracted. After silylation using BTMSA or an equivalent silylating reagent, samples are injected into a GC-MS and integrated peak intensities are analyzed. The sterol abundance is determined by comparison with authentic sterol reference standards.

[0091]

[0113] In some embodiments, the compounds are administered to mammals (e.g., rats and mice). Further screening can be performed using a brain slice assay to assess myelination in the mouse brain. Such assays are described, for example, in Bai et al., Neurosci Bull. 2013 Apr;29(2):239-50, Yang et al., Dev Biol. 2011 Feb;350(1):127-38, and Cho et al., Curr Neuropharmacol. 2007 Mar;5(1):19-33.

[0092]

[0114] In another embodiment, the compound is used to treat MOG35-55 induced chronic multiple sclerosis. Screening can be performed using an in vivo assay that assesses remyelination and reduction in clinical severity in an experimental autoimmune encephalomyelitis (EAE) rodent model.

[0093]

[0115] In another embodiment, the compound is administered to mice suffering from lysolecithin-induced focal myelin loss. Screening can be performed using an assay that evaluates in vivo myelination in a model, such as that described in Mi et al., S et al., Ann Neurol 65, 304-325 (2009).

[0094]

[0116] In certain embodiments, identified compounds capable of enhancing OPC differentiation are The compounds can modulate and / or inhibit the enzyme-mediated synthesis of one or more sterol intermediates in the cholesterol biosynthetic pathway of OPCs (see, e.g., Figure 15). In some embodiments, the compounds can modulate and / or inhibit the enzyme-mediated synthesis of one or more sterol intermediates in the cholesterol biosynthetic pathway of OPCs in an amount effective to promote and / or induce differentiation, proliferation, and / or maturation of oligodendrocyte precursor cells. For example, the compounds can inhibit the enzyme-mediated synthesis of one or more sterol intermediates in the cholesterol biosynthetic pathway of OPCs by at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% when compared to the amount of enzyme-mediated synthesis of one or more sterol intermediates in the cholesterol biosynthetic pathway of untreated OPCs or subjects.

[0095]

[0117] Cholesterol biosynthesis is initiated in the upstream step of the sterol intermediate lanosterol. Inhibiting the synthesis of lanosterol-containing steroid hormones has been shown to have little to no effect on OPC differentiation. Thus, in some embodiments, compounds identified by OPC HTS and GCMS may be compounds that can modulate and / or inhibit one or more enzyme-mediated conversion steps in the cholesterol biosynthetic pathway from lanosterol to cholesterol and / or between lanosterol and lanosterol in an amount effective to promote and / or induce differentiation, proliferation, and / or maturation of oligodendrocyte precursor cells. For example, a compound may inhibit one or more enzyme-mediated conversion steps in the cholesterol biosynthetic pathway from lanosterol to cholesterol and / or between lanosterol and lanosterol by at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% when compared to the amount of enzyme-mediated synthesis of one or more sterol intermediates in the cholesterol biosynthetic pathway from lanosterol to cholesterol in untreated OPCs or subjects.

[0096]

[0118] In certain embodiments, OPC HTS screening and GCMS Compounds identified by the present invention include compounds that inhibit or inhibit a phosphodiesterase (PDS) pathway in the cholesterol biosynthesis pathway in an amount effective to promote and / or induce differentiation, proliferation and / or maturation of oligodendrocyte precursor cells. There may be compounds that can modulate and / or inhibit the enzyme-mediated conversion of lanosterol to FF-MAS, FF-MAS to T-MAS, zymostenol to lathosterol, T-MAS to zymosterol, zymosterol to dehydrolasosterol, and / or desmosterol to cholesterol. For example, a compound can inhibit the enzyme-mediated conversion of lanosterol to FF-MAS, FF-MAS to T-MAS, zymostenol to lathosterol, T-MAS to zymosterol, zymosterol to dehydrolasosterol, and / or desmosterol to cholesterol in the cholesterol biosynthetic pathway by at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% when compared to the amount of enzyme-mediated conversion of lanosterol to FF-MAS, FF-MAS to T-MAS, zymostenol to lathosterol, T-MAS to zymosterol, zymosterol to dehydrolasosterol, and / or desmosterol to cholesterol in untreated OPCs or subjects. Examples of compounds that can inhibit the enzyme-mediated conversion of lanosterol to FF-MAS, FF-MAS to T-MAS, zymostenol to lanosterol, T-MAS to zymosterol, zymosterol to dehydrolanthosterol, and / or desmosterol to cholesterol in the cholesterol biosynthetic pathway include ketoconazole, 2-methylketoconazole, amorolfine, AY9944, EPZ005687, tamoxifen, benztropine, bexarotene, clemastine, FR171456, cis-N-cyclohexyl-N-ethyl-3-(3-chloro-4-cyclohexylphenyl)prop-2-enylamine, and ifenprodil.

[0097]

[0119] In some embodiments, the compound is zymostenol lathosterol. The compounds can modulate and / or inhibit enzyme (e.g., emopamil-binding protein)-mediated conversion of zymostenol to lathosterol in the cholesterol biosynthesis pathway of OPCs. Emopamil-binding protein (EBP, Δ8Δ7 isomerase, 3-beta-hydroxysteroid-delta(8),delta(7)-isomerase, also known as CDPX2, CHO2, CPX, or CPXD) is an enzyme responsible for one of the final steps in the production of cholesterol. In particular, it converts zymostenol to lathosterol, which is then modified by other enzymes to produce cholesterol (see, e.g., Figure 15). Thus, in some embodiments, compounds that can inhibit EBP-mediated conversion of zymostenol to lathosterol in the cholesterol biosynthesis pathway of OPCs can promote and / or induce the differentiation, proliferation, and / or maturation of oligodendrocyte precursor cells. For example, the compound can inhibit the EBP-mediated enzymatic conversion of zymostenol to desmosterol in the cholesterol biosynthetic pathway by at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% when compared to the amount of EBP-mediated conversion of desmosterol to cholesterol in untreated OPCs or subjects.

[0098]

[0120] In some embodiments, the EBs used in the methods described herein The P inhibitor compound can have the formula (I)

[0099] [ka]

[0100] (In the formula, (a)X 1 is a substituted or unsubstituted aryl, heteroaryl, cyclyl, or heterocyclyl; (b)Y 1 is a substituted or unsubstituted C1-C6 linear or branched alkyl; (c)Z 1 is CR'R", NR' or OR'; and (d) R', R", R 1 , R 2 , R 3 , or R 4 are each independently hydrogen, substituted or unsubstituted C1-C 24 Alkyl, C2-C 24 Alkenyl, C2-C 24 Alkynyl, C3-C 20 Aryl, heteroaryl, heterocycloalkenyl containing 5 to 6 ring atoms (wherein 1 to 3 ring atoms are independently selected from N, NH, N(C1-C6 alkyl), NC(O)(C1-C6 alkyl), O, and S), C6-C 24 Alkaryl, C6-C 24 Aralkyl, halo, -Si(C1-C3 alkyl), hydroxyl, sulfhydryl, C1-C 24 Alkoxy, C2-C 24 Alkenyloxy, C2-C 24 Alkynyloxy, C5-C 20 Aryloxy, acyl (C2-C 24 Alkylcarbonyl (--CO-alkyl) and C6-C 20 Arylcarbonyl (including -CO-aryl), acyloxy (-O-acyl), C2-C 24 Alkoxycarbonyl (-(CO)-O-alkyl), C6-C 20 Aryloxycarbonyl (-(CO)-O-aryl), C2-C 24 Alkylcarbonate (-O-(CO)-O-alkyl), C6-C 20 Arylcarbonato (-O-(CO)-O-aryl), carboxy (-COOH), carboxylato (-COO - ), carbamoyl (-(CO)-NH2), C1-C 24 Alkyl-carbamoyl(-(CO)-NH(C1-C 24 alkyl), arylcarbamoyl (-(CO)-NH-aryl), thiocarbamoyl (-(CS)-NH2), carbamido (-NH-(CO)-NH2), cyano (-CN), isocyano (-N + C - ), cyanate (-O-CN), isocyanate (-ON +=C - ), isothiocyanato (-S-CN), azido (-N=N + =N - ), formyl (--(CO)--H), thioformyl (--(CS)--H), amino (--NH2), C1 to C 24 Alkylamino, C5-C 20 Arylamino, C2-C 24 Alkylamide (-NH-(CO)-alkyl), C6-C 20 Arylamide (—NH—(CO)-aryl), imino (—CR═NH, where R is hydrogen, C1-C 24 Alkyl, C5-C 20 Aryl, C6-C 24 Alkaryl, C6-C 24 aralkyl, etc.), alkylimino (-CR=N(alkyl), where R=hydrogen, alkyl, aryl, alkaryl, aralkyl, etc.), arylimino (-CR=N(aryl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (-NO2), nitroso (-NO), sulfo (-SO2-OH), sulfonato (-SO2-O - ), C1~C 24 Alkyl sulfanyl (-S-alkyl; also called "alkylthio"), aryl sulfanyl (-S-aryl; also called "arylthio"), C1-C 24 Alkylsulfinyl (-(SO)-alkyl), C5-C 20 Arylsulfinyl (-(SO)-aryl), C1-C 24 Alkylsulfonyl (-SO2-alkyl), C5-C 20 Arylsulfonyl (-SO2-aryl), sulfonamide (-SO2-NH2, -SO2NY2 (where Y is independently H, allyl, or alkyl), phosphono (-P(O)(OH)2), phosphonato (-P(O)(O - )2), phosphinato (-P(O)(O - )), phospho (-PO2), phosphino (--PH2), polyalkyl ether, phosphate, phosphate ester, amino acid, or another moiety predicted to have a positive or negative charge at physiological pH groups, and combinations thereof, and wherein R ’ and R ” may be joined to form a cyclic or polycyclic ring, where the ring is substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocyclyl).

[0101]

[0121] In some embodiments, the compound having formula (I) includes a compound having formula (II): There may be a compound having

[0102] [ka]

[0103] (In the formula, (a)Y 1 is a substituted or unsubstituted C1-C6 linear or branched alkyl; (b)Z 1 is CR'R", NR' or OR'; (c) R', R", R 1 , R 2 , R 3 , R 4 and R 5 are each independently hydrogen, substituted or unsubstituted C1-C 24 Alkyl, C2-C 24 Alkenyl, C2-C 24 Alkynyl, C3-C 20 Aryl, heteroaryl, heterocycloalkenyl containing 5-6 ring atoms (wherein 1-3 ring atoms are independently selected from N, NH, N(C1-C6 alkyl), NC(O)(C1-C6 alkyl), O, and S), C6-C 24 Alkaryl, C6-C 24 Aralkyl, halo, -Si(C1-C3 alkyl), hydroxyl, sulfhydryl, C1-C 24 Alkoxy, C2-C 24 Alkenyloxy, C2-C 24 Alkynyloxy, C5-C 20 Aryloxy, acyl (C2-C 24Alkylcarbonyl (--CO-alkyl) and C6-C 20 Arylcarbonyl (including -CO-aryl), acyloxy (-O-acyl), C2-C 24 Alkoxycarbonyl (-(CO)-O-alkyl), C6-C 20 Aryloxycarbonyl (-(CO)-O-aryl), C2-C 24 Alkylcarbonate (-O-(CO)-O-alkyl), C6-C 20 Arylcarbonato (-O-(CO)-O-aryl), carboxy (-COOH), carboxylato (-COO - ), carbamoyl (-(CO)-NH2), C1-C 24 Alkyl-carbamoyl(-(CO)-NH(C1-C 24 alkyl), arylcarbamoyl (-(CO)-NH-aryl), thiocarbamoyl (-(CS)-NH2), carbamido (-NH-(CO)-NH2), cyano (-CN), isocyano (-N + C - ), cyanate (-O-CN), isocyanate (-ON + =C - ), isothiocyanato (-S-CN), azido (-N=N + =N - ), formyl (--(CO)--H), thioformyl (--(CS)--H), amino (--NH2), C1 to C 24 Alkylamino, C5-C 20 Arylamino, C2-C 24 Alkylamide (-NH-(CO)-alkyl), C6-C 20 Arylamide (—NH—(CO)-aryl), imino (—CR═NH, where R is hydrogen, C1-C 24 Alkyl, C5-C 20 Aryl, C6-C 24 Alkaryl, C6-C 24aralkyl, etc.), alkylimino (-CR=N(alkyl), where R=hydrogen, alkyl, aryl, alkaryl, aralkyl, etc.), arylimino (-CR=N(aryl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (-NO2), nitroso (-NO), sulfo (-SO2-OH), sulfonato (-SO2-O - ), C1~C 24 Alkyl sulfanyl (-S-alkyl; also called "alkylthio"), aryl sulfanyl (-S-aryl; also called "arylthio"), C1-C 24 Alkylsulfinyl (-(SO)-alkyl), C 5~C 20 Arylsulfinyl (-(SO)-aryl), C1-C 24 Alkylsulfonyl (-SO2-alkyl), C5-C 20 Arylsulfonyl (-SO2-aryl), sulfonamide (-SO2-NH2, -SO2NY2 (where Y is independently H, allyl, or alkyl), phosphono (-P(O)(OH)2), phosphonato (-P(O)(O - )2), phosphinato (-P(O)(O - )), phospho (—PO2), phosphino (—PH2), polyalkyl ether, phosphate, phosphate ester, amino acid or a group incorporating another moiety that is expected to have a positive or negative charge at physiological pH, combinations thereof, and wherein R′ and R″ may be joined to form a cyclic or polycyclic ring, where the ring is substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocyclyl; and (d) where n is 1 to 5).

[0104]

[0122] In another embodiment, the compound having formula (I) includes a compound having formula (IIIa): There may be compounds that have

[0105] [ka]

[0106] (In the formula, (a)Y 1 is a substituted or unsubstituted C1-C6 linear or branched alkyl; (b)Z 2 and Z 3 are each independently selected from the group consisting of NR′ or (CHR′)n1, where Z 2 or Z 3 is NR'; (c) R', R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 are each independently hydrogen, oxygen, substituted or unsubstituted C1-C 24 Alkyl, C2-C 24 Alkenyl, C2-C 24 Alkynyl, C3-C 20 Aryl, heteroaryl, heterocycloalkenyl containing 5 to 6 ring atoms (wherein 1 to 3 ring atoms are independently selected from N, NH, N(C1-C6 alkyl), NC(O)(C1-C6 alkyl), O, and S), C6-C 24 Alkaryl, C6-C 24 Aralkyl, halo, -Si(C1-C3 alkyl), hydroxyl, sulfhydryl, C1-C 24 Alkoxy, C2-C 24 Alkenyloxy, C2-C 24 Alkynyloxy, C5-C 20 Aryloxy, acyl (C2-C 24 Alkylcarbonyl (--CO-alkyl) and C6-C 20 Arylcarbonyl (including -CO-aryl), acyloxy (-O-acyl), C2-C 24 Alkoxycarbonyl (-(CO)-O-alkyl), C6-C 20 Aryloxycarbonyl (-(CO)-O-aryl), C2-C 24 Alkylcarbonate (-O-(CO)-O-alkyl), C6-C 20Arylcarbonato (-O-(CO)-O-aryl), carboxy (-COOH), carboxylato (-COO - ), carbamoyl (-(CO)-NH2), C1-C 24 Alkyl-carbamoyl(-(CO)-NH(C1-C 24 alkyl), arylcarbamoyl (-(CO)-NH-aryl), thiocarbamoyl (-(CS)-NH2), carbamido (-NH-(CO)-NH2), cyano (-CN), isocyano (-N + C - ), cyanate (-O-CN), isocyanate (-ON + =C - ), isothiocyanato (-S-CN), azido (-N=N + =N - ), formyl (--(CO)--H), thioformyl (--(CS)--H), amino (--NH2), C1 to C 24 Alkylamino, C5-C 20 Arylamino, C2-C 24 Alkylamide (-NH-(CO)-alkyl), C6-C 20 Arylamide (-NH-(CO)-aryl), amine (-CR=NH, where R is hydrogen, C1-C 24 Alkyl, C5-C 20 Aryl, C6-C 24 Alkaryl, C6-C 24 aralkyl, etc.), alkylimino (-CR=N(alkyl), where R=hydrogen, alkyl, aryl, alkaryl, aralkyl, etc.), arylimino (-CR=N(aryl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (-NO2), nitroso (-NO), sulfo (-SO2-OH), sulfonato (-SO2-O - ), C1~C 24 Alkyl sulfanyl (-S-alkyl; also called "alkylthio"), aryl sulfanyl (-S-aryl; also called "arylthio"), C1-C 24 Alkylsulfinyl (-(SO)-alkyl), C5-C 20 Arylsulfinyl (-(SO)-aryl), C1-C24 Alkylsulfonyl (-SO2-alkyl), C5-C 20 Arylsulfonyl (-SO2-aryl), sulfonamide (-SO2-NH2, -SO2NY2 (where Y is independently H, allyl, or alkyl), phosphono (-P(O)(OH)2), phosphonato (-P(O)(O - )2), phosphinato (-P(O)(O - )), phospho (-PO2), phosphino (--PH2), polyalkyl ether, phosphate, phosphate ester, amino acid or a group incorporating another moiety that is predicted to have a positive or negative charge at physiological pH, or combinations thereof; (d) where n is 1 to 5 and n1 is 1 to 4; and (e) The dashed line represents, independently at each occurrence, a double bond or a single bond.

[0107]

[0123] In yet another embodiment, the compound having formula (I) includes a compound having formula (III) There may be compounds with b)

[0108] [ka]

[0109] (In the formula, (a);R 1 , R 2 , R 3 , R 4 , R 5 , R 8 , and R 9 are each independently hydrogen, substituted or unsubstituted C1-C 24 Alkyl, C2-C 24 Alkenyl, C2-C 24 Alkynyl, C3-C 20 Aryl, heteroaryl, heterocycloalkenyl containing 5 to 6 ring atoms (wherein 1 to 3 ring atoms are independently selected from N, NH, N(C1-C6 alkyl), NC(O)(C1-C6 alkyl), O, and S), C6-C 24 Alkaryl, C6-C 24Aralkyl, halo, -Si(C1-C3 alkyl), hydroxyl, sulfhydryl, C1-C 24 Alkoxy, C2-C 24 Alkenyloxy, C2-C 24 Alkynyloxy, C5-C 20 Aryloxy, acyl (C2-C 24 Alkylcarbonyl (--CO-alkyl) and C6-C 20 Arylcarbonyl (including -CO-aryl), acyloxy (-O-acyl), C2-C 24 Alkoxycarbonyl (-(CO)-O-alkyl), C6-C 20 Aryloxycarbonyl (-(CO)-O-aryl), C2-C 24 Alkylcarbonate (-O-(CO)-O-alkyl), C6-C 20 Arylcarbonato (-O-(CO)-O-aryl), carboxy (-COOH), carboxylato (-COO - ), carbamoyl (-(CO)-NH2), C1-C 24 Alkyl-carbamoyl(-(CO)-NH(C1-C 24 alkyl), arylcarbamoyl (-(CO)-NH-aryl), thiocarbamoyl (-(CS)-NH2), carbamido (-NH-(CO)-NH2), cyano (-CN), isocyano (-N + C - ), cyanate (-O-CN), isocyanate (-ON + =C - ), isothiocyanato (-S-CN), azido (-N=N + =N - ), formyl (--(CO )--H), thioformyl (--(CS)--H), amino (--NH2), C1-C 24 Alkylamino, C5-C 20 Arylamino, C2-C 24 Alkylamide (-NH-(CO)-alkyl), C6-C 20 Arylamide (—NH—(CO)-aryl), imino (—CR═NH, where R is hydrogen, C1-C 24 Alkyl, C5-C 20Aryl, C6-C 24 Alkaryl, C6-C 24 aralkyl, etc.), alkylimino (-CR=N(alkyl), where R=hydrogen, alkyl, aryl, alkaryl, aralkyl, etc.), arylimino (-CR=N(aryl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (-NO2), nitroso (-NO), sulfo (-SO2-OH), sulfonato (-SO2-O - ), C1~C 24 Alkyl sulfanyl (-S-alkyl; also called "alkylthio"), aryl sulfanyl (-S-aryl; also called "arylthio"), C1-C 24 Alkylsulfinyl (-(SO)-alkyl), C5-C 20 Arylsulfinyl (-(SO)-aryl), C1-C 24 Alkylsulfonyl (-SO2-alkyl), C5-C 20 Arylsulfonyl (-SO2-aryl), sulfonamide (-SO2-NH2, -SO2NY2 (where Y is independently H, allyl, or alkyl), phosphono (-P(O)(OH)2), phosphonato (-P(O)(O - )2), phosphinato (-P(O)(O - )), phospho (—PO2), phosphino (—PH2), polyalkyl ether, phosphate, phosphate ester, amino acid or a group incorporating another moiety that is predicted to have a positive or negative charge at physiological pH, or combinations thereof; and (b) n is 1 to 5 and m is 1 to 3).

[0110]

[0124] Examples of compounds of formula (I) have the formula:

[0111] [ka]

[0112]

[0125] Another embodiment of the compound having formula (I) is a compound having formula (IVa): There is something

[0113] [ka]

[0114] (In the formula, (a);R 1 , R 2 , R 3 , R 4 , R 5 , and R 10 are each independently hydrogen, substituted or unsubstituted C1-C 24 Alkyl, C2-C 24 Alkenyl, C2-C 24 Alkynyl, C3-C 20 Aryl, heteroaryl, heterocycloalkenyl containing 5 to 6 ring atoms (wherein 1 to 3 ring atoms are independently selected from N, NH, N(C1-C6 alkyl), NC(O)(C1-C6 alkyl), O, and S), C6-C 24 Alkaryl, C6-C 24 Aralkyl, halo, -Si(C1-C3 alkyl)3, hydroxy Sulfhydryl, C1-C 24 Alkoxy, C2-C 24 Alkenyloxy, C2-C 24 Alkynyloxy, C5-C 20 Aryloxy, acyl (C2-C 24 Alkylcarbonyl (--CO-alkyl) and C6-C 20 Arylcarbonyl (including -CO-aryl), acyloxy (-O-acyl), C2-C 24 Alkoxycarbonyl (-(CO)-O-alkyl), C6-C 20 Aryloxycarbonyl (-(CO)-O-aryl), C2-C 24 Alkylcarbonate (-O-(CO)-O-alkyl), C6-C 20 Arylcarbonato (-O-(CO)-O-aryl), carboxy (-COOH), carboxylato (-COO - ), carbamoyl (-(CO)-NH2), C1-C 24 Alkyl-carbamoyl(-(CO)-NH(C1-C24 alkyl), arylcarbamoyl (-(CO)-NH-aryl), thiocarbamoyl (-(CS)-NH2), carbamido (-NH-(CO)-NH2), cyano (-CN), isocyano (-N + C - ), cyanate (-O-CN), isocyanate (-ON + =C - ), isothiocyanato (-S-CN), azido (-N=N + =N - ), formyl (--(CO)--H), thioformyl (--(CS)--H), amino (--NH2), C1 to C 24 Alkylamino, C5-C 20 Arylamino, C2-C 24 Alkylamide (-NH-(CO)-alkyl), C6-C 20 Arylamide (—NH—(CO)-aryl), imino (—CR═NH, where R is hydrogen, C1-C 24 Alkyl, C5-C 20 Aryl, C6-C 24 Alkaryl, C6-C 24 aralkyl, etc.), alkylimino (-CR=N(alkyl), where R=hydrogen, alkyl, aryl, alkaryl, aralkyl, etc.), arylimino (-CR=N(aryl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (-NO2), nitroso (-NO), sulfo (-SO2-OH), sulfonato (-SO2-O - ), C1~C 24 Alkyl sulfanyl (-S-alkyl; also called "alkylthio"), aryl sulfanyl (-S-aryl; also called "arylthio"), C1-C 24 Alkylsulfinyl (-(SO)-alkyl), C5-C 20 Arylsulfinyl (-(SO)-aryl), C1-C 24 Alkylsulfonyl (-SO2-alkyl), C5-C 20Arylsulfonyl (-SO2-aryl), sulfonamide (-SO2-NH2, -SO2NY2 (where Y is independently H, allyl, or alkyl), phosphono (-P(O)(OH)2), phosphonato (-P(O)(O - )2), phosphinato (-P(O)(O - )), phospho (—PO2), phosphino (—PH2), polyalkyl ether, phosphate, phosphate ester, amino acid or a group incorporating another moiety that is predicted to have a positive or negative charge at physiological pH, or combinations thereof; and (b) n is 1 to 5 and m is 1 to 3).

[0115]

[0126] Yet another embodiment of the compound of formula (I) is a compound of formula (IVb): There are compounds that

[0116] [ka]

[0117] (In the formula, (a);R 2 , R 3 , R 4 , R 5 , and R 10 are each independently hydrogen, substituted or unsubstituted C1-C 24 Alkyl, C2-C 24 Alkenyl, C2-C 24 Alkynyl, C3-C 20 Aryl, heteroaryl, 5-6 ring atoms (wherein 1-3 ring atoms are independently N, NH, N(C1-C6 alkyl), NC(O)(C1-C6 alkyl) heterocycloalkenyl, including C6-C 24 Alkaryl, C6-C 24 Aralkyl, halo, -Si(C1-C3 alkyl), hydroxyl, sulfhydryl, C1-C 24 Alkoxy, C2-C 24 Alkenyloxy, C2-C 24 Alkynyloxy, C5-C20 Aryloxy, acyl (C2-C 24 Alkylcarbonyl (--CO-alkyl) and C6-C 20 Arylcarbonyl (including -CO-aryl), acyloxy (-O-acyl), C2-C 24 Alkoxycarbonyl (-(CO)-O-alkyl), C6-C 20 Aryloxycarbonyl (-(CO)-O-aryl), C2-C 24 Alkylcarbonate (-O-(CO)-O-alkyl), C6-C 20 Arylcarbonato (-O-(CO)-O-aryl), carboxy (-COOH), carboxylato (-COO - ), carbamoyl (-(CO)-NH2), C1-C 24 Alkyl-carbamoyl(-(CO)-NH(C1-C 24 alkyl), arylcarbamoyl (-(CO)-NH-aryl), thiocarbamoyl (-(CS)-NH2), carbamido (-NH-(CO)-NH2), cyano (-CN), isocyano (-N + C - ), cyanate (-O-CN), isocyanate (-ON + =C - ), isothiocyanato (-S-CN), azido (-N=N + =N - ), formyl (--(CO)--H), thioformyl (--(CS)--H), amino (--NH2), C1 to C 24 Alkylamino, C5-C 20 Arylamino, C2-C 24 Alkylamide (-NH-(CO)-alkyl), C6-C 20 Arylamide (—NH—(CO)-aryl), imino (—CR═NH, where R is hydrogen, C1-C 24 Alkyl, C5-C 20 Aryl, C6-C 24 Alkaryl, C6-C 24aralkyl, etc.), alkylimino (-CR=N(alkyl), where R=hydrogen, alkyl, aryl, alkaryl, aralkyl, etc.), arylimino (-CR=N(aryl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (-NO2), nitroso (-NO), sulfo (-SO2-OH), sulfonato (-SO2-O - ), C1~C 24 Alkyl sulfanyl (-S-alkyl; also called "alkylthio"), aryl sulfanyl (-S-aryl; also called "arylthio"), C1-C 24 Alkylsulfinyl (-(SO)-alkyl), C5-C 20 Arylsulfinyl (-(SO)-aryl), C1-C 24 Alkylsulfonyl (-SO2-alkyl), C5-C 20 Arylsulfonyl (-SO2-aryl), sulfonamide (-SO2-NH2, -SO2NY2 (where Y is independently H, allyl, or alkyl), phosphono (-P(O)(OH)2), phosphonato (-P(O)(O - )2), phosphinato (-P(O)(O - )), phospho (—PO2), phosphino (—PH2), polyalkyl ether, phosphate, phosphate ester, amino acid or a group incorporating another moiety that is predicted to have a positive or negative charge at physiological pH, or combinations thereof; and (b) n is 1 to 5 and m is 1 to 3).

[0118]

[0127] Examples of compounds of formula (I) have the formula:

[0119] [ka]

[0120]

[0128] In some embodiments, OP for use in the methods described herein. EBP inhibitor compounds identified by C screening and / or GCMS include , EPZ005687, tamoxifen, benztropine, clemastine, and ifenprodil.

[0121]

[0129] Additional examples of EBP inhibitor compounds for use in the methods described herein include: Then,

[0122] [ka]

[0123] Possible agents include hydroxyzine, p-fluorohexahydro-silazifenidol, enclomiphene, raloxifene, nafoxidine, levormeloxifene, clomiphene, toremifene, raloxifene, bazedoxifene, lasofoxifene, perphenazine, fluphenazine, trifluoperazine, prochlorperazine, hydrazine, triparanol, droloxifene, idoxifene, mixproxifene (TAT59), clozapine, pramoxine, TMB-8, vesamicol, diphenidol analogs, L-745,870, 7-keto-cholesterol, and 7-hydroxy-cholesterol.

[0124]

[0130] In another embodiment, EBP for use in the methods described herein. The inhibitor compound can be a benzene derivative compound. Benzene derivatives for use in the methods described herein include, for example, those described in U.S. Patent No. 5,354,781, the subject matter of which is incorporated herein by reference in its entirety. Such benzene derivative compounds have the general formula:

[0125] [ka]

[0126] (Wherein, R1 is 、 is a hydrogen or halogen atom; R2 is cyclohexyl or phenyl; R3 is (C3-C6)cycloalkyl; R4 is hydrogen, (C1-C6) alkyl or (C3-C6) cycloalkyl; A is -CO-CH2-, -CH(CI)-CH2-, -CH(OH)-CH2-, -CH2-CH2-, -CH=CH- and -C≡C-. or a pharmaceutical salt thereof.

[0127]

[0131] In some embodiments, the benzene derivative includes a compound of formula

[0128] [ka]

[0129] (cis-N-cyclohexyl-N-ethyl-3-(3-chloro-4-cyclohexylphenyl)prop-2-enylamine), as well as small molecules having analogs, derivatives and pharmaceutical salts thereof.

[0130]

[0132] In some embodiments, for use in the methods described herein EBP inhibitor compounds include those of the formula:

[0131] [ka]

[0132] There may be cis and trans isomers of compounds having the formula:

[0133] In certain embodiments, EBs for use in the methods described herein P inhibitor compounds include those of the formula:

[0133] [ka]

[0134] There may be cis isomers of compounds having the formula: EBP inhibitor compounds for use in the methods described herein also include ,

[0135] [ka]

[0136] and analogs thereof. Examples of Tasin-1 analogs include those of the formula:

[0137] [ka]

[0138] [ka]

[0139] [ka]

[0140] [ka]

[0141] [ka]

[0142] [ka]

[0143] [ka]

[0144] [ka]

[0145] There may be a compound having the formula: Additional EBP inhibitor compounds for use in the methods described herein include , pyridylethanol(phenylethyl)amine derivatives. Pyridylethanol(phenylethyl)amine derivatives for use in the methods described herein include, for example, those described in U.S. Patent No. 7,560,474 B2, the subject matter of which is incorporated herein by reference in its entirety. Such compounds have the general formula (I):

[0146] [ka]

[0147] (wherein n is an integer of 1 to 4, and R1 is a hydrogen atom, a hydroxyl group, or a lower C 1~6 R2 is a hydrogen atom or a linear or branched lower C 1~6 is an alkyl group, and X is hydrogen, fluorine, chlorine, bromine, a hydroxyl group, a trifluoromethyl group, 3,4-di-Cl, 2,4-di-Cl, or a lower C 1~6 alkoxy group), its enantiomers, diastereoisomers or racemates, or physiologically acceptable acid addition salts thereof.

[0148] Examples of pyridylethanol(phenylethyl)amine derivatives include: 1-(3-pyridyl)-2-(N-(2-phenylethyl)-N-propylamino)ethanol and dihydrobromide salts of formula II:

[0149] [ka]

[0150] 1-(3-pyridyl)-2-(N-(2-(3,4-dichlorophenyl)ethyl)-N-methylamino)ethanol and dihydrobromide salt of formula III:

[0151] [ka]

[0152] 1-(3-pyridyl)-2-(N-(2-(3,4-dichlorophenyl)ethyl)-N-propylamino)ethanol and dihydrobromide salt of formula IV:

[0153] [ka]

[0154] and 1-(4-pyridyl)-2-(N-(2-(3,4-dichlorophenyl)ethyl)-N-methylamino)ethanol and dihydrobromide salt of formula V:

[0155] [ka]

[0156] There is. Additional examples of EBP inhibitor compounds for use in the methods described herein include: Examples include compounds having the formula:

[0157] [ka]

[0158] wherein R is selected from benzyl, (5-iodofuran-2yl)methyl, 3-chlorobenzyl, (furan-3-yl)methyl, pent-4-en-1yl, 2,4-dichlorobenzyl, and 2,3-dichlorobenzyl;

[0159] [ka]

[0160] wherein R is selected from methyl, benzyl, and pent-4-en-1yl; and

[0161] [ka]

[0162] . Additional EBP inhibitors for use in the methods described herein include: The compound may have the formula

[0163] [ka]

[0164] . Further examples of EBP inhibitor compounds for use in the methods described herein include: Examples include those described in U.S. Pat. No. 6,489,481 B1, the subject matter of which is incorporated herein by reference in its entirety.

[0165] In certain embodiments, compounds for use in the methods described herein The substances can include compounds that can modulate and / or inhibit EBP and sterol C14 reductase (DHCR14, ANG1; DHCR14A; NET47; also known as delta(14)-sterol reductase), an enzyme that catalyzes the reduction of sterol intermediates to zymostenol during cholesterol biosynthesis. Examples of compounds that can modulate and / or inhibit both EBP and sterol C14 reductase include:

[0166] [ka]

[0167] and analogs thereof. Additional compounds for use in the methods described herein include sterols. Compounds capable of modulating and / or inhibiting cholin C14 reductase include ziprasidone, ifenprodil, 2-methylketoconazole and AY9944.

[0168] Sterol C14 reductase for use in the methods described herein Inhibitors may also include Corydalis Turtschaninowii Besser extract derivatives. Turtschaninowii Besser) is described in U.S. Pat. No. 6,255,317 B1, the subject matter of which is incorporated herein by reference in its entirety, and has the general formula:

[0169] [ka]

[0170] (wherein R1 and R2 may be the same or different and represent a hydroxyl group or an alkoxy group having 1 to 4 carbon atoms, or both R1 and R2 represent a methylenedioxy group; R3 represents a hydrogen atom; R4 and R5 may be the same or different and represent a hydroxyl group, a hydroxyethylamino group, or an alkoxy group having 1 to 4 carbon atoms; R6 represents a hydrogen atom, an alkyl group having 1 to 8 carbon atoms, an alkenyl group having 3 to 8 carbon atoms, or an alkyl group having 1 to 7 carbon atoms. a cycloalkylalkyl group having 1 to 4 carbon atoms, a haloalkyl group having 1 to 4 carbon atoms, an ethoxycarbonyl group, an ethoxycarbonylmethyl group, a hydroxycarbonylmethyl group, an ethoxycarbonylethyl group, or a 2-valerolactonyl group; and Z represents a halogen atom.

[0171] Sterol C14 reductase for use in the methods described herein Inhibitors may also include 5,6-dihydrodibenzeno[a,g]quinolizinium derivatives and salts thereof. Such compounds are described in U.S. Patent No. 6,030,979, the subject matter of which is incorporated herein by reference in its entirety, and have the general formula:

[0172] [ka]

[0173] (wherein R1 and R2 may be the same or different and represent a hydroxyl group or an alkoxy group having 1 to 4 carbon atoms, or R1 and R2 taken together represent a methylenedioxy group; R3 represents a hydroxyl group or an alkoxy group having 1 to 4 carbon atoms; R4 represents a hydrogen atom, an alkyl group having 1 to 8 carbon atoms, or an alkenyl group having 3 to 8 carbon atoms; X represents an inorganic acid ion, an organic acid ion, or a halide, more specifically, nitrate, sulfate, acetate, tartrate, maleate, succinate, citrate, fumarate, aspartate, salicylate, glycerate, ascorbate, fluoride, chloride, iodide, or bromide; Z represents an alkyl group having 5 to 12 carbon atoms, or an alkenyl group having 4 to 6 carbon atoms, an N-benzotriazolyl group, a quinolinyl group, a furyl group, a substituted furyl group, or a group represented by the formula

[0174] [ka]

[0175] (wherein Z1, Z2, Z3, Z4, and Z5 may be the same or different and represent a hydrogen atom, a halogen, an alkyl group having 1 to 5 carbon atoms, a trifluoromethyl group, a phenyl group, a substituted phenyl group, a nitro group, an alkoxy group having 1 to 4 carbon atoms, a methylenedioxy group, a trifluoromethoxy group, a hydroxyl group, a benzyloxy group, a phenoxy group, a vinyl group, a benzenesulfonylmethyl group, or a methoxycarbonyl group; and A and B may be the same or different and represent carbon or nitrogen).

[0176] In certain embodiments, compounds for use in the methods described herein CYP51, an enzyme that catalyzes the conversion of lanosterol to 4,4-dimethylcholesta-8(9),14,24-trien-3β-ol during cholesterol biosynthesis. (or lanosterol 14α-methyldehydrogenase). Examples of compounds that can regulate and / or inhibit CYP51 include (LK-935).

[0177] [ka]

[0178] Non-imidazole CYP51 inhibitors, such as

[0179] [ka]

[0180] where R1 represents an alkyl group having 1 to 3 carbon atoms; and R2 represents hydrogen, (3,4)-diCL, (3,4)-diF, (3,4)-diOMe, (3,4)-diOH, 4-NO2, 4-CF3, or 3-CF3. Additional CYP51 inhibitors can include ketoconazole and its derivatives, econazole, isoconazole, bifonazole, clotrimazole, miconazole, butoconazole, medroxyprogesterone acetate, and fulvestrant.

[0181] In certain embodiments, a small amount of a pharmaceutical composition for use in the methods described herein is These compounds have been identified as being able to increase sterol intermediate levels and enhance oligodendrocyte and myelin formation by inhibiting cholesterol biosynthetic enzymes ranging from CYP51 to EBP in a narrow therapeutic window. Examples of compounds that can increase sterol intermediate levels and enhance myelin formation by inhibiting cholesterol biosynthetic enzymes ranging from CYP51 to EBP include the CYP51 inhibitors bifonazole, clotrimazole, miconazole, butoconazole, ketoconazole, and fulvestrant; the sterol 14-reductase (TM7SF2 / LBR) inhibitors amorolfine, ifenprodil, and ziprasidone; and the EBP inhibitors EGFR, EGFR, and EGFR. P inhibitors include Tasin-1, tamoxifen, benztropine, clemastine, trans-U50488, EPZ005687, raloxifene, hydroxyzine, vesamicol, L-745,870, TMB-8, cis-N-cyclohexyl-N-ethyl-3-(3-chloro-4-cyclohexylphenyl)prop-2-enylamine, p-fluorohexahydro-silazifenidol, pramoxine, and toremifene (see Figure 14).

[0182] In some embodiments, for use in the methods described herein The compounds may include compounds capable of modulating and / or inhibiting NAD(P)-dependent steroid dehydrogenase-like (NSDHL, also known as 3β-hydroxysteroid dehydrogenase / C4 decarboxylase). NSDHL is a steroid hormone involved in the cholesterol pathway. It is located in the lanosterol synthase, distal to the NAD+ receptor, which is involved in the C-4 demethylation of the sterol precursor to the 4α-carboxysterol intermediate. + In certain embodiments, NSDHL inhibitor compounds for use in the methods described herein include those having the formula

[0183] [ka]

[0184] (FR171456), as well as analogs, derivatives and pharmaceutical salts thereof. In some embodiments, for use in the methods described herein The compounds include compounds that can regulate and / or inhibit DHCR24 (24-dehydrocholesterol reductase), an enzyme that catalyzes the reduction of the delta-24 double bond of sterol intermediates during cholesterol biosynthesis. For example, the compounds for use in the methods described herein include compounds that can inhibit the DHCR24-mediated conversion of desmosterol to cholesterol in the cholesterol biosynthesis pathway in an amount effective to promote and / or induce the differentiation, proliferation and / or maturation of oligodendrocyte precursor cells. For example, the compounds can inhibit the DHCR24-mediated conversion of desmosterol to cholesterol in the cholesterol biosynthesis pathway by at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% when compared to the amount of DHCR24-mediated conversion of desmosterol to cholesterol in untreated OPCs or subjects.

[0185] The compounds described herein can be prepared using standard synthetic techniques known to those skilled in the art. may be synthesized using methods known in the art in combination with those described herein. Additionally, solvents, temperatures, and other reaction conditions presented herein may be varied according to the practice and knowledge of one of ordinary skill in the art.

[0186] Starting materials used to synthesize the compounds described herein were obtained from Aldric Starting materials can be obtained from commercial sources such as Sigma Chemical Co. (Milwaukee, Wis.), Sigma Chemical Co. (St. Louis, Mo.), or can be synthesized. The compounds described herein, and other related compounds with different substituents, can be found in, for example, March, ADVANCED ORGANIC CHEMISTRY, 4th Edition, (Wiley 1992); Carey and Sundberg, ADVANCED ORGANIC CHEMISTRY 4th edition, volumes A and B (Plenum 2000, 2001), and Green and Wuts, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, 3rd ed. (Wiley 1999), all of which are incorporated by reference in their entireties.

[0187] In another embodiment, the Δ8,9 The at least one agent that enhances and / or induces accumulation of unsaturated sterol intermediates can be an agent that reduces or inhibits expression of an enzyme in the cholesterol biosynthetic pathway, such as EBP, C14 reductase, or CYP51 expression in a tissue or cell of a subject in need thereof. "Expression" refers to the overall flow of information from a gene to producing a gene product (typically a protein, optionally post-translationally modified, or functional / structural RNA).

[0188] In some embodiments, the agent includes EBP, C14 reductase in cells. There may be an RNAi construct that inhibits or reduces the expression of CYP51 or CYP52. The RNAi construct comprises double-stranded RNA that can specifically block the expression of target genes. "RNA interference" or "RNAi" is a term that was first applied to a phenomenon observed in plants and insects, where double-stranded RNA (dsRNA) specifically blocks gene expression in a post-transcriptional manner.

[0189] As used herein, the term "dsRNA" refers to an siRNA molecule, or other RNA molecules, such as hairpin RNA segments, that contain double-stranded strand characteristics and can be processed into siRNA in the cell.

[0190] The term "loss of function" refers to the genetic alteration that is inhibited by the targeted RNAi method. When referring to a gene, "reduced expression" refers to a decrease in gene expression levels compared to levels in the absence of the RNAi construct.

[0191] As used herein, the phrase "mediates RNAi" refers to any RNA The term refers to the ability to distinguish whether a protein should be degraded by the RNAi process, e.g., degradation occurs in a sequence-specific manner rather than by a sequence-independent dsRNA response, e.g., a PKR response.

[0192]

[0156] As used herein, the term "RNAi construct" refers to a RNAi construct herein is a general term used as a general term, and includes small interfering RNA (siRNA), hairpin RNA and other RNA species that can be cleaved in vivo to form siRNA.RNAi construct herein also includes the transcription product that can form dsRNA or hairpin RNA in cells, and / or the expression vector (also referred to as RNAi expression vector) that can produce the transcription product that can produce siRNA in vivo.

[0193]

[0157] "RNAi expression vector" (referred to herein as "dsRNA-encoding plasmid"). ") refers to a replicable nucleic acid construct used to express (transcribe) an RNA that produces an siRNA portion in cells in which the construct is expressed. Such a vector includes a transcription unit that includes the assembly of (1) a genetic element that has a regulatory role in gene expression, such as a promoter, operator, or enhancer, operably linked to (2), (2) a "coding" sequence that is transcribed to produce double-stranded RNA (two RNA portions that anneal in the cell to form siRNA, or a single hairpin RNA that can be processed into siRNA), and (3) appropriate transcription start and stop sequences.

[0194]

[0158] The selection of promoters and other control elements generally depends on the objective of the gene, such as OPC. The specificity of a vector varies with the intended host cell. In general, expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids," which refer to circular double-stranded DNA loops that, in their vector form, are not bound to the chromosome. In the present specification, the terms "plasmid" and "vector" can be used interchangeably, as the plasmid is the most commonly used form of vector. However, this application will refer to other forms of expression vectors which serve equivalent functions and which are known in the art, and are described herein and hereinafter.

[0195]

[0159] The RNAi construct comprises a molecule related to the gene to be inhibited (i.e., the "target" gene). The nucleotide sequence of the siRNA duplex comprises a nucleotide sequence that hybridizes to at least a portion of the nucleotide sequence of the RNA transcript under physiological conditions in cells.The double-stranded strand RNA only needs to be sufficiently similar to natural RNA to have the ability to mediate RNAi.Therefore, this embodiment allows for sequence variations that may be expected due to genetic mutation, strain polymorphism, or evolutionary divergence.The number of nucleotide mismatches that can be tolerated between the target sequence and the RNAi construct sequence is no more than 1 in 5 base pairs, or no more than 1 in 10 base pairs, or no more than 1 in 20 base pairs, or no more than 1 in 50 base pairs.Mismatches in the center of the siRNA duplex are the most serious and can substantially disable the cleavage of target RNA.In contrast, the nucleotide at the 3' end of the siRNA strand that is complementary to the target RNA does not significantly contribute to the specificity of target recognition.

[0196]

[0160] Sequence identity can be determined using sequence comparison and alignment algorithms known in the art. The double-stranded region of RNA may be functionally defined as the nucleotide sequence that can hybridize with a portion of the target gene transcript. Alternatively, the ...

[0197]

[0161] Production of RNAi constructs can be by chemical synthesis methods or by recombinant nucleic acid technology. This can be achieved by using a modified nucleotide sequence. Endogenous RNA polymerase in the treated cells may mediate in vivo transcription, or cloned RNA polymerases may be used for in vitro transcription. The RNAi construct may contain modifications to either the phosphate-sugar backbone or the nucleoside to, for example, reduce susceptibility to cellular nucleases, improve bioavailability, improve formulation properties, and / or alter other pharmacokinetic properties. For example, the phosphodiester linkages of natural RNA may be modified to include at least one nitrogen or sulfur heteroatom. Modifications in the RNA structure may be tailored to enable specific genetic inhibition while simultaneously avoiding a general response to dsRNA. Similarly, bases may be modified to block the activity of adenosine deaminase. RNAi constructs may be produced enzymatically or by partial or total organic synthesis, and modified ribonucleotides may be introduced by in vitro enzymatic or organic synthesis.

[0198] Methods for chemically modifying RNA molecules are applicable to modifying RNAi constructs. (See, e.g., Nucleic Acids Res, 25:776-780; J Mol Recog 7:89-98; Nucleic Acids Res 23:2661-2668; Antisense Nucleic Acid Drug Dev 7:55-61.) By way of example only, the backbone of the RNAi construct can be modified with phosphorothioate, phosphoramidate, phosphodithioate, chimeric methylphosphonate-phosphodiester, peptide nucleic acid, 5-propynyl-pyrimidine-containing oligomer, or sugar modification (e.g., 2'-substituted ribonucleoside, a-configuration).

[0199]

[0163] The structure of the double strand may consist of a single self-complementary RNA strand or two The double-stranded RNA may be formed by complementary RNA strands. RNA duplex formation may be initiated either inside or outside the cell. RNA may be introduced in an amount that allows delivery of at least one copy per cell. Higher doses of double-stranded strand material (for example, at least 5, 10, 100, 500 or 1000 copies per cell) may result in more effective inhibition, while lower doses may be useful for certain applications. Inhibition is sequence-specific, in that the nucleotide sequence corresponding to the double-stranded region of RNA is the target of genetic inhibition.

[0200] In certain embodiments, the subject RNAi constructs are "small interfering RNAs" or are "siRNAs." These nucleic acids are approximately 19-30 nucleotides in length, and even more preferably 21-23 nucleotides in length, corresponding in length, for example, to fragments generated by nuclease "dicing" of longer double-stranded RNA. It is understood that siRNAs bring together nuclease complexes and guide the complexes to the target mRNA by pairing specific sequences. Consequently, the target mRNA is degraded by the nucleases in the protein complex. In certain embodiments, the 21-23 nucleotide siRNA molecules contain a 3' hydroxyl group.

[0201] The siRNA molecules described herein can be obtained using a number of techniques known to those skilled in the art. siRNA can be synthesized chemically or recombinantly produced by methods known in the art. For example, siRNA can be synthesized chemically or recombinantly produced by methods known in the art. For example, short sense and antisense RNA oligomers can be synthesized and annealed to form double-stranded RNA structures with 2-nucleotide overhangs at each end (Proc Natl Acad Sci USA, 98:9742-9747; EMBO J, 20:6877-88).Then, these double-stranded siRNA structures can be directly introduced into cells by passive uptake or by any of the following alternative delivery systems.

[0202] In certain embodiments, the siRNA construct comprises a longer double-stranded strand R dsRNA can be generated by processing dsRNA, for example, in the presence of the enzyme Dicer. In one embodiment, Drosophila is used as an in vitro system. In this embodiment, dsRNA is combined with a soluble extract from Drosophila embryos, resulting in a combination. The combination is maintained under conditions in which the dsRNA is processed into RNA molecules of about 21 to about 23 nucleotides.

[0203]

[0167] siRNA molecules can be purified using a number of techniques known to those of skill in the art. For example, gel electrophoresis can be used to purify siRNA. Alternatively, non-denaturing methods such as non-denaturing column chromatography can be used to purify siRNA. Additionally, chromatography (e.g., size exclusion chromatography), glycerol gradient centrifugation, and antibody-based affinity purification can be used to purify siRNA.

[0204] In certain embodiments, the RNAi construct comprises a hairpin structure (hairpin RNA). Hairpin RNAs are in the form of a gene that is referred to as a "hairpin RNA." Hairpin RNAs can be exogenously synthesized or can be formed in vivo by transcription from an RNA polymerase III promoter. Examples of the production and use of such hairpin RNAs for gene silencing in mammalian cells are described, for example, in Genes Dev, 2002, 16:948-58; Nature, 2002, 418:38-9; RNA, 2002, 8:842-50; and Proc Natl Acad Sci, 2002, 99:6047-52. Preferably, such hairpin RNAs are engineered in cells or animals to ensure continuous and stable suppression of the desired gene. It is known in the art that siRNAs can be produced by processing hairpin RNAs in cells.

[0205] In yet another embodiment, a plasmid is used to generate double-stranded RNA. For example, the RNAi construct is delivered as a transcription product.In this embodiment, the plasmid is designed to contain the "coding sequence" of each of the sense and antisense strands of the RNAi construct.The coding sequence can be, for example, the same sequence flanked by an inverted promoter, or can be two separate sequences under the transcription control of separate promoters.After the coding sequence is transcribed, complementary RNA is transcribed base pair to form double-stranded RNA.

[0206]

[0170] PCT Application WO 01 / 77350 discloses the same transgene in eukaryotic cells. The example of the vector for bidirectional transcription of transgene is described, so as to obtain both sense and antisense RNA transcripts of gene.Therefore, certain embodiments provide a recombinant vector with the following unique characteristics: The recombinant vector comprises a viral replicon, has two overlapping transcription units arranged in opposite directions, and is flanked by a transgene for the target RNAi construct, where the two overlapping transcription units cause both sense and antisense RNA transcription from the same transgene fragment in host cell.

[0207] In some embodiments, lentiviral vectors are used to Long-term expression of siRNAs, such as hairpin RNAs (shRNAs), can knock down the expression of RPTPs in cancer cells. Although there are some safety concerns regarding the use of lentiviral vectors for gene therapy, self-inactivating lentiviral vectors are considered excellent candidates for gene therapy because they readily transfect mammalian cells.

[0208]

[0172] For example, down-regulation of AKR1A1 expression by short hairpin RNA (shRNA) Controls can be generated using OligoEngine software (OligoEngine, Seattle, WA) to identify sequences as siRNA targets. Oligo sequences can be annealed, ligated into a linearized pSUPER RNAi vector (OligoEngine, Seattle, WA), and transformed into Escherichia coli (E. coli) strain DH5α cells. After selecting positive clones, the plasmids can be transfected into 293T cells by calcium precipitation. The collected viral supernatant containing the shRNA can then be used to infect mammalian cells to downregulate EBP, C14 reductase, or CYP51 enzymes.

[0209] In another embodiment, EBP, C14 reductase, or CYP51 inhibition The agent can comprise antisense oligonucleotides.Antisense oligonucleotides are relatively short nucleic acids that are complementary (or antisense) to the coding strand (sense strand) of mRNA that encodes a specific protein.Antisense oligonucleotides are typically RNA-based, but they can also be DNA-based.In addition, antisense oligonucleotides are often modified to enhance their stability.

[0210]

[0174] When these relatively short oligonucleotides bind to mRNA, they form double-stranded strands. It is believed that the stretching of RNA strands induces degradation of the message by endogenous RNAases. Antisense oligonucleotides are specifically designed to bind to the target region, and under these circumstances, they further interfere with the translation of messages. Despite the specific mechanism by which antisense oligonucleotides function, when administered to cells or tissues, they allow the degradation of mRNA encoding specific proteins. Thus, antisense oligonucleotides reduce the expression and / or activity of specific proteins (e.g., EBP, C14 reductase, or CYP51).

[0211]

[0175] An oligonucleotide is a single-stranded or double-stranded DNA or RNA or chimeric mixtures or derivatives or modified versions thereof. The oligonucleotides can be modified at the base moiety, sugar moiety, or phosphate backbone to improve, for example, the stability, hybridization, etc. of the molecule. Oligonucleotides may have other attached groups, such as peptides (e.g., for targeting host cell receptors) or agents that facilitate transport across cell membranes (see, e.g., Proc Natl Acad Sci 86:6553-6556; Proc Natl Acad Sci 84:648-652; PCT Application WO 88 / 09810, published December 15, 1988) or the blood-brain barrier (see, e.g., PCT Application WO 89 / 10134, published April 25, 1988), hybridization-triggered cleavage agents (see, e.g., BioTechniques 6:958-976), or intercalating agents (see, e.g., Pharm Res 5:539-549). To achieve this goal, oligonucleotides may be conjugated or linked to other molecules.

[0212] The oligonucleotides described herein may be synthesized by standard methods known in the art. Thus, for example, they may be synthesized using an automated DNA synthesizer (such as those available from Biosearch, Applied Biosystems, etc.). For example, phosphorothioate oligonucleotides may be synthesized by the method of Stein et al. (Nucl. Acids Res. 16:3209), and methylphosphonate oligonucleotides can be prepared by using a controlled pore glass polymer support (Proc Natl Acad Sci 85:7448-7451).

[0213]

[0177] The selection of an appropriate oligonucleotide can be performed by one skilled in the art. Given the nucleic acid sequence encoding a protein, a person skilled in the art can design antisense oligonucleotides that bind to the protein, and test these oligonucleotides in vitro or in vivo to confirm that they bind to the mRNA encoding the specific protein and mediate its degradation.To design an antisense oligonucleotide that specifically binds to a specific protein and mediates its degradation, it is important that the sequence recognized by the oligonucleotide is unique or substantially unique to that specific protein.For example, a sequence that is frequently repeated throughout a protein may not be an ideal choice for designing an oligonucleotide that specifically recognizes and degrades a specific message.A person skilled in the art can design an oligonucleotide and compare the sequence of the oligonucleotide with the nucleic acid sequences deposited in a publicly available database to confirm that the sequence is specific or substantially specific to a specific protein.

[0214]

[0178] Many methods have been developed to deliver antisense DNA or RNA to cells. For example, antisense molecules can be injected directly into a tissue site, or modified antisense molecules designed to target desired cells (e.g., antisense linked to a peptide or antibody that specifically binds to a receptor or antigen expressed on the surface of a target cell) can be administered systemically.

[0215] However, in certain cases, sufficient cellular activity to suppress translation of endogenous mRNAs may be achieved. It can be difficult to achieve intracellular concentrations of antisense oligonucleotides. Therefore, another approach utilizes recombinant DNA constructs in which antisense oligonucleotides are placed under the control of a strong Pol III or Pol II promoter. For example, a vector can be introduced in vivo so that it is taken up by cells and directs the transcription of antisense RNA. Such vectors can remain episomal or be integrated into chromosomes, as long as they can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by standard recombinant DNA engineering methods in the art. Vectors can be plasmid, viral, or other vectors known in the art that are used for replication and expression in mammalian cells.

[0216] Expression of the sequence encoding the antisense RNA is in mammalian, preferably human, cells. The promoter may be any promoter known in the art for acting in cells. Such promoters may be inducible or constitutive. Examples of such promoters include, but are not limited to, the SV40 early promoter region (Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Cell 22:787-797), the herpes thymidine kinase promoter (Proc Natl Acad Sci 78:1441-1445), and the regulatory sequences of the metallothionein gene (Nature 296:39-42). Recombinant DNA constructs can be prepared using certain types of plasmids, cosmids, YACs, or viral vectors to allow direct introduction into tissue sites. Alternatively, viral vectors can be used to selectively infect the desired tissue, although administration may be achieved by another route (e.g., systemically).

[0217] In some embodiments, the present invention provides a method for the treatment of cholesterol in the cholesterol biosynthesis pathway of OPC cells. Endogenous expression of enzymes that synthesize one or more sterol intermediates can be reduced in OPCs through the use of nucleases to edit (e.g., mutate) genes encoding the enzymes or their genetic control elements. Nucleases and related systems for use in the methods described herein can include, but are not limited to, zinc finger nucleases (ZFNs), TALE effectors (TALENs), CRISPR / Cas systems, or NgAgo systems. In some embodiments, endogenous genes encoding EBP, C14 reductase, and / or CYP51 enzymes, or their genetic control elements, can be edited using CRISPR / Cas9 guide RNAs. In certain embodiments, the nuclease can be a Class 2 CRISPR / Cas system. For example, the Class 2 CRISPR / Cas system can be a Type II Cas9-based CRISPR system or a Type V Cpf1-based CRISPR system.

[0218] In a further embodiment, the Δ8 The agent that enhances and / or induces accumulation of a Δ8,9-unsaturated sterol intermediate can comprise at least one Δ8,9-unsaturated sterol intermediate or derivative thereof of the cholesterol biosynthetic pathway in OPCs, wherein the Δ8,9-unsaturated sterol intermediate or derivative thereof enhances oligodendrocyte production from OPCs. In some embodiments, the Δ8,9-unsaturated sterol intermediate includes at least one of lanosterol, 14-dehydrozymostenol, FF-MAS, MAS-412, zymosterol, zymostenol, and derivatives thereof.

[0219] The Δ8,9 pathway of cholesterol biosynthesis in OPCs described herein Agents that enhance and / or induce the accumulation of -unsaturated sterol intermediates can be provided in the form of pharmaceutical compositions and administered to promote oligodendrocyte precursor differentiation and / or maturation in vivo. The pharmaceutical compositions can be administered to any subject capable of experiencing the beneficial effects of the oligodendrocyte precursor differentiation and / or maturation compounds of the present invention. Foremost among such animals are humans, although the invention is not intended to be so limited.

[0220] The pharmaceutical compositions for use in the methods of the present invention preferably contain a therapeutically effective amount or a salt thereof at a dosage in the range of 0.01 to 1,000 mg / kg of subject body weight, more preferably in the range of about 10 to 100 mg / kg of patient body weight.

[0221]

[0185] The overall dosage will depend on the subject's overall health, the subject's condition, the severity of the condition, and the findings of improvement. The therapeutically effective amount will depend on several factors, including the compound and the route of administration of the selected drug. Determining a therapeutically effective amount is within the ability of one skilled in the art. The exact compound, route of administration and dosage can be selected by an individual physician taking into account the condition of the subject.

[0222]

[0186] The present invention provides a method for promoting the differentiation and / or proliferation of oligodendrocyte precursors in a subject. The present invention provides a method for treating a disease in a subject by administering a therapeutically effective amount of a pharmaceutical compound according to the present invention to a subject in need thereof. As mentioned above, one or more of the compounds can be administered together with one or more non-toxic pharmaceutically acceptable carriers and / or diluents and / or adjuvants, and optionally other active ingredients.

[0223]

[0187] A "therapeutically effective amount" of a compound and its salts used in the methods of the present invention is The amount of the therapeutically effective amount of the compound varies depending on the mode of administration, the age and weight of the subject, and the condition of the subject being treated, and will ultimately be determined by one skilled in the art. The term "therapeutically effective amount" refers to an amount (dosage) effective for treating a subject with, for example, a neurodegenerative disease (e.g., multiple sclerosis).

[0224] In certain embodiments, the compounds described herein are useful in treating naive CNS neurons. The compound may be administered in an amount effective to promote myelination of CNS neurons in a subject by increasing the amount of myelin protein (e.g., MBP) by 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% when compared to the myelin protein level in the neuron or subject.

[0225] In another embodiment, the compounds described herein are administered to intact CNS neurons. The compound may be administered in an amount effective to promote survival of CNS neurons in a subject by increasing the number of surviving neurons by 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% when compared to the number of surviving neurons in the subject.

[0226] In some embodiments, the compounds described herein are administered to naive OPCs or or the like. Alternatively, the compound may be administered in an amount effective to enhance production of OPCs in the central nervous system of a subject by increasing OPC production by 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% when compared to OPC production in the subject.

[0227] In some embodiments, the compounds described herein are administered to naive OPCs or or the amount of OPC differentiation in the subject by increasing the amount of OPC differentiation by 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% when compared to the amount of OPC differentiation in the subject.

[0228] In some embodiments, the compounds described herein are administered to naive OPCs or Alternatively, the compound may be administered in an amount effective to modulate the cholesterol biosynthetic pathway in OPC cells of a subject by reducing the amount of cholesterol and / or one or more sterol intermediate synthesis in the OPCs by at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% when compared to the amount of cholesterol and / or one or more sterol intermediate synthesis in the subject.

[0229] As used herein, "treating" or "treatment" refers to "Treatment" refers to a reduction in the severity and / or frequency of symptoms, elimination of symptoms and / or underlying causes, prevention of the onset of symptoms and / or their underlying causes, and amelioration or correction of the disease. Such treatment does not require complete amelioration of the disease. For example, treatment of a subject exhibiting a neurodegenerative disease by administering an oligodendrocyte precursor differentiation compound of the invention can include inhibiting or causing regression of the disease. Furthermore, such treatment can be used in conjunction with other conventional treatments for neurodegenerative diseases known to those skilled in the art.

[0230] The pharmaceutical compositions of the present invention may be administered by any means that achieves their intended purpose. For example, administration can be by parenteral, subcutaneous, intravenous, intraarticular, intrathecal, intramuscular, intraperitoneal, or intradermal injection, or by transdermal, buccal, lingual, ocular routes, or via inhalation. Alternatively, or concurrently, administration can be by the oral route.

[0231] Pharmaceutical compound formulations for use in the above (and other) modes of administration include: These methods are known in the art and are described, for example, in Remington's Pharmaceutical Sciences (18th ed.), A. Gennaro, ed., 1990, Mack Publishing Company, Easton, Pa. (e.g., M.J. Rathbone, ed., Oral Mucosal Drug Delivery, Drugs and the Pharmaceutical Sciences Series, Marcel Dekker, Inc., NY, USA, 1996; M.J. Rathbone et al., eds., Modified-Release Drug Delivery Technology, Drugs and the Pharmaceutical Sciences Series, Marcel Dekker, Inc., NY, USA, 2003; Ghosh et al., eds., Drug Delivery to the Oral Cavity, Drugs and the Pharmaceutical Sciences Series, Marcel Dekker, Inc., NY, USA, 2005; and Mathiowitz et al., eds., Bioadhesive Drug Delivery Systems, Drugs and the Pharmaceutical Sciences Series, Marcel (See also Dekker, Inc., NY, USA, 1999). The compounds of the present invention can be formulated into pharmaceutical compositions comprising pharmaceutically acceptable, non-toxic excipients and carriers. Excipients are all components other than the active ingredient or ingredients present in a pharmaceutical formulation. 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 undesired biological side effects or undesired interactions with other medications. Suitable excipients and carriers are composed of materials that will not affect the bioavailability and performance of the drug."Excipients," as used throughout this specification, include, but are not limited to, surfactants, emulsifiers, emulsions, stabilizers, emollients, buffers, solvents, dyes, flavorings, binders, fillers, lubricants, and preservatives. Suitable excipients include those commonly known in the art, such as those described in "Handbook of Pharmaceutical Excipients," 4th Edition, Pharmaceutical Press, 2003.

[0232] The compounds can be administered to subjects to treat neurodegenerative diseases and disorders. Neurodegenerative diseases that may be considered for treatment by the methods of the present invention may result from, but are not limited to, inherited genetic abnormalities, stroke, heat stress, head and spinal trauma (blunt or infectious conditions), and / or bleeding occurring within the brain.

[0233] Neurodegenerative diseases contemplated for treatment by some aspects of the present invention may include myelin-related disorders. associated with demyelination, insufficient myelination and remyelination, or dysmyelination in a subject;The disorder may be any disease, condition (e.g., resulting from traumatic spinal cord injury and cerebral infarction), or disorder. As used herein, myelin-related disorders result from myelin loss resulting from myelination-related disorders or various neurotoxic insults. As used herein, "demyelination" refers to the process of demyelination, or the loss of the myelin sheath that insulates nerves, and is emblematic of several neurodegenerative autoimmune diseases, including multiple sclerosis, transverse myelitis, chronic inflammatory demyelinating polyneuropathy, and Guillain-Barré syndrome. Leukodystrophies result from inherited enzyme deficiencies that cause abnormal formation, destruction, and / or abnormal turnover of myelin sheaths within the CNS white matter. Both acquired and inherited myelin disorders share a poor prognosis and result in severe disability. Therefore, some embodiments of the present invention may include a method for treating a neurodegenerative autoimmune disease in a subject. Oligodendrocytes are required for neuronal remyelination. As used herein, the term "remyelination" refers to the regeneration of the myelin sheath of a nerve by replacing or restoring the function of myelin-producing cells.

[0234] Myelin-related diseases or conditions that may be treated or ameliorated by the methods of the present invention The disorders include diseases, disorders, or injuries associated with hypomyelinization or demyelination in brain cells, e.g., CNS neurons, of a subject, including, but not limited to, diseases and disorders in which the myelin surrounding neurons is absent, incomplete, improperly formed, or deteriorated. Such diseases include, but are not limited to, multiple sclerosis (MS), neuromyelitis optica (NMO), progressive multifocal leukoencephalopathy (PML), encephalomyelitis (EPL), central pontine myelin breakdown (CPM), adrenoleukodystrophy, Alexander disease, Pelizaeus-Merzbacher disease (PMD), vanishing white matter disease, Wallerian degeneration, optic neuritis, transverse myelitis, amyotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease, Parkinson's disease, spinal cord injury, traumatic brain injury, post-radiation injury, neurological complications of chemotherapy, stroke, acute ischemic optic neuropathy, vitamin E deficiency, isolated vitamin E deficiency syndrome, Bassen-Korn-Zweig syndrome, Marchia-Farba-Bignami syndrome, metachromatic leukodystrophy, trigeminal neuralgia, acute disseminated encephalitis, Guillain-Barré syndrome, Charcot-Marie-Tooth disease, Bell's palsy, and mental health disorders such as schizophrenia.

[0235] In some embodiments, a patient may be treated or improved by the methods of the present invention. Myelin-related diseases or disorders that may be of interest include leukodystrophies. Leukodystrophies are a group of progressive metabolic genetic disorders that affect the brain, spinal cord, and often peripheral nerves. Each type of leukodystrophy is caused by a specific genetic defect that leads to the abnormal development or destruction of the brain's myelin sheath. Each type of leukodystrophy affects a different part of the myelin sheath, resulting in various neurological problems. Exemplary leukodystrophies that may be treated or ameliorated by the methods of the invention include, but are not limited to, adult-onset autosomal dominant leukodystrophy (ADLD), Aicardi-Goutières syndrome, Alexander disease, CADASIL, Canavan disease, CARASIL, cerebrotendinous xanthomatosis, childhood ataxia and cerebral hypomyelination (CACH) / vanishing white matter disease (VWMD), Fabry disease, fucosidosis, GM1 gangliosidosis, Krabbe disease, L-2-hydroxyglutaric aciduria, macrocephalic leukoencephalopathy with subcortical cysts, metachromatic leukodystrophy, multiple sulfatase deficiency, Pelizaeus-Merzbacher disease (PMD), Pol III-associated leukodystrophy, Refsum disease, Salla disease (free sialic acid storage disease), Sjogren-Larsson syndrome, X-linked adrenoleukodystrophy, and Zellweger syndrome spectrum disorder.

[0236] Myelin-related diseases or conditions that may be treated or ameliorated by the methods of the present invention Disorders include diseases or disorders characterized by myelin defects. Insufficient myelination in the central nervous system is involved in a wide range of neurological disorders. One of these is a form of cerebral palsy in which a congenital defect in forebrain myelination contributes to the neurological pathology in children who present with periventricular leukomalacia (Goldman et al., 2008). Goldman, SA, Schanz, S., and Windrem, MS (2008). Stem cell-based strategies for treating pediatric disorders of myelin. Hum Mol Genet. 17, pp. 76–83. At the other end of the age spectrum, myelin loss and ineffective repair may contribute to the cognitive decline associated with aging (Kohama et al., 2011). Kohama, SG, Rosene, DL, and Sherman, LS (2011). Age (Dordr). Age-related changes in human and non-human primate white matter: from myelination disturbances to cognitive decline. It is therefore contemplated that effective compounds and methods that enhance myelination and / or remyelination may have substantial therapeutic benefit in halting disease progression and recovery function in MS and in a wide range of neurological disorders.

[0237] In some embodiments, the compounds of the present invention are useful for treating myelin-related disorders. In some embodiments, the compounds described herein can be administered to subjects who have and / or are not suspected of having myelin-dependent processes to enhance or promote myelin-dependent processes. can be administered to a subject to promote myelination of CNS neurons, enhancing cognition in cognitively healthy subjects, a process known to be myelin-dependent. In certain embodiments, the compounds described herein can be administered in combination with cognitive enhancement (nootropic) agents. Exemplary agents include any drug, supplement, or other substance that improves cognitive function, particularly executive function, memory, creativity, or motivation in healthy individuals. Non-limiting examples include racetams (e.g., piracetam, oxiracetam, and aniracetam), dietary supplements (e.g., bacopa monnieri, panax ginseng, ginkgo biloba, and GABA), stimulants (e.g., amphetamine medications, methylphenidate, eugeloic, xanthines, and nicotine), L-theanine, tolcapone, levodopa, atomoxetine, and desipramine.

[0238]

[0202] In one particular embodiment of the present invention, the treatment of multiple sclerosis in a subject is contemplated. The method includes administering to the subject a therapeutically effective amount of one or more oligodendrocyte differentiation-promoting compounds described above.

[0239]

[0203] Multiple sclerosis (MS) is the most common demyelinating disease. It is believed that the body's inability to repair myelin results in nerve damage, causing multiple sclerosis-associated symptoms and increasing disability. The myelin loss observed in MS is not always permanent, and remyelination has been demonstrated in early stages of the disease. It is believed that the methods of the present invention can promote the differentiation of oligodendrocyte precursor cells in a subject, thus triggering endogenous remyelination.

[0240] In another particular aspect of the present invention, loss of myelin (demyelination) in a subject is Contemplated herein is the treatment of a genetic myelin disorder resulting from a Δ8,9-unsaturated sterol intermediate of the cholesterol biosynthetic pathway in OPCs. The method comprises administering to a subject a therapeutically effective amount of one or more agents that enhance and / or induce accumulation of a Δ8,9-unsaturated sterol intermediate of the cholesterol biosynthetic pathway in the OPCs described above. In certain embodiments, the genetic myelin disorder is a leukodystrophy, such as, but not limited to, Pelizaeus-Merzbacher disease (PMD).

[0241] Another strategy for treating a subject suffering from a neurodegenerative disease or disorder is to use therapeutic The therapeutically effective amount of a compound described herein is administered in combination with a therapeutically effective amount of an additional oligodendrocyte differentiation and / or proliferation inducer and / or anti-neurodegenerative disease agent, such as L-dopa, cholinesterase inhibitors, anticholinergics, dopaminergics, steroids, and immunomodulatory agents, including interferons, monoclonal antibodies, and glatiramer acetate.

[0242]

[0206] Thus, in a further aspect of the present invention, oligodendrocytes as described herein The progenitor differentiation and / or proliferation inducing compounds can be administered as part of a combination therapy, along with adjunctive therapies for treating neurodegenerative and myelin-related disorders.

[0243] The phrase "combination therapy" refers to any combination of oligodendrocyte precursor differentiation-inducing agents described herein. The present invention encompasses the administration of a therapeutic agent as part of a specific treatment regimen intended to provide beneficial effects from the synergistic action of these therapeutic agents, as well as combinations of oligodendrocyte precursor differentiation-inducing compounds and therapeutic agents. When administered as a combination, the oligodendrocyte precursor differentiation-inducing compound and therapeutic agent can be formulated as separate compositions. The administration of these therapeutic agents in a typical combination occurs over a defined period of time (usually minutes, hours, days, or weeks, depending on the combination selected).

[0244] "Combination therapy" refers to the administration of these therapeutic agents in a sequential manner, i.e., each treatment administration of the therapeutic agents at different times, as well as administering the therapeutic agents in a substantially simultaneous manner; is intended to encompass the administration of at least two of the therapeutic agents. Substantially simultaneous administration can be achieved, for example, by administering to the subject a single capsule having a fixed ratio of each therapeutic agent, or multiple single capsules for each therapeutic agent. Sequential or substantially simultaneous administration of each therapeutic agent can be achieved by any suitable route, including, but not limited to, oral, intravenous, intramuscular, and direct absorption through mucosal tissue. The therapeutic agents can be administered by the same route or by different routes. For example, a first therapeutic agent of a selected combination can be administered by intravenous injection, while the other therapeutic agent of the combination can be administered orally. Alternatively, for example, all therapeutic agents can be administered orally, or all therapeutic agents can be administered by intravenous injection. The order in which the therapeutic agents are administered is marginally important. "Combination therapy" can also encompass the administration of the therapeutic agents described above in further combination with other biologically active ingredients (such as, but not limited to, second and different therapeutic agents) and non-drug therapies (e.g., surgery).

[0245] In another aspect of the present invention, oligodendrocyte differentiation and / or Therapeutic agents administered in combination therapy with the proliferation-inducing compound can include at least one anti-neurodegenerative agent, such as, but not limited to, an immunotherapeutic agent.

[0246] Immunotherapeutic agents for use in the methods of the present invention include those that target the immune components of the disease. Therapies targeting the underlying acute inflammatory response during acute attacks in minute and / or relapsing-remitting multiple sclerosis may be available. Examples include, but are not limited to, immunomodulatory agents such as interferon beta-1a and beta-1b (Avonex and Betaseron, respectively), natalizumab (Copaxone), glatiramer acetate (Copaxone), or mitoxantrone.

[0247] The present invention is further illustrated by the following examples, which are included in the appended claims. It is not intended to limit the scope. [Example]

[0248] In this example, remyelination is achieved by targeting an abundant stem cell population in the CNS. We show that this is mediated by oligodendrocyte progenitor cells (OPCs), the primary source of new myelin-forming oligodendrocytes. Loss of myelin-producing oligodendrocytes in the central nervous system (CNS) underlies numerous neurological disorders, including multiple sclerosis and diverse genetic disorders. Using a high-throughput chemical screening approach, we demonstrate that stimulating oligodendrocyte formation promotes myelination from OPCs and in We have identified small molecules that functionally enhance remyelination in vivo. Here, we demonstrate that a broad range of these pro-myelinating molecules functions not through their canonical targets but by directly inhibiting a narrow range of enzymes in the cholesterol biosynthetic pathway: CYP51, sterol 14-reductase, and EBP. We found that chemical or genetic inhibition of these enzymes led to the accumulation of Δ8,9-unsaturated sterol intermediates, which, when independently supplied to OPCs, enhanced the formation of new oligodendrocytes. Functional studies showed that small molecule inhibitors of CYP51, sterol 14-reductase, and EBP induced the accumulation of Δ8,9-unsaturated sterols in human brain tissue in vitro and in mouse brain tissue in vivo. At the same dose, these molecules also enhanced the rate of myelination in vivo in a mouse model of lysolecithin-induced focal myelin loss. Taken together, our results provide a unifying mechanism of action for most known small molecule enhancers of oligodendrogenesis and key specific targets for the development of optimal remyelination therapies.

[0249] method

[0213] Non-statistical methods were used to predetermine the sample size. small molecule The identity and purity of the small molecules were verified by LC / MS before use. The following compounds were purchased as solids from Sigma-Aldrich: ketoconazole, miconazole, clotrimazole, fluconazole, fulvestrant, ifenprodil, benztropine, liothyronine, bexarotene, tamoxifen, ospemifene, GSK343, trans-U50488, and cholesterol. The following compounds were purchased as solids from Cayman Chemicals: clemastine, AY9944, YM53601, and Ro-48-8071. The following compounds were obtained as solids from Janssen Pharmaceuticals: R-trans-ketoconazole and S-trans-ketoconazole. Mevastatin was purchased as solid from Selleck Chemicals. The following compounds were purchased from Selleck Chemicals as 10 mM DMSO solutions: bifonazole, butoconazole, amorolfine, toremifene, EPZ005687, EPZ6438, UNC1999, hydroxyzine, ziprasidone, p-fluorohexahydro-sila-difenidol (abbreviated as Sigma H127 in the figures), vesamicol, raloxifene, L-745,870, TMB-8, pramoxine, varespladib, tanshinone-I, levofloxacin, nateglinide, abiraterone, allopurinol, detomidine, rivastigmine, beta-carotene, BEZ-235, scopolamine, and homatropine. Pirenzepine and telenzepine were purchased from Sigma-Aldrich as 10 mM DMSO solutions. Cholesterol biosynthetic intermediates: lanosterol, zymosterol, zymostenol, lathosterol, desmosterol, 7-dehydrodesmosterol, and T-MAS were purchased as solids from Avanti Polar Lipids. 14-Dehydrozymostenol (cholesta-8,14-dien-β-ol) was provided by Franz Bracher, Ludwig-Maximilians University of Munich. Imidazole 124, TASIN-1, and MGI39 were synthesized as reported.

[0250] Preparation of mouse OPCs To rigorously evaluate the effects of small molecules and genetic interventions on OPCs, All treatments were assayed on two batches of OPCs derived from epiblast stem cells, and key results were confirmed using mouse primary OPCs. OPCs were generated from two individual EpiSC lines, EpiSC5 (yielding OPC-5 OPCs) and 129O1 (yielding OPC-1 OPCs). Unless otherwise noted, results for OPC-5 cells are shown in Figures 1-4, while results for OPC-1 are shown in Figures 5-11.

[0251] OPCs derived from EpiSCs were cultured using in vitro differentiation procedures and pre-culture. OPCs were obtained using the culture conditions described above. To ensure uniformity across all in vitro screening experiments, EpiSC-derived OPCs were sorted for purity at passage 5 by fluorescence-activated cell sorting using conjugated CD140a-APC (eBioscience, 17-1401; 1:80) and NG2-AF488 (Millipore, AB5320A4; 1:100) antibodies. Batches of sorted OPCs were expanded, aliquoted, and frozen. OPCs were thawed and passaged once in growth conditions before use in further assays. Cultures were periodically tested and shown to be mycoplasma-free.

[0252] To obtain primary mouse OPCs, whole brain tissue was collected from anesthetized mouse pups 2 days postnatal. The brains were placed on ice, placed in chilled DMEM / F12, and the cortices were isolated and the meninges were removed. The cortices were manually minced and dissected using a Tumor Dissociation Kit (Miltenyi). The cells were processed using a PBS and incubated at 37°C for 10 minutes. The cell suspension was filtered through a 70 μM filter and centrifuged at 200 × g for 4 minutes at room temperature. The cells were washed with DMEM / F12, centrifuged again, and plated into polyornithine- and laminin-treated flasks containing DMEM / F12 supplemented with N2Max, B27 (ThermoFisher), 20 ng / mL FGF, and 20 ng / mL PDGF. Small molecules were passaged once before treatment. The medium was changed every 48 hours.

[0253] In vitro phenotypic screening of OPCs OPCs derived from EpiSCs were cultured in growth medium (N2-MAX (R&D Syst ( Cells were grown and expanded in polyornithine (PO)- and laminin-coated flasks containing DMEM / F12 supplemented with N2-MAX (N2-MAX), B-27 (ThermoFisher), GlutaMax (Gibco), FGF2 (10 μg / mL, R&D systems, 233-FB-025), and PDGF-AA (10 μg / mL, R&D systems, 233-AA-050), then harvested and plated. Cells were seeded onto poly-D-lysine 96-well CellCarrier plates (PerkinElmer) coated with laminin (Sigma, L2020; 15 μg / mL) using a multichannel pipette. For experiments, a stock solution of 800,000 cells / mL in differentiation medium (DMEM / F12 supplemented with N2-MAX and B-27) was prepared and stored on ice for 2 h. Then, 40,000 cells per well were seeded in differentiation medium and allowed to attach for 30 minutes before drug addition. For dose-response testing of all molecules except sterols, a 1000x compound stock solution in dimethyl sulfoxide (DMSO) was added to the assay plate with a 0.1 μL solid-pin multiblot replicator (V&P Scientific; VP409) to obtain a final primary screening concentration of 1x. Sterols were added to the cells as an ethanol solution (0.2% final ethanol concentration). A positive control well (ketoconazole, 2.5 μM) and a DMSO vehicle control were included in each assay plate. Cells were incubated under standard conditions (37°C, 5% CO2) for 3 days and then fixed with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 20 minutes. Fixed plates were washed twice with PBS (200 μL per well), permeabilized with 0.1% Triton X-100, and blocked with 10% (v / v) donkey serum in PBS for 40 min. Cells were then labeled with MBP antibody (Abcam, ab7349; 1:200) for 16 h at 4 °C, followed by detection with an Alexa Fluor-conjugated secondary antibody (1:500) for 45 min. Nuclei were visualized by DAPI staining (Sigma; 1 μg / ml).During the washing steps, PBS was added using a multichannel pipette and aspiration was performed using a Biotek EL406 washer dispenser (Biotek) equipped with a 96-well aspiration manifold.

[0254] High-Content Imaging and Analysis The plates were then scanned using an Operetta high content imaging and analysis system ( Imaging was performed with a PerkinElmer (PerkinElmer Harmony and Columbus software), and a series of 6 fields were captured from each well, scoring an average of 1200 cells per well. Analysis (PerkinElmer Harmony and Columbus software) was performed on intact nuclei stained with DAPI; i.e., cells with a surface area of ​​300 μm 2 We began by identifying the larger traced nuclei. Each traced nuclear area was then magnified by 50% and cross-referenced with mature myelin protein (MBP) staining to identify oligodendrocyte nuclei, from which the percentage of oligodendrocytes was calculated. In some experiments, the total process length of MBP+ oligodendrocytes was calculated as described above.

[0255] High-throughput screening of 3,000 bioactive small molecules OPCs derived from EpiSCs were cultured on polyornithine- and laminin-coated flasks. The cells were grown and expanded in PBS, then harvested and plated on poly-D-lysine / laminar cells. Cells were dispensed into sterile, 384-well CellCarrier Ultra plates (PerkinElmer) coated with PEG-1 (Sigma, L2020; 4 μg / ml) in differentiation medium supplemented with noggin (R&D Systems; 100 ng / ml), neurotrophin 3 (R&D Systems; 10 ng / ml), cAMP (Sigma; 50 μM), and IGF-1 (R&D Systems; 100 ng / ml) using a Biotek EL406 microplate washer dispenser (Biotek) with a 5 μL dispense cassette (Biotek) to a final density of 12,500 cells per well. Cells were allowed to attach for 45 minutes before drug addition. A stock solution of the bioactive compound library at 3 mM in dimethyl sulfoxide (DMSO) was prepared in Abgene storage 384-well plates (ThermoFisher Scientific; AB1055). These were added to the assay plate using a 50 nL solid pin tool attached to a Janus automated workstation (PerkinElmer) to a final screening concentration of 2 μM. Cells were incubated at 37°C for 1 hour, and then T3 (Sigma; 40 ng / ml) was added to all wells except the negative control, which was replaced with FGF (20 ng / ml). The negative control and T3 alone were included in each assay plate. After 72 hours of incubation at 37°C, cells were fixed, washed, and stained similarly to the 96-well OPC assay procedure, except that all washing steps were performed using a Biotek EL406 microplate washer dispenser (Biotek) equipped with a 96-well vacuum manifold. Cells were stained with DAPI (Sigma; 1 μg / ml) and MBP antibody (Abcam, ab7349; 1:100). Plates were imaged with an Operetta high-content imaging and analysis system (PerkinElmer), capturing a series of four fields from each well and scoring an average of 700 cells per well. Analysis was performed as described above for high-content imaging and analysis. All plates for the primary screen were processed and analyzed simultaneously to minimize variability.Molecules that showed a >20% reduction in nuclei relative to DMSO control wells were removed from consideration, and hit compounds were determined based on the maximum fold increase in the percentage of MBP+ oligodendrocytes relative to DMSO controls within the same plate. When selecting lead hit compounds for further experimentation, molecules from previous screens, including imidazole antifungals and clemastine, were removed.

[0256] GC / MS-based sterol profiling OPCs derived from EpiSCs were cultured at 1 million cells per well in differentiation medium Cells were plated onto PDL- and laminin-coated 6-well plates containing 1000 mg of ... 2The mixture was dried under a stream of nitrogen and derivatized with 55 μl of bis(trimethylsilyl)trifluoroacetamide / trimethylchlorosilane to form the trimethylsilyl derivative. Following derivatization at 60°C for 20 minutes, 1 μl was analyzed by gas chromatography / mass spectrometry using an Agilent 5973 network mass selective detector equipped with a 6890 gas chromatography system and an HP-5MS capillary column (60 m x 0.25 μm x 0.25 mm). Samples were injected in splitless mode and analyzed using electron impact ionization. Ion fragment peaks were integrated, and sterol abundance was calculated. Quantification was performed relative to cholesterol-d7. The following m / z ion fragments were used to quantify each metabolite: cholesterol-D7 (465), FF-Mas (482), cholesterol (368), zymostenol (458), zymosterol (456), desmosterol (456, 343), 7-dehydrocholesterol (456, 325), lanosterol (393), lanosterol (458), and 14-dehydrozymostenol (456). Calibration curves were generated by injecting different concentrations of sterol standards and maintaining a fixed amount of cholesterol-D7. The human glioma cell line GBM528 was a gift from Jeremy Rich (Cleveland Clinic). Cortical organoids were prepared as previously described.

[0257] CYP51 enzyme assay CYP51 enzyme activity was measured using a reported method with the following slight modifications. The enzyme used was rat CYP51 (Cypex, Inc.); the reaction volume was 500 μl; the reaction time was 30 minutes; the lanosterol concentration was 50 μM; and the reaction was quenched with 500 μl of isopropanol. Finally, 15 μl of each reaction / isopropanol mixture was injected into a SCIEX Triple Quad 6500 LC-MS / MS system equipped with a Shimadzu UFLC-20AD HPLC and a Phenomenex Kinetix C18XB50×2.1×2.6 column at 40°C, using an APCI ion source in positive ion mode.

[0258] EBP enzyme assay EBP enzyme activity was measured using a published method with the following slight modifications. Active EBP was obtained from mouse microsomes, inhibitors were added, and zymostenol was added to a final concentration of 25 μM in a final reaction volume of 500 μl, and the reaction was incubated at 37°C for 2 hours. Sterols were extracted using 3 × 1 ml hexane, cholesterol-d7 was added to allow quantification, and the pooled organics were dried (NaSO) and evaporated under nitrogen gas. Samples were then silylated and analyzed using GC / MS as described above.

[0259] siRNA treatment Cell-permeable siRNAs were prepared by combining four individual mouse CYP51-targeting siRNAs. siRNAs were obtained as a pool, or a non-targeting control (Accell siRNAs, Dharmacon). For differentiation analysis, cells were plated in 96-well plates (as described above) and treated with 1 μM pooled siRNA suspended in RNase-free water diluted in differentiation medium (as described above). For sterol analysis, cells were plated at 300,000 cells per well in 6-well plates in standard differentiation medium supplemented with PDGF (R&D Systems, 20 ng / ml), neurotrophin 3 (R&D Systems; 10 ng / ml), cAMP (Sigma; 50 μM), IGF-1 (R&D Systems; 100 ng / ml), and noggin (R&D Systems; 100 ng / ml). At 24 hours, 1 μM siRNA was added to the medium. Cells were grown in siRNA-containing medium for an additional 3 days, with growth factor replenishment every 48 hours, before being harvested, processed, and analyzed by GC / MS as described above.

[0260] Local myelin loss, drug treatment and histological analysis

[0225] Local myelin loss in the dorsal columns of the spinal cord was induced by injecting a 1% LPC solution. Twelve-week-old C57BL / 6 female mice were anesthetized using isoflurane and underwent a T10 laminectomy. 1 μl of 1% LPC was injected into the dorsal column at a rate of 15 μl / h. On day 4, animals were assigned to treatment groups and then treated (two animals were excluded due to surgical complications). Between days 4 and 11 after laminectomy, animals received daily intraperitoneal injections of either vehicle or drug. Drugs were dissolved in DMSO or corn oil and then diluted with sterile saline for injection to a final dose of 2 mg / kg for tamoxifen and 10 mg / kg for ifenprodil. This experiment was performed in a blinded manner: compounds were coded to ensure that the researchers conducting the experiment were unaware of the treatment administered to each animal. 12 days after laminectomy, all animals were euthanized (n = 4–6 per group). Mice were anesthetized using a ketamine / xylazine rodent cocktail and then euthanized by transcardial perfusion with 4% PFA, 2% glutaraldehyde, and 0.1 M sodium cacodylate. Samples were osminated, stained globally with uranyl acetate, and embedded in EMbed 812, an Epon-812 substitute (EMS). 1 μm sections were cut, stained with toluidine blue, and visualized under a light microscope (Leica DM5500B). The number of myelinated axons per unit area was counted from sections at the center of each lesion and then averaged across treatment groups. Statistical significance was assessed using Mann-Whitney statistical analysis.

[0261] Analysis of mouse brain sterol levels

[0226] Male C57BL / 6 mice, aged 10-12 weeks, were given corn oil in sterile saline. Mice were injected daily for 3 days with 2 mg / kg tamoxifen, 10 mg / kg ifenprodil, or 10 mg / kg miconazole dissolved in DMSO (tamoxifen) or DMSO (ifenprodil, miconazole). Mice were anesthetized with isoflurane, perfused with phosphate-buffered saline, and blood was removed from the brain. Brains were collected and flash-frozen using liquid nitrogen. Samples were pulverized, and 50–100 milligrams of tissue were collected for further processing. Sterols were extracted using a modified Forch protocol. Briefly, samples were resuspended and homogenized in a 2:1 chloroform / methanol mixture. Cellular debris was removed by centrifugation at 4000 g for 10 minutes. The solution was dried under air, resuspended in hexane with cholesterol-D7 standards, and dried again. Lipids were derivatized with 70 μl of bis(trimethylsilyl)trifluoroacetamide; 2 μl was injected and analyzed by GC / MS as described above.

[0262] Estrogen-dependent cell proliferation assay Estrogen-dependent cell proliferation was measured as previously described with minor modifications. After 5 days of growth in estrogen-free medium (phenol red-free RPMI supplemented with 10% charcoal-stripped fetal bovine serum), cells were seeded at 2,500 cells / well in 96-well plates. The day after 3x drug-containing medium was added to triplicate wells, cells were grown for an additional 5 days at 37°C in a standard 5% CO2 humidified incubator. Total DNA per well was measured using an adaptation of the Labarca and Paigen method. At this point, the medium was removed, cells were washed once with 0.25x PBS, and 100 μl of distilled water was added. Plates were frozen and thawed to enhance cell lysis, and 200 μl of 10 μg / ml Hoechst 33258 (Sigma-Aldrich, St. Louis, MO) in 2 M NaCl, 1 mM EDTA, 10 mM Tris-HCl pH 7.4 was added. After 2 hours of incubation at room temperature, plates were read on a SpectraMax i3 fluorescent plate reader (Molecular Devices, Sunnyvale, CA) with excitation at 360 nm and emission at 460 nm. All values ​​were converted to micrograms of DNA per well using a standard curve derived from purified salmon test DNA.

[0263] Oligodendrocyte formation and imaging on electrospun microfibers

[0228] Mimetex Alignment Scaffold (Microfiber Plate, AMS 12-well plates containing BIO, AMS.TECL-006-1X, electrospun poly-L-lactide scaffolds, and 2 μM fiber diameter cell crown inserts were prepared as previously described. Briefly, inserts were sterilized with 70% ethanol, washed with PBS, and then coated with polyornithine and laminin. After laminin coating, EpiSC-derived OPCs were plated at 100,000 cells / mL in differentiation medium. After 24 hours, the medium was replaced with fresh medium containing small molecule treatment. The medium was replaced with fresh compound-containing medium every 48 hours for a total of 14 days. Plates were fixed with 4% PFA, permeabilized with 0.1% Triton X-100, and blocked with 10% (v / v) donkey serum in PBS for 60 minutes. Plates were stained for MBP (Abcam, ab7349; 1:100) and DAPI stain (Sigma; 5 μg / mL). After staining, the inserts were transferred into a new 12-well plate, covered with 2 mL of PBS, and then imaged using an Operetta High-Content Imaging and Analysis System. Plates were imaged using an Operetta High-Content Imaging and Analysis System (PerkinElmer), capturing a series of eight fields from each well and scoring an average of 45,000 cells per well. Analysis (PerkinElmer Harmony and Columbus software) identified intact DAPI-stained nuclei and calculated MBP signal intensity per cell per well. Microfiber insert tracking images were taken using a Leica DMi8 with a 20x Dry / NA 0.40 objective. Microfiber plate inserts were mounted using Flouromount-G (SouthernBiotech) and partially cured before adding a coverslip and removing the insert ring. Confocal images were obtained using a Leica SP8 confocal scanning microscope with a 40x oil immersion / NA 1.30 objective. Confocal stacks with 0.336 μm z-steps were taken at 1024 × 1024. Each fluorophore was excited sequentially and all contrast and brightness changes were applied consistently between images.

[0264] CYP51qPCR Cells were plated in 6-well plates at 500,000 cells per well. Cells were grown for 4 days in standard differentiation medium supplemented with PDGF, neurotrophin 3, cAMP, IGF-1, and noggin, as described above. At 24 hours, cells were treated with 1 μM siRNA. Growth factors were added every 48 hours. Three days after siRNA treatment, RNA was isolated using the RNeasy Mini Kit (Qiagen), and cDNA was generated using the High-Capacity RNA-to-cDNA™ Kit (Applied Biosystems). Relative RNA levels were detected by quantitative real-time PCR (Applied Biosystems 7300 Real-Time PCR System) using exon-spanning primers for actin B (Thermo-Fisher, Taqman, Mm02619580_g1) and CYP51 (Thermo-Fisher, Taqman, Mm00490968_m1). Cycle times and outliers were calculated using Applied Biosystems 7300 System Sequence Detection Software version 1.4.

[0265] Muscarinic receptor antagonism assay GeneBLAzerM1-NFAT-blaCHO-K1 cells (or M3 M5-NFAT-bla or M5-NFAT-blaCHO-K1 cells (ThermoFisher) were thawed in assay medium (DMEM, 10% dialyzed FBS, 25 mM HEPES pH 7.3, 0.1 mM NEAA). 10,000 cells / well were added to a 384-well TC-treated assay plate and incubated at 37°C for 16–24 hours. 4 μl of a 10× stock solution of antimuscarinic molecule was added to the plate and incubated for 30 minutes. 4 μl of 10× control agonist carbachol was added to wells containing the antimuscarinic molecule at a predetermined EC80 concentration. The plate was incubated for 5 hours, and then 8 μl of 1 μM substrate + Solution D loading solution was added to each well. The plate was incubated for 2 hours at room temperature before being read on a fluorescent plate reader.

[0266] result High-throughput phenotypic screening of novel oligodendrocytes from OPCs Remyelination has emerged as a promising pathway for identifying small molecules that enhance the production of oligodendrocytes. Multiple groups have identified screening hit compounds that demonstrate functional benefits in animal models of demyelination. However, translating these discoveries to humans has been hampered by a lack of knowledge of the functional targets of these molecules in enhancing oligodendrocyte formation. Previously, we used OPCs derived from mouse pluripotent stem cells to identify structurally diverse imidazole antifungal drugs as a robust class of hit compounds that stimulate the generation of new mouse and human oligodendrocytes and enhance remyelination in mouse disease models. Imidazole antifungals are known to mediate their effects in yeast by inhibiting sterol 14-α-methyldehydrogenase (CYP51), an enzyme essential for sterol biosynthesis in both fungal and mammalian cells. However, the mechanism of action of imidazole antifungal drugs in OPCs remains undefined.

[0267] CYP51 inhibition may be a potential therapeutic target for imidazoline in promoting oligodendrocyte generation from OPCs. To test whether azole antifungal drugs are responsible for their efficacy, we assembled a collection of nine azole-containing molecules with a wide range of potencies for mammalian CYP51 inhibition (Figure 1A). We used a mass spectrometry-based biochemical assay to identify similar IC values, ranging from 300 to 700 nM. 50We confirmed that FDA-approved imidazole antifungal agents (miconazole, clotrimazole, and ketoconazole) with known antifungal activity exhibited significant inhibition of rodent CYP51 in vitro (Figure 1B). Three close analogs of ketoconazole known to lack antifungal activity: the R-trans and S-trans diastereomers of ketoconazole and the truncated analog imidazole 124, were not significantly effective at inhibiting mammalian CYP51 (Figure 1A, B). Furthermore, we confirmed that fluconazole, a triazole antifungal agent that selectively targets yeast CYP51, did not inhibit mammalian CYP51 in vitro (Figure 1B). For each molecule in our panel, the potency of inhibiting mammalian CYP51 in vitro paralleled the enhanced formation of mature myelin basic protein-positive (MBP+) oligodendrocytes from OPCs derived from mouse epiblast stem cells (Figure 1C, D). These findings suggest that imidazole antifungals act through their canonical target, CYP51, to enhance oligodendrocyte formation.

[0268] CYP51 functions in cholesterol biosynthesis in mammalian cells. Given the known role of CYP51 in OPCs, we evaluated the functional inhibition of CYP51 in OPCs using gas chromatography / mass spectrometry (GC / MS) to measure cellular sterol levels. Mouse OPCs were treated with each azole-containing molecule for 24 hours, at which point they were lysed for analysis by GC / MS to quantify the levels of lanosterol, a CYP51 substrate, as well as downstream cholesterol levels. Lanosterol accumulated in OPCs only after treatment with each of the five active imidazole antifungals, reflecting the effects of these molecules on CYP51 function in our biochemical assays (Figures 1B, E, 5A). Notably, to exclude potential cell source or assay artifacts, we confirmed all effects of small molecules on oligodendrogenesis sterol levels using an independently isolated second batch of OPCs derived from mouse epiblast stem cells (Extended Data Figures 1b-d; see Methods for details on OPC induction). Furthermore, we confirmed the effects of azole molecules on oligodendrocyte formation and lanosterol levels using primary mouse OPCs (Figure 5E, F), an orthogonal image quantification approach measuring total process length (Figure 1G), and PLP1 as a second marker of oligodendrocyte formation (Figure 1H). For ketoconazole, the dose-response for lanosterol accumulation closely resembled the dose-response for enhanced oligodendrocyte formation (Figure 1F, 1C; compare Figure 5I, 5B). Among these structurally diverse azole-containing small molecules, the close correlation between CYP51 inhibition and enhanced oligodendrocyte formation suggests that CYP51 is a suitable target in OPCs.

[0269] Next, we used genetic manipulation and metabolite supplementation to induce the growth of oligodendrocytes. We independently confirmed the role of CYP51 in the formation of MBP+ oligodendrocytes. We used a cell-permeable siRNA reagent to dramatically reduce CYP51 transcript levels in OPCs by 80% (Figure 1G). Suppression of CYP51 resulted in a significant accumulation of lanosterol and increased MBP+ oligodendrocyte potency. Although enhanced formation occurred, this effect was smaller than that observed with ketoconazole treatment, likely due to the slow kinetics and incomplete target suppression of siRNA treatment (Figures 1H, I, 5J; confirmed with an independent batch of OPCs in Figures 5K, L). Furthermore, we directly treated OPCs with purified lanosterol and observed enhanced formation of MBP+ oligodendrocytes in a dose-response manner (confirmed in Figures 1J, 5M, 5N). This finding suggests that the accumulation of sterol intermediates may play a direct role in enhancing oligodendrocyte formation from OPCs.

[0270]

[0235] CYP51 inhibition is sufficient to induce oligodendrocyte formation, We used a chemical genetic approach to test whether altering other steps in cholesterol biosynthesis would have similar effects. Cholesterol biosynthesis is a long, complex, and regulated pathway, and many high-quality small molecule probes and approved drugs are available (Figure 2A, Figure 6). We collected selective small molecule inhibitors of eight enzymes spanning the cholesterol biosynthesis pathway and evaluated their effects on oligodendrocyte production and sterol levels in OPCs. We confirmed that inhibitors selectively targeting sterol metabolic enzymes resulted in the accumulation of the expected upstream sterol intermediates in OPCs, and we confirmed a reduction in the levels of one or both of the pathway's end products, desmosterol and cholesterol, for all probes (Figures 7A-D). We evaluated the effect of each of these eight pathway inhibitors on OPC oligodendrocyte differentiation. Only molecules targeting CYP51 (ketoconazole), sterol 14-reductase (amorolfine), and EBP (TASIN-1) enhanced MBP+ oligodendrocyte formation, whereas inhibitors of the remaining five pathway enzymes were ineffective (Figure 2B, Figure 7E, confirmed in Extended Data Figure 3F and validated with primary OPCs in Figure 7G,H). Treatment had minimal effects on total cell number during the 3-day assay (Figure 7E). Amorolfine and TASIN-1 were effective at doses below 100 nM and had the potency to accumulate 14-dehydrozymostenol and zymostenol, reflecting their potency in enhancing oligodendrocyte formation (Figure 2C, Figure 7I). Direct treatment of OPCs with 14-dehydrozymostenol and zymostenol enhanced MBP+ oligodendrocyte formation, whereas the remaining sterols associated with downstream steps of EBP, including cholesterol itself, had no effect (Figure 2D; Figures 7J, K). Overall, this chemical genetic analysis suggests that inhibiting the cholesterol biosynthetic pathway within a narrow therapeutic window spanning from CYP51 to EBP is sufficient to enhance oligodendrocyte formation.The mechanism of this effect is not mediated by a simple reduction in sterol levels, as we identified statin drugs and various other pathway inhibitors that deplete cholesterol and desmosterol levels, but they did not modulate OPC differentiation (Figure 2B, Figure 7A, B). Notably, the sterol intermediates that accumulate after inhibiting the step between CYP51 and EBP are unified by the presence of Δ8,9 unsaturation, suggesting a chemical basis for their functional effect in enhancing oligodendrocyte formation (Figure 6).

[0271] In parallel, we have identified over 3,000 bioactive small molecules and approved We achieved screening across a range of drugs at a uniform dose of 2 μM (Figure 8A). This library includes many previously screened approved drugs, as well as a wide range of unapproved drug candidates and well-annotated chemical probes. Among our hit compounds, we obtained nine imidazole antifungal agents and other molecules already annotated, including clemastine, as potentiating OPC differentiation (Extended Data Table 1). We also identified numerous confirmed hit compounds that could not be easily classified into distinct categories using known targets (Extended Data Table 1). Among molecules not yet reported to regulate OPC differentiation, our top hit was EPZ005687, a histone methyltransferase EZH2 inhibitor. EPZ005687 enhanced oligodendrogenesis in both OPCs derived from our mouse epiblast stem cells and mouse primary OPCs (Figure 8B–F). Surprisingly, we verified that three structurally analogous EZH2 inhibitors had no effect on OPC differentiation, suggesting that EPZ005687 has specific off-target effects beyond EZH2 (Figure 8C, D, F). We examined the effects of these four EZH2 inhibitors in our GC / MS-based sterol profiling assay and found that EPZ005687 uniquely led to the accumulation of zymosterol and zymostenol in OPCs, indicating EBP inhibition (Figure 8G-I). Among these four closely related EZH2 inhibitors, structural differences associated with the presence of morpholine and indazole rings likely allow EPZ005687 alone to inhibit EBP and enhance oligodendrocyte formation in OPCs.

[0272] [Table 1-1]

[0273] [Table 1-2]

[0274]

[0237] The inventors have identified the top 10 hits after EPZ005687. The compounds (excluding imidazole antifungals and other molecules identified in previously published screens) were further examined, and all 10 showed a significant reduction in sterol activity at the screening dose. We found that the molecules induced changes in the cytotoxicity profile (Figures 3A-B, 9A-B). Seven molecules inhibited EBP, two molecules inhibited sterol 14-reductase activity, and one molecule (fulvestrant) targeted CYP51 (Figures 9C-D). Four of these molecules: ziprasidone, ifenprodil, hydroxyzine, and raloxifene, have previously been shown to modulate sterol 14-reductase or EBP activity in CNS-derived cells. Of the 10 library molecules that we independently confirmed did not affect OPC oligodendrocyte differentiation at screening doses, none enhanced levels of 8,9-unsaturated sterol intermediates (Figures 3A-B, 9A-B). These data indicate that modulation of sterol synthesis appears to be the primary mechanism of action for enhancing oligodendrocyte formation among small molecules identified by high-throughput screening.

[0275] Cholesterol within our top screening hits Given the frequency of pathway modulators, we assessed whether any previously reported enhancers of remyelination also induce the accumulation of sterol intermediates. We assembled a collection of molecules reported to induce OPC differentiation across a variety of canonical targets: benztropine (muscarinic receptors), clemastine (H1 and muscarinic receptors), tamoxifen (estrogen receptors), U50488 (κ-opioid receptors), bexarotene (RXR), and liothyronine (thyroid hormone receptor). We identified the dose at which each molecule showed near-maximal upregulation of oligodendrocyte formation and then evaluated each molecule in our GC / MS sterol profiling assay (Figure 9E-G). Benztropine, clemastine, tamoxifen, and U50488 induced the accumulation of zymostenol and zymosterol and a decrease in basal sterol levels, indicating inhibition of EBP (Figures 3C-D, 9H). Tamoxifen has previously been shown in biochemical assays to inhibit EBP in cell culture models and in cancer patients undergoing chemotherapy. In contrast, liothyronine and bexarotene showed minimal effects on sterol levels, consistent with their known function as regulators of transcription factor function, confirming that many, but not all, treatments that enhance oligodendrogenesis result in the accumulation of sterol intermediates.

[0276] In OPCs after treatment with clemastine, tamoxifen, or other small molecules EBP inhibition in OPCs may result from direct targeting of EBP or reflect downstream consequences of each molecule inhibiting its canonical protein target. We evaluated direct inhibition of EBP in vitro using a GC / MS-based biochemical assay of EBP enzymatic activity. We observed clear inhibition by the selective EBP inhibitor TASIN-1, as well as by benztropine, clemastine, tamoxifen, and U50488, with more potent cellular EBP inhibitors exhibiting greater magnitude of inhibition (Figure 3E). We also annotated two molecules identified in our bioactivity screen, EPZ005687 and hydroxyzine, as directly inhibiting EBP enzymatic activity in this biochemical assay, suggesting that many enhancers of oligodendrogenesis directly target EBP in OPCs.

[0277]

[0240] The present inventors have developed a muscarinic receptor antagonist and selective estrogen receptor modulator. We sought additional evidence that drugs (SERMs) mediate enhanced oligodendrogenesis in OPCs by acting on EBPs as their functional targets. While clemastine and benztropine have been validated as inducers of OPC oligodendrocyte differentiation, previous studies suggested that many other muscarinic receptor antagonists do not share this functional property. Using our bioactivity screening data, we identified four muscarinic receptor antagonists with varying isoform selectivity. We independently confirmed that all four of these receptor antagonists did not enhance MBP+ oligodendrocyte formation at 2 μM (Figure 10A–C). However, independent cellular activity assays were also performed at 2 μM, demonstrating that these four molecules and clemastine exhibited comparable and nearly complete inhibition of muscarinic receptor M1, M3, and M5 isoforms, suggesting that muscarinic receptors are not functional targets in OPCs (Figure 10D). In contrast to clemastine and benztropine, which enhance 8,9-unsaturated sterol accumulation and directly inhibit EBP enzyme activity, profiling cellular sterol levels for muscarinic receptor antagonists that do not enhance oligodendrocyte formation revealed no effect on zymostenol or other sterol intermediates in OPCs and no inhibition of EBP, as observed in enzyme activity assays (Figure 3E, Figure 10E–F). These findings suggest that only muscarinic receptor antagonists that inhibit EBP can enhance oligodendrocyte formation.

[0278] The ability to inhibit EBP in OPCs also allows selective estrogen Enhanced oligodendrocyte formation is predicted among steroid receptor modulators (SERMs). Many SERMs have been shown to potently target EBP in other cell types, and the tertiary amine functionality of SERMs, such as tamoxifen and toremifene, is thought to mimic the sterol C8 cation-like transition state of EBP when protonated at physiological pH. We confirmed that toremifene inhibited EBP over a wide dose range and enhanced MBP+ oligodendrocyte formation (Figure 10G-L). Ospemifene is also an FDA-approved SERM that is structurally identical to toremifene, except that the tertiary amine functionality of toremifene is replaced with a primary alcohol. This change would prevent ospemifene from possessing a positive charge at physiological pH and should eliminate its EBP inhibitory activity. Indeed, ospemifene did not inhibit EBP in OPCs or enhance differentiation into MBP+ oligodendrocytes (Figure 10G-L). Notably, toremifene and ospemifene demonstrated comparable antiestrogenic efficacy in independent estrogen-dependent cell proliferation assays, suggesting that among structurally nearly identical SERMs, the ability to inhibit EBPs and not modulate estrogen receptors predicts enhanced oligodendrogenesis ( Figure 10L ).

[0279] Our results suggest that sterol modulation enhances oligodendrocyte formation in many To suggest a shared function for some (but not all) compounds, we tested the efficacy of small molecule combinations, demonstrating additive or non-additive effects. Combining the thyroid hormone agonist liothyronine with various sterol-modulating OPC differentiation-inducing treatments produced additive effects on oligodendrogenesis, indicating that these molecules likely function to enhance oligodendrogenesis through mechanisms other than thyroid hormone receptor signaling (Figures 11A and 11B). In contrast, combining maximally effective doses of ketoconazole with any of four previously reported OPC differentiation enhancers (benztropine, clemastine, tamoxifen, and U50488) did not enhance differentiation beyond the levels observed with ketoconazole alone (Figures 3F and 11C). This non-additive effect is consistent with these molecules sharing 8,9-unsaturated sterol accumulation as a common mechanism for inducing oligodendrogenesis. By blocking the pathway flow in CYP51 with ketoconazole, the EBP inhibitor can no longer lead to further sterol accumulation or enhance oligodendrogenesis.

[0280] Our in vitro OPC assay demonstrates that early OPC development leads to oligodendrocytes. Since this is only a model of differentiation events, we next tested whether modulating the sterol pathway also enhances subsequent oligodendrocyte maturation and myelination in vitro and in vivo. We cultured OPCs on microfibers for 14 days and assessed the effects of sterol pathway modulators on the ability of oligodendrocytes to track along and envelop axon-like substrates. The functions of ketoconazole (CYP51), amorolfine (sterol 14-reductase), and TASIN-1 (EBP), which accumulate sterol intermediates in OPCs, significantly enhanced MBP+ oligodendrocytes' ability to track along and envelop microfibers (Fig. 12A–C). Inhibition of other upstream or downstream enzymes in the pathway had no effect on oligodendrocyte maturation and microfiber ensheathment (Fig. 12A–B).

[0281]

[0244] Previously, the present inventors have reported on the imidazole antifungal agent miconazole, which targets CYP51. We established that miconazole penetrates the mouse blood-brain barrier and enhances remyelination in a mouse model of demyelination. To assess whether inhibition of other sterol pathway enzymes could also enhance in vivo remyelination, we selected one inhibitor of sterol 14-reductase (ifenprodil) and one inhibitor of EBP (tamoxifen) for further evaluation. Both ifenprodil and tamoxifen are known to cross the mouse blood-brain barrier. We first used GC / MS-based sterol profiling to test their engagement in vivo in the CNS. Miconazole (10 mg per kg body weight), ifenprodil (10 mg per kg), and tamoxifen (2 mg per kg) each induced significant accumulation of 8,9-unsaturated sterols in the brains of wild-type adult mice 3 days after intraperitoneal administration (Figure 4A). These data demonstrate that the sterol-modulating drugs miconazole, ifenprodil, and tamoxifen can functionally engage CYP51, sterol 14-reductase, and EBP, respectively, in the mouse CNS.

[0282] We have used lysolecithin injection to induce myelination in the dorsal column white matter of the adult spinal cord. We have previously demonstrated the positive effect of miconazole on remyelination using a well-established mouse model that generates focal lesions of dephosphorylated steroids. To test whether the accumulation of other 8,9-unsaturated sterols enhances remyelination in vivo, we treated lesioned mice with ifenprodil (10 mg per kg) or tamoxifen (2 mg per kg) by daily intraperitoneal injection. Treatment began 4 days after the lesion, and the effect on remyelination was quantified histologically 8 days later (Figure 4B). In vehicle-treated animals, sparsely distributed profiles of remyelinated axons characterized by thin myelin sheaths were detected primarily at the periphery of the lesion (Figure 4C). Concurrently, ultrastructural analysis revealed unmyelinated axons or axons singly wrapped in myelin (Figure 4D). In contrast, after 8 days of treatment with ifenprodil or tamoxifen, remyelination was widespread throughout the lesion (Figure 4C). In both central and peripheral regions of the lesion, the majority of axons were surrounded by a thin myelin sheath (Figure 4D). No significant differences in axon diameter were evident between unmyelinated and myelinated axons, and both small- and large-diameter axons showed equal myelination with both treatments. Collectively, these data indicate that small-molecule inhibitors of CYP51, sterol 14-reductase, and EBP can significantly enhance remyelination in mice.

[0283] Finally, we also investigated the effects of miconazole, ifenprodil, and tamoxifen on the efficacy of tamoxifen. We have established that the sterol-modulating activity of xifen is not limited to murine cells but extends to human cells and tissues. We performed sterol profiling in human glioma cell lines and established that these molecules lead to the accumulation of predicted sterol intermediates (Figure 13A). Similarly, miconazole and ifenprodil also induced 8,9-unsaturated sterol accumulation in cortical spheroids derived from human induced pluripotent stem cells. We further confirmed that these molecules are similarly involved in the sterol synthesis pathway in mouse and human cells and CNS tissues (Fig. 13B).

[0284]

[0247] Several groups have identified small molecule enhancers of oligodendrogenesis; A significant hurdle to clinically translating these findings into patients with white matter disease is our incomplete understanding of the functional targets of these molecules in OPCs. Here, we define a key mechanism shared by many small molecule enhancers of remyelination as increasing sterol intermediate levels by inhibiting a narrow therapeutic window spanning the cholesterol biosynthetic enzymes CYP51 and EBP. In total, we characterized 24 small molecules with broad canonical targets that both enhance myelination and increase sterol intermediate levels (Figure 14). No molecules have yet been identified that inhibit the step between CYP51 and EBP but are ineffective in enhancing oligodendrogenesis. Some of these molecules have already been shown to increase 8,9-unsaturated sterol levels in mouse CNS cells and in human patients. Supplying OPCs with 8,9-unsaturated sterols was sufficient to enhance oligodendrocyte formation, whereas depletion of cholesterol levels had no effect, suggesting that the accumulation of sterol intermediates plays a positive role in promoting oligodendrocyte formation from OPCs. Notably, the accumulation of 8,9-unsaturated sterol intermediates is observed during other cell state transitions, and alterations in membrane sterol composition can perturb membrane structure and signal transduction. Furthermore, other laboratories have independently shown that several molecules annotated herein as enhancing sterol intermediate levels reverse paralysis in mice exhibiting MS-like disease, in These findings suggest that altering sterol levels in vivo can regenerate functional myelin. Finally, our study demonstrates that modulating the sterol environment in OPCs can enhance oligodendrocyte formation and suggests novel therapeutic targets, potent inhibitors of these targets, and metabolite-based biomarkers to accelerate the development of optimal remyelination therapies.

[0285] The methods described herein may be carried out in a variety of ways, various of which are well known in the art. It is understood that modifications and variations may occur. It is also understood that any theory stated as to the mode of action should not be construed as limiting the invention in any manner, but is presented so that the method of the present invention may be more fully understood.

[0286] All publications and patents mentioned in the above specification are incorporated herein by reference. This document is hereby incorporated by reference.

Claims

1. 1. A pharmaceutical composition for inhibiting expression of CYP51 in oligodendrocyte precursor cells (OPCs) for promoting myelination in a subject in need thereof, comprising: (1) an antisense oligonucleotide targeting the CYP51, an interfering RNA targeting the CYP51, or a guide RNA and a CRISPR / Cas nuclease targeting the CYP51; or (2) A polynucleotide encoding the antisense oligonucleotide, the interfering RNA, or the guide RNA and the CRISPR / Cas nuclease. The pharmaceutical composition comprising:

2. 2. The pharmaceutical composition of claim 1, comprising the antisense oligonucleotide that targets CYP51 or the polynucleotide that encodes the antisense oligonucleotide that targets CYP51.

3. 3. The pharmaceutical composition of claim 1, wherein the antisense oligonucleotide is modified to improve stability.

4. 4. The pharmaceutical composition of any one of claims 1 to 3, wherein the subject has a myelin-related disorder selected from neuromyelitis optica (NMO), progressive multifocal leukoencephalopathy (PML), encephalomyelitis (EPL), central pontine myelin breakdown (CPM), adrenoleukodystrophy, Alexander disease, Pelizaeus-Merzbacher disease (PMD), vanishing white matter disease, Wallerian degeneration, optic neuritis, transverse myelitis, amyotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease, Parkinson's disease, spinal cord injury, traumatic brain injury, post-radiation injury, neurological complications of chemotherapy, stroke, acute ischemic optic neuropathy, vitamin E deficiency, isolated vitamin E deficiency syndrome, Bassen-Korn-Zweig syndrome, Marchia-Fava-Bignami syndrome, metachromatic leukodystrophy, trigeminal neuralgia, acute disseminated encephalitis, Guillain-Barré syndrome, Charcot-Marie-Tooth disease, Bell's palsy, and mental health disorders.

5. A pharmaceutical composition described in any one of claims 1 to 3, wherein the subject has a myelin-related disorder that is multiple sclerosis.

6. The pharmaceutical composition of any one of claims 1 to 5, wherein the polynucleotide encoding the antisense oligonucleotide, the interfering RNA, or the guide RNA is in a plasmid.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the subject is a human.

8. The pharmaceutical composition of any one of claims 1, 2, 4-5, and 7, wherein the interfering RNA is siRNA.

9. 1. An in vitro method for (i) enhancing oligodendrogenesis or (ii) promoting myelination, the method comprising administering to oligodendrocyte precursor cells (OPCs) an agent that inhibits expression of CYP51 in OPCs, the agent comprising: (1) an antisense oligonucleotide targeting the CYP51, an interfering RNA targeting the CYP51, or a guide RNA and a CRISPR / Cas nuclease targeting the CYP51; or (2) A polynucleotide encoding the antisense oligonucleotide, the interfering RNA, or the guide RNA and the CRISPR / Cas nuclease.

10. 10. The method of claim 9, wherein the agent comprises the antisense oligonucleotide that targets the CYP51 or the polynucleotide that encodes the antisense oligonucleotide that targets the CYP51.

11. 11. The method of claim 9 or 10, wherein the antisense oligonucleotide is modified to improve stability.

12. The method of any one of claims 9 to 11, wherein the method enhances oligodendrogenesis and promotes myelination.

13. 13. The method of any one of claims 9 to 12, wherein the polynucleotide encoding the antisense oligonucleotide, the interfering RNA, or the guide RNA is in a plasmid.

14. The method of any one of claims 9 to 12, wherein the interfering RNA is an siRNA.

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