Compositions and methods for treating brain injury

Abstract

Disclosed herein are methods of treating brain injury (e.g., a stroke) in a subject in need thereof.

Description

[0001] Docket No.: UCH-35225

[0002] UCLA Ref. No.: [UCLA 2020-937-2] WO

[0003] COMPOSITIONS AND METHODS FOR TREATING BRAIN INJURY

[0004] RELATED APPLICATION

[0005] This application claims the benefit of U.S. Provisional Application No. 63 / 704,828, filed on October 8, 2024, the contents of which are fully incorporated by reference herein.

[0006] STATEMENT OF GOVERNMENT SUPPORT

[0007] This invention was made with government support under Grant Number NS083740, awarded by the National Institutes of Health. The government has certain rights in the invention.

[0008] BACKGROUND

[0009] Each year more than 750,000 people in the United States suffer from a stroke, of which approximately 87% are ischemic strokes. Critically, injuries to the brain or spinal cord from stroke produce loss of behavioral function and limited recovery. Axonal degeneration is a core feature resulting from ischemic stroke. The degree of axon loss within the corticospinal tract is a major determinant of functional recovery for stroke patients, however the mechanisms of axon degeneration after ischemic injury are not known. Several pathways contributing to axon degeneration are found to overlap in part with those that drive axon regeneration, suggesting that there may be potential convergence between mechanisms regulating axonal degeneration and attempted regeneration after injury. Thus, understanding the mechanism of axonal degeneration may also reveal regulatory processes critical for neural repair. Accordingly, there is an ongoing unmet need for new therapies to prevent or reverse axonal degeneration and / or promote axon regeneration in patients suffering from a stroke.

[0010] SUMMARY OF THE INVENTION

[0011] In certain aspects, the present disclosure provides methods of treating a brain injury, a spinal cord injury, or a neurodegenerative disease in a subject in need thereof comprising administering a compound selected from Docket No.: UCH-35225

[0012] UCLA Ref. No.: [UCLA 2020-937-2] WO Docket No.: UCH-35225

[0013] UCLA Ref No.: [UCLA 2020-937-2] WO thereof, to the subj ect.

[0014] In further aspects, the present disclosure provides methods of promoting axon regeneration in a subject in need thereof comprising administering a compound selected from Docket No.: UCH-35225

[0015] UCLA Ref. No.: [UCLA 2020-937-2] WO

[0016] Docket No.: UCH-35225

[0017] UCLA Ref. No.: [UCLA 2020-937-2] WO thereof, to the subj ect.

[0018] BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIGs. 1A-1F show that loss of Sarml results in persistent axonal survival after white matter stroke. FIG. 1A contains images showing focal white matter stroke with concurrent retrograde tracer administration (Fluororuby (FR)) targets subcortical white matter (right) and differentially labels transcallosal distal axonal projections 7d after stroke between wild-type (WT) (upper left) and Sarml- / - mice (lower left). Numbered white boxes denote corresponding topographic location along the corpus callosum. Asterisks denote lateral edge of stroke lesion. Scale bars = 100 pm; FIG. IB shows representative confocal microscopy (right) and electron Docket No.: UCH-35225

[0020] UCLA Ref. No.: [UCLA 2020-937-2] WO microscopy images (left) from the midline corpus callosum of WT and Sarml- / - brain tissues at 7d after white matter stroke. Asterisks denote lateral edge of stroke lesion. Scale bars = 80 pm (right), 1 pm (left). FIG. 1C shows mean neurite FR intensities per unit distance ipsilateral to the stroke lesion at 7d post-stroke (*** / ?<0.001 by Student t-test, n = 9 per genotype). Error bars = SEM. FIG. ID shows representative confocal microscopy images of WT and Sarml- / - brain tissues at 28d after white matter stroke. FIG. IE shows mean neurite FR intensities per unit distance ipsilateral to the stroke lesion at 28d post-stroke (*** / ?<0.001 by Student t-test, n = 9 per genotype). Error bars = Standard Error of Mean (SEM). FIG. IF shows quantification of myelinated axons / FOV in WT and Sarml- / - axons per midline callosal cross section (n = 4 / genotype / timepoint). Error bars = SEM.

[0021] FIGs. 2A-2C show that loss of SARM1 promotes neuronal survival after focal white matter stroke. FIG. 2A shows schematic representation of Ultimate Three-Dimensional Imaging of Solvent-Cleared Organs (uDISCO) clearing of brain tissue slabs (3 mm) in WT and Sarml- / - mice at 7d after subcortical stroke results in volumetric visualization of FR+ cortical neurons that also label for NeuN. FIG. 2B shows representative optical slices from reconstructed 3D image from WT (upper) and Sarml- / - (lower) mice demonstrates retrograde labeling of stroke-injured surviving neuronal cells in overlying sensorimotor cortex. FIG. 2C is a bar graph showing density of surviving FR+ cortical neurons measured using semiautomated 3D cell counter plugin in Image J (*p = 0.04 by Student’s t-test, n = 4 / genotype) (C). Error bars = SEM. Scale bars = 500 pm (left); 100 pm (right).

[0022] FIGs. 3A-3F show that genetic deletion of Sarml activates axonogenesis and synaptogenesis in stroke-injured cortical neurons. FIG. 3A shows schematic model for Magnetic- Activated Cell Sorting-Fluorescence Activated Cell Sorting (MACS-FACS) sequence (seq) cell isolation workflow in wild-type (WT) and Sarml-I- mice. FIG. 3B shows the log fold-change (logFC) expression of cell-type marker (left) and cortical layer (right) genes demonstrating enrichment of deep cortical layer neurons by MACS-FACS-seq. FIG. 3C shows a volcano plot of 891 differentially expressed genes (DEGs) (False Discovery Rate (FDR)<0.05) between Sarml-I- and WT neurons seven days after subcortical stroke. FIG. 3D shows a heat-map of logFC expression changes in known Sarml pathway genes between Sarml-I- and WT neurons after stroke (all FDR>0.05). FIG. 3E shows Reduce + Visualize Gene Ontology (REVIGO) semantic clustering map of gene ontology terms derived from stroke-induced Sarml-I- DEG profile showing five major clusters of activity (bold) (bar = - log 10 / ?-value). FIG. 3F depicts STRING protein-protein interaction network derived from Docket No.: UCH-35225

[0023] UCLA Ref. No.: [UCLA 2020-937-2] WO genes in the axonogenesis (PPI enrichment p = 3.88e'08) and synaptogenesis (PPI enrichment p = 8.84e'08) clusters.

[0024] FIGs. 4A-4G show functional genomics screen for pharmacologic targets to replicate axonogenesis and synaptogenesis programs identified in stroke-injured Sarml- / - cortical neurons. FIG. 4A shows a volcano plot of top differentially regulated genes in Sarml - / - stroke- injured cortical neurons used in CLUE-10 (The Broad Institute, Boston, MA) query to identify compounds with combined CMap threshold score > 140. FIG. 4B shows a bar plot showing the number of compounds with CMap score > 140 by pharmacologic class and blood-brain barrier permeability (right) with schematic of filtering strategy for compound library generation (right). FIG. 4C shows a polar plot of neurite outgrowth (% mean neurite outgrowth at subnanomolar doses) by each compound tested vs. control cortical neurons (three replicates / drug / dose). FIG. 4D shows the dose effect of neurite outgrowth for two hit compounds (HL017 - Eugenol; HL013 - 5-methoxytryptamine) ( / ?<0.0001 by two-way ANOVA; *adjusted / ?<0.01 for individual dose comparisons vs. control) with representative Calcein-AM imaging. Horizontal heatmaps of logICso (pM) across five cell lines are shown. FIG. 4E shows a heatmap of up-regulated gene ontology (GO) terms vs. vehicle identified by ATAC-seq in drug-treated neurons plotted vs. GO terms from Sarml -I- axonogenesis and synaptogenesis clusters (top). Top up- and down-regulated transcription factor motifs enriched in drug-treated cortical neurons are also shown (bottom). FIG. 4F shows a heatmap of up- regulated GO terms vs. vehicle identified by ATAC-seq in drug-treated neurons plotted vs. GO terms from Sarml -I- axonogenesis and synaptogenesis clusters FIG. 4G shows RRHO heatmaps of ATAC-seq signatures of drug-treated El 8 mouse cortical neurons comparing growth-promoting (HL013 and HL017) and non-growth promoting (HL011) compounds. The top and left regions represent ascending drug concentrations. The R values are as follows: HL011 versus HL013 (r=0.41053), HL011 versus HL017 (r=0.53185), and HL013 versus HL017 (r=0.63957).

[0025] FIG. 5 shows MACS-FACS isolation of stroke-injured cortical neurons. Seven days following subcortical ischemic stroke, sensorimotor cortex overlying the stroke lesion was dissected and subjected to MACS to remove non-neuronal cells. Resulting cell suspension was labeled with anti-NCAM-488 and NCAM+ / fluororuby+ cells were selected by FACS in both wild-type and SARM1 knockout mice. Equal numbers of NCAM+ / FR+ cells were sorted in both groups (TT = 0.87, F(i,28) = 0.03 by two- ANOVA; adjusted 7i= 0.98 for genotype effect). Docket No.: UCH-35225

[0026] UCLA Ref. No.: [UCLA 2020-937-2] WO

[0027] FIG. 6 shows expression levels of hallmark genes from molecular program clusters in Sarml-I- stroke-injured cortical neurons. Box plot of normalized read counts (logic) between wild-type (WT) and Sarml-I- is shown for selected differentially expressed genes (FDR<0.05) from each of the five major gene ontology clusters identified by REVIGO in MACS-FACS isolated stroke-injured cortical neurons.

[0028] FIG. 7 shows genes within the axonogenesis and synaptogenesis clusters. Heat maps of logFC for genes (all FDR<0.05) within the axonogenesis (left) and synaptogenesis (right) gene ontology clusters enriched in Sarml-I- stroke-injured cortical neurons.

[0029] FIG. 8 shows a bar plot of percentage mean neurite outgrowth at peak subnanomolar doses for hit compound Ellipticine and analogs versus vehicle control primary mouse cortical neurons after 48 hours of exposure (three replicates / drug / dose) (**** / ?<0.0001 by one way ANOVA). Drug nomenclature: HL004 = Ellipticine, HL004 A = Chlorophenyl, HL004 B = Morpholin, and HL004C = Telenzepine.

[0030] FIG. 9 shows a bar plot of percentage mean neurite outgrowth at peak submicromolar doses for each compound tested versus vehicle control human induced pluripotent stem cell (iPSC) derived neurons (three replicates / drug / dose) (**** / ?<0.0001 by one way ANOVA). Drug nomenclature: HL001 = Benocyclidine, HL002 = Piperlongumine, HL003 = Withaferin- a, HL004 = Ellipticine, HL005 = Vicriviroc, HL006 = 1 -monopalmitin, HL007 = Levosulpiride, HL009 = Bufalin, HL010 = Propranolol, HL011 = Digitoxin, HL012 = Digoxin, HL013 = Methoxytryptamine, HL014 = Tyrphostin AG- 126, HL015 = Amonafide, HL016 = Homoharringtonine, HL017 = Eugenol, HL018 = Alisertib, HL004A = Chlorophenyl, HL004B = Morpholin, HL004C = Telenzepine.

[0031] FIG. 10A shows Calcein-AM imaging of control results of human iPSC.

[0032] FIG. 10B shows Calcein-AM imaging of HLOO 13 -treated human iPSC.

[0033] FIGs. 11A-11C show rank rank hypergeometric overlay (RRHO) heatmaps of ATAC- seq signatures of drug-treated human induced pluripotent stem cell derived neurons comparing growth-promoting and a non-growth promoting (HL011) compounds. The heatmap shows ascending drug concentration from bottom right (low drug concentration) to top left (high drug concentration). FIG. HA shows results for HL011, HL004, HL004A, HL004B, HL004C, HLOO 17, and HLOO 18 versus HL011, HL004, and HL004A. The R values for HL011 are as follows: HL011 (Upper limit = 60 mM) versus HL004 (Upper limit = 1, mM) (r=-0.08664), HL011 versus HL004A (Upper limit = 5 mM) (r=0.37885), HL011 versus HL004B (Upper limit = 2.5 mM) (r=0.20500), HL011 versus HL004C (Upper limit = 7 mM) (r=0.48808), Docket No.: UCH-35225

[0034] UCLA Ref. No.: [UCLA 2020-937-2] WO

[0035] HL011 versus HL017 (Upper limit = 8 mM) (r=0.55818), HL011 versus HL018 (Upper limit = 6 mM) (r=0.039737). The rvalues for HL004 are as follows: HL004 versus HL004A (Upper limit = 2 mM) (r=0.21922), HL004 versus HL004B (Upper limit = 6 mM) (r=0.46107), HL004 versus HL004C (Upper limit = 2.5 mM) (r=0.21860), HL004 versus HL017 (Upper limit = 2 mM) (r=0.09995), HL004 versus HL018 (Upper limit = 3.5 mM) (r=0.33382). The r values for HL004A are as follows: HL004A versus HL004B (Upper limit = 5 mM) (r=0.45153), HL004A versus HL004C (Upper limit = 5 mM) (r=0.43792), HL004A versus HL017 (Upper limit = 7 mM) (r=0.50724), HL004A versus HL018 (Upper limit = 2.5 mM) (r=0.28739). FIG. 11B shows results for HL011, HL004, HL004A, HL004B, HL004C, HL0017, and HL0018 versus HL004B and HL004C. The r values for HL004B are as follows: HL004B versus HL004C (Upper limit = 4 mM) (r=0.39704), HL004B versus HL017 (Upper limit = 5 mM) (r=0.39381), HL004B versus HL018 (Upper limit = 4 mM) (r=0.41177). The r values for HL004C are as follows: HL004C versus HL017 (Upper limit = 6 mM) (r=0.47953), HL004C versus HL018 (Upper limit = 6 mM) (r=0.45676). FIG. 11C shows results for HL011, HL004, HL004A, HL004B, HL004C, HL0017, and HL0018 versus HL017 and HL018. The R value for HL017 are as follows: HL0017 versus HL0018 (Upper limit = 5 mM) (r=0.40630).

[0036] DETAILED DESCRIPTION OF THE INVENTION

[0037] Inhibition of Sarml not only mitigates anterograde axonal degeneration and promotes neuronal survival after ischemic stroke, but also surprisingly facilitates a regenerative program in cortical neurons that is encoded at the epigenetic level. The finding that this epigenetic signature can be recapitulated with a low-dose pharmacologic agent supports the concept that a regenerative capacity for axonal outgrowth in cortical neurons after stroke is programmed into the genome but is actively restricted by pro-degenerative regulators, such as Sarml. Well-timed inhibition of Sarml to relieve this regulation, or direct activation of the downstream, druggable epigenetic program, represents a novel strategy to mitigate axonal degeneration and concurrently promote axon regeneration after stroke.

[0038] In one aspect, the present disclosure provides methods of treating a brain injury, a spinal cord injury, or a neurodegenerative disease in a subject in need thereof comprising administering a compound selected from Docket No.: UCH-35225

[0039] UCLA Ref. No.: [UCLA 2020-937-2] WO Docket No.: UCH-35225

[0040] UCLA Ref No.: [UCLA 2020-937-2] WO thereof, to the subj ect.

[0041] In one aspect, the present disclosure provides methods of treating a brain injury, a spinal cord injury, or a neurodegenerative disease in a subject in need thereof comprising administering a compound selected from Docket No.: UCH-35225

[0042] UCLA Ref. No.: [UCLA 2020-937-2] WO pharmaceutically acceptable salt thereof, to the subject.

[0043] Cl

[0044] In some embodiments, the compound is Docket No.: UCH-35225

[0045] UCLA Ref. No.: [UCLA 2020-937-2] WO

[0046] In some embodiments, the compound

[0047] In some embodiments, the compound pharmaceutically acceptable salt thereof. salt thereof. Docket No.: UCH-35225

[0048] UCLA Ref. No.: [UCLA 2020-937-2] WO

[0049] In some embodiments, the compound is acceptable salt thereof.

[0050] In some embodiments, the compound is or a pharmaceutically acceptable salt thereof.

[0051] In some embodiments, the compound pharmaceutically acceptable salt thereof.

[0052] In some embodiments, the compound acceptable salt thereof. Docket No.: UCH-35225

[0053] UCLA Ref. No.: [UCLA 2020-937-2] WO

[0054] In some embodiments, the compound acceptable salt thereof.

[0055] In some embodiments, the compound is

[0056] O

[0057] OH , or a pharmaceutically acceptable salt thereof.

[0058] In some embodiments, the compound pharmaceutically acceptable salt thereof. pharmaceutically acceptable salt thereof. Docket No.: UCH-35225

[0059] UCLA Ref. No.: [UCLA 2020-937-2] WO

[0060] In some embodiments, the compound pharmaceutically acceptable salt thereof.

[0061] In some embodiments, the compound acceptable salt thereof. pharmaceutically acceptable salt thereof.

[0062] In some embodiments, the compound i pharmaceutically acceptable salt thereof.

[0063] In some embodiments, the compound pharmaceutically acceptable salt thereof. Docket No.: UCH-35225

[0064] UCLA Ref. No.: [UCLA 2020-937-2] WO

[0065] In some embodiments, the compound pharmaceutically acceptable salt thereof.

[0066] In some embodiments, the compound acceptable salt thereof

[0067] In certain embodiments, the method is a method of treating a brain injury. In certain preferred embodiments, the brain injury is a stroke (e.g., an ischemic stroke).

[0068] In another aspect, the present disclosure provides methods of promoting axon regeneration in a subject in need thereof comprising administering a compound selected from Docket No.: UCH-35225

[0069] UCLA Ref. No.: [UCLA 2020-937-2] WO Docket No.: UCH-35225

[0070] UCLA Ref No.: [UCLA 2020-937-2] WO thereof, to the subj ect.

[0071] In another aspect, the present disclosure provides methods of promoting axon regeneration in a subject in need thereof comprising administering a compound selected from Docket No.: UCH-35225

[0072] UCLA Ref. No.: [UCLA 2020-937-2] WO pharmaceutically acceptable salt thereof, to the subject. acceptable salt thereof.

[0073] In some embodiments, the compound acceptable salt thereof.

[0074] In some embodiments, the compound pharmaceutically acceptable salt thereof.

[0075] In some embodiments, the compound pharmaceutically acceptable salt thereof. Docket No.: UCH-35225

[0076] UCLA Ref. No.: [UCLA 2020-937-2] WO salt thereof.

[0077] In some embodiments, the compound is pharmaceutically acceptable salt thereof.

[0078] In some embodiments, the compound pharmaceutically acceptable salt thereof.

[0079] In some embodiments, the compound i pharmaceutically acceptable salt thereof.

[0080] In some embodiments, the compound pharmaceutically acceptable salt thereof. Docket No.: UCH-35225

[0081] UCLA Ref. No.: [UCLA 2020-937-2] WO

[0082] In some embodiments, the compound acceptable salt thereof.

[0083] In some embodiments, the compound pharmaceutically acceptable salt thereof.

[0084] In some embodiments, the compound is

[0085] O

[0086] OH , or a pharmaceutically acceptable salt thereof.

[0087] In some embodiments, the compound pharmaceutically acceptable salt thereof. Docket No.: UCH-35225

[0088] UCLA Ref. No.: [UCLA 2020-937-2] WO

[0089] In some embodiments, the compound pharmaceutically acceptable salt thereof.

[0090] In some embodiments, the compound pharmaceutically acceptable salt thereof.

[0091] In some embodiments, the compound acceptable salt thereof.

[0092] In some embodiments, the compound IS or a pharmaceutically acceptable salt thereof.

[0093] In some embodiments, the compound i pharmaceutically acceptable salt thereof. Docket No.: UCH-35225

[0094] UCLA Ref. No.: [UCLA 2020-937-2] WO

[0095] In some embodiments, the compound pharmaceutically acceptable salt thereof.

[0096] In some embodiments, the compound acceptable salt thereof.

[0097] In some embodiments, the compound pharmaceutically acceptable salt thereof.

[0098] Pharmaceutical Compositions

[0099] The compositions and methods of the present disclosure may be utilized to treat an individual in need thereof. In certain embodiments, the individual is a mammal such as a human, or a non-human mammal. When administered to an animal, such as a human, the composition or the compound is preferably administered as a pharmaceutical composition comprising, for example, a compound described herein and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters. In preferred embodiments, when such pharmaceutical compositions are for human administration, particularly for invasive routes of administration (i.e., routes, such as injection or implantation, that circumvent transport or diffusion through an epithelial barrier), the aqueous solution is Docket No.: UCH-35225

[0100] UCLA Ref. No.: [UCLA 2020-937-2] WO pyrogen-free, or substantially pyrogen-free. The excipients can be chosen, for example, to effect delayed release of an agent or to selectively target one or more cells, tissues or organs. The pharmaceutical composition can be in dosage unit form such as tablet, capsule (including sprinkle capsule and gelatin capsule), granule, lyophile for reconstitution, powder, solution, syrup, suppository, injection or the like. The composition can also be present in a transdermal delivery system, e.g., a skin patch. The composition can also be present in a solution suitable for topical administration, such as a lotion, cream, or ointment.

[0101] A pharmaceutically acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize, increase solubility or to increase the absorption of a compound such as a compound described herein. Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. The choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent, depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition can be a self-emulsifying drug delivery system or a self-microemulsifying drug delivery system. The pharmaceutical composition (preparation) also can be a liposome or other polymer matrix, which can have incorporated therein, for example, a compound described herein. Liposomes, for example, which comprise phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.

[0102] The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0103] The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which 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 carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; Docket No.: UCH-35225

[0104] UCLA Ref. No.: [UCLA 2020-937-2] WO

[0105] (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

[0106] A pharmaceutical composition (preparation) can be administered to a subject by any of a number of routes of administration including, for example, orally (for example, drenches as in aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingually); subcutaneously; transdermally (for example as a patch applied to the skin); and topically (for example, as a cream, ointment or spray applied to the skin). The compound may also be formulated for inhalation. In certain embodiments, a compound may be simply dissolved or suspended in sterile water. Details of appropriate routes of administration and compositions suitable for same can be found in, for example, U.S. Pat. Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and 4,172,896, as well as in patents cited therein.

[0107] The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.

[0108] Methods of preparing these formulations or compositions include the step of bringing into association an active compound, such as a compound described herein, with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound described herein with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product. Docket No.: UCH-35225

[0109] UCLA Ref. No.: [UCLA 2020-937-2] WO

[0110] Formulations suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), lyophile, powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and / or as mouth washes and the like, each containing a predetermined amount of a compound described herein as an active ingredient. Compositions or compounds may also be administered as a bolus, electuary or paste.

[0111] To prepare solid dosage forms for oral administration (capsules (including sprinkle capsules and gelatin capsules), tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and / or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; (10) complexing agents, such as, modified and unmodified cyclodextrins; and (11) coloring agents. In the case of capsules (including sprinkle capsules and gelatin capsules), tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

[0112] A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surfaceactive or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. Docket No.: UCH-35225

[0113] UCLA Ref. No.: [UCLA 2020-937-2] WO

[0114] The tablets, and other solid dosage forms of the pharmaceutical compositions, such as dragees, capsules (including sprinkle capsules and gelatin capsules), pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and / or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.

[0115] Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, lyophiles for reconstitution, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, cyclodextrins and derivatives thereof, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3- butylene glycol, oils (in particular, cottonseed, groundnut, com, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

[0116] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0117] Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

[0118] Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound Docket No.: UCH-35225

[0119] UCLA Ref. No.: [UCLA 2020-937-2] WO may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required.

[0120] The ointments, pastes, creams and gels may contain, in addition to an active compound, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

[0121] Powders and sprays can contain, in addition to an active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[0122] Transdermal patches have the added advantage of providing controlled delivery of a compound described herein to the body. Such dosage forms can be made by dissolving or dispersing the active compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel.

[0123] The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrastemal injection and infusion. Pharmaceutical compositions suitable for parenteral administration comprise one or more active compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

[0124] Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions described herein include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper Docket No.: UCH-35225

[0125] UCLA Ref. No.: [UCLA 2020-937-2] WO fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0126] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.

[0127] In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

[0128] Injectable depot forms are made by forming microencapsulated matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissue.

[0129] For use in the methods described herein, active compounds can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.

[0130] Methods of introduction may also be provided by rechargeable or biodegradable devices. Various slow release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinaceous biopharmaceuticals. A variety of biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form an implant for the sustained release of a compound at a particular target site. Docket No.: UCH-35225

[0131] UCLA Ref. No.: [UCLA 2020-937-2] WO

[0132] Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0133] The selected dosage level will depend upon a variety of factors including the activity of the particular compound or combination of compounds employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound(s) being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compound(s) employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0134] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the pharmaceutical composition or compound at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. By “therapeutically effective amount” is meant the concentration of a compound that is sufficient to elicit the desired therapeutic effect. It is generally understood that the effective amount of the compound will vary according to the weight, sex, age, and medical history of the subject. Other factors which influence the effective amount may include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and, if desired, another type of therapeutic agent being administered with the compound described herein. A larger total dose can be delivered by multiple administrations of the agent. Methods to determine efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison’s Principles of Internal Medicine 13 ed., 1814-1882, herein incorporated by reference).

[0135] In general, a suitable daily dose of an active compound used in the compositions and methods described herein will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.

[0136] If desired, the effective daily dose of the active compound may be administered as one, two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In certain embodiments, the active Docket No.: UCH-35225

[0137] UCLA Ref. No.: [UCLA 2020-937-2] WO compound may be administered two or three times daily. In preferred embodiments, the active compound will be administered once daily.

[0138] The patient receiving this treatment is any animal in need, including primates, in particular humans; and other mammals such as equines, cattle, swine, sheep, cats, and dogs; poultry; and pets in general.

[0139] In certain embodiments, compounds described herein may be used alone or conjointly administered with another type of therapeutic agent.

[0140] The present disclosure includes the use of pharmaceutically acceptable salts of compounds described herein in the compositions and methods described herein. In certain embodiments, contemplated salts described herein include, but are not limited to, alkyl, dialkyl, trialkyl or tetra-alkyl ammonium salts. In certain embodiments, contemplated salts include, but are not limited to, L-arginine, benenthamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, IH-imidazole, lithium, L- lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, l-(2- hydroxyethyljpyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts. In certain embodiments, contemplated salts include, but are not limited to, Na, Ca, K, Mg, Zn or other metal salts. In certain embodiments, contemplated salts include, but are not limited to, 1- hydroxy -2 -naphthoic acid, 2,2-dichloroacetic acid, 2-hydroxy ethanesulfonic acid, 2- oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, 1- ascorbic acid, 1-aspartic acid, benzenesulfonic acid, benzoic acid, (+)-camphoric acid, (+)- camphor- 10-sulfonic acid, capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane- 1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, d-glucoheptonic acid, d-gluconic acid, d-glucuronic acid, glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, 1- malic acid, malonic acid, mandelic acid, methanesulfonic acid , naphthalene-l,5-disulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, proprionic acid, 1 -pyroglutamic acid, salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, 1-tartaric acid, thiocyanic acid, p- toluenesulfonic acid, trifluoroacetic acid, and undecylenic acid salts. Docket No.: UCH-35225

[0141] UCLA Ref. No.: [UCLA 2020-937-2] WO

[0142] The pharmaceutically acceptable acid addition salts can also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, and the like. Mixtures of such solvates can also be prepared. The source of such solvate can be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or adventitious to such solvent.

[0143] Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0144] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal-chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0145] Definitions

[0146] Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclature used in connection with, and techniques of, chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics and protein and nucleic acid chemistry, described herein, are those well known and commonly used in the art.

[0147] The methods and techniques of the present disclosure are generally performed, unless otherwise indicated, according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout this specification. See, e.g. “Principles of Neural Science”, McGraw-Hill Medical, New York, N.Y. (2000); Motulsky, “Intuitive Biostatistics”, Oxford University Press, Inc. (1995); Lodish et al., “Molecular Cell Biology, 4th ed.”, W. H. Freeman & Co., New York (2000); Griffiths et al., “Introduction to Genetic Analysis, 7th ed ”, W. H. Freeman & Co., N.Y. (1999); and Gilbert et al., “Developmental Biology, 6th ed.”, Sinauer Associates, Inc., Sunderland, MA (2000). Docket No.: UCH-35225

[0148] UCLA Ref. No.: [UCLA 2020-937-2] WO

[0149] Chemistry terms used herein, unless otherwise defined herein, are used according to conventional usage in the art, as exemplified by “The McGraw-Hill Dictionary of Chemical Terms”, Parker S., Ed., McGraw-Hill, San Francisco, C.A. (1985).

[0150] All of the above, and any other publications, patents and published patent applications referred to in this application are specifically incorporated by reference herein. In case of conflict, the present specification, including its specific definitions, will control.

[0151] The term “agent” is used herein to denote a chemical compound (such as an organic or inorganic compound, a mixture of chemical compounds), a biological macromolecule (such as a nucleic acid, an antibody, including parts thereof as well as humanized, chimeric and human antibodies and monoclonal antibodies, a protein or portion thereof, e.g., a peptide, a lipid, a carbohydrate), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. Agents include, for example, agents whose structure is known, and those whose structure is not known.

[0152] A “patient,” “subject,” or “individual” are used interchangeably and refer to either a human or a non-human animal. These terms include mammals, such as humans, primates, livestock animals (including bovines, porcines, etc.), companion animals (e.g., canines, felines, etc.) and rodents (e.g., mice and rats).

[0153] “Treating” a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0154] The term “preventing” is art-recognized, and when used in relation to a condition, such as a local recurrence (e.g., pain), a disease such as cancer, a syndrome complex such as heart failure or any other medical condition, is well understood in the art, and includes administration of a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition. Thus, prevention of cancer includes, for example, reducing the number of detectable cancerous growths in a population of patients receiving a prophylactic treatment relative to an untreated control population, and / or delaying the appearance of detectable cancerous growths in a treated Docket No.: UCH-35225

[0155] UCLA Ref. No.: [UCLA 2020-937-2] WO population versus an untreated control population, e.g., by a statistically and / or clinically significant amount.

[0156] “Administering” or “administration of’ a substance, a compound or an agent to a subject can be carried out using one of a variety of methods known to those skilled in the art. For example, a compound or an agent can be administered, intravenously, arterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, ocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebrally, and transdermally (by absorption, e.g., through a skin duct). A compound or agent can also appropriately be introduced by rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, which provide for the extended, slow or controlled release of the compound or agent. Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods.

[0157] Appropriate methods of administering a substance, a compound or an agent to a subject will also depend, for example, on the age and / or the physical condition of the subject and the chemical and biological properties of the compound or agent (e.g., solubility, digestibility, bioavailability, stability and toxicity). In some embodiments, a compound or an agent is administered orally, e.g., to a subject by ingestion. In some embodiments, the orally administered compound or agent is in an extended release or slow release formulation, or administered using a device for such slow or extended release.

[0158] As used herein, the phrase “conjoint administration” refers to any form of administration of two or more different therapeutic agents such that the second agent is administered while the previously administered therapeutic agent is still effective in the body (e.g., the two agents are simultaneously effective in the patient, which may include synergistic effects of the two agents). For example, the different therapeutic compounds can be administered either in the same formulation or in separate formulations, either concomitantly or sequentially. Thus, an individual who receives such treatment can benefit from a combined effect of different therapeutic agents.

[0159] A “therapeutically effective amount” or a “therapeutically effective dose” of a drug or agent is an amount of a drug or an agent that, when administered to a subject will have the intended therapeutic effect. The full therapeutic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations. The precise effective amount needed for a subject will depend upon, for example, the subject’s size, Docket No.: UCH-35225

[0160] UCLA Ref. No.: [UCLA 2020-937-2] WO health and age, and the nature and extent of the condition being treated, such as cancer or MDS. The skilled worker can readily determine the effective amount for a given situation by routine experimentation.

[0161] As used herein, the terms “optional” or “optionally” mean that the subsequently described event or circumstance may occur or may not occur, and that the description includes instances where the event or circumstance occurs as well as instances in which it does not. For example, “optionally substituted alkyl” refers to the alkyl may be substituted as well as where the alkyl is not substituted.

[0162] The phrase “pharmaceutically acceptable” is art-recognized. In certain embodiments, the term includes compositions, excipients, adjuvants, polymers and other materials and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0163] “Pharmaceutically acceptable salt” or “salt” is used herein to refer to an acid addition salt or a basic addition salt which is suitable for or compatible with the treatment of patients.

[0164] The term “pharmaceutically acceptable acid addition salt” as used herein means any non-toxic organic or inorganic salt of any base compounds described herein. Illustrative inorganic acids which form suitable salts include hydrochloric, hydrobromic, sulfuric and phosphoric acids, as well as metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Illustrative organic acids that form suitable salts include mono-, di-, and tricarboxylic acids such as glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic, phenylacetic, cinnamic and salicylic acids, as well as sulfonic acids such as p-toluene sulfonic and methanesulfonic acids. Either the mono or di-acid salts can be formed, and such salts may exist in either a hydrated, solvated or substantially anhydrous form. In general, the acid addition salts of compounds described herein are more soluble in water and various hydrophilic organic solvents, and generally demonstrate higher melting points in comparison to their free base forms. The selection of the appropriate salt will be known to one skilled in the art. Other non-pharmaceutically acceptable salts, e.g., oxalates, may be used, for example, in the isolation of compounds described herein for laboratory use, or for subsequent conversion to a pharmaceutically acceptable acid addition salt.

[0165] The term “pharmaceutically acceptable basic addition salt” as used herein means any non-toxic organic or inorganic base addition salt of any acid compounds described herein or Docket No.: UCH-35225

[0166] UCLA Ref. No.: [UCLA 2020-937-2] WO any of their intermediates. Illustrative inorganic bases which form suitable salts include lithium, sodium, potassium, calcium, magnesium, or barium hydroxide. Illustrative organic bases which form suitable salts include aliphatic, alicyclic, or aromatic organic amines such as methylamine, trimethylamine and picoline or ammonia. The selection of the appropriate salt will be known to a person skilled in the art.

[0167] Many of the compounds useful in the methods and compositions of this disclosure have at least one stereogenic center in their structure. This stereogenic center may be present in a R or a S configuration, said R and S notation is used in correspondence with the rules described in Pure Appl. Chem. (1976), 45, 11-30. The disclosure contemplates all stereoisomeric forms such as enantiomeric and diastereoisomeric forms of the compounds, salts, prodrugs or mixtures thereof (including all possible mixtures of stereoisomers). See, e.g., WO 01 / 062726.

[0168] Furthermore, certain compounds which contain alkenyl groups may exist as Z (zusammen) or E (entgegen) isomers. In each instance, the disclosure includes both mixture and separate individual isomers.

[0169] “Prodrug” or “pharmaceutically acceptable prodrug” refers to a compound that is metabolized, for example hydrolyzed or oxidized, in the host after administration to form the compound of the present disclosure. Typical examples of prodrugs include compounds that have biologically labile or cleavable (protecting) groups on a functional moiety of the active compound. Prodrugs include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated, or dephosphorylated to produce the active compound. Examples of prodrugs using ester or phosphoramidate as biologically labile or cleavable (protecting) groups are disclosed in U.S. Patents 6,875,751, 7,585,851, and 7,964,580, the disclosures of which are incorporated herein by reference. The prodrugs of this disclosure are metabolized to produce a compound described herein. The present disclosure includes within its scope, prodrugs of the compounds described herein. Conventional procedures for the selection and preparation of suitable prodrugs are described, for example, in “Design of Prodrugs” Ed. H. Bundgaard, Elsevier, 1985.

[0170] The phrase “pharmaceutically acceptable carrier” as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filter, diluent, excipient, solvent or encapsulating material useful for formulating a drug for medicinal or therapeutic use. Docket No.: UCH-35225

[0171] UCLA Ref. No.: [UCLA 2020-937-2] WO

[0172] The term “Log of solubility”, “LogS” or “logS” as used herein is used in the art to quantify the aqueous solubility of a compound. The aqueous solubility of a compound significantly affects its absorption and distribution characteristics. A low solubility often goes along with a poor absorption. LogS value is a unit stripped logarithm (base 10) of the solubility measured in mol / liter.

[0173] EXAMPLES

[0174] The invention now being generally described, it will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments of the present invention and are not intended to limit the invention.

[0175] Example 1: Exemplary Activity of the Compounds of the Disclosure

[0176] Axonal degeneration after ischemic injury is mediated by Sarml.

[0177] Methods

[0178] All animal studies presented here were approved by the UCLA and Stanford University Animal Research Committees, both accredited by the AAALAC. Mice were housed under respective institutional regulation with a 12-h dark-light cycle. All mice used in the study were male between the ages of 3-6 months. Strain matched wild-type C57BL / 6 mice (Jackson Labs, Strain #000667) and Sarml- / - (Jackson Labs, Strain #018069) backcrossed and bred in C57BL / 6 background x 3 generations were used for all experiments unless otherwise stated. White matter ischemic stroke

[0179] Subcortical white matter ischemic injury was induced as previously described (Hinman et al, 2013; Nunez et al, 2016) using focal injections into subcortical white matter x 3 of the iNOS inhibitor L-N5-(l -Iminoethyl) ornithine, Dihydrochloride (LNiO; 27 mg / mL, Millipore) added at 1 : 1 ratio with 20% fluororuby (Fluorochrome LLC) dissolved in saline to label stroke- injured axons and connected cortical neurons. Animals were sacrificed at 7 and 28 days after stroke, and freshly dissected and transcardially perfused with 4% PFA and prepared for tissue sections as previously described (Hinman et al, 2013).

[0180] Cortical neuron virus labeling

[0181] Stock adeno-associated virus encoding EGFP with the hSyn neuronal promoter (AAV- DJ-hsyn-EGFP) was obtained from the Stanford Gene Vector and Viral Core (GVVC-AAV- 127). AAV-DJ-hsyn-EGFP was stereotactically injected into the cortex of WT and Sarml-I- Docket No.: UCH-35225

[0182] UCLA Ref. No.: [UCLA 2020-937-2] WO mice. A 1 / / L microsyringe (Hamilton, Cinnaminson, NJ) was used inject the virus (2 / / L;lE+12v.g. / mL) into the cortex at anticipated location of the infarct in the cortex with the following stereotactic coordinates: -0.26mm posterior and +1.3mm lateral with respect to the bregma, and with depth of -2.17mm from the surface of the cortex. To allow sufficient gene expression, all injections were completed 2 weeks before stroke surgery.

[0183] Docket No.: UCH-35225

[0184] UCLA Ref. No.: [UCLA 2020-937-2] WO

[0185] Distal middle cerebral artery occlusion (dMCAO) stroke dMCAO was induced as described previously. In brief, C57 / BL6 mice were anesthetized by isoflurane inhalation (2% isoflurane in 100% oxygen) and an incision was made to expose the right temporalis muscle. A pocket was created in the temporalis muscle and the right middle cerebral artery (MCA) was identified through the skull underneath. A microdrill was used to penetrate the skull and expose the underlying MCA. The meninges were then cut and the vessel was cauterized using a high temperature Bovie thermo-cauterizer. After complete occlusion of the underlying MCA was visually confirmed, the wound was closed using surgical adhesive. Throughout surgery, the animal body temperature was maintained at 37°C using a feedback controlled heating blanket. Immunofluorescence and confocal imaging

[0186] Fluororuby-labeled WT and Sarml- / - stroke-injured brains were harvested, perfused and cryosectioned at 40 / / m in a -22C cryostat and then stored in cryoprotectant at -20C. For staining, tissue sections containing stroke were removed from cryoprotectant and washed in PBS and incubated for 30 min in 10 mM sodium citrate buffer. After cooling and washing, sections were blocked in PBTDS and tissue was incubated overnight in anti-NF-H (Sigma, 1 :500) for neurites, anti-NeuN (Abeam, 1 :500) for neuronal cell bodies, and anti-CD68 (Abeam, 1 : 1000) for macrophages (to delineate stroked region in the cortex after dMCAO) in PBTDS. Corresponding secondary antibodies were added (1 :500) including Donkey antiRabbit 488, Alexa Fluor 555-conjugated goat anti-rabbit IgG (1 :500; Abeam), Donkey antiMouse 647 (Jackson ImmunoResearch) and counterstained with DAPI. Tissue was mounted onto glass slides and dehydrated in ethanol and xylenes and covered with DPX and a coverslip. Imaging was conducted on a Nikon C2 confocal microscope. Three 60X images were taken on each tissue section in the region of interest containing stroke-injured and non-stroke injured axons or cortical neurons. For imaging of thalamic neurons, Axonal morphology and integrity evaluated in Fiji. The axons were measured by manual tracing beginning at infarct core and moving medially towards midline of corpus callosum in 50um increments. Fluorescence intensity within the corpus callosum as manually traced in each ROI was quantified using ImageJ software (NIH). For all confocal imaging of thalamic neurons, anatomical landmarks were used to identify the thalamus.

[0187] This included first the selection of stroked tissue sections (marked by +CD68 staining) with the hippocampus in view. This is followed by identification of the region of intersection Docket No.: UCH-35225

[0188] UCLA Ref. No.: [UCLA 2020-937-2] WO between the hemispheric midline and the corpus callosum. Thalamic region was then characterized in all sections as 2 fields of view (FOV) lateral to the midline and 3 FOV inferior to the corpus callosum on a 40X mag view. u-DISCO tissue clearing and neuronal density measurement

[0189] At 7 days after white matter stroke, brain tissues of WT and sarml- / - animals (n = 4 in each cohort) were transcardially perfused with 4% PFA and post-fixed overnight at 4C. Tissue slabs 3 mm in thickness and spanning the region of stroke were generated that included left and right cortical regions. Tissues were cleared using uDISCO as previously described (Pan, Cai et al. 2016). Briefly, tissues were optically cleared by serial incubation in increasing concentrations of tert-butanol (Acros Organics) followed by immersion in benzyl alcohol (Sigma-Aldrich) / benzyl benzoate (Sigma-Aldrich) / diphenyl ether (Alfa Aesar) (BABB-D) solution until transparent. The tissues were then immediately imaged on a Leica SP5 laser confocal microscope. Cell density was analyzed and quantified using Imaris software. A 3D grid was applied to images in the contralateral cortical hemisphere and density of FR+ neurons within each field of view was measured. Computer-generated labeling of each cell body based on cell body size, diameter and circularity was performed to aid in identification and quantification of the neuronal cells.

[0190] Stroke-injured neuron isolation by MACS-FACS

[0191] At 7 days after focal white matter stroke, regions of cortex overlying subcortical white matter stroke lesions were dissected and mechanically digested following a single-cell suspension protocol using Neurocult dissociation kit (STEMCELL). For magnetic bead cell sorting (MACS), neuronal enrichment kit microbeads and CD 11b microbeads (Miltenyi Biotec) were added to the suspension before applying to a MACS column to negatively select non-neuronal cells. After collecting all neuronal cells, cells were further labeled with anti- NCAM antibody (Abeam) followed by anti-rabbit Alexa 488 secondary antibody to label cortical neurons and sorted by FACS for fluororuby and NCAM+ / Alexa488+ neurons at the UCLA Flow Cell Cytometry Core. Total RNA was collected from sorted cells using conventional RNA preparation kit (RNeasy, Qiagen).

[0192] Gene expression profiling

[0193] Isolated RNA from MACS-FACS isolated cortical neurons was normalized by FACS cell counts. Harvested RNA samples at 0.5ng / ul concentration were validated and quality- controlled using TapeStation 2200 bioanalyzer (Agilent Technology). cDNA library creation performed by the UCLA Neuroscience Genomic Core (UNGC) using the NuGEN Ovation Docket No.: UCH-35225

[0194] UCLA Ref. No.: [UCLA 2020-937-2] WO

[0195] Ultralow system with KAPA HyperPrep and sequencing on the Illumina HiSeq 4000 platform. Quality of sequencing was assessed using FastQC, followed by read alignment with STAR aligner. Differential gene expression performed using DESeq2. After read-count normalization, differentially expressed genes (DEGs) with FDR<0.05 were compared. Gene ontology analysis was performed by comparing to the whole mouse genome using GOrilla (Eden, Navon et al. 2009). Resulting gene ontology terms (p<0.05) were used as input to REVIGO (Supek, Bosnjak et al. 2011). Semantic clustering of gene ontology terms derived from REVIGO were plotted via MatLab to create a gene ontology map.

[0196] Protein-protein interaction network analysis

[0197] Protein-protein network interactions were evaluated using the STRING Db resource in 2019. Conserved genes in both the axonogenesis and synaptic regulation GO terms were used as input criteria against the whole genome with the following search parameters: evidence for network edges; all sources; high confidence 0.7; no interactors).

[0198] Functional genomics screen

[0199] The top 150 differentially expressed genes by log-fold change and the top 150 down- regulated genes by log-fold change were used as individual gene expression (LI 000) query inputs for CLUE-IO in two separate trials using the latest Touchstone database (2018). Queries were compared to reference perturbagens in cell lines including human melanoma (A375), human non-small cell carcinoma (A549), human lung adenocarcinoma (HCC515), human liver cancer (HEPG2), human colorectal adenocarcinoma (HT29), human breast cancer (MCF7), human prostate cancer (PC3), immortalized human kidney (HA1E), and human adherent, epithelial prostate cancer (VCAP). Compound lists were generated for each trial producing a total of 2428 compounds. CMap scores were summated and compounds with a summated enrichment score 140 were curated further based on actual or predicted blood brain barrier permeability by literature search, identifying 18 viable candidates labeled with identifiers: HL001-018.

[0200] Cell culture

[0201] Primary mouse cortical neurons (Thermo Fisher Scientific, Catalog #A15586) were plated at a density of 5000 cells / well and cultured in Neurobasal media (Gibco, Ref #21103- 049) supplemented with B27 (Gibco, Ref #17504044) and Glutamax (Gibco, Ref #35050-061) in black 384 well flat bottom microClear cell culture microplates (Greiner Bio One, Cat #781091). Prior to plating, the culture plates were coated with 50 / / g / mL of Poly-D-Lysine Docket No.: UCH-35225

[0202] UCLA Ref. No.: [UCLA 2020-937-2] WO solution (Cat #A3890401). Plates were incubated overnight in 37°C, 5% CO2 humidified incubator. Poly-D-Lysine solution was aspirated prior to washing wells 3x with sterile dH2O and aspirating wells completely to dry. Neurons were cultured for seven days with half-media exchanges every 2-3 days. Chemical ischemia was induced with 3 hrs of rotenone exposure (25pM in 0.5% DMSO) as previously described. For drug toxicity measurement, four additional cells lines (293T, THP1, HepG2, and HUVEC) were cultured in normal growth conditions for 48-72 hours in CELLSTAR uClear 384 well plates at seeding density of 2000 cells / well in respective growth medias.

[0203] Pharmacologic screen

[0204] The final compound library set was 17 compounds due to elimination of Alprazolam as a DEA Schedule 4 drug. All compounds were diluted in 0.5% DMSO (Sigma, Cat #276855) at 10 pM. Diluted compounds were pinned to 384 well plates with 25 / / L of cell media per well with pin size of 250nL using BioMek FX (Beckman Coulter, CA). DIV7 cultured neurons plated in 384 well plates were exposed to compounds across a 20-step serial dilution (24pM- 50 11 M) for 48 hours in a 37°C, 5% CO2 humidified incubator without media exchange. Cellular toxicity was established using propidium iodide (Invitrogen, Cat #P3566) assay. To determine the effects of compound exposure on neurite outgrowth, cells were exposed for 48 hrs, washed three times, and loaded with 1 / / M Calcein-AM (Sigma, Cat #17783-1MG) and Hoechst (Invitrogen, Ref #H3570) before a single well FOV was captured on an ImageXpress Confocal (Molecular Devices, CA) using a lOx objective. All experiments were conducted in triplicate. Total neurite outgrowth was measured via a MetaXpress (Molecular Devices, CA) neurite outgrowth analysis algorithm. Neurite outgrowth was set to detect cell bodies with approximate width of at least 20 / / m and outgrowth with maximal width of 5 / / m and length of at least 100 m with intensity of 1000 grey scales over background. Mean neurite outgrowth per drug exposure was determined by normalizing to control (w=30).

[0205] Assay for transposase-accessible chromatin with high-throughput sequencing

[0206] Primary mouse cortical neurons (Lonza, Cat #M-CX-300) were thawed and seeded in a 24 well TC treated plate coated with Poly-D-Lysine and Laminin. Neurons were grown in 24 wells at seeding density of ~475k cells / well. Cells were fed every 2-3 days with half media changes. On day 6 of cell culture, neurons showed axonal processes and were exposed to (8) treatment conditions: PMCN, Vehicle, HL004, HL009, HL011, HL012, HL013, and HL017. Each condition was run in triplicates and total treatment time was 48 hours. After drug Docket No.: UCH-35225

[0207] UCLA Ref. No.: [UCLA 2020-937-2] WO incubation, the neurons were directly lysed on the plate with AT AC -RSB -Lysis buffer and collected with a cell scraper to count ~50k nuclei from each well per condition on a hemocytometer with trypan blue staining. The isolated nuclei were prepared for transposition by incubating with the transposase enzyme Nextera Tn5 Transposase (Illumina, REF #20034210) and then purified with Qiagen MinElute PCR Purification kit (Qiagen, REF #28004).

[0208] Transposed DNA was eluted in 10 L and subjected to PCR amplification and library generation in the UCLA Neurogenetics and Genomics Core Facility. Resulting DNA sequences were aligned to the mouse genome (mm 10) using Burrows-Wheeler Aligner mem with default parameters. Duplicates removed with Picard software. Reads were pre-shifted by 75 bp prior to peak calling using MACS2. Gene ontology analysis was performed using Homer and conserved motifs across the vehicle control, negative control, and 5 drug conditions were thresholded to remove motifs enriched in vehicle and HL011 (no growth control). The remaining motifs were sorted by significance to identify top hits related to drug treatment. Rank-rank hypergeometric overlap (RRHO) analysis

[0209] Gene accessibility signatures derived from ATAC-seq were compared using the RRHO algorithm. Each signature was processed as a ranked list using different expressions for two classes of compounds. Signature inputs were pre ranked gene lists with peak ID, gene name, rank 1, rank 2, metric 1, and metric 2. Gene lists were pre ranked greatest to least logFC according to the first compound in the comparison serving as rank 1 and metric 1. For metric 1 and 2, the logFC values of compound comparisons were used with FDR 0.05. Step size of 100 was used to bin the ranked items to improve run time of calculating the hypergeometric distribution. R values were calculated as a correlation between the comparisons of two compounds.

[0210] Statistical analysis

[0211] Imaging analysis and data quantification were performed using Imaged (NIH) and Prism v7.0 software (GraphPad). Error bars shown in all graphs are standard error of the mean (SEM). Gene expression and gene accessibility values were normalized and compared using a false-discovery rate adjusted - value assuming significance at FDR < 0.05. A paired two-tailed t-test was used to compare the intensity of fluororuby-labelled WT and Sarml- / - axons, as well as the mean NeuN+ neurons in the thalamus between WT and Sarml- / - animals. Unpaired two- tailed t-test used to compare neuron counts in 3D. Docket No.: UCH-35225

[0212] UCLA Ref. No.: [UCLA 2020-937-2] WO uDISCO analysis

[0213] One-way ANOVAs were used to verify neuronal and layer-specific enrichment. Neurite outgrowth assay data were compared using a two-way ANOVA for drug effect and concentration. All judgements of statistical significance were performed using post-hoc adjustments for multiple comparisons with a starting assumption of a=0.05.

[0214] To understand the mechanism of axonal loss in stroke, it was sought to determine if Sarml activity is required for axonal degeneration after ischemic injury. An in vivo model of ischemic white matter stroke was employed and axonal survival between Nz / viU-null (Sarml- / -) and wildtype (WT) mice was compared. Focal ischemic injury to the white matter is achieved via stereotactic delivery of an irreversible eNOS inhibitor L-N5-(l -Iminoethyl) ornithine, Dihydrochloride (L-Nio) to antero-lateral region of the corpus callosum to generate a local vasoconstrictive and ischemic environment in the white matter. A retrograde fluorescent tracer (fluororuby; FR) concomitantly delivered at the same time as L-Nio-induced ischemic injury aids in labeling and identifying stroke-injured neurons and axonal projections (FIG. 1A). Mean neurite fluorescence of FR+ callosal axon projections distal to the subcortical stroke are preserved throughout the corpus callosum in Sarml- / -mice while they are significantly attenuated in WT mice at 7 days (mean neurite fluorescence 20.78 in WT vs 255.70 in Sarml- / - mice; / ?<0.0001) and at 28 days (mean 10.59 in WT vs 79.53 in Sarml- / -mice; / ?<0.0001) after ischemic stroke (FIGs. 1B-1E).

[0215] In ultrastructural electron microscopy samples from the midline corpus callosum, Sarml- / - mice demonstrate relative preservation of axons with increased numbers of intact and organized peri- and endoneural structures, with a trend towards increased myelinated axons per ultrastructural field of view during the first week after stroke (3d: 66.9±20.8 vs. 74.3± 11.7; 7d: 69.9±23.3 vs. 114.6±21.4; =0.35 for interaction by two-way ANOVA; FIGs. IB & IF). Consistent with findings in other neuronal injury models, these results show that loss of Sarml activity robustly confers long-distance and long-acting (up to 28 days) axonal preservation after ischemic stroke.

[0216] Sarml activity is required for degeneration of cortical neurons distant but connected to the stroke lesion after subcortical ischemic stroke.

[0217] As subcortical ischemic stroke is uniquely associated with selective neuronal degeneration in the cortex, how delaying axonal degeneration in subcortical regions after stroke may impact cortical neuronal survival was further investigated. Tissue clearing of stroke- Docket No.: UCH-35225

[0218] UCLA Ref. No.: [UCLA 2020-937-2] WO injured brains using the previously described uDISCO technique was performed to resolve FR+ stroke-injured neurons between WT and Sarml- / - mice (FIG. 2B). Using 3D analysis, a significant increase was identified in the density of stroke-injured FR+ cortical neurons in Sarml- / - animals compared to WT mice at 7 days after ischemic white matter stroke (mean cell density in WT vs. Sarm 7-7-49.2±3.48 cells vs. 81.8±11.5 cells per 3xl06pm3of cortical tissue, respectively; p = 0.043 by unpaired t-test) (FIGS. 2A-2C).

[0219] To further determine whether the neuronal protective effect from loss of Sarml is conserved across different stroke models, the survival of subcortical thalamic neurons was compared between wildtype and sarml- / - animals following cortical ischemia as achieved by permanent distal middle cerebral artery occlusion (dMCAO). It was found that loss of Sarml similarly improved the survival of subcortical thalamic neurons 7 days after cortical dMCAO stroke, as evidenced by the significantly greater number of thalamic NeuN+ cells in sarml- / - mice than that in WT mice (mean thalamic NeuN+ cells 31.25 ± 4.07 in wildtype vs 46.50 ± 5.24 in sarml- / - mice; / ?=0.002) (FIG. 2D-E). Thus, the absence of Sarml significantly ameliorated stroke-induced neuronal degeneration in two different models of stroke injury. Enhancement of neuronal survival in the axonally protective Sarml- / - mice after ischemic stroke occurs via transcriptional regulation.

[0220] To determine the effect of Sarml deletion on gene expression by surviving neurons after ischemic injury, MACS-FACS-seq was utilized to transcriptionally profile stroke-injured cortical neurons of Sarml- / - and WT mice at 7 days post-stroke (FIG. 3A). Similar numbers of cells (5,788±3,171 vs. 4,207±l,743 FR+ / NCAM+ cells; =0.68 by unpaired t-test; FIG. 5) that enrich for deep cortical layer neurons without contamination of other non-neuronal cell types (neuronal enrichment p=0.00042 by one-way ANOVA, F(4,ii) = 12.68. FIG. 3B) were sorted, using a FR+ / NCAM+ MACS-FACS isolation strategy.

[0221] Differential gene expression (DEG) analysis (FDR<0.05) between Sarml-I- and WT stroke-injured cortical neurons identified a unique set of 891 genes (621 up-regulated, 270 down-regulated) (FIG. 3C). The top 150 up-regulated genes in Nw / iU-null neurons are functionally correlated with neuronal differentiation, axonal outgrowth, synaptic transmission, calcium signaling, and neuronal metabolism. Notably, genes implicated in the defined Sarml signaling pathways were not significantly altered in Sarml-I- mice compared to WT (FIG. 3D), indicating that the transcriptional response of cortical neurons to ischemic axonal injury in the absence of Sarml does not directly involve activation of known Sarml pathways. Docket No.: UCH-35225

[0222] UCLA Ref. No.: [UCLA 2020-937-2] WO

[0223] REVIGO was utilized to organize the gene ontology terms derived from the DEG profile and five clusters of molecular programs enriched in stroke-injured cortical neurons in the absence of Sarml were identified (FIG. 3E): cellular transport,' RNA stability,' tissue remodeling, synaptic regulation, and axonogenesis . Consistent with an anticipated impact on retrograde axon-to-cell body signaling in the absence of Sarml, the cellular transport cluster is enriched for GO terms including protein localization to cell periphery (GO: 1990778), and vesicle-mediated transport (G0:0016192), driven in part by strong up-regulation of genes such as Ezr (logFC = 3.11), and Tulp3 (logFC = 3.83) with recognized roles in these processes (FIG. 6). Similarly, the RNA stability cluster is marked by GO terms and genes implicated in the regulation of RNA stability, a key feature of retrograde axon signaling (GO:0061157 - Rbm24,' G0:0043488 -Khsrp). Unexpectedly, two clusters (axonogenesis and synaptic regulation) that suggest a pro-growth, regenerative state in stroke-injured cortical neurons is induced in the absence of Sarml were also identified. These clusters feature numerous differentially expressed genes implicated in neuron differentiation (Bell la, Tbrl, Napll2), axonogenesis (Cdk5rl, Itga4, Serpinil), and synaptic formation (Crtcl, Itsnl, Prkcz) (Table 1, FIGs. 6 & 7). Notably, the genes within these regenerative clusters demonstrate a high degree of connectivity with each other and tightly associate within a predicted protein-protein interaction network utilizing the recognized pro-growth molecule Cdc42 as a hub. This suggests that in addition to its local activity in regulating axon degeneration after stroke, Sarml also indirectly inhibits a robust pro-growth regenerative state within axonally-injured cortical neurons. Thus, loss of Sarml in stroke-injured cortical neurons results in selective transcriptional expression of genes with known, pro-growth functions including axonal transport, growth and repair.

[0224] Docket No.: UCH-35225

[0225] UCLA Ref. No.: [UCLA 2020-937-2] WO

[0226] Table 1. Differentially regulated genes in axonogenesis and synaptogenesis clusters. Docket No.: UCH-35225

[0227] UCLA Ref. No.: [UCLA 2020-937-2] WO

[0228] Bold indicates genes shared in both clusters. All GO terms were significant at / ?<7.28xl0'4.

[0229] Differential gene expression threshold defined at FDR<0.05.

[0230] Identification of small molecule compounds that promote axonal sprouting after stroke.

[0231] It was further reasoned that the regenerative program manifested by loss of Sarml in cortical neurons could be exploited to identify novel small molecule therapeutic strategies to promote axonal sprouting after ischemic stroke. A functional genomics screen was conducted using the top 150 differentially regulated genes from N / / 77U- -stroke-injured cortical neurons (FIG. 4A). Using the CLUE-IO bioinformatics database, 2428 perturbagens were identified capable of mimicking or inhibiting the transcriptional signature of Sarml- / - stroke-injured cortical neurons. A structured filtering strategy (FIG. 4B), resulted in a compound library of 18 molecules with known or predicted blood-brain barrier permeability. Numerous pharmacologic drugs of different classes, including adrenergic and androgen receptor antagonists, neurotransmitter (e.g., dopamine (DA), serotonin or 5-hydroxytryptamine (5-HT)) agonists and antagonists, as well as enzymatic inhibitors and DNA modulators were among the identified compound library (FIG. 4B).

[0232] Using a high-throughput drug screen design employing murine El 8 cultured cortical neurons, the extent of neurite outgrowth promoted by a library of 17 compounds (excluding one Schedule II FDA-regulated drug) was analyzed. Beginning at DIV7, cortical neurons were exposed to library compounds for 48 hours across a 20-step dilution curve. Immediately after drug exposure, mean neurite outgrowth was measured at doses below 100 nM and significant neurite outgrowth (>2X control neurons) was identified in 5 of 18 compounds (hypergeometric p = 0.024; FIG. 4C).

[0233] Notably, for the majority of hit compounds, drug effects on neurite outgrowth were prominent at the low end of the dilution curve. Among the hit compounds, HL017, the androgen receptor antagonist eugenol, demonstrated robust neurite outgrowth in the sub-nanomolar Docket No.: UCH-35225

[0234] UCLA Ref. No.: [UCLA 2020-937-2] WO range while having a fairly high IC50 for toxicity in primary cortical neurons ( / ?<0.0001 by two- way ANOVA, F(2i, 682)=19.32, loglCso(PMCN) =17.72 pM, FIG. 4D). Similarly, HL013, the serotonin (5-HT) receptor agonist 5-methoxytryptamine, also drives neurite outgrowth at extremely low doses while having a favorable toxicity profile in primary cortical neurons ( / UO.OOOl by two-way ANOVA, F(2i,682) =13.47, loglCso(PMCN) = -1.37pM, FIG. 4D).

[0235] Thus, using a functional genomics screen for neurite outgrowth, multiple existing compounds capable of mimicking the regenerative profile of Sarm 7-null stroke-injured cortical neurons were identified, indicating a conserved pro-growth pathway in the CNS regulated by Sarml.

[0236] To determine if these hit compounds could also drive neurite outgrowth after ischemic injury, chemical ischemia was induced by treatment of primary cortical neurons with vehicle or rotenone for 3 hrs and treated with HL013 and HL017 followed by measurement of neurite outgrowth. In this chemical ischemia model, both HL013 and HL017 compounds significantly increased neurite outgrowth after ischemic injury as compared to vehicle treated neurons ( / 2=O.OOO5 by ordinary two-way ANOVA, F(2, 8)=22.74. Thus, using functional genomics multiple compounds were identified that independently mimic the pro-regenerative molecular profile of stroke-injured sarml- / - neurons, and moreover are sufficient to drive neurite outgrowth following ischemic injury in vitro.

[0237] Molecular candidates recapitulate the pro-regenerative molecular program in Sarml- / - cortical neurons by modulating chromatin accessibility

[0238] Because the mechanism of action of all five hit compounds was substantially different from each other and their maximal effect on neurite outgrowth was most prominent at low dose, it was postulated that these compounds may be working to drive axonal outgrowth via a shared mechanism. Given that these compounds were all identified using the CLUE-IO resource employing a design to mimic a proregenerative transcriptional profile, the most reasonable shared mechanism among these compounds was via epigenetic regulation of the neuronal genome. To establish the mechanism, El 8 wild-type cortical neurons were exposed to the maximally effective dose driving neurite outgrowth for 48 hrs and performed ATAC-seq to assess genome wide chromatin accessibility and expression changes of accessible genes.

[0239] Compared to vehicle controls and a screened compound that did not drive neurite outgrowth in vitro (HL011), all five hit compounds generate a similar epigenetic signature of differentially accessible genes (DAGs) in cortical neurons (FIG. 4E). The key significantly enriched GO terms comprising the axonogenesis and synaptic regulation clusters identified in Docket No.: UCH-35225

[0240] UCLA Ref. No.: [UCLA 2020-937-2] WO

[0241] Sanni - / - stroke-injured cortical neurons by RNA-seq are conserved in the up-regulated GO terms derived from DAGs by ATAC-seq across all five hit compounds that promote neurite outgrowth in vitro compared to vehicle-treated cells (FIG. 4F). To statistically define similarities in genome-wide chromatin accessibility driven by hit compounds, gene accessibility profiles were compared between non-growth promoting hit compounds (HL011) and growth promoting hit compounds (HL013, HL017) using the rank-rank hypergeometric overlap (RRHO) algorithm (FIG. 4G). RRHO heatmaps confirm increasing similarity in gene accessibility signatures among hit compounds showed that growth-promoting compounds HL013 and HL017 demonstrate the highest level of genome-wide similarity (r=0.64, / ?<0.0001) while comparisons between non-growth promoting HL011 and HL013 (r=0.41, / ?<0.0001) and HL017 (r=0.53, / ?<0.0001) were significant but not as strong indicating a coordinated epigenetic signature driven by growth-promoting hit compounds. Notably, motif analysis demonstrates 8 upregulated and 2 down-regulated transcription factor recognition motifs that were conserved across all hit compounds and enriched compared to vehicle and non-growth promoting compounds (HL011). The corresponding transcription factors (RBPJ, Asci, E2A) containing these conserved motifs all have recognized roles in neuronal- and axonogenesis, thus further reflecting an epigenetic control of a pro-growth molecular program by Sanni in cortical neurons.

[0242] Selected comments

[0243] Using a characterized in vivo murine model of focal white matter stroke, it is demonstrated in Example 1 that loss of Sarml significantly promotes long distance and long- acting (up to 28 days— the latest time point after stroke evaluation) structural preservation of the axons after focal ischemic stroke to the white matter. Thus, data demonstrate Sarml is required for axonal degeneration from ischemic injury, and that axonal degradation following ischemia, similar to multiple types of traumatic and chemotoxic mechanisms of CNS injuries, converge on a Sarml -dependent mechanism. Thus, interventions aimed at inhibiting or abrogating Sarml activity present a viable and effective strategy to mitigate axonal degeneration after ischemic stroke.

[0244] It is further demonstrated herein that the absence of Sarml also led to significantly increased number of surviving neurons following ischemia in two different models of stroke. This includes increased cortical neuron survival after subcortical white matter stroke, and increased survival of subcortical neurons after a cortical stroke via permanent occlusion of the distal MCA. In both cases, the protected neurons from ischemia due to the absence of Sarml Docket No.: UCH-35225

[0245] UCLA Ref. No.: [UCLA 2020-937-2] WO are distant from but anatomically connected to the actual stroke lesion. This suggests that a likely mechanism by which loss of Sarml mitigates somal degeneration is by preventing the activation, translocation or retrograde transport of axonal injury signal(s) to the soma that then trigger eventual degeneration of the cell body. This is further supported by the fact that in both the white matter stroke and the cortical dMCAO stroke models selective labeling of neuronal cell bodies take place distal to the region where either the fluorescent dye or the reporter viral construct was injected. This further indicates that long-distance labeling of neuronal cell bodies necessitates retrograde axonal transport of the FR dye or retrograde transport of reporter construct from the distal axonal terminal to the neuronal soma. The finding of improved survival of neuronal cell bodies distant from site of ischemic injury due to absence of Sarml supports a mechanism by which sarml- / - dependent axonal preservation prevents retrograde axonal signaling from taking place to trigger secondary neuronal cell body degeneration after ischemic stroke.

[0246] Indeed, multiple studies have identified c-Jun N-terminal kinases (JNK), a member of the MAPK family, to be retrogradely transported to the soma and alter somal transcription of known injury response molecules following traumatic peripheral nerve as well as optic nerve injuries. Moreover, downstream MAPK kinases including MAPK3, DLK and LZK are implicated in activating JNK1 - 3 and their canonical target JUN, which leads to transcriptional changes that ultimate culminate in BAX activation and neuronal apoptosis. Curiously, loss of Sarml delayed axonal degeneration, but did not prevent DLK / JNK pathway activation or delay RGC cell death in traumatic optic nerve crush. In contrast, in stroke-injured FACS-captured cortical neurons limited transcriptional activation of Mapk kinases was observed in Sarml- / - mice compared to wild-type animals. Thus, while Sarml mediates a common mechanism of axon degeneration across multiple injury mechanisms, there may be heterogeneity and injuryspecific activation and transport of injury signals following axonal injury. This may be resolved from further studies ascertaining whether the JNK / MAPK pathway are activated after focal white matter injury in our model, and whether a causal relationship exists between Sarml activity and activation of retrograde axonal injury signals in ischemic stroke. Moreover, in probing for molecular basis of sarml- / - mediated neuronal protection, there was an unexpected identification of a restricted subset of DEGs in stroked-injured sarml- / - neurons that cluster to pro-growth functional groups. Unique molecular signatures were observed in stroke-injured Docket No.: UCH-35225

[0247] UCLA Ref. No.: [UCLA 2020-937-2] WO neurons deficient in Sarml that favor a pro-growth pattern of expression that support poststroke neural survival and repair.

[0248] Notably, transcriptomic analyses between sarml- / - and WT stroke-injured cortical neurons did not show enrichment or alteration of DEGs that are normally known to participate in previously defined Sarml signaling pathway. This indicates that the transcriptional response of cortical neurons to ischemic axonal injury in the absence of Sarml involves expression changes of novel pathways that are distinct from known Sarml targets.

[0249] As recovery and neural repair after stroke is ultimately mediated by the development of new axonal connections between disconnected brain regions, the findings herein suggest that in addition to its local activity in regulating axon degeneration after stroke, Sarml and / or its downstream target(s) normally inhibit a robust pro-growth regenerative program in cortical neurons after stroke. In fact, several pathways contributing to axon degeneration overlap in part with those that drive axon regeneration, suggesting a convergence of mechanisms that regulate axonal degeneration and attempted regeneration after injury. In the present example, the expression of pro-regenerative genes promoted by the loss of the degenerative molecule Sarml supports a similar mechanistic overlap between axonal degeneration and inhibition of regenerative programs for neural repair after stroke and raises the prospect that both axonal degeneration and axonal remodeling after stroke injury are regulated by Sarml activity. However, whether this pro-regenerative program induced by loss of Sarml is directly mediated by activity of Sarml itself or indirectly by downstream targets of Sarml remains unclear. Mechanistically, it was further shown that this pro-growth program in cortical neurons that is normally inhibited by Sarml activity is regulated at the level of the epigenome. Molecular compounds were identified from functional genomics screen that recapitulate a similar progrowth genetic program favoring axonogenesis as that seen in sarml- / - stroke injured neurons, and the compounds were confirmed to independently promote axonal outgrowth in vitro, even in the presence of Sarml . Although the pro-growth molecular compounds individually target different mechanisms, the presence of conserved motifs that is similar between DAGs in ATAC-seq and bulk RNAseq between WT and sarml- / - neurons indicates a highly conserved, de novo epigenetic pro-growth program in cortical neurons that is druggable by modulating differential accessibility to chromatin at defined loci. Thus, the present study indicates that pharmacologic interventions to modulate Sarml activity itself, or to phenocopy the same progrowth expression profile as sarml- / - in cortical neurons after stroke may concurrently Docket No.: UCH-35225

[0250] UCLA Ref. No.: [UCLA 2020-937-2] WO attenuate axon degeneration and promote reparative capacity of cortical neurons after ischemia, and points to a potential new approach to enhance neural repair after stroke by targeting Sarml .

[0251] Herein it has been demonstrated that loss of Sarml not only mitigates anterograde axonal degeneration and promotes neuronal survival after ischemic stroke, but surprisingly also facilitates a regenerative program in cortical neurons that is encoded at the epigenetic level. The finding that this epigenetic signature can be recapitulated with multiple low dose pharmacologic agents supports the concept that a regenerative capacity for axonal outgrowth in cortical neurons after stroke is programmed into the genome but actively restricted by pro- degenerative regulators such as Sarml. Well-timed inhibition of Sarml to relieve this regulation, or direct activation of this downstream, druggable epigenetic program may represent a novel strategy to mitigate axonal degeneration and promote axon repair after stroke.

[0252] Example 2: Additional Characterization of the Compounds of the Disclosure

[0253] In vitro data of human neurons

[0254] Compounds of the disclosure were tested through in vitro experiments where multiple screened compounds were capable of driving axon outgrowth.

[0255] Methods

[0256] Neurite outgrowth was measured at submicromolar concentrations for both mouse and human cells. Compounds were dosed over 48 hours in three replicates per drug per dose. Results

[0257] Fig. 8 shows the growth results versus control for mouse cortical neurons, and Fig. 9 shows the growth results versus control for human iPSC neurites. Fig. 8 shows a bar plot of percentage mean neurite outgrowth at peak subnanomolar doses for HL004A, HL004B, and HL004C versus vehicle control primary mouse cortical neurons after 48 hours of exposure (three replicates / drug / dose) (**** / ?<0.0001 by one way ANOVA). Fig. 9 shows a bar plot of percentage mean neurite outgrowth at peak submicromolar doses for each compound tested versus vehicle control human iPSC derived neurons (three replicates / drug / dose).

[0258] Human iPSC Calcein-AM imaging is shown in Fig. 10A and Fig. 10B. Fig. lOA shows a control result, and Fig. 10B shows the effect of HL013 after 48 hours of dosing. Further results are shown in Table 2, which details R values of neurite growth upon treatment with compounds of the disclosure. Fig. 11 shows visualization of the rank rank hypergeometric overlay (RRHO) heatmaps of ATAC-seq signatures of compound-treated human induced Docket No.: UCH-35225

[0259] UCLA Ref. No.: [UCLA 2020-937-2] WO pluripotent stem cell derived neurons comparing growth-promoting and a non-growth promoting (HL011) compound. The compounds of the disclosure demonstrate effectiveness in stimulating neurite outgrowth in human iPSC-derived neurons. The mechanism of action of the growth-stimulating compounds drives a distinct epigenetic signature in human iPSCs. The result is discovery of compounds that can stimulate axon or neurite outgrowth by regulating a coordinated genetic target.

[0260] Table 2. Neurite growth analysis of compounds in human iPSC assay.

[0261] Other in vitro testing has shown that after a chemical ischemic injury, HL013 and HL017 can still promote neurite (axon) outgrowth.

[0262] INCORPORATION BY REFERENCE

[0263] All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0264] EQUIVALENTS

[0265] While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Claims

Docket No.: UCH-35225UCLA Ref. No.: [UCLA 2020-937-2] WOWe claim:

1. A method of treating a brain injury, a spinal cord injury, or a neurodegenerative disease in a subject in need thereof comprising administering a compound selected fromDocket No.: UCH-35225UCLA Ref. No.: [UCLA 2020-937-2] WODocket No.: UCH-35225UCLA Ref No.: [UCLA 2020-937-2] WOsalt thereof, to the subj ect.Cl2. The method of claim 1, wherein the compound is selected fromDocket No.: UCH-35225UCLA Ref. No.: [UCLA 2020-937-2] WOpharmaceutically acceptable salt thereof, to the subject.

3. The method of claim 1 or claim 2, wherein the method is a method of treating a brain injury.

4. The method of claim 3, wherein the brain injury is a stroke.

5. The method of claim 3, wherein the brain injury is an ischemic stroke.

6. The method of claim 3, wherein the brain injury is an ischemic brain injury.

7. The method of claim 3, wherein the brain injury is a traumatic brain injury.

8. The method of claim 1 or claim 2, wherein the method is a method of treating a neurodegenerative disease.

9. The method of claim 8, wherein the neurodegenerative disease is Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, amyotrophic lateral sclerosis, cerebellar ataxia, frontotemporal dementia, or Huntington’s disease.

10. The method of any one of claims 1-9, wherein the method promotes axon regeneration.Docket No.: UCH-35225UCLA Ref. No.: [UCLA 2020-937-2] WO11. A method of promoting axon regeneration in a subject in need thereof comprisingDocket No.: UCH-35225UCLA Ref. No.: [UCLA 2020-937-2] WOsalt thereof, to the subj ect.Docket No.: UCH-35225UCLA Ref. No.: [UCLA 2020-937-2] WOCl12. The method of claim 11, wherein the compound is selected frompharmaceutically acceptable salt thereof, to the subject.

13. The method of any one of claims 1-12, wherein the compoundor a pharmaceutically acceptable salt thereof.Docket No.: UCH-35225UCLA Ref. No.: [UCLA 2020-937-2] WO14. The method of any one of claims 1-12, wherein the compoundor a pharmaceutically acceptable salt thereof.

15. The method of any one of claims 1-12, wherein the compound is17. The method of any one of claims 1-12, wherein the compound issalt thereof.Docket No.: UCH-35225UCLA Ref. No.: [UCLA 2020-937-2] WO18. The method of any one of claims 1-12, wherein the compound ispharmaceutically acceptable salt thereof.

19. The method of any one of claims 1-12, wherein the compounda pharmaceutically acceptable salt thereof.

20. The method of any one of claims 1-12, wherein the compound isor a pharmaceutically acceptable salt thereof.

21. The method of any one of claims 1-12, wherein the compoundor a pharmaceutically acceptable salt thereof.

22. The method of any one of claims 1-12, wherein the compoundor a pharmaceutically acceptable salt thereof.Docket No.: UCH-35225UCLA Ref. No.: [UCLA 2020-937-2] WO23. The method of any one of claims 1-12, wherein the compound is24. The method of any one of claims 1 and 3-11, wherein the compound isOH or a pharmaceutically acceptable salt thereof.

25. The method of any one of claims 1 and 3-11, wherein the compound is pharmaceutically acceptable salt thereof. y one of claims 1 and 3-11, wherein the compound ispharmaceutically acceptable salt thereof.Docket No.: UCH-35225UCLA Ref. No.: [UCLA 2020-937-2] WO27. The method of any one of claims 1 and 3-11, wherein the compound is29. The method of any one of claims 1 and 3-11, wherein the compound is30. The method of any one of claims 1 and 3-11, wherein the compound ispharmaceutically acceptable salt thereof.Docket No.: UCH-35225UCLA Ref. No.: [UCLA 2020-937-2] WO31. The method of any one of claims 1 and 3-11, wherein the compound ispharmaceutically acceptable salt thereof.

32. The method of any one of claims 1 and 3-11, wherein the compound is armaceutically acceptable salt thereof. ny one of claims 1 and 3-11, wherein the compound ispharmaceutically acceptable salt thereof.

Images