Modulators of mTORC1 activity and uses thereof

Abstract

The present invention relates to compounds and methods useful for selectively modulating mTORC1 activity.The present invention describes compounds comprising a lithium salt and an mTORC1 activator in stoichiometric ratio or co-crystal form.Such mTORC1 activator can be an amino acid as described in published PCT application WO2017 / 070518.In some embodiments, the compounds comprising a lithium salt and an mTORC1 activator have improved efficacy due to the synergistic effect of lithium and mTORC1 activator.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 421,288, filed November 1, 2022, the contents of which are hereby incorporated by reference herein. [Background technology]

[0002] Background of the Invention Depression in individuals with either major depressive disorder (MDD) or bipolar disorder (BD) is a major cause of disease burden. In published PCT application WO2017 / 070518, small molecule mTORC1 activators were found to be beneficial in depressive disorders and other CNS-related disorders. It has been suggested that activation of mTORC1 and subsequent synaptogenesis in the prefrontal cortex (PFC) mediates the rapid antidepressant effects of small molecule mTORC1 activators. Through activation of the mTOR signaling pathway, administration of mTORC1 activators increased synaptic protein levels and dendritic spine density. Separately, the mood stabilizer lithium is known to have anti-suicidal properties and shows promise in treating other neurological and neurodegenerative diseases. Chiu et al. (International Journal of Neuropsychopharmacology, 2015, pp. 1–13) also showed that mice pretreated with subtherapeutic lithium (600 mg / L) exhibited an antidepressant-like response to ineffective ketamine (2.5 mg / kg, i.p.) exposure in the forced swim test. Both the antidepressant-like effect and the restoration of dendritic spine density in the medial prefrontal cortex of stressed mice induced by a single ketamine (50 mg / kg) injection were sustained by at least two weeks of post-ketamine treatment with 1200 mg / L lithium. These benefits of lithium treatment were associated with activation of the mammalian target of rapamycin / brain-derived neurotrophic factor signaling pathway in the prefrontal cortex. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. 2017 / 070518 [Non-patent literature]

[0004] [Non-Patent Document 1] International Journal of Neuropsychopharmacology, 2015, 1-13 Summary of the Invention

[0005] There is an urgent and compelling unmet medical need for more effective treatments for mTORC1-related diseases, disorders, or conditions. DETAILED DESCRIPTION OF THE INVENTION

[0006] Detailed Description of Certain Embodiments 1. Overview of Certain Embodiments of the Invention: Among the various aspects of the present invention may be noted the provision of compositions containing a stoichiometric ratio of a lithium salt and an organic molecule.

[0007] The present invention describes a compound comprising a lithium salt and an mTORC1 activator in a stoichiometric ratio or in a co-crystal form.Such an mTORC1 activator can be an amino acid as described in published PCT application WO2017 / 070518.In some embodiments, the compound comprising a lithium salt and an mTORC1 activator has improved efficacy due to the synergistic effect of lithium and the mTORC1 activator.In some embodiments, such derivatives are effective at a lower therapeutic dose than either of the single agents administered alone.

[0008] In some embodiments, a compound comprising a lithium salt and an mTORC1 activator is described, the compound being a cocrystal comprising a lithium salt and an mTORC1 activator in a stoichiometric ratio. Such an mTORC1 activator may be an amino acid as described in published PCT application WO2017 / 070518. In some embodiments, the lithium salt and the mTORC1 activator as a cocrystal have improved efficacy due to the synergistic effect of lithium and the mTORC1 activator. In some embodiments, such derivatives are effective at a lower therapeutic dose than either of the single agents administered alone.

[0009] U.S. Patent No. 10,100,066 ("the '066 patent"), filed October 21, 2016 as U.S. Patent Application No. US15 / 331,362 and published as U.S. Patent Application Publication No. US2017 / 0114080 ("the '080 publication"), each of which is incorporated herein by reference in its entirety, describes certain mTORC1 modulating compounds. Such compounds include Compound I: [ka] Includes:

[0010] Compound I, (S)-2-amino-5,5-difluoro-4,4-dimethylpentanoic acid, is described in both the '066 patent and the '080 publication. The synthesis of Compound I is described in detail in Example 90 of the '066 patent and the '080 publication.

[0011] It has now been found that compounds comprising lithium chloride and Compound I, such as salts or co-crystals comprising lithium chloride and Compound I, and compositions thereof, are useful for treating, preventing, and / or reducing the risk of diseases, disorders, or conditions mediated by mTORC1.

[0012] One aspect of the present invention is that compound I has the structure: [ka] or a solvate or hydrate thereof.

[0013] One aspect of the present invention is further directed to pharmaceutical compositions comprising a compound having formula LiCl*Compound I, or a solvate or hydrate thereof. The present invention is further directed to dosage unit forms comprising a compound having formula LiCl*Compound I, or a solvate or hydrate thereof. The compound having formula LiCl*Compound I may be one or more solvates or salts or co-crystals that may also include water molecules in the crystal lattice.

[0014] The present invention is further directed to a method for preparing a salt or co-crystal comprising a stoichiometric ratio of a lithium salt and an organic compound. The method comprises dissolving the lithium salt and the organic compound in a solvent and evaporating or cooling the solvent. In one embodiment, the stoichiometric ratio of the organic compound to the lithium salt is 1:1, respectively.

[0015] Other aspects and objects of the invention will be in part apparent and in part pointed out hereinafter.

[0016] In some embodiments, compound I has the structure: [ka] or a solvate or hydrate thereof.

[0017] In some embodiments, the compound having the formula LiCl*Compound I is a co-crystal.

[0018] In some embodiments, the lithium and Compound I are in a stoichiometric ratio of about 1 to about 1.

[0019] In some embodiments, a pharmaceutical composition is provided that includes a compound having the formula LiCl*Compound I and a pharmaceutically acceptable carrier, adjuvant, or vehicle.

[0020] In some embodiments, the pharmaceutical composition is formulated for oral administration.

[0021] In some embodiments, the pharmaceutical composition further comprises an additional therapeutic agent.

[0022] In some embodiments, methods are provided for treating, preventing, and / or reducing the risk of a disease, disorder, or condition mediated by mTORC1 in a patient, comprising administering to a patient in need thereof an effective amount of a compound having the formula LiCl*Compound I or a pharmaceutical composition comprising a compound having the formula LiCl*Compound I.

[0023] In some embodiments, the disease, disorder, or condition mediated by mTORC1 is depression, bipolar disorder, schizophrenia, chronic unpredictable stress, autism, lysosomal storage disease, Batten disease, cystinosis, Fabry disease, mucolipidosis, mental retardation, anorexia, bulimia, anemia, neutropenia, headache, alcoholism, post-traumatic stress disorder (PTSD), epilepsy, diabetes, liver disease, kidney damage, arthritis, skin conditions such as seborrhea, hyperthyroidism, asthma, Huntington's disease, Graves' disease, herpes simplex, movement disorders such as tardive dyskinesia, Tourette's syndrome, cyclic vomiting, Meniere's disease, tingling or formication sensations in the skin (paresthesia), or aggressive behavior in attention deficit hyperactivity disorder (ADHD).

[0024] In some embodiments, provided are methods of treating treatment-resistant depression in a patient in need thereof, comprising administering to the patient an effective amount of a compound having the formula LiCl*Compound I or a pharmaceutical composition comprising a compound having the formula LiCl*Compound I. 2.Definition:

[0025] As used herein, the following definitions shall apply unless otherwise indicated: For purposes of the present invention, chemical elements are defined as defined in the Periodic Table of the Elements, CAS edition, Handbook of Chemistry and Physics, pp. 75-78. th In addition, the general principles of organic chemistry are identified in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999 and "March's Advanced Organic Chemistry", 5 th Ed., Ed.: Smith, MB and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are hereby incorporated by reference herein.

[0026] In certain embodiments, the present invention provides compound I: [ka] is provided as its lithium chloride salt or co-crystal.

[0027] In certain embodiments, the present invention provides compound I: [ka] as its lithium chloride co-crystal. 4. Use, Formulation and Administration Pharmaceutically acceptable compositions

[0028] According to another embodiment, the present invention provides a composition comprising Compound I and lithium chloride and a pharmaceutically acceptable carrier, adjuvant, or vehicle. According to another embodiment, the present invention provides a composition comprising Compound I as a lithium chloride salt or co-crystal and a pharmaceutically acceptable carrier, adjuvant, or vehicle.

[0029] The amount of compound I and lithium chloride in the compositions of the present invention is such that it is effective to measurably modulate or activate mTORC1 in biological sample or patient.In certain embodiments, the amount of compound I and lithium chloride in the compositions of the present invention is such that it is effective to measurably modulate or activate mTORC1 in biological sample or patient.In certain embodiments, compound I and lithium chloride form a cocrystal.

[0030] In certain embodiments, the compositions of the invention are formulated for administration to a patient in need of such a composition, hi some embodiments, the compositions of the invention are formulated for oral administration to a patient.

[0031] The term "patient", as used herein, means an animal, preferably a mammal, and most preferably a human.

[0032] The term "pharmaceutically acceptable carrier, adjuvant, or vehicle" refers to a non-toxic carrier, adjuvant, or vehicle that does not destroy the pharmacological activity of the compound with which it is formulated. Pharmaceutically acceptable carriers, adjuvants, or vehicles that may be used in the compositions of the present invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphate, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, salts or electrolytes such as zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylate, wax, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat.

[0033] The compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir. The term "parenteral," as used herein, includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques. Preferably, the compositions are administered orally, intraperitoneally, or intravenously. Sterile injectable forms of the compositions of the present invention may be aqueous or oily suspensions. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. Sterile injectable preparations may also be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents, such as solutions in 1,3-butanediol. Acceptable vehicles and solvents that may be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally used as solvents or suspending media.

[0034] For this purpose, any bland fixed oil may be used, including synthetic mono- or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils such as olive oil or castor oil, especially polyoxyethylated versions. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, such as carboxymethylcellulose or similar dispersants, which are commonly used in the formulation of pharmaceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants, such as Tween® and Span®, and other emulsifiers or bioavailability enhancers commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for formulation purposes.

[0035] The pharmaceutically acceptable composition of the present invention can be orally administered in any orally acceptable dosage form, including but not limited to capsules, tablets, aqueous suspensions or solutions.For tablets for oral use, commonly used carriers include lactose and corn starch.Lubricants such as magnesium stearate are also typically added.For oral administration in capsule form, useful diluents include lactose and dried corn starch.When aqueous suspensions are required for oral use, active ingredient is combined with emulsifiers and suspending agents.If desired, certain sweeteners, flavorings or coloring agents can also be added.

[0036] Alternatively, the pharmaceutically acceptable compositions of the present invention may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature, and therefore melts in the rectum to release the drug. Such materials include cocoa butter, beeswax, and polyethylene glycol.

[0037] The pharmaceutically acceptable compositions of this invention may also be administered topically, particularly when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs.

[0038] Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Topical transdermal patches may also be used.

[0039] For topical application, the provided pharmaceutically acceptable composition may be formulated into a suitable ointment containing the active ingredient suspended or dissolved in one or more carriers.Carriers for topical administration of compositions containing Compound I and lithium chloride include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying wax and water.Alternatively, the provided pharmaceutically acceptable composition may be formulated into a suitable lotion or cream containing the active ingredient suspended or dissolved in one or more pharmaceutically acceptable carriers.Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

[0040] For ophthalmic use, the provided pharmaceutically acceptable compositions may be formulated as a micronized suspension in isotonic, pH-adjusted, sterile saline, with or without a preservative such as benzylalkonium chloride, or as a solution, preferably in isotonic, pH-adjusted, sterile saline. Alternatively, for ophthalmic use, the pharmaceutically acceptable compositions may be formulated into an ointment such as petrolatum.

[0041] The pharmaceutically acceptable compositions of this invention may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulation, and may be prepared as solutions in saline employing benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, fluorocarbons, and / or other conventional solubilizing or dispersing agents.

[0042] Most preferably, the pharmaceutically acceptable composition of the present invention is formulated for oral administration. Such formulations may be administered with or without food. In some embodiments, the pharmaceutically acceptable composition of the present invention is administered without food. In other embodiments, the pharmaceutically acceptable composition of the present invention is administered with food.

[0043] The amount of the composition containing Compound I and lithium chloride that may be combined with the carrier materials to produce a single dosage form will vary depending on the host being treated and the particular mode of administration. Preferably, the provided compositions should be formulated so that a dosage of between 0.01 and 100 mg / kg body weight / day of the inhibitor can be administered to a patient receiving these compositions.

[0044] It should also be understood that the specific administration and treatment regimen for any particular patient will depend on a variety of factors, including the activity of the specific compound used, age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, and the judgment of the treating physician and the severity of the particular disease being treated. The amount of Compound I and the lithium chloride salt in the composition will also depend on the specific compounds in the composition. Uses and Pharmaceutically Acceptable Compositions

[0045] Compositions comprising Compound I and lithium chloride are generally useful for modulating or activating mTORC1. In some embodiments, Compound I, or a composition thereof, as a lithium chloride salt or cocrystal, is a modulator of mTORC1. In some embodiments, Compound I, or a composition thereof, as a lithium chloride salt or cocrystal, is a selective modulator of mTORC1. In some embodiments, Compound I, or a composition thereof, as a lithium chloride salt or cocrystal, is an activator of mTORC1.

[0046] The activity of a composition comprising Compound I and lithium chloride as a modulator or activator of mTORC1 may be assayed in vitro, in vivo, or in a cell system. In vitro assays include assays that determine the modulation or activation of mTORC1. Detailed conditions for assaying compounds utilized in the present invention as modulators or activators of mTORC1 are provided in the Examples below.

[0047] As used herein, the terms "treatment," "treat," and "treating" refer to ameliorating, alleviating, delaying the onset of, or inhibiting the progression of a disease or disorder as described herein, or one or more symptoms thereof. In some embodiments, treatment may be administered after one or more symptoms appear. In other embodiments, treatment may be administered in the absence of symptoms. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and / or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example, to prevent or delay their recurrence.

[0048] Compositions comprising Compound I and lithium chloride are modulators or activators of mTORC1 and are therefore useful for treating one or more disorders associated with the activity of mTORC1. Accordingly, in certain embodiments, the present invention provides methods for treating an mTORC1-mediated disorder, comprising administering to a patient in need thereof a composition comprising Compound I and lithium chloride, or a pharmaceutically acceptable composition thereof.

[0049] As used herein, the term "mTORC1-mediated" disorder, disease, and / or condition means any disease or other deleterious condition in which mTORC1 is known to play a role. Accordingly, another embodiment of the present invention relates to treating or reducing the severity of one or more diseases in which mTORC1 is known to play a role.

[0050] In some embodiments, the method of activating mTORC is used to treat or prevent depression. (See Ignacio et al., (2015) Br J Clin Pharmacol. Nov 27.) Accordingly, in some embodiments, the present invention provides a method of treating or preventing depression in a patient in need thereof, comprising administering to the patient Compound I, or a pharmaceutically acceptable composition thereof, as a lithium chloride salt or cocrystal. In some embodiments, the depression is major depressive disorder ("MDD"). Accordingly, in some embodiments, the present invention provides a method of treating or preventing major depressive disorder in a patient in need thereof, comprising administering to the patient Compound I, or a pharmaceutically acceptable composition thereof, as a lithium chloride salt or cocrystal. In some embodiments, the depression is treatment-resistant depression ("TRD"). Accordingly, in some embodiments, the present invention provides a method of treating or preventing treatment-resistant depression in a patient in need thereof, comprising administering to the patient Compound I, or a pharmaceutically acceptable composition thereof, as a lithium chloride salt or cocrystal. In some embodiments, the treatment-resistant depression is resistant to a first-line treatment. In some embodiments, the treatment-resistant depression is resistant to a second-line treatment.

[0051] In some embodiments, the present invention provides a method for treating depression in a patient in need thereof, wherein the patient experiences a 50% reduction in depression scale score. In some embodiments, the patient experiences a 50% reduction in depression scale score within less than six weeks of administering the compound or pharmaceutically acceptable composition. In some embodiments, the patient experiences a 50% reduction in depression scale score within less than four weeks of administering the compound or pharmaceutically acceptable composition. In some embodiments, the patient experiences a 50% reduction in depression scale score within two weeks of administering the compound or pharmaceutically acceptable composition. In some embodiments, the patient experiences a 50% reduction in depression scale score within two weeks of administering the compound or pharmaceutically acceptable composition. In some embodiments, the patient experiences a 50% reduction in depression scale score within one week of administering the compound or pharmaceutically acceptable composition. In some embodiments, the patient experiences a 50% reduction in depression scale score within seven days of administering the compound or pharmaceutically acceptable composition. In some embodiments, the patient experiences a 50% reduction in depression scale score within 6 days of administering the compound or pharmaceutically acceptable composition. In some embodiments, the patient experiences a 50% reduction in depression scale score within 5 days of administering the compound or pharmaceutically acceptable composition. In some embodiments, the patient experiences a 50% reduction in depression scale score within 4 days of administering the compound or pharmaceutically acceptable composition. In some embodiments, the patient experiences a 50% reduction in depression scale score within 3 days of administering the compound or pharmaceutically acceptable composition. In some embodiments, the patient experiences a 50% reduction in depression scale score within 2 days of administering the compound or pharmaceutically acceptable composition. In some embodiments, the patient experiences a 50% reduction in depression scale score within 1 day of administering the compound or pharmaceutically acceptable composition. In some embodiments, the patient experiences a 50% reduction in depression scale score within 24 hours of administering the compound or pharmaceutically acceptable composition.In some embodiments, the depression scale score is selected from the Montgomery-Asberg Depression Rating Scale (MADRS), the Hamilton Depression Rating Scale (HAMD-6), the Inventory of Depressive Symptoms (IDS-SR), and the Clinical Global Impression-Severity Scale (CGI-S).

[0052] In some embodiments, the present invention provides a method of treating depression in a patient in need thereof, comprising orally administering Compound I, or a pharmaceutically acceptable composition thereof, as a lithium chloride salt or cocrystal, to the patient, wherein the patient experiences a reduction in depression scale score comparable to that of ketamine administered by ip injection. In some embodiments, the reduction in depression scale score occurs from a single oral administration. In some embodiments, the reduction in depression scale score occurs from multiple oral administrations.

[0053] In some embodiments, the method of activating mTORC1 is used to induce rapid-onset antidepressant activity. Accordingly, in some embodiments, the present invention provides a method of inducing rapid-onset antidepressant activity in a patient suffering from TRD in need thereof, comprising administering to the patient Compound I, or a pharmaceutically acceptable composition thereof, as a lithium chloride salt or cocrystal. In some embodiments, rapid-onset antidepressant activity occurs within two weeks of administration of the compound or composition. In some embodiments, rapid-onset antidepressant activity occurs within one week of administration of the compound or composition. In some embodiments, rapid-onset antidepressant activity occurs within seven days of administration of the compound or composition. In some embodiments, rapid-onset antidepressant activity occurs within six days of administration of the compound or composition. In some embodiments, rapid-onset antidepressant activity occurs within five days of administration of the compound or composition. In some embodiments, rapid-onset antidepressant activity occurs within four days of administration of the compound or composition. In some embodiments, rapid-onset antidepressant activity occurs within three days of administration of the compound or composition. In some embodiments, the rapid-onset antidepressant activity occurs within two days of administration of the compound or composition. In some embodiments, the rapid-onset antidepressant activity occurs within one day of administration of the compound or composition. In some embodiments, the rapid-onset antidepressant activity occurs within less than 24 hours of administration of the compound or composition.

[0054] In some embodiments, the present invention provides a method of inducing long-lasting, sustained antidepressant activity in a patient suffering from depression, comprising administering Compound I, as the lithium chloride salt or a co-crystal, or a pharmaceutically acceptable composition thereof, to the patient. In some embodiments, the patient in need thereof is suffering from TRD. In some embodiments, the long-lasting, sustained antidepressant activity persists for at least 24 hours after a single administration of Compound I, as the lithium chloride salt or a co-crystal, or a pharmaceutically acceptable composition thereof. In some embodiments, the long-lasting, sustained antidepressant activity persists for more than one day. In some embodiments, the long-lasting, sustained antidepressant activity persists for at least two days. In some embodiments, the long-lasting, sustained antidepressant activity persists for at least three days. In some embodiments, the long-lasting, sustained antidepressant activity persists for at least four days. In some embodiments, the long-lasting, sustained antidepressant activity persists for at least five days. In some embodiments, the long-lasting, sustained antidepressant activity persists for at least six days. In some embodiments, the long-lasting, sustained antidepressant activity persists for at least seven days.

[0055] In some embodiments, the present invention provides methods of inducing rapid onset, long-lasting, and sustained antidepressant activity.

[0056] In some embodiments, the present invention provides a method of inducing a positive behavioral response in a subject, comprising administering to the subject Compound I, or a pharmaceutically acceptable composition thereof, as a lithium chloride salt or cocrystal. In some embodiments, the positive behavioral response is correlated with improved mood. In some embodiments, the positive behavioral response is correlated with reduced anxiety. In some embodiments, the positive behavioral response is consistent with improved mood. In some embodiments, the positive behavioral response is correlated with improved ability to cope with stress.

[0057] In some embodiments, the present invention provides a method of inducing a rapid onset positive behavioral response in a subject, the method comprising administering to the subject Compound I, or a pharmaceutically acceptable composition thereof, as a lithium chloride salt or co-crystal. In some embodiments, the positive behavioral response occurs within 24 hours of administration. In some embodiments, the positive behavioral response occurs within 1 day of administration. In some embodiments, the positive behavioral response occurs within 2 days of administration. In some embodiments, the positive behavioral response occurs within 3 days of administration. In some embodiments, the positive behavioral response occurs within 4 days of administration. In some embodiments, the positive behavioral response occurs within 5 days of administration. In some embodiments, the positive behavioral response occurs within 6 days of administration. In some embodiments, the positive behavioral response occurs within 7 days of administration. In some embodiments, the positive behavioral response occurs within 1 week of administration.

[0058] In some embodiments, the present invention provides a method of inducing a long-lasting, sustained, positive behavioral response in a subject, comprising administering to the patient Compound I, or a pharmaceutically acceptable composition thereof, as a lithium chloride salt or cocrystal. In some embodiments, the long-lasting, sustained, positive behavioral response lasts for more than one day. In some embodiments, the long-lasting, sustained, positive behavioral response lasts for at least two days. In some embodiments, the long-lasting, sustained, positive behavioral response lasts for at least three days. In some embodiments, the long-lasting, sustained, positive behavioral response lasts for at least four days. In some embodiments, the long-lasting, sustained, positive behavioral response lasts for at least five days. In some embodiments, the long-lasting, sustained, positive behavioral response lasts for at least six days. In some embodiments, the long-lasting, sustained, positive behavioral response lasts for at least seven days.

[0059] In some embodiments, the present invention provides methods for eliciting rapid-onset, long-lasting, and sustained positive behavioral responses.

[0060] In some embodiments, the present invention provides a method of improving and / or reversing behavioral and synaptic deficits caused by chronic unpredictable stress (CUS) in a patient in need thereof, comprising administering to the patient Compound I, or a pharmaceutically acceptable composition thereof, as a lithium chloride salt or cocrystal. In some embodiments, the method improves and / or reverses behavioral deficits caused by CUS. In some embodiments, the method improves and / or reverses synaptic deficits caused by CUS. In some embodiments, the synaptic deficit caused by CUS is a decrease in postsynaptic protein expression. In some embodiments, the decrease in postsynaptic protein expression is a decrease in expression of GLUR1 or PSD95.

[0061] In some embodiments, the method of activating mTORC1 is used to treat or prevent a form of autism. (See Novarino et al., (2012) Science 19 Oct, 338:6105, pp. 394-397.) Thus, in some embodiments, the present invention provides a method for treating or preventing a form of autism in a subject in need thereof, comprising administering to the subject Compound I or a pharmaceutically acceptable composition thereof as a lithium chloride salt or cocrystal. In some embodiments, the autism is a genetic form of autism.

[0062] In some embodiments, the present invention provides a method for treating a genetic form of autism in a patient in need thereof, comprising administering to the patient Compound I or a pharmaceutically acceptable composition thereof as a lithium chloride salt or cocrystal. SHANK3 haploinsufficiency causes neurological features of Phelan-McDermid syndrome (PMDS), including a high risk of autism spectrum disorder (Bidinosti et al. (2016) Science Reports 351, 1199-1203). Downregulation of mTORC1 in SHANK3-deficient neurons is due to enhanced phosphorylation and activation of the serine / threonine protein phosphatase 2A (PP2A) regulatory subunit B56b by its kinase, Cdc2-like kinase 2 (Bidinosti et al. (2016) Science Reports 351, 1199-1203). SHANK3 mutant mice exhibit autistic traits (Yang et al. (2012) The Journal of Neuroscience 32, 6525-6541). Patients with autistic traits and motor delay carry deleterious homozygous mutations in the SLC7A5 gene. Solute carrier transporter 7a5 (SLC7A5), a large neutral amino acid transporter localized at the blood-brain barrier (BBB), plays an essential role in maintaining normal brain BCAA levels. Intracerebroventricular administration of leucine improves abnormal behavior in adult mutant mice (Tarlungeanu et al. (2016) Cell 167, 1481-1494).

[0063] In some embodiments, the present invention provides a method of treating a lysosomal storage disease or disorder ("LSD") in a patient in need thereof, comprising administering Compound I, as the lithium chloride salt or co-crystal, or a pharmaceutically acceptable composition thereof, to the patient. LSDs are a group of inherited metabolic disorders resulting from defects in lysosomal function. Lysosomal storage disorders are usually caused by impaired lysosomal function as a result of a deficiency in a single enzyme required for the metabolism of lipids, glycoproteins (sugar-containing proteins), or so-called mucopolysaccharides. In some embodiments, the present invention provides a method of treating a lipid storage disorder in a patient in need thereof, comprising administering Compound I, as the lithium chloride salt or co-crystal, or a pharmaceutically acceptable composition thereof, to the patient. In some embodiments, the lipid storage disorder is selected from sphingolipidosis (e.g., gangliosidosis, Gaucher disease, Niemann-Pick disease, or metachromatic leukodystrophy). In some embodiments, the present invention provides a method of treating a gangliosidosis (e.g., Tay-Sachs disease or leukodystrophy). In some embodiments, the present invention provides a method of treating a mucopolysaccharidosis in a patient in need thereof, comprising administering to the patient Compound I, as a lithium chloride salt or a co-crystal, or a pharmaceutically acceptable composition thereof. In some embodiments, the mucopolysaccharidosis is Hunter syndrome or Hurler disease.

[0064] In some embodiments, the present invention provides a method for treating JNCL (Batten disease) in a patient in need thereof, comprising administering to the patient Compound I or a pharmaceutically acceptable composition thereof as a lithium chloride salt or cocrystal. JNCL is caused by a deletion of exons 7 and 8 of the CLN3 gene, resulting in a non-functional protein. Battenin, the full-length protein encoded by CLN3, is a transmembrane protein localized in late endosomes and lysosomes, where it has been shown to help regulate pH, amino acid balance, and vesicle trafficking (Pearce et al. (1999) Nature Genetics 22, 1; Fossale et al. (2004) BMC Neuroscience 10, 5). mTOR activation requires intracellular nutrients provided by autophagy, which is reduced due to the lack of functional battenin in in vitro and in vivo models of JNCL (Cao et al. (2006) Journal of Biological Chemistry 281, 29).

[0065] In some embodiments, the present invention provides a method for treating cystinosis in a patient in need thereof, comprising administering to the patient Compound I or a pharmaceutically acceptable composition thereof as a lithium chloride salt or cocrystal. Cystinosis is an autosomal recessive disease affecting individuals with two mutated alleles of the CTNS gene (CSTN gene). The lysosomal cystine transporter cystinosin is defective in cystine efflux from lysosomes, resulting in cystine crystal formation in renal epithelial tubules and loss of kidney function. Studies have shown defective or reduced mTORC1 signaling in cells lacking CSTN and mislocalized mTOR (Ivanova et al. (2016) J Inherit Metab Dis. 39(3), 457-64; Andrzejewska et al. (2016) J Am Soc Nephrol. 27(6), 1678-1688e). These defects could not be rescued by cysteamine (Ivanova et al. (2016) J Inherit Metab Dis. 39(3), 457-64; Andrzejewska et al. (2016) J Am Soc Nephrol. 27(6), 1678-1688e). Cystinosine has also been found to bind to mTORC1 pathway components, v-ATPase, Rag, and regulatory factors (Andrzejewska et al. (2016) J Am Soc Nephrol. 27(6), 1678-1688e). CTNS-deficient cells exhibit increased autophagosome numbers and reduced chaperone-mediated autophagy (Napolitano et al. (2015) EMBO Mol Med. 7(2), 158-74).

[0066] In some embodiments, the present invention provides a method for treating Fabry disease in a patient in need thereof, comprising administering to the patient Compound I or a pharmaceutically acceptable composition thereof as a lithium chloride salt or cocrystal. In Fabry disease, alpha-galactosidase deficiency leads to lysosomal accumulation of globotriaosylceramide lipids. Decreased mTOR activity and increased autophagy have been observed in vitro and in vivo in a cell model of Fabry disease in which alpha-galactosidase has been knocked down with shRNA (Liebau et al. (2013) PLoS 8, e63506). Hyperactive autophagy has also been observed in the brains of mice in which alpha-galactosidase has been knocked out (Nelson et al. (2014) Acta Neuropathologica Communications 2, 20).

[0067] In some embodiments, the present invention provides a method for treating mucolipidosis type IV (MLIV) in a patient in need thereof, comprising administering to the patient Compound I or a pharmaceutically acceptable composition thereof as a lithium chloride salt or cocrystal. In MLIV, mutations in the TRPML1 lysosomal Ca(2+) channel cause impaired lysosomal membrane trafficking. MLIV knockout in Drosophila resulted in upregulated autophagy and reduced mTOR activity, both of which could be reversed by genetically activating mTORC1 or by feeding the animals a high-protein diet (Wong et al. (2012) Curr Biol. 22(17), 1616-1621). Enhanced autophagy was also observed in fibroblasts derived from MLIV patients (Vergarajauregui et al. (2008) Human Molecular Genetics 17, 2723-2737).

[0068] In some embodiments, the present invention provides a method of treating mental retardation in a patient in need thereof, comprising administering to the patient Compound I, or a pharmaceutically acceptable composition thereof, as a lithium chloride salt or cocrystal. In homo sapiens, cereblon mutations are associated with a mild form of autosomal recessive non-syndromic mental retardation. In a mouse cereblon knockout model of mental retardation, loss of cereblon activates AMPK, inhibits mTOR, and reduces protein translation in the cerebellum (Lee et al. (2014) J. Biol. Chem. 289, 23343-52; Xu et al. (2013) J. Biol. Chem. 288, 29573-85).

[0069] In some embodiments, the present invention provides a method for increasing neuronal protein expression in a subject, comprising administering to the subject Compound I, or a pharmaceutically acceptable composition thereof, as a lithium chloride salt or cocrystal. In some embodiments, the increase in neuronal protein expression occurs in postsynaptic neurons. In some embodiments, the increase in neuronal protein expression comprises an increase in expression of brain-derived neurotrophic factor (BDNF). In some embodiments, the increase in neuronal protein expression comprises an increase in expression of glutamate receptor 1 (GluR1). In some embodiments, the increase in neuronal protein expression comprises an increase in expression of synapsin. In some embodiments, the increase in neuronal protein expression comprises an increase in expression of PSD95.

[0070] In some embodiments, the present invention provides a method of increasing synaptogenesis in a subject, comprising administering to the subject Compound I, or a pharmaceutically acceptable composition thereof, as a lithium chloride salt or cocrystal. In some embodiments, increasing synaptogenesis comprises synaptic remodeling. In some embodiments, increasing synaptogenesis comprises dendritic spine induction. In some embodiments, dendritic spine induction causes an increase in dendritic spine density. In some embodiments, the dendritic spines are thin spines. In some embodiments, the dendritic spines are mushroom spines.

[0071] In some embodiments, the present invention provides a method of enhancing synaptic function in a subject, comprising administering to the subject Compound I, or a pharmaceutically acceptable composition thereof, as a lithium chloride salt or cocrystal. In some embodiments, enhancing synaptic function in the subject comprises an increase in excitatory postsynaptic currents (EPSCs).

[0072] In some embodiments, the present invention provides a method of treating a disorder of the central nervous system (CNS), comprising administering to said patient Compound I, as a lithium chloride salt or cocrystal, or a pharmaceutically acceptable composition thereof. In some embodiments, the CNS disorder is bipolar disorder, depression, or schizophrenia.

[0073] In some embodiments, the present invention provides a method of treating an eating disorder, comprising administering to said patient Compound I, as a lithium chloride salt or cocrystal, or a pharmaceutically acceptable composition thereof. In some embodiments, the eating disorder is anorexia or bulimia.

[0074] In some embodiments, the present invention provides a method of treating a hematological disorder, comprising administering to said patient Compound I, as a lithium chloride salt or cocrystal, or a pharmaceutically acceptable composition thereof. In some embodiments, the hematological disorder is anemia and low white blood cell count (neutropenia).

[0075] In some embodiments, the present invention provides a method of treating headache, alcoholism, post-traumatic stress disorder (PTSD), epilepsy, diabetes, liver disease, kidney damage, arthritis, skin conditions such as seborrhea, hyperthyroidism, asthma, Huntington's disease, Graves' disease, herpes simplex, movement disorders such as tardive dyskinesia, Tourette's syndrome, cyclic vomiting syndrome, Meniere's disease, tingling or "running" sensations in the skin (paresthesia), and aggressive behavior in attention deficit hyperactivity disorder (ADHD), comprising the step of administering to said patient Compound I, as a lithium chloride salt or a co-crystal, or a pharmaceutically acceptable composition thereof.

[0076] The pharmaceutically acceptable compositions of the present invention can be administered to humans and other animals orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powder, ointment, or drops), buccal, oral or nasal spray, etc., depending on the severity of the infection being treated. In certain embodiments, Compound I as the lithium chloride salt or cocrystal may be administered orally or parenterally, one or more times daily, at dosage levels of about 0.01 mg / kg to about 200 mg / kg, or about 0.01 mg / kg to about 50 mg / kg, preferably about 1 mg / kg to about 25 mg / kg, of the subject's body weight per day to obtain the desired therapeutic effect.

[0077] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.In addition to active compounds, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan, and mixtures thereof.In addition to inert diluents, oral compositions may also contain auxiliary substances such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and perfumes.

[0078] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, can be formulated according to known techniques using suitable dispersing or wetting agents and suspending agents. Sterile injectable preparations can also be sterile injectable solutions, suspensions, or emulsions in non-toxic parenterally acceptable diluents or solvents, such as solutions in 1,3-butanediol. Acceptable vehicles and solvents that can be used include water, Ringer's solution (USP), and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally used as solvents or suspending media. For this purpose, any non-irritating, fixed oil can be used, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

[0079] Injectable preparations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium before use.

[0080] To prolong the effect of Compound I as a lithium chloride salt or cocrystal, it is often desirable to slow the absorption of the compound from subcutaneous or intramuscular injection. This may be accomplished by using a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the compound then depends on its dissolution rate, which in turn may depend on crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered compound form can be achieved by dissolving or suspending the compound in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the compound in biodegradable polymers such as polylactide-polyglycolide. The compound release rate can be controlled depending on the ratio of compound to polymer and the nature of the particular polymer used. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations can also be prepared by entrapping the compound in liposomes or microemulsions that are compatible with body tissues.

[0081] Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing Compound I as the lithium chloride salt or co-crystal with a suitable non-irritating excipient or carrier such as cocoa butter, polyethylene glycol or a suppository wax which is solid at ambient temperature but liquid at body temperature and therefore melts in the rectum or vaginal cavity and releases the active compound.

[0082] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and / or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid; b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and gum arabic; c) humectants such as glycerol; d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; e) solution retarders such as paraffin; f) absorption accelerators such as quaternary ammonium compounds; g) humectants such as, for example, cetyl alcohol and glycerol monostearate; h) absorbents such as kaolin and bentonite clay; and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

[0083] Solid compositions of a similar type may also be used as fillers for soft and hard-filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols, etc. Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulation art. They may optionally contain opacifying agents, and may also be composed so that they release the active ingredient only, or preferentially, in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type may also be used as fillers for soft and hard-filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols, etc.

[0084] The active compound may also be in microencapsulated form with one or more excipients as described above. Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings, release-controlling coatings, and other coatings well known in the pharmaceutical formulation arts. In such solid dosage forms, the active compound may be mixed with at least one inert diluent, such as sucrose, lactose, or starch. As is common practice, such dosage forms may also contain additional substances other than inert diluents, such as tableting lubricants and other tableting aids, such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets, and pills, the dosage forms may also contain buffering agents. They may optionally contain opacifying agents, and may be of a composition that releases the active ingredient only or preferentially in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

[0085] Dosage forms for topical or transdermal administration of Compound I as the lithium chloride salt or cocrystal include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches. The active ingredient is mixed under sterile conditions with a pharmaceutically acceptable carrier and any necessary preservatives or buffers, as needed. Ophthalmic formulations, ear drops, and eye drops are also contemplated within the scope of the present invention. In addition, the present invention contemplates the use of transdermal patches, which have the added advantage of providing controlled delivery of the compound to the body. Such dosage forms can be made by dissolving or dispersing the compound in the appropriate medium. Absorption enhancers can also be used to increase the flux of the compound through the skin. The rate can be controlled by providing a rate-controlling membrane or by dispersing the compound in a polymer matrix or gel.

[0086] According to one embodiment, the present invention relates to a method of modulating mTORC1 activity in a biological sample, comprising contacting said biological sample with Compound I as a lithium chloride salt or a co-crystal, or a composition comprising said compound.

[0087] According to one embodiment, the present invention relates to a method for selectively modulating mTORC1 activity in a biological sample, comprising contacting said biological sample with Compound I as a lithium chloride salt or a co-crystal, or a composition comprising said compound.

[0088] According to one embodiment, the present invention relates to a method of activating mTORC1 in a biological sample, comprising contacting the biological sample with Compound I as a lithium chloride salt or a co-crystal, or a composition comprising said compound.

[0089] The term "biological sample," as used herein, includes, but is not limited to, a cell culture or extract thereof; a biopsy or extract thereof obtained from a mammal; and blood, saliva, urine, stool, semen, tears, or other bodily fluids or extracts thereof.

[0090] Another embodiment of the invention relates to a method of modulating mTORC1 activity in a patient, comprising administering to said patient Compound I, as a lithium chloride salt or a co-crystal, or a composition comprising said compound.

[0091] Another embodiment of the invention relates to a method of selectively modulating mTORC1 activity in a patient, comprising administering to the patient Compound I, as a lithium chloride salt or a co-crystal, or a composition comprising said compound.

[0092] Another embodiment of the invention relates to a method of activating mTORC1 in a patient, comprising administering to the patient Compound I, as a lithium chloride salt or a co-crystal, or a composition comprising said compound.

[0093] In another embodiment, the present invention provides a method for treating an mTORC1-mediated disorder in a patient in need thereof, comprising administering to the patient Compound I, as a lithium chloride salt or co-crystal, or a pharmaceutically acceptable composition thereof. Such disorders are described in detail herein.

[0094] Depending on the particular condition, or disease, being treated, additional therapeutic agents, which are normally administered to treat that condition, may also be present in the compositions of this invention. As used herein, additional therapeutic agents that are normally administered to treat a particular disease, or condition, are known as "appropriate for the disease, or condition, being treated."

[0095] In some embodiments, Compound I as a lithium chloride salt or co-crystal is administered in combination with an antidepressant. Antidepressant therapeutic agents are well known to those skilled in the art and include selective serotonin reuptake inhibitors ("SSRIs," e.g., sertraline, escitalopram, citalopram, fluvoxamine, fluoxetine, paroxetine), antidepressants (e.g., bupropion, venlafaxine, mirtazapine, duloxetine, amitriptyline, imipramine, selegiline, nortriptyline, trazodone, desvenlafaxine, and aripiprazole).

[0096] In some embodiments, Compound I as the lithium chloride salt or co-crystal is administered in combination with an additional therapeutic agent or process useful for treating one or more LSDs. In some embodiments, Compound I as the lithium chloride salt or co-crystal is administered in combination with enzyme replacement therapy, chemical chaperone therapy, bone marrow transplantation, substrate synthesis inhibition therapy, α-L-iduronidase, recombinant human N-acetylgalactosamine-4-sulfatase (arylsulfatase B), an inhibitor of glycosphingolipid biosynthesis, N-butyldeoxynojirimycin (miglustat), a hydrophobic iminosugar, or an inhibitor of α-galactosidase A (e.g., 1-deoxy-galactonojirimycin).

[0097] These additional agents may be administered separately from the compound-containing compositions of the present invention as part of a multiple-dose regimen. Alternatively, the agents may be part of a single dosage form mixed together with Compound I as the lithium chloride salt or co-crystal in a single composition. When administered as part of a multiple-dose regimen, the two active agents may be presented simultaneously, sequentially, or within a certain time period of each other, typically within 5 hours of each other.

[0098] As used herein, the terms "combination," "combined," and related terms refer to simultaneous or sequential administration of therapeutic agents according to the present invention. For example, Compound I as a lithium chloride salt or cocrystal may be administered simultaneously or sequentially with another therapeutic agent in separate unit dosage forms, or together in a single unit dosage form. Thus, the present invention provides a single unit dosage form comprising Compound I as a lithium chloride salt or cocrystal, an additional therapeutic agent, and a pharmaceutically acceptable carrier, adjuvant, or vehicle.

[0099] The amounts of both Compound I as the lithium chloride salt or co-crystal and the additional therapeutic agent (in those compositions containing an additional therapeutic agent as described above) that may be combined with carrier materials to produce a single dosage form will vary depending on the host treated and the particular mode of administration. Preferably, the compositions of the present invention should be formulated to allow for the administration of a dosage of between 0.01 and 100 mg / kg body weight / day of Compound I as the lithium chloride salt or co-crystal.

[0100] In those compositions containing an additional therapeutic agent, the additional therapeutic agent and Compound I as a lithium chloride salt or co-crystal may act synergistically. Thus, the amount of additional therapeutic agent in such compositions is less than the amount required in a monotherapy utilizing only that therapeutic agent. In such compositions, the additional therapeutic agent can be administered at a dose of between 0.01 and 1,000 μg / kg body weight / day.

[0101] The amount of additional therapeutic agent present in the compositions of the invention is no more than the amount that would normally be administered in a composition comprising that therapeutic agent as the only active agent. Preferably, the amount of additional therapeutic agent in the compositions disclosed herein ranges from about 50% to 100% of the amount that would normally be present in a composition comprising that agent as the only therapeutically active agent.

[0102] Compound I, or a pharmaceutical composition thereof, as a lithium chloride salt or cocrystal, may also be incorporated into compositions for coating implantable medical devices, such as prostheses, artificial valves, vascular grafts, stents, and catheters. For example, vascular stents are used to overcome restenosis (re-narrowing of the blood vessel wall after injury). However, patients using stents or other implantable devices are at risk of thrombus formation or platelet activation. These undesirable effects may be prevented or mitigated by pre-coating the device with a pharmaceutically acceptable composition containing a kinase inhibitor. An implantable device coated with Compound I, as a lithium chloride salt or cocrystal, is another embodiment of the present invention.

[0103] All features of each of the aspects of the invention apply mutatis mutandis to all other aspects.

[0104] In order that the invention described herein may be more fully understood, the following examples are set forth. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting this invention in any way. [Example]

[0105] Example As shown in the Examples below, in certain exemplary embodiments, Compound I, as a lithium chloride salt or co-crystal, is prepared according to the following general procedure: While the general method illustrates the synthesis of certain specific compounds of the invention, it will be understood that the following general method, as well as other methods known to those of skill in the art, can be applied as described herein. General procedure for compound preparation

[0106] Compound I was prepared according to the methods described in US Pat. No. 10,100,066, which is incorporated herein by reference in its entirety.

[0107] The preparation of Compound I as a lithium chloride co-crystal is described below. Example 1 Compound I as lithium chloride salt or cocrystal

[0108] 2 g of Compound I (11.04 mmol) and 3.51 g of lithium chloride (82.8 mmol, 7.5 equivalents) were dissolved in 250 ml (125 vol) of water to give a colorless solution. The resulting solution was concentrated to 10 vol at atmospheric pressure for approximately 5 hours. The solution was then cooled to room temperature over a period of 1.5 hours. Precipitation was observed during the cooling process. The solid material was isolated by filtration and dried under vacuum overnight to give a white solid (950 mg; 38.5% yield). The solid was characterized as Compound I as a lithium chloride cocrystal using various techniques. The results are summarized in Table 1 below. [Table 1]

[0109] HPLC assay, ICP, and chloride-based stoichiometry reveal a 1:1 ratio of Compound I to LiCl, as summarized in Table 2 below. [Table 2] General procedure for in vivo testing

[0110] Animal Use: Male Sprague Dawley rats (Charles River Laboratories, Wilmington, MA) weighing 175-200 g are group-housed upon arrival (Yale University, New Haven CT) and allowed to acclimate for 5 days before the start of experimental studies. Rats are provided with food and water ad libitum, except during protocol-specified fasting periods. Animals are monitored daily for clinical signs. A qualified veterinarian oversees all rodent procedures. All personnel receive training from the Yale animal care and use committee (IACUC). All animal procedures are performed at Yale University in strict accordance with the National Institutes of Health IACUC and approved by the Yale Animal Care and Use Committee.

[0111] Behavioral analysis using the female urine sniffing test (FUST): The FUST is performed 24 hours after dosing according to published procedures (Malkesman, O. et al., Biol Psychiatry 67(9): 864-71 (2010)). Briefly, rats are habituated to a cotton swab soaked in tap water for 60 minutes in their home cage. Rats are then exposed to a second cotton swab soaked in tap water, and 45 minutes later, they are exposed to a third cotton swab infused with fresh rat urine from an 11- to 14-week-old female rat in estrus. The total time (seconds) spent sniffing the cotton-tipped applicator is quantified for each animal over a 5-minute period.

[0112] Behavioral analysis using Locomotor Activity Assessment (LMA): LMA is assessed in an open field equipped with an automated activity meter consisting of parallel rows of infrared beams according to published procedures (Warner-Schmidt, JL & Duman, RS PNAS 104(11): 4647-52 (2007)). The number of beam breaks is recorded for each animal at 30-minute intervals.

[0113] Behavioral analysis using the Novelty Suppressed Feeding Test (NSFT): The NSFT is performed as previously described (Warner-Schmidt, JL & Duman, RS PNAS 104(11): 4647-52 (2007)). Rats are fasted in their home cage for 20 hours, and then placed in a Plexiglas open field (76.5 cm × 76.5 cm × 40 cm) with a small amount of food in the center. The animals are allowed to explore the open field for 8 minutes, and the latency (seconds) to eat is recorded.

[0114] Behavioral analysis using the sucrose preference test (SPT): Rats were habituated to a palatable 1% sucrose solution for 48 hours to develop neophobia avoidance. Rats were treated with Compound I or Veh as the lithium chloride salt or cocrystal at the end of day 0, and the SPT was performed 24 hours after administration on day 1. For the SPT, rats were deprived of water for 6 hours and exposed to two bottles containing equal volumes of 1% sucrose or water for 60 minutes. Sucrose preference was defined as the ratio of the volume of sucrose water consumed to the total water consumed during the 1-hour test (e.g., a ratio of 1 indicates that the rat consumed only 1% sucrose, while a ratio of 0.5 indicates that the rat drank equal amounts of 1% sucrose and water).

[0115] Chronic Unpredictable Stress (CUS) Condition: As described (Li, N. et al., Biol Psychiatry 69(8): 754-61 (2011)), rats were exposed to 12 unpredictable stressors in a fluctuating order to prevent habituation. The following 12 stressors were applied (two per day for 25 days): cage rotation, lights on, lights off, cold stress, isolation, swim stress, food and water deprivation, wet bedding, stroboscope, tilted cage, odor exposure, and group housing. Animals in the non-stressed (NS) group were housed normally without the application of external stressors. Both NS and CUS rats were handled and weighed weekly.

[0116] Marmoset Human Threat Test (HTT): Marmosets are exposed to the presence of human observers periodically over an extended period of time. Such chronic stimulation is known to increase plasma cortisol, and the subsequent increased hypothalamic-pituitary-adrenal function contributes to the pathophysiology of depression. Example A Behavioral changes in novelty suppression and female urine sniffing tests following a single administration of Compound I or ketamine as the lithium chloride salt or cocrystal.

[0117] Study Design: Male Sprague-Dawley rats weighing between 175 and 200 g were randomized into four study groups after a 5-day acclimation period. On study day 0, rats in groups 1 and 2 were administered a single intraperitoneal (ip) dose of either saline (Sal) or ketamine (Ket). Rats in groups 3 and 4 were administered a single oral gavage dose of Compound I vehicle (Veh, 0.5% methylcellulose / 0.1% Tween®-80) or Compound I (160 mg / kg) as the lithium chloride salt or cocrystal. All rats were subjected to FUST on day 1, 24 hours after administration. On day 2, 48 hours after administration, the LMA of all rats was measured in an open field. The rats were then fasted for 20 hours and subjected to NSFT 72 hours after administration.

[0118] Preparation of test articles: Ket (Sigma, Cat. No. K1884) is dissolved in Sal at a concentration of 10 mg / mL. A volume of 1 ml / kg of Sal or Ket is injected i.p. into groups 1 and 2, respectively. Compound I as the lithium chloride salt or co-crystal is prepared by dissolving it in Veh (0.5% methylcellulose / 0.1% Tween®-80) at a concentration of 50 mg / mL. A dose volume based on the animal's weight (3.2 mL / kg) of Veh or Compound I as the lithium chloride salt or co-crystal is administered by oral gavage to the study animals in groups 3 and 4, respectively. The test articles are prepared on the day of administration. Example B Comparative effects of a single dose of Compound I and ketamine administered as lithium chloride salt or co-crystal on the mTORC1 signaling pathway and synaptic protein expression in synaptosomal preparations derived from rat prefrontal cortex

[0119] Study Design: Male Sprague-Dawley rats weighing between 175 and 200 g were randomized into eight study groups after a 5-day acclimation period. On study day 0, rats in groups 3 and 7 received a single dose of Sal, while groups 4 and 8 received a single dose of Ket (10 mg / kg), each via ip injection. Rats in groups 1 and 5 received a single dose of Veh, while groups 2 and 6 received a single dose of Compound I (160 mg / kg) as the lithium chloride salt or cocrystal, each via oral gavage. One hour after administration, rats in groups 1–4 were sacrificed by conscious decapitation, followed by harvesting of the PFC. Crude synaptosomes were prepared from the PFC, and the three mTORC1 substrates, pmTOR, pp70S6K, and p4E-BP1, as well as the corresponding total protein loading controls (mTOR, p70S6K, and GAPDH), were quantified by Western blot. 24 hours after administration, rats from groups 5 to 8 were sacrificed by conscious decapitation and the PFC was harvested. Crude synaptosomes were prepared from the PFC, and synaptic proteins (GluR1 and PSD95), as well as the total protein loading control (GAPDH), were quantified by Western blot.

[0120] Formulation of Ket and Compound I as lithium chloride salt or co-crystal for administration: Ket (Sigma, Cat. No. K1884) is dissolved in Sal at a concentration of 10 mg / mL. A volume of 1 mL / kg is injected i.p. Compound I as lithium chloride salt or co-crystal is prepared by dissolving in Veh at a concentration of 50 mg / mL. The dose volume based on the animal's weight (3.2 mL / kg) is administered by oral gavage. The test article is prepared on the day of administration.

[0121] Prefrontal cortex synaptosome preparation: Brains were removed from all groups of rats and rinsed in PBS. The PFC was harvested and homogenized at 4°C in homogenization buffer (0.32 M sucrose, 20 mM HEPES pH 7.4, 1 mM EDTA, 5 mM NaF, 1 mM NaVO3, and protease inhibitor cocktail (Roche; #19543200)). The homogenate was centrifuged at 2,800 rpm for 10 min at 4°C, after which the supernatant was removed and recentrifuged at 12,000 rpm for 10 min at 4°C. The resulting pellet containing crude synaptosomes was resuspended in lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton® X-100, 0.1% SDS, 2 mM EDTA, 1 mM NaVO, 5 mM NaF, and protease inhibitor cocktail) and sonicated on ice for 20 s at 50% amplitude. Protein concentration was determined by Bradford assay, and all samples were mixed with loading buffer (60 mM Tris-HCl pH 6.8, 20 mM DTT, 2% SDS, 10% glycerol, 5% β-mercaptoethanol, and 0.01% bromophenol blue) and stored at -20 °C until WB analysis.

[0122] Western blot analysis: Western blot analysis of GluR1, PSD95, and GAPDH was performed as previously described. Briefly, synaptosome preparations (15 µg of total protein) were loaded onto a 10-15% SDS PAGE gel for electrophoresis and transferred to a polyvinylidene difluoride (PVDF) membrane in transfer buffer (10X premixed electrophoresis buffer containing 25 mM Tris, 192 mM glycine, pH 8.3; Bio-Rad). The PVDF membrane was blocked with blocking buffer (2% BSA in PBS-T (10 mM phosphate, pH 7.4, 2.7 mM KCl, 137 mM NaCl, and 0.1% Tween®-20)) for 1 hour at room temperature, followed by incubation with the following primary antibodies in blocking buffer: rabbit anti-pmTOR (Cell Signaling; #5536) at 1:1000, rabbit anti-mTOR (Cell Signaling; #2972) at 1:1000, rabbit anti-pp70S6K (Cell Signaling; #9205) at 1:1000, rabbit anti-p70S6K (Cell Signaling; #2708) at 1:1000, rabbit anti-p4E-BP1 (Cell Signaling; #2855) at 1:1000, rabbit anti-GluR1 (Cell Signaling; #2855) at 1:1000. The blots were incubated overnight at 4°C with 1:1000 rabbit anti-synapsin 1 (Cell Signaling; #13185), 1:1000 rabbit anti-PSD95 (Cell Signaling; #9644), and 1:1000 rabbit anti-GAPDH (Cell Signaling; #5174). The next day, the membranes were washed three times in PBS-T buffer and incubated for 1 hour with 1:5000–1:10000 horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (Vector Laboratories Inc.). After the final three washes with PBS-T buffer, bands were detected using enhanced chemiluminescence. The blots were then incubated in stripping buffer (2% SDS, 100 mM β-mercaptoethanol, 50 mM Tris-HCl pH 6.8) at 50–55°C for 30 minutes, followed by three washes with PBS-T buffer.The stripped blots were then incubated in blocking solution for 1 hour with primary antibodies against total levels of each protein or GAPDH as a loading control. Densitometric analysis of phospho- and total immunoreactivity for each protein was performed using NIH Image J software. The resulting densitometric readings were used to generate ratios of phospho-proteins to their respective total protein levels or GAPDH, as indicated. The resulting ratios were further normalized to the Sal- or Veh-treated control groups for each protein. Example C Effects of a single oral dose of Compound I as lithium chloride salt or cocrystal on the mTORC1 signaling pathway in multiple regions of the rat brain

[0123] Study Design: Male rats weighing between 175 and 200 g were randomized into two study groups after a 5-day acclimation period. Group 1 received a single dose of Veh via oral gavage, and Group 2 received a single dose of Compound I (160 mg / kg, prepared in Veh) as a lithium chloride salt or cocrystal via oral gavage. One hour after administration, rats were sacrificed by conscious decapitation, and plasma was collected for analysis of Compound I exposure as a lithium chloride salt or cocrystal, in addition to isolation of the PFC, hippocampus, striatum, neocortex, and cerebellum by microdissection. Total protein extracts were prepared from the collected tissues and subjected to WB analysis, followed by quantitative analysis of selected mTORC1 substrates.

[0124] Formulation of Compound I as lithium chloride salt or co-crystal (160 mg / ml): Compound I as lithium chloride salt or co-crystal is prepared by dissolving in Veh at a concentration of 160 mg / mL. A dose volume based on the animal's weight (10 mL / kg) is administered by oral gavage to study animals in Group 2. The test article is prepared on the day of administration.

[0125] Western blot analysis: Synaptosome preparations (15 μg total protein) were loaded and separated on a NuPAGE 4-12% Bis-Tris gel and transferred to a PVDF membrane (Immobilon-FL PVDF membrane, Millipore) using CAPS buffer (10 mM 3-(cyclohexylamino)-1-propanesulfonic acid, 12.5% ​​ethanol pH=10). After transfer, the membrane was incubated in Odyssey blocking buffer (Licor) for 1 hour at room temperature. After blocking, the membrane was incubated with primary antibody overnight at 4°C. The primary antibody used was rabbit anti-rabbit antibody at 1:1000 in Odyssey blocking buffer. S400 / 440 pS6 (Cell Signaling; #5364) and mouse anti-α-tubulin (Sigma; #T5168) at 1:10,000. The next day, membranes were washed three times in 1X TBS-Tween® (25 mM Tris, pH 7.4, 3.0 mM KCl, 140 mM NaCl, and 0.05% Tween®-20) and incubated with dye-conjugated secondary antibodies (goat anti-mouse IRdye680 and goat anti-rabbit IRdye800 from LI-COR) at 1:20,000 in Odyssey blocking buffer for 30 minutes, followed by three washes in 1X TBS-Tween®. Signals were quantified using an Odyssey infrared imaging system (LI-COR Bioscience). The resulting densitometry readings were used to generate a ratio of phospho-protein to α-tubulin. The resulting ratios were further normalized to the vehicle-treated control group.

[0126] Prefrontal cortex synaptosome preparation: One hour after administration, rats were sacrificed by conscious decapitation, and plasma and brains were collected. Brains were removed from each group and rinsed in PBS. PFC, striatum, hippocampus, neocortex, and cerebellum were collected and homogenized at 4°C in homogenization buffer (0.32 M sucrose, 20 mM HEPES pH 7.4, 1 mM EDTA, 5 mM NaF, 1 mM NaVO3, and protease inhibitor cocktail (Roche; #19543200)). The homogenate was centrifuged at 2,800 rpm for 10 minutes at 4°C, after which the supernatant was removed and recentrifuged at 12,000 rpm for 10 minutes at 4°C. The resulting pellets were resuspended in lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton® X-100, 0.1% SDS, 2 mM EDTA, 1 mM NaVO, 5 mM NaF, and protease inhibitor cocktail) and sonicated on ice for 20 s at 50% amplitude. Total protein concentration was determined by Bradford assay, and all samples were mixed with loading buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 5% glycerol, 5% β-mercaptoethanol, and 0.01% bromophenol blue) and stored at -20 °C until WB analysis.

[0127] Compound analysis: For determination of compound levels in plasma, proteins are precipitated from 50 μL of the resulting tissue homogenate containing an internal standard (tolbutamide) in 150 μL of acetonitrile, followed by centrifugation at 3000 rpm for 10 minutes. 100 microliters of the resulting supernatant is added to 100 μL of water, mixed thoroughly, and injected into an LC-MS / MS system using the following program to assess compound levels: Phenomenex LUX cellulose column (4.6 x 150 mm, 5 μm) Mobile phase A - 0.1% formic acid in water Mobile phase B - 0.1% formic acid in acetonitrile ·gradient: ○ Initial -40%A ○2 minutes - 40% A ○2.1 min - 2% A ○3 minutes - 2% A ○3.1 min - 40% A ○4 minutes - 40%A ·Flow rate 0.8mL / min Column temperature: 40°C ·Sciex 5500 Triple Quad Mass Spec Example D Effects of a single oral administration of Compound I or leucine as lithium chloride salt or cocrystal on the mTORC1 signaling pathway in rat brain and selected peripheral organs

[0128] Study Design: Male rats weighing between 175 and 200 g are randomized into three study groups after a 5-day acclimation period. Test articles are administered by oral gavage. One hour after administration, rats are sacrificed by conscious decapitation, and plasma, brain, and selected peripheral tissues are collected for compound levels and Western blot analysis. Tissues are prepared for Western blot to quantify the mTORC1 substrate pS6 as a measure of mTORC1 activity.

[0129] Test article preparation: Compound I and leucine (Leu, Sigma; #L8912) as lithium chloride salts or co-crystals are prepared by dissolving them in Veh (0.5% methylcellulose / 0.1% Tween®-80) at concentrations of 16 mg / mL and 100 mg / mL, respectively. The dose volume (10 mL / kg) based on the animal's weight is administered by oral gavage. The test article is prepared on the day of administration.

[0130] Tissue preparation: One hour after administration, rats are sacrificed by decapitation under conscious control, and plasma, brain, and surrounding tissues are collected and immediately frozen in liquid nitrogen. Tissues are thawed and homogenized twice for 1 minute at 4°C in lysis buffer (cell lysis buffer: 1% Triton® X-100, 50 mM HEPES pH 7.4, 100 mM NaCl, 2 mM EDTA, 10 mM beta-glycerophosphate, 10 mM sodium pyrophosphate, and one protease inhibitor tablet per 50 mL fresh solution) using an MP homogenizer. The lysate is then sonicated on ice for 20 seconds at 50% amplitude. Protein concentrations are determined by Bradford assay and all samples are mixed with loading buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 5% glycerol, 5% β-mercaptoethanol and 0.01% bromophenol blue) and stored at −20° C. until WB analysis.

[0131] Western blot (WB) analysis: Equal amounts of each sample (15 μg total protein) were loaded and separated on a NuPAGE 4-12% Bis-Tris gel. The gel was then transferred to a PVDF membrane (Immobilon-FL PVDF membrane, Millipore) using CAPS buffer (10 mM 3-(cyclohexylamino)-1-propanesulfonic acid, 12.5% ​​ethanol pH = 10). After transfer, the membrane was incubated in Odyssey blocking buffer (Licor) for 1 hour at room temperature. After blocking, the membrane was incubated with primary antibodies overnight at 4°C. The primary antibodies used were rabbit anti-S400 / 440pS6 (Cell Signaling; #5364) at 1:1000, mouse anti-GAPDH (Sigma; #G8795) at 1:1000, and mouse anti-α-tubulin (Sigma; #T5168) at 1:10000 in Odyssey blocking buffer. The next day, the membranes were washed three times in 1X TBS-Tween® (25 mM Tris, pH 7.4, 3.0 mM KCl, 140 mM NaCl, and 0.05% Tween®-20) and incubated with dye-linked secondary antibodies (goat anti-mouse IRdye680 and goat anti-rabbit IRdye800 from LI-COR) at 1:20,000 in Odyssey blocking buffer for 30 minutes, followed by three washes in 1X TBS-Tween®. Signals were quantified using an Odyssey infrared imaging system (LI-COR Bioscience). The resulting densitometry readings were used to generate ratios of phospho-protein to α-tubulin or GAPDH. The resulting ratios were further normalized to the vehicle-treated control group.

[0132] Compound analysis: For determination of compound levels in tissue preparations, 70% isopropyl alcohol at a 3:1 v:w (µL:mg) ratio is added to the tissue sample, followed by homogenization in a beadbeater (Biospec). Proteins are precipitated from 50 µL of the resulting tissue homogenate in 150 µL of acetonitrile containing an internal standard (tolbutamide), followed by centrifugation at 3000 rpm for 10 minutes. 100 microliters of the resulting supernatant is added to 100 µL of water, mixed thoroughly, and injected into an LC-MS / MS system using the following program to assess compound levels: Phenomenex LUX cellulose column (4.6 x 150 mm, 5 μm) Mobile phase A - 0.1% formic acid in water Mobile phase B - 0.1% formic acid in acetonitrile ·gradient: ○ Initial -40%A ○2 minutes - 40% A ○2.1 min - 2% A ○3 minutes - 2% A ○3.1 min - 40% A ○4 minutes - 40%A ·Flow rate 0.8mL / min Column temperature: 40°C ·Sciex 5500 Triple Quad Mass Spec Example E Effects of a single dose of Compound I as lithium chloride salt or co-crystal on sucrose preference and novel environment feeding suppression tests and synaptic protein expression

[0133] Study Design: Male rats weighing between 175 and 200 g were randomized into four study groups after a 5-day acclimation period. On day minus 20 of the study, two groups of rats were subjected to CUS for 25 days, while two groups of rats were housed normally and served as the NS group. On day 21 of the CUS protocol, rats received a single dose of either Veh or compound I (160 mg / kg) as a lithium chloride cocrystal via oral gavage (day 0). SPT and NSFT were performed 24 and 48 hours after administration, respectively (days 1 and 2). Following completion of behavioral testing, a second dose of compound I or Veh as a lithium chloride salt or cocrystal was administered on day 5 after the 25-day CUS protocol, and rats were sacrificed by conscious decapitation 24 hours later. Crude synaptosomes were prepared from the PFC, and synaptic proteins, GluR1 and PSD95, were quantified by WB.

[0134] Formulation of Compound I as lithium chloride salt or co-crystal (50 mg / mL): Compound I as lithium chloride salt or co-crystal is prepared by dissolving in Veh to a concentration of 50 mg / mL. The solution is administered by oral gavage to rats in groups 2 and 4 at a volume of 10 mL / kg, resulting in a final dose of 160 mg / kg. An equivalent volume of Veh is administered to groups 1 and 3.

[0135] Prefrontal cortex synaptosome preparation: Brains were excised from rats and rinsed in PBS. The PFC was harvested and homogenized at 4°C in homogenization buffer (0.32 M sucrose, 20 mM HEPES pH 7.4, 1 mM EDTA, 5 mM NaF, 1 mM NaVO3, and protease inhibitor cocktail (Roche; #19543200)). The homogenate was centrifuged at 2,800 rpm for 10 min at 4°C, after which the supernatant was removed and recentrifuged at 12,000 rpm for 10 min at 4°C. The resulting pellets were resuspended in lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton® X-100, 0.1% SDS, 2 mM EDTA, 1 mM NaVO, 5 mM NaF, and protease inhibitor cocktail) and sonicated on ice for 20 s at 50% amplitude. Protein concentrations were determined by Bradford assay, and all samples were mixed with loading buffer (60 mM Tris-HCl pH 6.8, 20 mM DTT, 2% SDS, 10% glycerol, 5% β-mercaptoethanol, and 0.01% bromophenol blue) and stored at -20 °C until WB analysis.

[0136] Western blot analysis: Western blot analysis for GluR1, PSD95, and GAPDH was performed as previously described (Li, N. et al., Science 329(5994): 959-964 (2010)). Briefly, synaptosomes (15 μg of protein) were loaded onto a 10-15% SDS-PAGE gel for electrophoresis and transferred to a polyvinylidene difluoride (PVDF) membrane in transfer buffer (10X premixed electrophoresis buffer containing 25 mM Tris, 192 mM glycine, pH 8.3; Bio-Rad). PVDF membranes were blocked with blocking buffer (2% BSA in PBS-T (10 mM phosphate, pH 7.4, 2.7 mM KCl, 137 mM NaCl, and 0.1% Tween®-20)) for 1 hour at room temperature, and then incubated overnight at 4°C with primary antibodies: rabbit anti-GluR1 (Cell Signaling; #13185) at 1:1000, rabbit anti-PSD95 (Cell Signaling; #9644) at 1:1000, and rabbit anti-GAPDH (Cell Signaling; #5174) at 1:1000 in blocking buffer. The next day, the membranes were washed three times in PBS-T buffer and incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (Vector Laboratories Inc.) at 1:5000–1:10000 for 1 hour. After three final washes with PBS-T buffer, bands were detected using enhanced chemiluminescence. The blots were then incubated in stripping buffer (2% SDS, 100 mM β-mercaptoethanol, 50 mM Tris pH 6.8) at 50-55°C for 30 min, followed by three washes with PBS-T buffer. The stripped blots were then kept in blocking solution for 1 h and incubated with a primary antibody against GAPDH as a loading control. Densitometry analysis of total immunoreactivity for each protein was performed using NIH Image J software. The resulting densitometry readings were used to generate ratios of total protein to GAPDH. The resulting ratios were further normalized to the NS-Veh group for each protein. Example F The pharmacological activity of Compound I as lithium chloride salt or cocrystal in the forced swimming and novel environment feeding suppression tests after a single oral dose in rats depends on mTORC1 activation

[0137] Study Design: Male rats weighing between 175 and 200 g are randomized into three study groups after a 5-day acclimation period. All rats are surgically implanted with bilateral IT cannulae in the PFC 2 weeks prior to dosing. On the day of dosing, all treatment groups receive bilateral IT infusions (0.5 μL / side) containing either rapamycin® vehicle (Veh-R, 10% DMSO) or rapamycin (R, 0.01 nmol / μL), previously shown to fully inhibit mTORC1 activity. Thirty minutes after the intrathecal infusion, either Compound I vehicle (Veh-NV, 0.5% methylcellulose / 0.1% Tween®-80) or Compound I (160 mg / kg) as the lithium chloride salt or cocrystal is administered by oral gavage. Each treatment group is evaluated at designated times after oral administration in a 24-hour FST (day 1), a 48-hour LMA (day 2), and a 72-hour NSFT (day 3, after a 20-hour fast). LMA is measured to exclude gross changes in general locomotor activity.

[0138] Preparation of test article: Rapamycin (Cell Signaling; #9904) is prepared in a solution of 10% DMSO (Veh-R) to a final concentration of 10 μM. R or Veh-R is administered bilaterally into the medial PFC via IT injection 30 minutes before treatment with compound I or Veh-NV as lithium chloride salt or co-crystal by oral gavage (0.005 nmol / 0.5 μL per side). Compound I as lithium chloride salt or co-crystal is prepared by dissolving in Veh (0.5% methylcellulose / 0.1% Tween®-80) at a concentration of 50 mg / mL. The dose volume (3.2 mL / kg) based on the animal's body weight is administered to the study animals in groups 2 and 3 by oral gavage.

[0139] Surgical procedure and rapamycin administration: Rats were stereotactically implanted with a guide cannula (22 GA) into the medial PFC (coordinates: +3.2 AP, ±1.0 ML from bregma, -3.5 DV from dura). The surgical procedure was performed under anesthesia with Nembutal (55 mg / kg i.p.). Postoperative care consisted of perioperative administration of carprofen (5 mg / kg) and topical triple antibiotics. After a 2-week recovery period, R (0.01 nmol in 1 μL for PFC injection) or Veh-R was delivered at a rate of 0.25 μL / min through an injection cannula (26 GA) protruding 0.5 mm from the guide cannula, 30 min prior to oral administration of compound I or Veh-NV as the lithium chloride salt or cocrystal. The dose of rapamycin was selected based on previous reports demonstrating effective and selective inhibition of mTORC1 activity. Example G Duration of behavioral changes in the forced swimming test and novel environment food suppression test after a single administration of Compound I or ketamine as the lithium chloride salt or cocrystal

[0140] Study Design: Male rats weighing between 175 and 200 g are randomized into six study groups after a 5-day acclimation period. A single dose of all test articles is administered on day 0, with behavioral testing performed 3, 7, and 10 days later. Groups 1 and 2 receive Compound I (160 mg / kg by oral gavage) and Ket (10 mg / kg by ip injection) as the lithium chloride salt or co-crystal, respectively, on day 0 and are subjected to the FST on day 3. Rats in groups 3 and 4 receive a single dose of Compound I vehicle (Veh) or Compound I (160 mg / kg) as the lithium chloride salt or co-crystal, respectively, on day 0 by oral gavage. Rats in groups 5 and 6 receive a single dose of Ket vehicle (Sal) or Ket (10 mg / kg) by ip injection, respectively, on day 0. Rats in groups 3 to 6 are subjected to the FST on day 7 and the NSFT on day 10. All rats in groups 3 to 6 were fasted for 20 hours the night before NSFT. Example H Physiological changes in layer V pyramidal neurons after a single administration of compound I as lithium chloride salt or cocrystal

[0141] Study Design: Male rats weighing between 175 and 200 g are randomized into two study groups after a 5-day acclimation period. On study day 0, rats receive a single dose of either NV vehicle (Veh, 0.5% methylcellulose / 0.1% Tween®-80) or Compound I (160 mg / kg) as the lithium chloride salt or cocrystal by oral gavage. On day 1, rats are sacrificed 24 hours after administration, and brain slices are prepared and subjected to whole-cell patch-clamp recording of layer V pyramidal neurons in the PFC.

[0142] Test article preparation: Compound I, either as the lithium chloride salt or as a co-crystal, is prepared by dissolving it in Veh (0.5% methylcellulose / 0.1% Tween®-80) at a concentration of 50 mg / mL. The dose volume (3.2 mL / kg) based on the animal's weight is administered to the study animals by oral gavage. The test article is prepared on the day of administration.

[0143] Brain slice preparation: Brain slices were prepared according to published procedures (Liu, RJ et al., J. Neurosci. 22(21): 9453-9464 (2002)). Briefly, rats were anesthetized with chloral hydrate (400 mg / kg, i.p.) in accordance with protocols approved by the Yale Animal Care and Use Committee. After decapitation, brains were rapidly removed and placed in ice-cold (4°C) artificial cerebrospinal fluid (ACSF) in which sucrose (252 mM) was substituted for NaCl (sucrose-ACSF) to prevent cell swelling. Blocks of tissue containing the PFC were cut, and coronal slices (400 μm) were cut in sucrose-ACSF with a vibrating blade tissue slicer (Leica VT1000S). After placement of the slices in a submerged recording chamber, the bath temperature was raised to 32°C. A known concentration of drug dissolved in ACSF is applied via a stopcock at a fast flow rate (approximately 4 mL / min) and reaches the slice within 7–10 seconds. Standard ACSF (pH = 7.35) is equilibrated with 95% O2 / 5% CO2 and contains 128 mM NaCl, 3 mM KCl, 2 mM CaCl2, 2 mM MgSO4, 24 mM NaHCO3, 1.25 mM NaH2PO4, and 10 mM D-glucose. A recovery period of approximately 1–2 h is allowed before the start of recording.

[0144] Electrophysiological recordings: Pyramidal neurons in layer V were visualized by infrared differential interference contrast (IR / DIC) video microscopy using an Olympus BX50WI microscope (×60 IR lens) with infrared video microscopy (Olympus) according to published procedures (Lambe, EK & Aghajanian, GK Neuron 40(1):139-150 (2003)). Low-resistance patch pipettes (3-5 MΩ) were pulled from patch clamp glass tubing (Warner Instruments) using a Flaming-Brown Horizontal Puller (model P-97; Sutter Instruments). Pipettes were filled with the following solution: 115 mM K-gluconate, 5 mM KCl, 2 mM MgCl, 2 mM Mg-ATP, 2 mM NaATP, 10 mM Na-phosphocreatine, 0.4 mM NaGTP, and 10 mM HEPES, pH 7.33. Neurobiotin (0.3%) is added to the pipette solution to mark the cells for subsequent imaging. Whole-cell recordings are performed with an Axoclamp-2B amplifier (Axon Instruments). Output signals are low-pass filtered at 3 kHz, amplified ×100 by Cyberamp, digitized at 15 kHz, and acquired using pClamp9.2 / Digidata1320 software (Axon Instruments). Series resistance, monitored throughout the experiment, is typically between 4 and 8 MΩ. To minimize series resistance error, cells are discarded if the series resistance exceeds 10 Ω. Postsynaptic currents are studied in continuous single-electrode voltage clamp mode (3000 Hz low-pass filter), clamped near the resting potential (75 mV ± 5 mV) to minimize holding currents. After recording is complete, slices are transferred to 4% paraformaldehyde in 0.1 M phosphate buffer and stored overnight at 4 °C. Sections are then treated with streptavidin conjugated with Alexa 594 (1:1000; Invitrogen) for visualization of neurobiotin in labeled cells.

[0145] Spine density analysis: Labeled neurons in layer V of the anterior cingulate gyrus (Cg1) and prelimbic mPFC (Cg3) were imaged with a two-photon Ti:sapphire laser scanning system (810 nm; Mai Tai, Spectra Physics, Mountain View, California) coupled to a direct-detection Radiance 2000 BioRad laser scanner (Zeiss Micromaging, Thornwood, New York) mounted on an Olympus BX50WI microscope using a 60x (0.9 numerical aperture) water immersion objective for analysis of spine density and morphology. This includes the total number of spines on the proximal and distal tufts of layer V neurons, as well as the diameter of the spine head, an indicator of spine maturation. The length of the apical tuft branch segment was determined within the 3D matrix of each Z-stack using Neurolucida 10.2 (MicroBrightField). Analysis of spine density and spine head diameter was performed on raw image stacks (2-5 optical sections, 1 μm apart) using the Autospine module in Neurolucida Explorer (version 10.2). Spine density and scission (beading) were sampled in three zones: the tip of the tufted branch approaching the pia mater, the mid-dendritic process approximately midway between the pia mater and the apical trunk branch, and the proximal tufted dendrite slightly distal to the branching. Results are expressed in terms of total dendrite length, spine density, and scission density. Results are expressed in terms of spine density per 10 μm. Example I Behavioral changes in the forced swimming and novelty environment food suppression tests following daily administration of Compound I as the lithium chloride salt or cocrystal or every other day administration of ketamine

[0146] Study Design: Male Sprague-Dawley rats weighing between 175 and 200 g were randomized into four study groups after a 5-day acclimation period. Rats were subjected to a pre-swim on study day minus 1. Starting on study day 0, rats in group 2 were administered a dose of Ket (10 mg / kg) every other day (days 0, 2, 4, and 6), each administered by ip injection. Rats in group 1 were administered a daily dose of Veh by oral gavage for 7 days (days 0-6). Rats in groups 3 and 4 were administered a daily dose of Compound I (40 or 80 mg / kg) as the lithium chloride salt or cocrystal, each administered by oral gavage for 7 days (days 0-6). All rats were subjected to the FST on day 7, 24 hours after the last dose. On day 8, 48 hours after the last dose, the LMA of all rats was measured in the open field. The rats are then fasted for 20 hours and subjected to NSFT on the 9th day (72 hours after the last dose).

[0147] Preparation of test articles: Ket (Sigma, catalog number K1884) is dissolved in Sal at a concentration of 10 mg / mL. An injection volume (ip) of 1 mL / kg of Ket is administered to Group 2. Compound I as the lithium chloride salt or co-crystal is prepared by dissolving it in Veh (0.5% methylcellulose / 0.1% Tween®-80) at a concentration of 50 mg / mL. A dose volume (3.2 mL / kg) based on the animal's body weight of Veh or Compound I as the lithium chloride salt or co-crystal is administered daily by oral gavage to the study animals in Groups 1, 3, and 4, respectively. The test articles are prepared on the day of administration. Example J Marmoset human threat testing

[0148] Study design: Marmosets (Callithrix jacchus) are paired and randomly assigned to treatment groups. 24 hours before the human threat test (HTT), animals are treated with vehicle, ketamine (0.3 mg / kg; im), or Compound I (160 mg / kg; po) as the lithium chloride salt or cocrystal. The following day, the same animals are treated with either vehicle (sc) or chloriazepoxide (1 mg / kg; sc). Animals are then monitored for the number of threat postures over a 2-minute period in the presence of a human observer. Locomotor activity, measured by the number of observed jumps, is monitored over the same period.

[0149] While the inventors have described several embodiments of the invention, it will be apparent that the inventors' basic examples may be modified to provide other embodiments that utilize the compounds and methods of the invention. It will therefore be understood that the scope of the invention is defined by the appended claims rather than by the specific embodiments that have been represented by way of example.

Claims

1. Compound I has the structure: 【Transformation 6】 The compound having the formula LiCl*Compound I, or a solvate or hydrate thereof.

2. 10. The compound of claim 1, wherein the LiCl*Compound I is a co-crystal.

3. 2. The compound of claim 1, wherein lithium and compound I are in a stoichiometric ratio of about 1 to about 1.

4. 10. A pharmaceutical composition comprising a compound of claim 1 and a pharmaceutically acceptable carrier, adjuvant, or vehicle.

5. 5. The pharmaceutical composition of claim 4, formulated for oral administration.

6. 5. The pharmaceutical composition of claim 4, further comprising an additional therapeutic agent.

7. A method for treating, preventing, and / or reducing the risk of a disease, disorder, or condition mediated by mTORC1 in a patient, comprising administering an effective amount of a compound described in claim 1 or a pharmaceutical composition described in claim 4 to a patient in need of treatment, prevention, and / or reduction of the risk of a disease, disorder, or condition mediated by mTORC1.

8. 8. The method of claim 7, wherein the disease, disorder, or condition mediated by mTORC1 is depression, bipolar disorder, schizophrenia, chronic unpredictable stress, autism, lysosomal storage disease, Batten disease, cystinosis, Fabry disease, mucolipidosis, mental retardation, anorexia, bulimia, anemia, neutropenia, headache, alcoholism, post-traumatic stress disorder (PTSD), epilepsy, diabetes, liver disease, kidney damage, arthritis, skin conditions such as seborrhea, hyperthyroidism, asthma, Huntington's disease, Graves' disease, herpes simplex, movement disorders such as tardive dyskinesia, Tourette's syndrome, cyclic vomiting, Meniere's disease, tingling or formication sensations in the skin (paresthesia), or aggressive behavior in attention deficit hyperactivity disorder (ADHD).

9. A method for treating treatment-resistant depression in a patient in need thereof, comprising administering to the patient an effective amount of a compound of claim 1 or a pharmaceutical composition of claim 4.

10. A method for treating major depressive disorder in a patient in need thereof, comprising administering to the patient an effective amount of a compound of claim 1 or a pharmaceutical composition of claim 4.

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