Dual orexin receptor antagonists and histone deacetylase inhibitors for treating haploinsufficiency neurodevelopmental disorders

Abstract

The present disclosure relates to methods for treating haploinsufficiency neurodevelopmental disorders associated with genes selected from TSC1, TSC2, NSD1, KMT2D, KDM6A, CREBBP, EP300, KANSL1 and RAI1, comprising administration of dual orexin receptor antagonists (DORAs) and / or orexin receptor antagonists (ORAs). The present disclosure further provides methods for treating tuberous sclerosis complex (TSC) by administration of DORAs and / or selective histone deacetylase inhibitors.

Description

Mintz Ref.: 064944-502001 WODUAL OREXIN RECEPTOR ANTAGONISTS AND HISTONE DEACETYLASE INHIBITORS FOR TREATING HAPLOINSUFFICIENCY NEURODEVELOPMENTAL DISORDERSCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 738,972 filed December 26, 2024 which is incorporated by reference herein in its entirety.FIELD

[0002] The present invention relates to methods of treating a haploinsufficiency neurodevelopmental disorder by use of dual orexin receptor antagonists and / or orexin receptor antagonists, and to methods of treating Tuberous Sclerosis Complex by use of dual orexin receptor antagonists and / or histone deacetylase inhibitors.BACKGROUND

[0003] Tuberous Sclerosis Complex (TSC), also known as Tuberous Sclerosis, is a rare genetic disease that manifests with seizures, autism, and cognitive deficits. In addition, it can cause non-cancerous (benign) tumors to grow in the brain and several areas of the body, including the spinal cord, nerves, eyes, lung, heart, kidneys, and skin. Mutations in the TSC1 gene or the TSC2 gene may cause TSC. HDAC inhibition has been suggested as a potential therapeutic rescue strategy, (see Basu T, O'Riordan KJ, Schoenike BA, et al. Histone deacetylase inhibitors restore normal hippocampal synaptic plasticity and seizure threshold in a mouse model of Tuberous Sclerosis Complex. Sci Rep. 2019;9(1):5266. Published 2019 Mar 27. doi:10.1038 / s41598-019-41744-7).

[0004] Orexin-A and orexin-B are neuropeptides produced by the hypothalamus that stimulate wakefulness. A Dual Orexin Receptor Antagonist (DORA) is a type of drug that suppresses wakefulness by blocking the action of orexin neuropeptides, promoting both REM and non-REM sleep. The first FDA approved DORA, Suvorexant, is used to improve sleep quality.Mintz Ref.: 064944-502001 WOSUMMARY

[0005] The present invention provides a method of administering a dual orexin receptor antagonist or a histone deacetylase inhibitor (e.g., a selective histone deacetylase inhibitor) for the treatment of haploinsufficiency neurodevelopmental disorders.

[0006] Upregulation of any of TSC 1 or TSC2 may be beneficial as many patients have mutations in only one allele (haploinsufficiency), which is sufficient to cause the condition. For example, it has been demonstrated that loss of one allele of TSC2 is sufficient to cause some morphological and physiological changes in human neurons, (see Winden KD, Sundberg M, Yang C, et al. Biallelic Mutations in TSC2 Lead to Abnormalities Associated with Cortical Tubers in Human iPSC-Derived Neurons. J Neurosci. 2019;39(47):9294-9305, incorporated by reference herein in its entirety). Data included herein identifies dual orexin receptor antagonists as upregulators of TSC2 and demonstrates phenotypic rescue.

[0007] Kabuki Syndrome is a haploinsufficiency neurodevelopmental disorder primarily caused by mutations in the KMT2D gene (see Lavery et al. KMT2D Haploinsufficiency in Kabuki Syndrome Impairs Differentiation of Neural Crest Cells. FASEB J 34(S1) (2020), incorporated by reference herein in its entirety).

[0008] Sotos Syndrome is a haploinsufficiency neurodevelopmental disorder most commonly caused by mutations or deletions in the NSD1 gene. (See Kurotaki et al. Haploinsufficiency of NSD1 causes Sotos Syndrome. Nat Genet 30(4) (2002), incorporated by reference herein in its entirety).

[0009] Smith-Magenis Syndrome (SMS) is a haploinsufficiency neurodevelopmental disorder most commonly caused by mutations or deletions in the RAI1 gene, which encodes a transcriptional regulator essential for neuronal development and circadian rhythm control. (See Slager et al. Mutations in RAI1 associated with Smith-Magenis syndrome. Am J Med Genet A 117(3) (2003), incorporated by reference herein in its entirety) Recent studies indicate that targeted rescue of RAI1 activity can ameliorate phenotypic features of SMS, supporting the feasibility of gene -based or epigenetic interventions. (See Chang et al. rAAV-CRISPRa therapy corrects Rail haploinsufficiency and rescues selective disease features in Smith-Magenis syndrome mice. J Biol Chem 299(1) (2022), incorporated by reference herein in its entirety).

[0010] Koolen-de Vries Syndrome (KdVS) is a disorder associated with KANSL1 expression. Haploinsufficiency of KANSL1 (caused by different types of mutations in the KANSL1 gene) is sufficient to cause the 17q21.31 microdeletion syndrome (also known as Koolen-de Vries Syndrome),Mintz Ref.: 064944-502001 WOa multisystem disorder characterized by intellectual disability, hypotonia and distinctive facial features, (see Koolen et al. Mutations in the chromatin modifier gene KANSL1 cause the 17q21.31 microdeletion syndrome. Nat Genet 44, 639-641 (2012), incorporated by reference herein in its entirety). Increasing gene product of the healthy allele is a therapeutic strategy for rescuing or alleviating the disease symptoms (see Li et al. Kansll haploinsufficiency impairs autophagosomelysosome fusion and links autophagic dysfunction with Koolen-de Vries syndrome in mice. Nat Commun 13, 931 (2022), linking autophagic dysfunction with Koolen-de Vries syndrome, and demonstrating that rescue of KANSL1 or a key downstream gene target rescues the impaired autophagic activity in KANSL1 -deficient cells).

[0011] Rubinstein-Taybi Syndrome (RTS) is a haploinsufficiency neurodevelopmental disorder, in most cases caused by different types of mutations in either the CREBBP gene (or the EP300 gene), (see Roelfsema et al. Genetic heterogeneity in Rubinstein-Taybi syndrome: mutations in both the CBP and EP300 genes cause disease. Am J Hum Genet. 2005;76(4):572-580 and Petrij et al. Rubinstein-Taybi syndrome caused by mutations in the transcriptional co-activator CBP. Nature. 1995 Jul 27;376(6538):348-51; each of which is incorporated by reference herein in its entirety) Rescue of CREBBP has been demonstrated to rescue part of the RTS phenotype, (see Korzus E. Rubinstein-Taybi Syndrome and Epigenetic Alterations. Adv Exp Med Biol. 2017;978:39-62, incorporated by reference herein in its entirety).BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIGs.1A-1F show mRNA expression of TSC2 (FIG. 1A), RAI1 (FIG. 1B), NSD1 (FIG. 1C), KMT2D (FIG. 1D), CREBBP (FIG. 1E), and KANSL1 (FIG. 1F) of Almorexant treated wild type glutamatergic neurons in a hit confirmation experiment. All replicates as well as mean with standard deviation shown for drug treatment data, while only mean with standard deviation shown for DMSO (280 replicates). Data normalized to GAPDH housekeeping gene.

[0013] FIGs.2A-2C show results of multielectrode array experiments following application of histone deacetylase inhibitors and dual orexin receptor antagonists, over a range of doses administered chronically (started prior to the establishment of hyperexcitability phenotype), in TSC2- / - neurons.

[0014] FIGs.3A-3D show images of MEA plates prior to and following drug treatment. Pan-histone deacetylase inhibitors (Dacinostat and Panobinostat) cause cell death following chronic treatment across indicated doses, whereas selective histone deacetylase inhibitors (BRD6688 and BRD4884) didMintz Ref.: 064944-502001 WOnot cause cell death in the experiments disclosed herein. Dual orexin receptor antagonists do not cause cell death at the range of doses chronically tested.DETAILED DESCRIPTION

[0015] It has been unexpectedly found that dual orexin receptor antagonists increase TSC2 gene expression, which is relevant for the treatment of Tuberous Sclerosis Complex. Dual orexin receptor antagonists, as well as selective histone deacetylase inhibitors, also decrease neuronal metrics such as weighted mean firing rate (WMFR) - see FIGs.2A-2C. In some embodiments, the present invention features methods of treating Tuberous Sclerosis Complex by use of dual orexin receptor antagonists. In some embodiments, the present invention features methods of treating Tuberous Sclerosis Complex by use of selective histone deacetylase inhibitors. In some embodiments, the present invention features methods of treating Tuberous Sclerosis Complex by use of class 1 histone deacetylase inhibitors with selectivity bias towards histone deacetylase 2 (HDAC2).

[0016] Definitions

[0017] The term “and / or” as used herein means that the listed items are present, or used, individually or in combination. In effect, this term means that “at least one of’ or “one or more” of the listed items is used or present. The term “and / or” with respect to pharmaceutically acceptable salts and / or solvates thereof means that the compounds of the disclosure exist as individual salts and hydrates, as well as a combination of, for example, a solvate of a salt of a compound of the disclosure.

[0018] As used in the present disclosure, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise. For example, an embodiment including “a compound” should be understood to present certain aspects with one compound, or two or more additional compounds.

[0019] In embodiments comprising an “additional” or “second” component, such as an additional or second compound, the second component as used herein is chemically different from the other components or first component. A “third” component is different from the other, first, and second components, and further enumerated or “additional” components are similarly different.

[0020] As used in this disclosure and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"),Mintz Ref.: 064944-502001 WO"including" (and any form of including, such as "include" and "includes") or "containing" (and any form of containing, such as "contain" and "contains"), are inclusive or open-ended and do not exclude additional, unrecited elements or process steps.

[0021] The term “administered” as used herein means administration of a therapeutically effective amount of one or more compounds or compositions of the disclosure to a cell, tissue, organ or subject.

[0022] The term “pharmaceutically acceptable salt” means either an acid addition salt or a base addition salt which is suitable for, or compatible with, the treatment of subjects.

[0023] The term “solvate” as used herein means a compound, or a salt of a compound, wherein molecules of a suitable solvent are incorporated in the crystal lattice.

[0024] “Isomerism” means compounds that have identical molecular formulae but differ in the sequence of bonding of their atoms or in the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are termed “stereoisomers.” Stereoisomers that are not mirror images of one another are termed “diastereoisomers,” and stereoisomers that are non-superimposable mirror images of each other are termed “enantiomers” or sometimes optical isomers. A mixture containing equal amounts of individual enantiomeric forms of opposite chirality is termed a “racemic mixture.”

[0025] An “metabolite” is a chemical entity that is formed through the metabolic transformation of a parent compound (typically a drug) within a living organism in vivo). A metabolite may be an active metabolite, i.e., a metabolite that retains pharmacological activity and contributes to the therapeutic or toxic effects of the parent compound. In some cases, the metabolite may exhibit greater potency, selectivity, or a different pharmacokinetic profile than the original drug. In some embodiments, the term “active metabolite” is used to refer to synthetically produced compounds that have the same chemical structure as a metabolite formed through metabolic transformation within a living organism. Metabolites may be formed by one or more Phase I and / or Phase II biotransformations, including, without limitation, reduction, oxidative deamination, deoximation / oxime hydrolysis, N oxidation, glucuronidation, sulfation, amino acid conjugation. As used herein, the term “metabolite” also includes salts, solvates, hydrates, polymorphs, stereoisomers, and tautomers of the metabolite, or metabolites of salts, solvates, hydrates, polymorphs, stereoisomers, and tautomers of the parent compound.

[0026] “Tautomer” is one of two or more structural isomers that exist in equilibrium and is readily converted from one isomeric form to another. This conversion results in the formal migration of aMintz Ref.: 064944-502001 WOhydrogen atom accompanied by a switch of adjacent conjugated double bonds. Tautomers exist as a mixture of a tautomeric set in solution. In solutions where tautomerization is possible, a chemical equilibrium of the tautomers will be reached. The exact ratio of the tautomers depends on several factors, including temperature, solvent and pH. The concept of tautomers that are interconvertable by tautomerizations is called tautomerism.

[0027] Of the various types of tautomerism that are possible, two are commonly observed. In ketoenol tautomerism a simultaneous shift of electrons and a hydrogen atom occur. Ring-chain tautomerism arises as a result of the aldehyde group (-CHO) in a sugar chain molecule reacting with one of the hydroxy groups (-OH) in the same molecule to give it a cyclic (ring-shaped) form as exhibited by glucose.

[0028] Common tautomeric pairs are: ketone-enol, amide-nitrile, lactam-lactim, amide-imidic acid tautomerism in heterocyclic rings (e.g., in nucleobases such as guanine, thymine and cytosine), imineenamine and enamine-enamine. An example of keto-enol equilibria is between pyridin-2(lH)-ones and the corresponding pyridin-2-ols, as shown below.pyridin-2(1H)-one pyridin-2-ol

[0029] It is to be understood that the compounds of the present invention may be depicted as different tautomers. It should also be understood that when compounds have tautomeric forms, all tautomeric forms are intended to be included in the scope of the present invention, and the naming of the compounds does not exclude any tautomer form. It will be understood that certain tautomers may have a higher level of activity than others.

[0030] The term “crystal polymorphs”, “polymorphs” or “crystal forms” means crystal structures in which a compound (or a salt or solvate thereof) can crystallize in different crystal packing arrangements, all of which have the same elemental composition. Different crystal forms usually have different X-ray diffraction patterns, infrared spectral, melting points, density hardness, crystal shape, optical and electrical properties, stability and solubility. Recrystallization solvent, rate ofMintz Ref.: 064944-502001 WOcrystallization, storage temperature, and other factors may cause one crystal form to dominate. Crystal polymorphs of the compounds can be prepared by crystallization under different conditions.

[0031] The term “treating” or “treatment” as used herein and as is well understood in the art, means an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results 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, diminishment of the reoccurrence of disease, and remission (whether partial or total), whether detectable or undetectable. “Treating” and “treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. “Treating” and “treatment” as used herein also include prophylactic treatment. For example, a subject with early cancer can be treated to prevent progression, or alternatively a subject in remission can be treated with a compound or composition of the disclosure to prevent recurrence. Treatment methods comprise administering to a subject a therapeutically effective amount of one or more of the compounds of the disclosure and optionally consist of a single administration, or alternatively comprise a series of administrations.

[0032] The term “inhibit” or “inhibition” as used herein refers to any decrease in an enzyme’s activity in the presence of one or more compounds of the disclosure compared to a control (for example, otherwise identical conditions except for the absence of one of more compounds of the disclosure).

[0033] As used herein, the term “effective amount” or “therapeutically effective amount” means an amount of one or more compounds of the disclosure that is effective, at dosages and for periods of time necessary to achieve the desired result.

[0034] As used herein, “Dual orexin receptor antagonist” or “DORA” means a compound that functionally antagonizes both orexin- 1 receptor (OX1R) and orexin-2 receptor (OX2R). Non-limiting examples include almorexant, filorexant, daridorexant, lemborexant, suvorexant, fazamorexant, vornorexant, SB-649868, and pharmaceutically acceptable salts, solvates, stereoisomers, prodrugs, and metabolites thereof.

[0035] As used herein, “Orexin receptor antagonist” or “ORA” means a compound that functionally antagonizes at least one orexin receptor subtype, i.e., orexin- 1 receptor (OX1R) and / or orexin-2 receptor (OX2R). ORAs include compounds that functionally antagonize one orexin, e.g., selective orexin receptor antagonists (SORA), which can be OXIR-selective antagonists or OX2R-selective antagonists; and dual orexin receptor antagonists (DORA).Mintz Ref.: 064944-502001 WO

[0036] As used herein, the term “HDAC inhibitor” refers to a compound that inhibits the enzymatic activity of one or more histone deacetylases (HDACs). For example, an HDAC inhibitor referred to herein may inhibit the enzymatic activity of one or more HDAC isoforms (e.g., HDAC 1, HDAC 2, etc.) or members of an HDAC class (e.g., Class I, Class II). An HDAC inhibitor may have activity against more than one isoform. For example, an “HDAC 1 inhibitor” may inhibit HDAC 1 and may also inhibit other HDAC isoforms. In other words, an “HDAC 1 inhibitor” may include selective and non-selective inhibitors of HDAC 1.

[0037] A “selective HDAC inhibitor” (e.g., “class I selective HDAC inhibitor” or “HDAC 1 selective / \HDAC inhibitor” refers to a comp 00ound that exhibits inhibitory activity more so against one HDAC isoform (e.g., HDAC 1 or HDAC 2) or? a 7 / =\ v specific HDAC class (e.g., Class I, Class II), with substantially reduced activity against other HDAC isoforms or classes. Selectivity may be determined by comparing IC50 or Ki values, with or without regard to kinetic profiles.

[0038] In some embodiments, non-limiting examples of dual orexin receptor antagonists include those listed in Table 1, as well as the pharmaceutically acceptable salt, free acid, or free base thereof:

[0039] Table 1: Exemplary dual orexin receptor antagonistsAlmorexantFilorexantMintz Ref.: 064944-502001 WODaridorexantLemborexanto 0'p" LL o!_ _I ziSuvorexant qr ZIA°, / p AoAP L°op _o p \ Fazamorexant T|" A A LLVornorexantSB-649868f ihny \ O -YNY^ ° °soFMintz Ref.: 064944-502001WO

[0040] In some embodiments, non-limiting examples of histone deacetylase inhibitors, including certain selective histone deacetylase inhibitors, include those listed in Table 2, as well as pharmaceutically acceptable salts, free acids, or free bases thereof:

[0041] Table 2: Exemplary histone deacetylase inhibitorsBRD6688, NH2£ 1 AN£HO poBRD4884oTZ NH^ 5-2oRomidepsin-1p ar°YNV HNA>oEntinostatMintz Ref.: 064944-502001 WO Tacedinaline j? A1 J-.. JHNH2HSantacruzamate AAA °0Mocetinostat ANA zx / x(f V N N Ai A NH21H1 A U i. Y ACXD101 / N-N\A'" ASn H px-k0Xj Givinostat 0JU zOH A I J B AAUAHBRD4161 oZ^N^NHX=> f jrMintz Ref.: 064944-502001 WOBRD3386BRD7232 0A \ ikl A TH^NH2I / UM o nrfl — )o= Tucidinostat >° / =\ a<) z\ / — / / X _ozxNS32 T r if J o kA rAbexinostat \PracinostatHH T / > — \0Resminostat o V., / 0 0A Jk A’H CH3[T A N v N 1 \ H A. J =H3AJMintz Ref.: 064944-502001 WOFimepinostatCUDC-101o1.o, z XJIu n xN,. O.^ AHO A^ zzO \ JO N^,zModified Compounds of the Invention / / 4 / \

[0042] A modified compound of any one of\ ozz- s~ 'uch compounds including a modification having anz= / — / \improved, e.g., enhanced, greater, pharmaceutical solubility, stability, bioavailability and / or therapeutic index as compared to the unmodified compound is also contemplated. The examples of Mmodifications include but are n oot limited to the prodrug derivatives, and isotopically-labeled / compounds, e.g., deuterium-enriched compounds.

[0043] Prodrug derivatives: prodrugs, upon administration to a subject, will be converted in vivo into active compounds of the present invention (Nature Reviews of Drug Discovery, 2008, 7:255). It is noted that in many instances, the prodrugs themselves also fall within the scope of the range of compounds according to the present invention. The prodrugs of the compounds of the present invention can be prepared by standard organic reaction, for example, by reacting with a carbamylating agent (e.g., 1, 1 -acyloxyalkylcarbonochloridate, para-nitrophenyl carbonate, or the like) or an acylating agent. Further examples of methods and strategies of making prodrugs are described in Bioorganic and Medicinal Chemistry Letters, 1994, 4:1985.

[0044] Certain isotopically-labelled compounds of the various Formulae (e.g., those labeled with3H and14C) are useful in compound and / or substrate tissue distribution assays. Tritiated (i.e.,3H) and carbon- 14 (i.e.,14C) isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium (i.e.,2H) may afford certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements) and hence may be preferred in some circumstances. Isotopically labelledMintz Ref.: 064944-502001 WOcompounds of the various Formulae can generally be prepared by following procedures analogous to those disclosed in the Schemes and / or in the Examples herein below, by substituting an appropriate isotopically labelled reagent for a non-isotopically labelled reagent.

[0045] Deuterium-enriched compounds: deuterium (D or2H) is a stable, non-radioactive isotope of hydrogen and has an atomic weight of 2.0144. Hydrogen naturally occurs as a mixture of the isotopesXH (hydrogen or protium), D (2H or deuterium), and T (3H or tritium). The natural abundance of deuterium is 0.015%. One of ordinary skill in the art recognizes that in all chemical compounds with an H atom, the H atom actually represents a mixture of H and D, with about 0.015% being D. Thus, compounds with a level of deuterium that has been enriched to be greater than its natural abundance of 0.015%, should be considered unnatural and, as a result, novel over their nonenriched counterparts.

[0046] The present disclosure is intended to include all isotopes of atoms occurring in the present compounds. Isotopes include those atoms having the same atomic number but different mass numbers. In particular one, some, or all hydrogens may be deuterium. Radioactive isotopes may be used, for instance for structural analysis or to facilitate tracing the fate of the compounds or their metabolic products after administration. By way of general example and without limitation, isotopes of hydrogen include deuterium and tritium and isotopes of carbon include C-13 and C-14.

[0047] It should be recognized that the compounds of the present invention may be present and optionally administered in the form of salts, and solvates. For example, it is within the scope of the present invention to convert the compounds of the present invention into and use them in the form of their pharmaceutically acceptable salts derived from various organic and inorganic acids and bases in accordance with procedures well known in the art.

[0048] When the compounds of the present invention possess a free base form, the compounds can be prepared as a pharmaceutically acceptable acid addition salt by reacting the free base form of the compound with a pharmaceutically acceptable inorganic or organic acid, e.g., hydrohalides such as hydrochloride, hydrobromide, hydroiodide; other mineral acids such as sulfate, nitrate, phosphate, etc.; and alkyl and monoarylsulfonates such as ethanesulfonate, toluenesulfonate and benzenesulfonate; and other organic acids and their corresponding salts such as acetate, tartrate, maleate, succinate, citrate, benzoate, salicylate and ascorbate. Further acid addition salts of the present invention include, but are not limited to: adipate, alginate, arginate, aspartate, bisulfate, bisulfite, bromide, butyrate, camphorate, camphorsulfonate, caprylate, chloride, chlorobenzoate, cyclopentanepropionate, digluconate, dihydrogenphosphate, dinitrobenzoate, dodecylsulfate, fumarate, galacterate (from mucic acid),Mintz Ref.: 064944-502001 WOgalacturonate, glucoheptaoate, gluconate, glutamate, glycerophosphate, hemisuccinate, hemisulfate, heptanoate, hexanoate, hippurate, 2-hydroxyethanesulfonate, iodide, isethionate, iso-butyrate, lactate, lactobionate, malonate, mandelate, metaphosphate, methanesulfonate, methylbenzoate, monohydrogenphosphate, 2-naphthalenesulfonate, nicotinate, oxalate, oleate, pamoate, pectinate, persulfate, phenylacetate, 3 -phenylpropionate, phosphonate and phthalate. It should be recognized that the free base forms will typically differ from their respective salt forms somewhat in physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free base forms for the purposes of the present invention.

[0049] When the compounds of the present invention possess a free acid form, a pharmaceutically acceptable base addition salt can be prepared by reacting the free acid form of the compound with a pharmaceutically acceptable inorganic or organic base. Examples of such bases are alkali metal hydroxides including potassium, sodium and lithium hydroxides; alkaline earth metal hydroxides such as barium and calcium hydroxides; alkali metal alkoxides, e.g., potassium ethanolate and sodium propanolate; and various organic bases such as ammonium hydroxide, piperidine, diethanolamine and N-methylglutamine. Also included are the aluminum salts of the compounds of the present invention. Further base salts of the present invention include, but are not limited to: copper, ferric, ferrous, lithium, magnesium, manganic, manganous, potassium, sodium and zinc salts. Organic base salts include, but are not limited to, salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, e.g., arginine, betaine, caffeine, chloroprocaine, choline, N, N'-dibenzylethylenediamine (benzathine), dicyclohexylamine, diethanolamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, iso-propylamine, lidocaine, lysine, meglumine, N-methyl-D-glucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethanolamine, triethylamine, trimethylamine, tripropylamine and tris-(hydroxymethyl)-methylamine (tromethamine). It should be recognized that the free acid forms will typically differ from their respective salt forms somewhat in physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free acid forms for the purposes of the present invention.

[0050] In one aspect, a pharmaceutically acceptable salt is a hydrochloride salt, hydrobromide salt, methanesulfonate, toluenesulfonate, acetate, fumarate, sulfate, bisulfate, succinate, citrate, phosphate,Mintz Ref.: 064944-502001 WOmaleate, nitrate, tartrate, benzoate, bicarbonate, carbonate, sodium hydroxide salt, calcium hydroxide salt, potassium hydroxide salt, tromethamine salt, or mixtures thereof.

[0051] Compounds of the present invention that comprise tertiary nitrogen-containing groups may be quaternized with such agents as (C1-4) alkyl halides, e.g., methyl, ethyl, iso-propyl and tert-butyl chlorides, bromides and iodides; di-(Ci-4) alkyl sulfates, e.g., dimethyl, diethyl and diamyl sulfates; alkyl halides, e.g., decyl, dodecyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; and aryl (Ci-4) alkyl halides, e.g., benzyl chloride and phenethyl bromide. Such salts permit the preparation of both water- and oil-soluble compounds of the invention.

[0052] Amine oxides, also known as amine-N-oxide and N-oxide, of anti-cancer agents with tertiary nitrogen atoms have been developed as prodrugs (Mai. Cancer Therapy, 2004 March; 3(3):233-244). Compounds of the present invention that comprise tertiary nitrogen atoms may be oxidized by such agents as hydrogen peroxide (H2O2), Caro's acid or peracids like meta-Chloroperoxybenzoic acid (mCPBA) to from amine oxide.

[0053] Pharmaceutical Compositions

[0054] The invention encompasses pharmaceutical compositions comprising the compound of the present invention and pharmaceutical excipients, as well as other conventional pharmaceutically inactive agents. Any inert excipient that is commonly used as a carrier or diluent may be used in compositions of the present invention, such as sugars, polyalcohols, soluble polymers, salts and lipids. Sugars and polyalcohols which may be employed include, without limitation, lactose, sucrose, mannitol, and sorbitol. Illustrative of the soluble polymers which may be employed are polyoxyethylene, poloxamers, polyvinylpyrrolidone, and dextran. Useful salts include, without limitation, sodium chloride, magnesium chloride, and calcium chloride. Lipids which may be employed include, without limitation, fatty acids, glycerol fatty acid esters, glycolipids, and phospholipids.

[0055] In addition, the pharmaceutical compositions may further comprise binders (e.g., acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone), disintegrating agents (e.g., cornstarch, potato starch, alginic acid, silicon dioxide, croscarmellose sodium, crospovidone, guar gum, sodium starch glycolate, Primogel), buffers (e.g., tris-HCL, acetate, phosphate) of various pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acidMintz Ref.: 064944-502001 WOsalts), protease inhibitors, surfactants (e.g., sodium lauryl sulfate), permeation enhancers, solubilizing agents (e.g., glycerol, polyethylene glycerol, cyclodextrins), a glidant (e.g., colloidal silicon dioxide), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite, butylated hydroxyanisole), stabilizers (e.g., hydroxypropyl cellulose, hydroxypropylmethyl cellulose), viscosity increasing agents (e.g., carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum), sweeteners (e.g., sucrose, aspartame, citric acid), flavoring agents (e.g., peppermint, methyl salicylate, or orange flavoring), preservatives (e.g., Thimerosal, benzyl alcohol, parabens), lubricants (e.g., stearic acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate), flow-aids (e.g., colloidal silicon dioxide), plasticizers (e.g., diethyl phthalate, triethyl citrate), emulsifiers (e.g., carbomer, hydroxypropyl cellulose, sodium lauryl sulfate, methyl cellulose, hydroxyethyl cellulose, carboxymethylcellulose sodium), polymer coatings (e.g., poloxamers or poloxamines), coating and film forming agents (e.g., ethyl cellulose, acrylates, polymethacrylates) and / or adjuvants.

[0056] In one embodiment, the pharmaceutical compositions are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U. S. Pat. No. 4,522,811.

[0057] Additionally, the invention encompasses pharmaceutical compositions comprising any solid or liquid physical form of the compound of the invention. For example, the compounds can be in a crystalline form, in amorphous form, and have any particle size. The particles may be micronized, or may be agglomerated, particulate granules, powders, oils, oily suspensions or any other form of solid or liquid physical form.

[0058] When compounds according to the present invention exhibit insufficient solubility, methods for solubilizing the compounds may be used. Such methods are known to those of skill in this art, and include, but are not limited to, pH adjustment and salt formation, using co-solvents, such as ethanol, propylene glycol, polyethylene glycol (PEG) 300, PEG 400, DMA (10-30%), DMSO (10-20%), NMPMintz Ref.: 064944-502001 WO(10-20%), using surfactants, such as polysorbate 80, polysorbate 20 (1-10%), cremophor EL, Cremophor RH40, Cremophor RH60 (5-10%), Pluronic F68 / Poloxamer 188 (20-50%), Solutol HS 15 (20-50%), Vitamin E TPGS, and d-a-tocopheryl PEG 1000 succinate (20-50%), and using advanced approaches such as micelle, addition of a polymer, nanoparticle suspensions, and liposome formation.

[0059] A wide variety of administration methods may be used in conjunction with the compounds of the present invention. Compounds of the present invention may be administered or coadministered topically, orally, intraperitoneally, intravenously, intraarterially, transdermally, sublingually, intramuscularly, rectally, transbuccally, intranasally, liposomally, via inhalation, vaginally, intraoccularly, via local delivery (for example by catheter or stent), subcutaneously, intraadiposally, intraarticularly, intrathecally, transmucosally, pulmonary, or parenterally, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal; by implant of a depot or reservoir, for example, subcutaneously or intramuscularly.

[0060] The compounds according to the invention may also be administered or coadministered in slow release dosage forms. Compounds may be in gaseous, liquid, semi-liquid or solid form, formulated in a manner suitable for the route of administration to be used. For oral administration, suitable solid oral formulations include tablets, capsules, pills, granules, pellets, sachets and effervescent, powders, and the like. Suitable liquid oral formulations include solutions, suspensions, dispersions, syrups, emulsions, oils and the like. For parenteral administration, reconstitution of a lyophilized powder is typically used.

[0061] Suitable doses of the compounds for use in treating the diseases or disorders described herein can be determined by those skilled in the relevant art. Therapeutic doses are generally identified through a dose ranging study in humans based on preliminary evidence derived from the animal studies. Doses must be sufficient to result in a desired therapeutic benefit without causing unwanted side effects. Mode of administration, dosage forms and suitable pharmaceutical excipients can also be well used and adjusted by those skilled in the art. All changes and modifications are envisioned within the scope of the present patent application.Mintz Ref.: 064944-502001 WOEXPERIMENTAL

[0062] Example 1: Increase of TSC2, RAI1, NSD1, KMT2D, CREBBP, and KANSL1 expression, via a dual orexin receptor antagonist, in wild type glutamatergic neurons.

[0063] In a primary screen, the dual orexin antagonists, Almorexant (HY- 10805), Filorexant (HY-15653), and Lemborexant (HY-16725) (purchased from MedChemExpress) were tested in wild type glutamatergic neurons. Almorexant and Filorexant both increased TSC2 and NSD1 mRNA in wild type glutamatergic neurons (24 hours). Both compounds increased TSC2 mRNA by fold changes representing greater than 2 standard deviations of 279 DMSO values (Filorexant fold change of 1.7 at 0.5uM and Almorexant fold change of >5 at 5uM). In addition, Lemborexant and Almorexant increased NSD1, KMT2D, CREBBP, and KANSL mRNA by fold changes ranging from 1.4 to 6.5 (depending on the compound and concentration), indicating that dual orexin receptor antagonists thematically drive the upregulation of specific genes. The effects of Almorexant on mRNA expression ofTSC2 and additional genes (RAI1, NSD1, KMT2D, CREBBP and KANSL 1) were further evaluated in a hit confirmation experiment, described below.Hit confirmation experimentCell culture conditions - hiPSC-derived glutamatergic neurons

[0064] The hiPSC-derived glutamatergic neurons (iCell GlutaNeurons, Fujifilm CDI, cat. #R1034, Lot. 106779) were thawed according to the manufacturer protocol and seeded at a density of 20,000 cells / well in 384-well black plates (Greiner bio-one, cat. #781091) coated with 0.07% PEI (Sigma, cat. #181978) and 10 pg / mL Laminin (Sigma, cat. #L2020).

[0065] The iCell GlutaNeurons were maintained in the BrainPhys Neuronal Medium (StemCell Technologies, cat. #05790), iCell Neural Supplement B (Fujifilm CDI, cat. #M1029), iCell Nervous System Supplement (Fujifilm CDI, cat. #M1031), IX N-2 supplement (Thermo Fisher, cat. #17502048), 1 pg / mL Laminin (Sigma, cat. #L2020), 1% Penicillin-Streptomycin (BioWhittaker, cat. #DE17-602E).

[0066] The iCell GlutaNeurons were kept in culture for 14 days, performing 50% media changes in semi-automation every other day, in order to allow their maturation.Mintz Ref.: 064944-502001 WOExperimental conditions - hiPSC-derived glutamatergic neurons

[0067] After 13 days of in vitro culture (DIV) the iCell GlutaNeurons were incubated with 280 compounds, under sterile conditions in semi-automation and incubated for 24 hours.

[0068] 280 compounds in total were tested at four (4) concentrations each, with inter-plate triplicate data points in order to analyze their effect on the expression of select target genes in iCell GlutaNeurons. One housekeeping gene (GAPDH) was used as normalization control. A one-step TaqMan approach was used.

[0069] In particular:Compounds were divided into two groups, and each group was tested at 4 different concentrations as follows:o Group 1: 5pM-l.58pM-0.5pM-0.16pMo Group 2: 1.58pM-0.5pM-0.16pM-0.05pMCells were washed with 40 pL of ice-clod PBS, then lysed with a qPCR compatible lysis buffer (20uL)The 1-step RT-qPCR reaction was assembled in a final volume of 5 pL, with 2 pL of 1:10 diluted lysate, TaqMan probes (Life Technologies) and Luna® Universal Probe One-Step RT- qPCR Kit (NEB) as recommended by the manufacturer, using these probes (for the select target gene referenced above):Gene Symbol gene ID TaqMan Assay IDTSC2 7249 Hs01020387_m1RAI1 10743 Hs01554690_m1NSD1 64324 Hs00328315_m1KMT2D 8085 Hs00912419_m1CREBBP 1387 Hs00932878_m1KANSL1 284058 Hs00393805_m1GAPDH 2597 Hs02758991_gl

[0070] RT-qPCR was run in thermocycler (QuantStudio 12S Flex, Life Technologies) with the following program: 10 min at 55°C, 2 min at 95°C, followed by 40 cycles of 10 seconds at 95°C andMintz Ref.: 064944-502001 WO60 seconds at 60°C. Data were analyzed with the Transcriptomic module of GeneData Analyser software. Results are summarized in FIG. 1A, representing TSC2 mRNA expression of Almorexant in wild type glutamatergic neurons in a hit confirmation experiment. Additionally, FIGs. 1B-1F show mRNA expression of RAI1 (FIG. 1B), NSD1 (FIG. 1C), KMT2D (FIG. 1D), CREBBP (FIG. 1E), and KANSL1 (FIG. 1F) of Almorexant treated wild type glutamatergic neurons. All replicates as well as mean with standard deviation shown for drug treatment data, while only mean with standard deviation shown for DMSO (280 replicates). Data normalized to GAPDH housekeeping gene.Mintz Ref.: 064944-502001 WO

[0071] Example 2: TSC2 iPSC-derived neuron multielectrode array experiment with Rapamycin treatment controliPSC lines

[0072] The subject recruited for this study was consented at Boston Children’s Hospital with the approved institutional review board protocol number P00008224. Informed consent was obtained from the participant or the participant’s parent if the participant was unable to provide consent. The iPSC line used in this study was previously characterized, including descriptions of reprogramming methods and quality control characterization (Ebrahimi-Fakhari et al., 2016; Sundberg et al., 2018; Winden et al., 2019). Briefly, the TSC2- / - iPSC line used in this study was originally derived from a female patient with TSC who was found to have heterozygous expression levels of TSC2. The iPSCs were derived by episomal expression of Oct4, Sox2, Klf4 and L-Myc. The second allele was knocked down using TALEN-based gene editing of the TSC2+ / - line to generate the biallelic TSC2- / - line.

[0073] The TSC2- / - iPSCs were previously reported to express the pluripotency markers NANOG, TRA1-60, OCT4, and SSEA4 and exhibit a normal karyotype (Sundberg et al., 2018). For routine cell culture, the iPSCs were cultured in StemFlex medium (ThermoFisher #A3349401 ) on Geltrex-coated plates (ThermoFisher #A1413301) and were passaged once a week, or when the cells reached 70% confluence, with Gentle Cell Dissociation Reagent (STEMCELL Technologies #07174). A low-passage stock was expanded and karyotyped, and all neuronal cultures were derived from iPSCs within 10 passages of the normal karyotype.Neuronal differentiation

[0074] TSC2- / - iPSC-derived cortical neurons were differentiated in replicate batches, defined as independent plating of starting material iPSCs and use of reagents to differentiate iPSCs into neurons. The NGN2-induced cortical neurons were differentiated following previously published methods (Winden et al., 2019; Zhang et al., 2013) that are also described here. To establish a line of iPSCs expressing the NGN2 vector, on differentiation day -2, iPSCs were dissociated with Accutase (Innovative Cell Technologies #AT104) and plated as single cells at 90,000 cells / cm2 in mTeSR-1 media supplemented with 10μM ROCK inhibitor (Y-27631, Cayman Chemical #10005583), onMintz Ref.: 064944-502001 WOGeltrex-coated 12-well plates. While iPSCs were routinely cultured with StemFlex (as noted above), the NGN2 differentiation was optimized with mTeSR-1 media, so the switch was made to this media prior to initiating differentiation.

[0075] One day after re-plating as single cells, on differentiation day -1, iPSCs were transduced with lentiviral vectors expressing NGN2 and rtTA and 8μg / ml polybrene (Sigma-Aldrich #TR-1003-G). The lentiviral particles were produced and concentrated at the Viral Core at Boston Children’s Hospital and were added to the culture at an MOI of 5 for each vector. Both plasmids are available at Addgene with the following IDs: 52047 and 20342. To initiate neuronal induction on differentiation day 0, NGN2 expression was induced via treatment with 2μg / ml doxycycline (Millipore #324385). Cells then underwent selection for expression of the NGN2 vector via treatment with 1μg / ml puromycin (Invitrogen #ant-pr-1) for 24-48 hours. Longer puromycin treatment was chosen when non-neuronal clusters appeared to remain after the first 24 hours of puromycin treatment. This selection helps to generate a relatively pure population of neurons.

[0076] For the first 2 days of differentiation, the following growth factors and supplements were added to the N2 medium [DMEM / F-12 with GlutaMax, lx N2 supplement, lx non-essential amino acids]: 10ng / ml BDNF (Peprotech #450-02), 10ng / ml NT3 (Peprotech #450-03), and 0.2mg / L laminin (ThermoFisher #23017-015). After day 2, the cells were fed with B27 media [Neurobasal-A, lx B27 supplement, lx GlutaMax] containing the following growth factors and supplements every other day until differentiation day 6: 10ng / ml BDNF, 10ng / ml NT3, 0.2mg / mL laminin, 2μg / ml doxycycline, and 2μM Ara-C (Sigma- Aldrich #C1768).

[0077] For morphological and MEA analyses, NGN2 neurons were dissociated on day 6 with >=16 units / mL papain (Worthington #LK003178) supplemented with >=166 Kunits / mL DNase I (Worthington #LK003172) for 20 minutes at 37°C. On day 6, astrocytes (NCardia Astro.4U) were added in co-culture with the neurons according to the densities for each endpoint listed further below. Cells were cultured in B27 media containing 33.4mM glucose and 27.3mM sodium bicarbonate, supplemented with 0.1mg / mL transferrin thereafter with fresh BDNF, NT3 and laminin added at final concentrations listed above, every time media change was performed.Plating for multi-electrode array (MEA) analysisMintz Ref.: 064944-502001 WO

[0078] On day 6 of the differentiation protocol for each replicate batch, the neurons were plated onto three full 48-well CytoView MEA plates (M768-tMEA-48B, Axion Biosystems). These plates were coated with 0.1% PEI (#408727, Sigma) and 5pg / mL laminin. Cortical neurons were plated at 75,000 neurons and 11,250 astrocytes (NCardia, Astro.4U) per well. To plate the neurons, laminin was aspirated off the wells one well at a time and the neuron and glia mixture was added to the well in a 20μl droplet. Following 30 minutes of incubation at room temperature, the wells were flooded with 400μL of media and moved to the incubator. The media at the time of re-plating was supplemented with Rock Inhibitor (Y-27632 #10005583, Cayman) and laminin (10μg / mL) to promote attachment. A half media change was completed 24 hours after plating to dilute the rock inhibitor and half media changes continued every Monday / Wednesday / Friday thereafter. The first day of MEA plating was considered day 1 for data analyses.MEA recording and analysis

[0079] Spontaneous neuronal activity was recorded three times per week for 30 days with an Axion Biosystems MaestroPro MEA System. Raw spike data were sampled at 12.5 kHz, digitized, and analyzed using Axion Integrated Studio software with a 200 Hz high-pass and 3000 Hz low-pass filter and with an adaptive spike detection threshold set at 6 times the SD for each electrode with 1 s binning. The burst detector was set to detect bursts at a threshold of 50 spikes within 100 ms and network bursts required 35% of the electrodes to burst simultaneously. The window size for synchrony calculation was set at 20 ms. After recording, the data were analysed using the Axion Biosystems Neurostatistics Compiler software which classified spikes, bursts, and network activity according to these thresholds. These summary files were collated and analysed using GraphPad Prism.

[0080] FIGs 2A-2C show results of multielectrode array experiments following application of histone deacetylase inhibitors and dual orexin receptor antagonists, over a range of doses administered chronically (started prior to the establishment of hyperexcitability phenotype), in TSC2- / - neurons. Rapamycin treatment and DMSO used as controls. Data presents mean with SEM (standard error of the mean), and includes ordinary one-way ANOVA analysis, with statistical significance shown (where applicable). Data shown across multiple timepoints (noting that the day number refers to the number of days following MEA plating, where the first day of plating is considered “day 1”). Experiment conducted on two separate plates (plates A and B), with DMSO and rapamycin drug control wells on both plates. All compounds were included on each MEA plate, with differing dosesMintz Ref.: 064944-502001 WObetween plates (lower doses for each compound were placed on plate A, while higher doses for each compound were placed on plate B). Each treatment condition included six replicates, with selected replicates excluded based on management’s view of potential outliers.

[0081] Data demonstrates TSC2 hyperexcitability phenotype, as measured by weighted mean firing rate (WMFR), and additionally that Rapamycin drug control prevents such hyperexcitability (see Muncy J, Butler IJ, Koenig MK. Rapamycin reduces seizure frequency in tuberous sclerosis complex. J Child Neurol. 2009;24(4):477. doi:10.1177 / 0883073808324535) (noting further that Rapamycin has demonstrated positive results in a clinical context). BRD6688 consistently showed a reduced WMFR in the nanomolar dose range, indicating potential therapeutic benefit as a selective histone deacetylase inhibitor (Class I, favoring HDAC2). BRD4884 also demonstrated some reduction of WMFR in the nanomolar range, indicating potential therapeutic benefit as well, as a Class I histone deacetylase inhibitor. Dual orexin receptor antagonists showed a reduced WMFR at select doses, also indicating potential therapeutic benefit and a link between TSC2 upregulation (per FIG.1A) and TSC2 phenotype improvement.

[0082] Images of MEA plates taken one to three times per week with 10X objective on Sartorius IncuCyteS3 live imaging instrument. Note that the date / time stamp on the images represents days in vitro (DIV), which differs from the dating convention referenced above. See FIGs. 3A-3D, showing images of MEA plates prior to and following drug treatment. Pan-histone deacetylase inhibitors (Dacinostat and Panobinostat) cause cell death following chronic treatment across indicated doses, whereas selective histone deacetylase inhibitors (BRD6688 and BRD4884) did not cause cell death in the experiments disclosed herein. Dual orexin receptor antagonists do not cause cell death at the range of doses chronically tested.Compound treatment

[0083] Cells were treated with varying concentrations of compounds every Monday, Wednesday and Friday days during a 15-day treatment window (commencing on day 8, a Monday, following neuron plating on MEA plates). Recordings occurred 24 hours after treatment. DMSO treatment was used as control in each plate. Stock vials of rapamycin and unknown compounds were stored at -20°C, in the dark and were equilibrated to room temperature prior to use. The compounds were provided in DMSO at lOmM stock concentration. Dilutions were made according to the chart below. Before addition of the compounds, the media in each well were brought to half the final volume and the 2x compoundMintz Ref.: 064944-502001 WOstocks prepared in culture media (see Table 3) were then carefully added to bring the final volume to 400μL per well.Mintz Ref.: 064944-502001 WOTable 3: Dilutionsul media final ul uL tot ul B to kept in well Finalf ] Stock[ ] ul Stock DMSO “A” tot “A”[ ] ul A to ul media vol for “B”[ ] add per each iinal[ ] volume Compound (nM) (uM) for “A” for “A” vol (uM) make B for “B” B (uM) well well (uM) (ul) Rapamycin 20 10000 1 499.00 500.00 20 3.2 1596.8 1600 0.04 200 200 0.02 400 Dacinostat 150 10000 1.5 98.50 100.00 150 3.2 1596.8 1600 0.3 200 200 0.15 400 Panobinostat 150 10000 1.5 98.50 100.00 150 3.2 1596.8 1600 0.3 200 200 0.15 400 Almorexant 0.5 10000 2 38.00 40.00 500 3.2 1596.8 1600 1 200 200 0.5 400 Filorexant 0.5 10000 2 38.00 40.00 500 3.2 1596.8 1600 1 200 200 0.5 400 BRD6688 150 10000 1.5 98.50 100.00 150 3.2 1596.8 1600 0.3 200 200 0.15 400 BRD4884 150 10000 1.5 98.50 100.00 150 3.2 1596.8 1600 0.3 200 200 0.15 400 DMSO 0 0 0 0.00 0.00 0 3.2 1596.8 1600 0 200 200 0 400 ControlRapamycin 20 10000 1 499.00 500.00 20 3.2 1596.8 1600 0.04 200 200 0.02 400 Dacinostat 0.5 10000 2 38.00 40.00 500 3.2 1596.8 1600 1 200 200 0.5 400 Panobinostat 0.5 10000 2 38.00 40.00 500 3.2 1596.8 1600 1 200 200 0.5 400 Almorexant 3 10000 12 28.00 40.00 3000 3.2 1596.8 1600 6 200 200 3 400 Filorexant 3 10000 12 28.00 40.00 3000 3.2 1596.8 1600 6 200 200 3 400 BRD6688 0.5 10000 2 38.00 40.00 500 3.2 1596.8 1600 1 200 200 0.5 400 BRD4884 0.5 10000 2 38.00 40.00 500 3.2 1596.8 1600 1 200 200 0.5 400 DMSO 0 0 0 0.00 0.00 0 3.2 1596.8 1600 0 200 200 0 400ControlMintz Ref.: 064944-502001 WO

[0084] Note for Table 3: The stock of each compound was first diluted in DMSO with a serial dilution to make the series “A ” dilutions of each concentration in DMSO. From there, series A was diluted in media to create series B dilutions. The same volume of each compound in series A was added to series B to ensure the final concentration of DMSO was the same across all compounds. The B series was added to the wells to reach the final concentrations. [ ] = concentration

[0085] Rapamycin (S1039) and Panobinostat (S1030) were purchased from Selleck Chemicals. Dacinostat (HY-13606), Almorexant (HY-10805), Filorexant (HY-15653), BRD6688 (HY-117709) and BRD4884 (HY-102083) were purchased from MedChemExpress.

[0086] Enumerated EmbodimentsEmbodiment 1. A method for treating Tuberous Sclerosis Complex (TSC), the method comprising administering to a subject in need thereof an effective amount of a selective histone deacetylase inhibitor or a dual orexin receptor antagonist (DORA).Embodiment 2. A method of reducing neuronal hyperexcitability of a subject with Tuberous Sclerosis Complex (TSC), the method comprising administering to a subject in need thereof an effective amount of a selective histone deacetylase inhibitor or a dual orexin receptor antagonist (DORA), thereby reducing neuronal hyperexcitability.Embodiment 3. The method of embodiment 1 or embodiment 2, wherein the method comprises administering to a subject in need thereof an effective amount of a selective histone deacetylase inhibitor.Embodiment 4. The method of embodiment 1 or embodiment 2, wherein the method comprises administering to a subject in need thereof an effective amount of a dual orexin receptor antagonist (DORA).Embodiment 5. The method of embodiment 1 or embodiment 2, wherein the method comprises administering to a subject in need thereof an effective amount of a selective histone deacetylase inhibitor and a dual orexin receptor antagonist (DORA).Embodiment 6. The method of any one of the preceding embodiments wherein the histone deacetylase inhibitor is a class 1 histone deacetylase inhibitor.Mintz Ref.: 064944-502001 WOEmbodiment 7. The method of any one of the preceding embodiments wherein the histone deacetylase inhibitor is a class 1 histone deacetylase inhibitor which inhibits histone deacetylase 2 (HDAC2).Embodiment 8. The method of any one of the preceding embodiments wherein the histone deacetylase inhibitor is an HDAC2-selective histone deacetylase inhibitor.Embodiment 9. The method of embodiment 1 wherein the histone deacetylase inhibitor is an HDAC 1 and HDAC2 histone deacetylase inhibitor.Embodiment 10. The method of any one of the preceding embodiments, wherein the histone deacetylase inhibitor is BRD6688Embodiment 11. The method of any one of the preceding embodiments, wherein the histone deacetylase inhibitor is BRD4884.Embodiment 12. The method of any one of the preceding embodiments, wherein the histone deacetylase inhibitor is Romidepsin.Embodiment 13. The method of any one of the preceding embodiments, wherein the histone deacetylase inhibitor is Entinostat.Embodiment 14. The method of any one of the preceding embodiments, wherein the histone deacetylase inhibitor is Tacedinaline.Embodiment 15. The method of any one of the preceding embodiments, wherein the histone deacetylase inhibitor is Santacruzamate A.Embodiment 16. The method of any one of the preceding embodiments, wherein the histone deacetylase inhibitor is Mocetinostat.Embodiment 17. The method of any one of the preceding embodiments, wherein the histone deacetylase inhibitor is CXD101.Embodiment 18. The method of any one of the preceding embodiments, wherein the histone deacetylase inhibitor is Givinostat.Embodiment 19. The method of any one of the preceding embodiments, wherein the histone deacetylase inhibitor is BRD4161.Embodiment 20. The method of any one of the preceding embodiments, wherein the histone deacetylase inhibitor is BRD3386.Embodiment 21. The method of any one of the preceding embodiments, wherein the histone deacetylase inhibitor is BRD7232.Mintz Ref.: 064944-502001 WOEmbodiment 22. The method of any one of the preceding embodiments, wherein the histone deacetylase inhibitor is Tucidinostat.Embodiment 23. The method of any one of the preceding embodiments, wherein the histone deacetylase inhibitor is Abexinostat.Embodiment 24. The method of any one of the preceding embodiments, wherein the histone deacetylase inhibitor is Pracinostat.Embodiment 25. The method of any one of the preceding embodiments, wherein the histone deacetylase inhibitor is Resminostat.Embodiment 26. The method of any one of the preceding embodiments, wherein the histone deacetylase inhibitor is Fimepinostat.Embodiment 27. The method of any one of the preceding embodiments, wherein the histone deacetylase inhibitor is CUDC-101.Embodiment 28. The method of any one of the preceding embodiments, wherein the method modulates expression of TSC 1 or TSC2.Embodiment 29. The method of any one of the preceding embodiments, wherein the method increases expression of TSC 1.Embodiment 30. The method of any one of the preceding embodiments, wherein the method increases expression of TSC2.Embodiment 31. The method of any one of the preceding embodiments, wherein the method causes a substantial reduction of weighted mean firing rate for a plurality of neurons.Embodiment 32. The method of any one of the preceding embodiments, wherein the DORA is Almorexant.Embodiment 33. The method of any one of the preceding embodiments, wherein the DORA is Filorexant.Embodiment 34. The method of any one of the preceding embodiments, wherein the DORA is Daridorexant.Embodiment 35. The method of any one of the preceding embodiments, wherein the DORA is Lemborexant.Embodiment 36. The method of any one of the preceding embodiments, wherein the DORA is Suvorexant.Embodiment 37. The method of any one of the preceding embodiments, wherein the DORA is Fazamorexant.Mintz Ref.: 064944-502001 WOEmbodiment 38. The method of any one of the preceding embodiments, wherein the DORA is Vornorexant.Embodiment 39. The method of any one of the preceding embodiments, wherein the DORA is SB-649868.Equivalents

[0087] While the present invention has been described in conjunction with the specific embodiments set forth above, many alternatives, modifications and other variations thereof will be apparent to those of ordinary skill in the art. All such alternatives, modifications and variations are intended to fall within the spirit and scope of the present invention.

Claims

Mintz Ref.: 064944-502001 WOCLAIMSWhat is claimed:

1. A method of treating a neurodevelopmental disorder in a subject in need thereof, wherein the disorder is associated with haploinsufficiency of a gene, the method comprising administering to the subject a therapeutically effective amount of a dual orexin receptor antagonist (DORA).

2. A method of treating a neurodevelopmental disorder in a subject in need thereof, wherein the disorder is associated with haploinsufficiency of a gene, the method comprising administering to the subject a therapeutically effective amount of an orexin receptor antagonist (ORA).

3. The method of claim 1 or 2, wherein the gene is selected from the group consisting of tuberous sclerosis complex subunit 1 (TSC1), tuberous sclerosis complex subunit 2 (TSC2), nuclear receptor binding SET domain protein (NSD1), lysine methyltransferase 2D (KMT2D), lysine demethylase 6A (KDM6A), CREB binding lysine acetyltransferase (CREBBP), EP300 lysine acetyltransferase (EP300), KAT8 regulatory NSL complex subunit 1 (KANSL1), Retinoic Acid Induced 1 (RAI1), and combinations thereof.

4. The method of any one of claims 1-3, wherein the disorder is tuberous sclerosis complex (TSC).

5. The method of any one of claims 1-3, wherein the disorder is selected from the group consisting of Sotos syndrome, Kabuki syndrome, Rubinstein-Taybi syndrome, Koolen-de Vries syndrome, and Smith-Magenis syndrome.

6. The method of any one of claims 1–4, wherein the subject has a germline or somatic heterozygous loss-of-function variant in Tuberous Sclerosis 1 (TSC1) and / or Tuberous Sclerosis 2 (TSC2).

7. The method of any one of claims 1-6, wherein the haploinsufficiency neurodevelopmental disorder is associated with a chromatin-relevant gene encoding a regulator of histone methylation and / or acetylation.Mintz Ref.: 064944-502001 WO8. The method of claim 7, wherein the relevant gene is selected from the group consisting of NSD1, KMT2D, KDM6A, CREBBP, EP300, and KANSL1.

9. The method of any one of claims 1-2, wherein the disorder is Smith-Magenis syndrome and wherein the gene is RAI110. The method of any one of claims 1-9, wherein treating the disorder comprises reducing neuronal hyperexcitability and / or reducing seizure frequency and / or seizure burden in the subject.

11. A method of reducing neuronal hyperexcitability of a subject with Tuberous Sclerosis Complex (TSC), the method comprising administering to a subject in need thereof a therapeutically effective amount of a selective histone deacetylase inhibitor or a dual orexin receptor antagonist (DORA).

12. The method of claim 10 or 11, wherein reducing neuronal hyperexcitability comprises a substantial reduction in weighted mean firing rate (WMFR) for a plurality of neurons, as measured using multielectrode array recordings.

13. The method of any one of claims 1, and 3-12, wherein the DORA is selected from the group consisting of almorexant, filorexant, daridorexant, lemborexant, suvorexant, fazamorexant, vornorexant, SB-649868, and pharmaceutically acceptable salts, solvates, stereoisomers, prodrugs, and metabolites thereof.

14. The method of any one of claims 2-10, wherein the ORA is an OXIR-selective antagonist.

15. The method of any one of claims 2-10, wherein the ORA is an OX2R-selective antagonist.

16. The method of any one of claims 1-15, wherein the method further comprises coadministering a therapeutically effective amount of an HDAC inhibitor.

17. A method of treating tuberous sclerosis complex (TSC) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a histone deacetylase (HDAC) inhibitor.Mintz Ref.: 064944-502001 WO18. The method of any one of claims 1-4, 6-7, or 10-17, wherein the method treats and / or prevents one or more symptoms associated with TSC.

19. The method of claim 18, wherein the one or more symptoms associated with TSC are selected from the group consisting of neurological symptoms, renal symptoms, pulmonary symptoms, cardiac symptoms, and cutaneous symptoms.

20. The method of claim 19, wherein the neurological symptom is selected from the group consisting of epilepsy, seizures, cortical tubers, subependymal nodules, subependymal giant cell astrocytoma (SEGA), TSC-associated neuropsychiatric disorders (TAND), autism spectrum disorder, intellectual disabilities, and developmental delays.

21. The method of claim 19, wherein the renal symptom is selected from group consisting of angiomyolipoma, multiple renal cysts, polycystic kidney disease, and renal cell carcinoma.

22. The method of claim 19, wherein the pulmonary symptom is selected from a group consisting of lymphangioleiomyomatosis (LAM), and multifocal micronodular pneumocyte hyperplasia (MMPH).

23. The method of claim 19, wherein the cardiac symptom is cardiac rhabdomyoma.

24. The method of claim 19, wherein the cutaneous symptom is selected from the group consisting of facial angiofibromas, ungual fibromas, fibrous cephalic plaques, shagreen patches, and hypomelanotic macules.

25. The method of claim 19, wherein the one or two symptoms associated with TSC are oral fibromas, or retinal astrocytic hamartomas.

26. The method of any one of claims 17-25, further comprising measuring neuronal activity and / or gene expression in a biological sample from the subject before and after administration of the HDAC inhibitor, wherein a change in neuronal activity and / or gene expression (including TSC-relevant network activity and / or histone acetylation markers) indicates effectiveness of the treatment.

27. The method of any one of claims 17-26, wherein the HDAC inhibitor increases histone acetylation and reverses a hypoacetylation phenotype associated with TSC.Mintz Ref.: 064944-502001 WO28. The method of any one of claims 16-27, wherein the HDAC inhibitor is a class I HDAC inhibitor.

29. The method of any one of claims 16-28, wherein the HDAC inhibitor is a class I HDAC inhibitor which inhibits HDAC2.

30. The method of any one of claims 16-29, wherein the HDAC inhibitor is a class I selective HDAC inhibitor.

31. The method of any one of claims 16-30, wherein the HDAC inhibitor is an HDAC2-selective inhibitor.

32. The method of any one of claims 16-30, wherein the HDAC inhibitor is an HDAC1 and HDAC2 inhibitor.

33. The method of any one of claims 16-29, wherein the HDAC inhibitor is selected from the group consisting of BRD6688, BRD4884, Romidepsin, Entinostat, Tacedinaline, Santacruzamate A, Mocetinostat, CXD101, Givinostat, BRD4161, BRD3386, BRD7232, Tucidinostat, Abexinostat, Pracinostat, Resminostat, Fimepinostat, CUDC-101, and pharmaceutically acceptable salts, solvates, stereoisomers, prodrugs, and metabolites thereof.

34. The method of any one of claims 4, 6-7 or 10-33, wherein the method further comprises coadministering a therapeutically effective amount of an mTOR inhibitor.

35. The method of any one of claims 1-16, wherein the method further comprises coadministering a therapeutically effective amount of an ATPase inhibitor.

36. The method of claim 35, wherein the ATPase inhibitor is selected from the group consisting of lanatoside C, digoxin, digitoxin, deslanoside, ouabain, strophanthidin, oleandrin, resibufogenin, cinobufotalin, istaroxime, istaroxime hydrochloride, PST2915, PST2922, PST3093, and pharmaceutically acceptable salts, solvates, stereoisomers, prodrugs, and metabolites thereof.

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