Modulators of mTORC1 activity and uses thereof

Abstract

The present invention relates to compounds and methods useful for selectively modulating mTORC1 activity.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 421,288, filed on November 1, 2022, the content of which is hereby incorporated by reference. Background Art

[0003] Depression in individuals suffering from either major depressive disorder (MDD) or bipolar disorder (BD) is a major cause of the disease burden. In the published PCT application WO 2017 / 070518, it was found that small molecule mTORC1 activators are beneficial for depression and other CNS-related diseases. Activation of mTORC1 in the prefrontal cortex (PFC) and subsequent synaptogenesis are thought to mediate the rapid antidepressant effects of administered small molecule mTORC1 activators. By activating the mTOR signaling pathway, administration of mTORC1 activators increases synaptic protein levels and dendritic spine density.

[0004] In addition, it is well known that the mood stabilizer lithium has anti-suicidal properties and shows promise for treating other neurological and neurodegenerative diseases.

[0005] Chiu et al. (International Journal of Neuropsychopharmacology, 2015, 1 - 13) also demonstrated that in the forced swim test, mice pretreated with subtherapeutic (600 mg / L) lithium showed an antidepressant-like response to ineffective ketamine (2.5 mg / kg, intraperitoneal) challenge. The antidepressant-like effects induced by a single injection of ketamine (50 mg / kg) in stressed mice and the restoration of dendritic spine density in the medial prefrontal cortex were both sustained by post-treatment with 1200 mg / L lithium for at least 2 weeks. These benefits of lithium treatment are associated with activation of the mammalian target of rapamycin / brain-derived neurotrophic factor signaling pathway in the prefrontal cortex.

[0006] There is an urgent and unmet medical need for more effective treatments for diseases, disorders, or conditions related to mTORC1. Detailed Description

[0007] 1. General description of certain embodiments of the invention:

[0008] In various aspects of the present invention, it can be noted that there are provided compositions containing a stoichiometric ratio of a lithium salt and an organic molecule.

[0009] In the present invention, a compound comprising a lithium salt in stoichiometric ratio or eutectic form and an mTORC1 activator is described. Such mTORC1 activators can be amino acids as described in the published PCT application WO 2017 / 070518. In some embodiments, due to the synergistic effect of lithium and the mTORC1 activator, the compound comprising the lithium salt and the mTORC1 activator has enhanced efficacy. In some embodiments, such derivatives are efficacious at a lower therapeutic dose than either single agent administered alone.

[0010] In some embodiments, a compound comprising a lithium salt and an mTORC1 activator is described, which compound is a eutectic comprising a stoichiometric ratio of lithium salt and mTORC1 activator. Such mTORC1 activators can be amino acids as described in the published PCT application WO 2017 / 070518. In some embodiments, due to the synergistic effect of lithium and the mTORC1 activator, the lithium salt and mTORC1 activator in eutectic form have enhanced efficacy. In some embodiments, such derivatives are efficacious at a lower therapeutic dose than either single agent administered alone.

[0011] U.S. Patent No. 10,100,066 (“the '066 patent”) (which was filed as U.S. Patent Application Serial No. U.S. 15 / 331,362 on October 21, 2016 and published as U.S. Patent Application Publication No. U.S. 2017 / 0114080 (“the '080 publication”), the entire content of each document being incorporated herein by reference) describes certain mTORC1 modulating compounds. Such compounds include Compound I:

[0012]

[0013] Compound I, namely (S)-2-amino-5,5-difluoro-4,4-dimethylvaleric 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.

[0014] It has now been found that compounds comprising lithium chloride and Compound I (such as salts or eutectics comprising lithium chloride and Compound I) and their compositions can be used to treat, prevent mTORC1-mediated diseases, disorders or conditions and / or reduce the risk of said diseases, disorders or conditions.

[0015] One aspect of the present invention is a compound having the formula LiCl*Compound I, or a solvate or hydrate thereof, wherein Compound I has the following structure:

[0016]

[0017] One aspect of the present invention further relates to a pharmaceutical composition comprising a compound of formula LiCl* Compound I, or a solvate or hydrate thereof. The present invention further relates to a dosage unit form comprising a compound of formula LiCl* Compound I, or a solvate or hydrate thereof. The compound of formula LiCl* Compound I can be a salt or a co-crystal, and the crystal lattice of the salt or co-crystal may further include one or more solvates or water molecules.

[0018] The present invention further relates to a method for preparing a salt or a co-crystal, the 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.

[0019] Other aspects and objects of the present invention will be in part apparent and in part pointed out hereinafter.

[0020] In some embodiments, there is provided a compound of formula LiCl* Compound I, or a solvate or hydrate thereof, wherein Compound I has the following structure:

[0021]

[0022] In some embodiments, the compound of formula LiCl* Compound I is a co-crystal.

[0023] In some embodiments, the stoichiometric ratio of lithium to Compound I is about 1 to about 1.

[0024] In some embodiments, there is provided a pharmaceutical composition comprising a compound of formula LiCl* Compound I and a pharmaceutically acceptable carrier, adjuvant or vehicle.

[0025] In some embodiments, the pharmaceutical composition is formulated for oral administration.

[0026] In some embodiments, the pharmaceutical composition further comprises an additional therapeutic agent.

[0027] In some embodiments, there is provided a method for treating, preventing a disease, disorder or condition mediated by mTORC1 in a patient and / or reducing the risk of the disease, disorder or condition in the patient, the method comprising administering to a patient in need an effective amount of a compound of formula LiCl* Compound I or a pharmaceutical composition comprising a compound of formula LiCl* Compound I.

[0028] In some embodiments, the diseases, disorders or conditions mediated by mTORC1 are depression, bipolar disorder, schizophrenia, chronic unpredictable stress, autism, lysosomal storage diseases, Batten disease, cystinosis, Fabry disease, mucolipidosis, mental retardation, anorexia, bulimia, anemia, neutropenia, headache, alcoholism, post-traumatic stress disorder (PTSD), epilepsy, diabetes, liver disease, kidney disease, 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, skin prickling or crawling sensations (paresthesia), or aggressive behavior in attention deficit hyperactivity disorder (ADHD).

[0029] In some embodiments, a method of treating treatment-resistant depression in a patient in need thereof is provided, the method 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.

[0030] 2. Definitions:

[0031] As used herein, unless otherwise indicated, the following definitions apply. For the purposes of the present invention, chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Edition. Additionally, the general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999; and "March's Advanced Organic Chemistry", 5th Edition, edited by Smith, M.B. and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are hereby incorporated by reference.

[0032] In certain embodiments, the present invention provides Compound I:

[0033]

[0034] The compound is in the form of its lithium chloride salt or eutectic.

[0035] In certain embodiments, the present invention provides Compound I:

[0036]

[0037] The compound is in the form of its lithium chloride eutectic.

[0038] 4. Uses, Formulations and Administrations

[0039] Pharmaceutically acceptable compositions

[0040] 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 in the form of its lithium chloride salt or eutectic, and a pharmaceutically acceptable carrier, adjuvant or vehicle.

[0041] The amounts of Compound I and lithium chloride in the compositions of the present invention are such that mTORC1 can be effectively and measurably modulated or activated in a biological sample or a patient. In certain embodiments, the amounts of Compound I and lithium chloride in the compositions of the present invention are such that mTORC1 can be effectively and measurably modulated or activated in a biological sample or a patient. In certain embodiments, Compound I and lithium chloride form a eutectic.

[0042] In certain embodiments, the compositions of the present invention are formulated for administration to a patient in need thereof. In some embodiments, the compositions of the present invention are formulated for oral administration to a patient.

[0043] As used herein, the term "patient" means an animal, preferably a mammal and most preferably a human.

[0044] 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 formulated therewith. Pharmaceutically acceptable carriers, adjuvants or vehicles that can 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), buffering substances (such as phosphates), glycine, sorbic acid, potassium sorbate, mixtures of partial glycerides of saturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts), colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polypropylene block polymers, polyethylene glycol and lanolin.

[0045] The compositions of the present invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or by an implanted reservoir. As used herein, the term "parenteral" includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intraliver, intralesional and intracranial injection or infusion techniques. Preferably, the compositions are administered orally, intraperitoneally or intravenously. The sterile injectable form of the compositions of the present invention may be aqueous or oleaginous suspensions. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a parenterally acceptable non-toxic diluent or solvent, such as a solution in 1,3-butanediol. Acceptable vehicles and solvents that may be employed include water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are commonly employed as a solvent or suspending medium.

[0046] For this purpose, any mild fixed oil may be employed, including synthetic mono- or di-glycerides of fatty acids. 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 in their polyoxylated forms. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersing agent, such as carboxymethylcellulose or similar dispersing agents commonly used in formulating pharmaceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants such as Tweens, Spans and other emulsifying agents or bioavailability enhancers commonly used in the preparation of pharmaceutically acceptable solid, liquid or other dosage forms may also be used for formulating purposes.

[0047] The pharmaceutically acceptable compositions of the present invention can be administered orally in any orally acceptable dosage form, including but not limited to capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, common carriers include lactose and corn starch. Lubricants, such as magnesium stearate, are usually also added. For oral administration in the form of capsules, useful diluents include lactose and dry corn starch. When an aqueous suspension is required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.

[0048] Alternatively, for rectal administration, the pharmaceutically acceptable compositions of the present invention can be administered in the form of a suppository. The suppository can be prepared by mixing the agent with a suitable non-irritating excipient which is solid at room temperature but liquid at rectal temperature and will thus melt in the rectum to release the drug. Such materials include cocoa butter, beeswax, and polyethylene glycol.

[0049] The pharmaceutically acceptable compositions of the present invention can also be administered topically, especially when the target of treatment includes areas or organs of diseases that are accessible by topical application, including the eye, skin, or lower intestine. Suitable topical formulations for each of these areas or organs are readily prepared.

[0050] Topical application to the lower intestine can be achieved in the form of a rectal suppository formulation (see above) or a suitable enema formulation. Topical transdermal patches can also be used.

[0051] For topical application, the provided pharmaceutically acceptable compositions can be formulated in a suitable ointment which contains 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 paraffin oil, white paraffin oil, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying wax, and water. Alternatively, the provided pharmaceutically acceptable compositions can be formulated in a suitable lotion or cream which contains 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 esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water.

[0052] For ophthalmic use, the provided pharmaceutically acceptable compositions can be formulated as a micronized suspension in isotonic, pH-adjusted sterile saline, or preferably as a solution in isotonic, pH-adjusted sterile saline (with or without a preservative such as benzalkonium chloride). Alternatively, for ophthalmic use, the pharmaceutically acceptable composition can be formulated in an ointment such as petrolatum.

[0053] The pharmaceutically acceptable compositions of the present invention can also be administered by nasal aerosol or by inhalation. Such compounds are prepared according to techniques well known in the art of pharmaceutical formulation and can be prepared as solutions in saline using benzyl alcohol or other suitable preservatives, absorption promoters for enhancing bioavailability, fluorocarbons, and / or other conventional solubilizers or dispersants.

[0054] Most preferably, the pharmaceutically acceptable compositions of the present invention are formulated for oral administration. Such formulations may be administered with or without food. In some embodiments, the pharmaceutically acceptable compositions of the present invention are not administered with food. In other embodiments, the pharmaceutically acceptable compositions of the present invention are administered with food.

[0055] The amounts of the compositions comprising Compound I and lithium chloride that can be combined with a carrier material to produce a single dosage form will vary depending on the subject being treated and the particular mode of administration. Preferably, the compositions provided should be formulated such that a dosage of the inhibitor between 0.01 - 100 mg / kg body weight / day can be administered to a patient receiving these compositions.

[0056] It should also be understood that the specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the activity of the specific compound employed, age, body weight, general health, gender, diet, time of administration, rate of excretion, drug combination, and the judgment of the treating physician, as well as the severity of the particular disease being treated. The amount of the compositions comprising Compound I and lithium chloride salt in the composition will also depend on the specific compound in the composition.

[0057] Use and pharmaceutically acceptable compositions

[0058] Compositions comprising Compound I and lithium chloride are generally useful for modulating or activating mTORC1. In some embodiments, Compound I or its composition in the form of a lithium chloride salt or co-crystal is a modulator of mTORC1. In some embodiments, Compound I or its composition in the form of a lithium chloride salt or co-crystal is a selective modulator of mTORC1. In some embodiments, Compound I or its composition in the form of a lithium chloride salt or co-crystal is an activator of mTORC1.

[0059] The activity of the compositions comprising Compound I and lithium chloride as a modulator or activator of mTORC1 can be determined in vitro, in vivo, or in cell lines. In vitro assays include assays for determining the modulation or activation of mTORC1. The detailed conditions for assaying the compounds used as modulators or activators of mTORC1 in the present invention are set forth in the following examples.

[0060] As used herein, the terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, delaying the onset of, or inhibiting the progression of a disease or disorder or one or more symptoms thereof as described herein. In some embodiments, treatment may be administered after the appearance of one or more symptoms. 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., based on a symptom history and / or based on genetic or other susceptibility factors). Treatment may also be continued after symptoms have subsided, for example, to prevent or delay their recurrence.

[0061] A composition comprising Compound I and lithium chloride is a modulator or activator of mTORC1 and, thus, can be used to treat one or more conditions associated with the activity of mTORC1. Accordingly, in certain embodiments, the present invention provides a method for treating an mTORC1-mediated condition, the method comprising the step of administering to a patient in need thereof a composition comprising Compound I and lithium chloride or a pharmaceutically acceptable composition thereof.

[0062] As used herein, the term “mTORC1-mediated” condition, disease, and / or disorder means any disease or other adverse disorder in which mTORC1 is known to play a role. Accordingly, another embodiment of the present invention relates to treating one or more diseases in which mTORC1 is known to play a role or reducing the severity thereof.

[0063] In some embodiments, methods of activating mTORC are used to treat or prevent depression. (See Ignácio et al., (2015) Br J Clin Pharmacol. Nov 27). Accordingly, in some embodiments, the present invention provides a method for treating or preventing depression in a patient in need thereof, the method comprising the step of administering to the patient Compound I in the form of a lithium chloride salt or co-crystal or a pharmaceutically acceptable composition thereof. In some embodiments, the depression is major depressive disorder (“MDD”). Accordingly, in some embodiments, the present invention provides a method for treating or preventing major depressive disorder in a patient in need thereof, the method comprising the step of administering to the patient Compound I in the form of a lithium chloride salt or co-crystal or a pharmaceutically acceptable composition thereof. In some embodiments, the depression is treatment-resistant depression (“TRD”). Accordingly, in some embodiments, the present invention provides a method for treating or preventing treatment-resistant depression in a patient in need thereof, the method comprising the step of administering to the patient Compound I in the form of a lithium chloride salt or co-crystal or a pharmaceutically acceptable composition thereof. In some embodiments, treatment-resistant depression is resistant to first-line treatment. In some embodiments, treatment-resistant depression is resistant to second-line treatment.

[0064] In some embodiments, the present invention provides a method for treating depression in a patient in need thereof, wherein the patient's depression scale score is reduced by 50%. In some embodiments, the patient's depression scale score is reduced by 50% within less than six weeks of administering the compound or pharmaceutically acceptable composition. In some embodiments, the patient's depression scale score is reduced by 50% within less than four weeks of administering the compound or pharmaceutically acceptable composition. In some embodiments, the patient's depression scale score is reduced by 50% within two weeks of administering the compound or pharmaceutically acceptable composition. In some embodiments, the patient's depression scale score is reduced by 50% within less than two weeks of administering the compound or pharmaceutically acceptable composition. In some embodiments, the patient's depression scale score is reduced by 50% within one week of administering the compound or pharmaceutically acceptable composition. In some embodiments, the patient's depression scale score is reduced by 50% within seven days of administering the compound or pharmaceutically acceptable composition. In some embodiments, the patient's depression scale score is reduced by 50% within six days of administering the compound or pharmaceutically acceptable composition. In some embodiments, the patient's depression scale score is reduced by 50% within five days of administering the compound or pharmaceutically acceptable composition. In some embodiments, the patient's depression scale score is reduced by 50% within four days of administering the compound or pharmaceutically acceptable composition. In some embodiments, the patient's depression scale score is reduced by 50% within three days of administering the compound or pharmaceutically acceptable composition. In some embodiments, the patient's depression scale score is reduced by 50% within two days of administering the compound or pharmaceutically acceptable composition. In some embodiments, the patient's depression scale score is reduced by 50% within one day of administering the compound or pharmaceutically acceptable composition. In some embodiments, the patient's depression scale score is reduced by 50% within twenty-four 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 Symptomatology-Self Report (IDS-SR), and the Clinical Global Impression-Severity (CGI-S).

[0065] In some embodiments, the present invention provides a method for treating depression in a patient in need thereof, the method comprising the step of orally administering to the patient Compound I in the form of a lithium chloride salt or a eutectic, or a pharmaceutically acceptable composition thereof, wherein the reduction in the depression scale score of the patient is comparable to that of ketamine administered by intraperitoneal injection. In some embodiments, the reduction in the depression scale score is produced by a single oral administration. In some embodiments, the reduction in the depression scale score is produced by multiple oral administrations.

[0066] In some embodiments, a method of activating mTORC1 is used to elicit rapid-onset antidepressant activity. Accordingly, in some embodiments, the present invention provides a method for eliciting rapid-onset antidepressant activity in a patient in need thereof suffering from TRD, the method comprising the step of administering to the patient Compound I in the form of a lithium chloride salt or a eutectic, or a pharmaceutically acceptable composition thereof. In some embodiments, the rapid-onset antidepressant activity occurs within two weeks of administering the compound or composition. In some embodiments, the rapid-onset antidepressant activity occurs within one week of administering the compound or composition. In some embodiments, the rapid-onset antidepressant activity occurs within seven days of administering the compound or composition. In some embodiments, the rapid-onset antidepressant activity occurs within six days of administering the compound or composition. In some embodiments, the rapid-onset antidepressant activity occurs within five days of administering the compound or composition. In some embodiments, the rapid-onset antidepressant activity occurs within four days of administering the compound or composition. In some embodiments, the rapid-onset antidepressant activity occurs within three days of administering the compound or composition. In some embodiments, the rapid-onset antidepressant activity occurs within two days of administering the compound or composition. In some embodiments, the rapid-onset antidepressant activity occurs within one day of administering the compound or composition. In some embodiments, the rapid-onset antidepressant activity occurs within less than twenty-four hours of administering the compound or composition.

[0067] In some embodiments, the present invention provides a method of eliciting a durable, sustained antidepressant activity in a patient in need suffering from depression, the method comprising the step of administering to the patient Compound I in the form of a lithium chloride salt or a eutectic, or a pharmaceutically acceptable composition thereof. In some embodiments, the patient in need suffers from TRD. In some embodiments, after a single administration of Compound I in the form of a lithium chloride salt or a eutectic, or a pharmaceutically acceptable composition thereof, the durable, sustained antidepressant activity persists for at least twenty-four hours. In some embodiments, the durable, sustained antidepressant activity persists for more than one day. In some embodiments, the durable, sustained antidepressant activity persists for at least two days. In some embodiments, the durable, sustained antidepressant activity persists for at least three days. In some embodiments, the durable, sustained antidepressant activity persists for at least four days. In some embodiments, the durable, sustained antidepressant activity persists for at least five days. In some embodiments, the durable, sustained antidepressant activity persists for at least six days. In some embodiments, the durable, sustained antidepressant activity persists for at least seven days.

[0068] In some embodiments, the present invention provides a method of eliciting a rapidly-acting, durable, and sustained antidepressant activity.

[0069] In some embodiments, the present invention provides a method of eliciting a positive behavioral response in an object, the method comprising the step of administering to the object Compound I in the form of a lithium chloride salt or a eutectic, or a pharmaceutically acceptable composition thereof. In some embodiments, the positive behavioral response is associated with an improvement in mood. In some embodiments, the positive behavioral response is associated with a reduction in anxiety. In some embodiments, the positive behavioral response corresponds to an improvement in mood. In some embodiments, the positive behavioral response is associated with an improved ability to cope with stress.

[0070] In some embodiments, the present invention provides a method of eliciting a rapidly-acting positive behavioral response in an object, the method comprising the step of administering to the object Compound I in the form of a lithium chloride salt or a eutectic, or a pharmaceutically acceptable composition thereof. In some embodiments, the positive behavioral response occurs within twenty-four hours of administration. In some embodiments, the positive behavioral response occurs within one day of administration. In some embodiments, the positive behavioral response occurs within two days of administration. In some embodiments, the positive behavioral response occurs within three days of administration. In some embodiments, the positive behavioral response occurs within four days of administration. In some embodiments, the positive behavioral response occurs within five days of administration. In some embodiments, the positive behavioral response occurs within six days of administration. In some embodiments, the positive behavioral response occurs within seven days of administration. In some embodiments, the positive behavioral response occurs within one week of administration.

[0071] In some embodiments, the present invention provides a method of eliciting a persistent, sustained positive behavioral response in a subject, the method comprising the step of administering to the subject Compound I in the form of a lithium chloride salt or a eutectic, or a pharmaceutically acceptable composition thereof. In some embodiments, the persistent, sustained positive behavioral response lasts for more than one day. In some embodiments, the persistent, sustained positive behavioral response lasts for at least two days. In some embodiments, the persistent, sustained positive behavioral response lasts for at least three days. In some embodiments, the persistent, sustained positive behavioral response lasts for at least four days. In some embodiments, the persistent, sustained positive behavioral response lasts for at least five days. In some embodiments, the persistent, sustained positive behavioral response lasts for at least six days. In some embodiments, the persistent, sustained positive behavioral response lasts for at least seven days.

[0072] In some embodiments, the present invention provides a method of eliciting a rapidly-acting, persistent, and sustained positive behavioral response.

[0073] In some embodiments, the present invention provides a method of improving and / or reversing behavioral and synaptic deficits in a patient in need thereof caused by chronic unpredictable stress (CUS), the method comprising the step of administering to the patient Compound I in the form of a lithium chloride salt or a eutectic, or a pharmaceutically acceptable composition thereof. 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 the expression of GLUR1 or PSD95.

[0074] In some embodiments, methods of activating mTORC1 are used to treat or prevent multiple forms of autism. (See Novarino et al., (2012) Science, October 19, 338:6105, pp. 394-397). Thus, in some embodiments, the present invention provides a method of treating or preventing a form of autism in a subject in need thereof, the method comprising the step of administering to the subject Compound I in the form of a lithium chloride salt or a eutectic, or a pharmaceutically acceptable composition thereof. In some embodiments, the autism is a genetic form of autism.

[0075] In some embodiments, the present invention provides a method of treating a patient in need of treatment for a genetic form of autism, the method comprising the step of administering to the patient a compound I in the form of a lithium chloride salt or a eutectic or a pharmaceutically acceptable composition thereof. Haploinsufficiency of SHANK3 is responsible for the neurological features of Phelan-McDermid syndrome (PMDS), which include 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 regulatory subunit B56b of serine / threonine protein phosphatase 2A (PP2A) by its kinase Cdc2-like kinase 2 (Bidinosti et al. (2016) Science Reports 351, 1199-1203). SHANK3 mutant mice exhibit autistic features (Yang et al. (2012) The Journal of Neuroscience 32, 6525–6541). Patients with autistic features and motor retardation carry a deleterious homozygous mutation in the SLC7A5 gene. Solute carrier transporter 7a5 (SLC7A5) is a large neutral amino acid transporter located at the blood-brain barrier (BBB), which plays a crucial role in maintaining normal levels of brain BCAA. Intracerebroventricular administration of leucine improves abnormal behavior in adult mutant mice (Tarlungeanu et al. (2016) Cell 167, 1481–1494).

[0076] In some embodiments, the present invention provides a method of treating a lysosomal storage disease or lysosomal storage disorder (“LSD”) in a patient in need thereof, the method comprising the step of administering to the patient compound I in the form of a lithium chloride salt or co-crystal, or a pharmaceutically acceptable composition thereof. LSD is a group of inherited metabolic disorders caused by lysosomal dysfunction. Lysosomal storage disorders are caused by lysosomal dysfunction, usually due to the lack of 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, the method comprising the step of administering to the patient compound I in the form of a lithium chloride salt or co-crystal, or a pharmaceutically acceptable composition thereof. In some embodiments, the lipid storage disorder is selected from sphingolipidoses (e.g., gangliosidosis, Gaucher disease, Niemann–Pick disease, or metachromatic leukodystrophy). In some embodiments, the present invention provides a method of treating 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, the method comprising the step of administering to the patient compound I in the form of a lithium chloride salt or co-crystal, or a pharmaceutically acceptable composition thereof. In some embodiments, the mucopolysaccharidosis is Hunter syndrome or Hurler disease.

[0077] In some embodiments, the present invention provides a method of treating juvenile neuronal ceroid lipofuscinosis (JNCL, Batten disease) in a patient in need thereof, the method comprising the step of administering to the patient compound I in the form of a lithium chloride salt or co-crystal, or a pharmaceutically acceptable composition thereof. JNCL is caused by the deletion of exons 7 and 8 of the CLN3 gene, rendering the protein non-functional. Battenin (the full-length protein encoded by CLN3) is a transmembrane protein localized to late endosomes and lysosomes, which has been shown to contribute to the regulation of pH, amino acid balance, and vesicle trafficking (Pearce et al. (1999) Nature Genetics 22, 1; Fossale et al. (2004) BMC Neuroscience 10, 5), and mTOR activation requires intracellular nutrients provided by autophagy, and in in vitro and in vivo models of JNCL, due to the lack of functional Battenin, the intracellular nutrients are reduced (Cao et al. (2006) Journal of Biological Chemistry 281, 29).

[0078] In some embodiments, the present invention provides a method for treating a patient in need thereof for cystinosis, the method comprising the step of administering to the patient a compound I in the form of a lithium chloride salt or a eutectic or a pharmaceutically acceptable composition thereof. Cystinosis is an autosomal recessive genetic disorder that affects subjects with two mutant alleles in the CSTN gene; the lysosomal cystine transporter cystinosin is defective in the efflux of cystine from lysosomes, leading to the formation of cystine crystals in renal epithelial tubules and loss of renal function. Studies have shown that in cells lacking CSTN and with mislocalized mTOR, mTORC1 signaling is defective or reduced (Ivanova et al. (2016) Journal of Inherited Metabolic Disease 39(3), 457-64; Andrzejewska et al. (2016) Journal of the American Society of Nephrology 27(6), 1678-1688e). Cysteamine is unable to rescue these defects (Ivanova et al. (2016) Journal of Inherited Metabolic Disease 39(3), 457-64; Andrzejewska et al. (2016) Journal of the American Society of Nephrology 27(6), 1678-1688e). It has also been found that cystinosin binds to the mTORC1 pathway components v-ATPase, Rags, and Ragulator (Andrzejewska et al. (2016) Journal of the American Society of Nephrology 27(6), 1678-1688e). CTNS-deficient cells exhibit an increase in the number of autophagosomes and a decrease in chaperone-mediated autophagy (Napolitano et al. (2015) EMBO Molecular Medicine 7(2), 158-74).

[0079] In some embodiments, the present invention provides a method for treating a patient in need thereof for Fabry disease, the method comprising the step of administering to the patient a compound I in the form of a lithium chloride salt or a eutectic or a pharmaceutically acceptable composition thereof. In Fabry disease, the lack of alpha-galactosidase results in the lysosomal accumulation of globotriaosylceramide lipids. In a Fabry cell model in which alpha-galactosidase was knocked down with shRNA, reduced mTOR activity and increased autophagy were observed in vitro and in vivo (Liebau et al. (2013) PLoS 8, e63506). Hyperactive autophagy was also observed in the brains of mice in which alpha-galactosidase was knocked out (Nelson et al. (2014) Acta Neuropathologica Communications 2, 20).

[0080] In some embodiments, the present invention provides a method for treating a patient in need thereof for mucolipidosis type IV (MLIV), the method comprising the step of administering to the patient compound I in the form of a lithium chloride salt or eutectic or a pharmaceutically acceptable composition thereof. In MLIV, mutations in the TRPML1 lysosomal Ca(2+) channel lead to lysosomal membrane trafficking disorders. MLIV knockout in Drosophila leads to upregulation of autophagy and reduced mTOR activity, both of which can 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). Increased autophagy has also been observed in fibroblasts from MLIV patients (Vergarajauregui et al. (2008) Human Molecular Genetics 17, 2723–2737).

[0081] In some embodiments, the present invention provides a method for treating mental retardation in a patient in need thereof, the method comprising the step of administering to the patient compound I in the form of a lithium chloride salt or eutectic or a pharmaceutically acceptable composition thereof. In humans, mutations in Cereblon are associated with a mild form of autosomal recessive non-syndromic mental retardation. In a knockout model of Cereblon in retarded mice, deletion 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).

[0082] In some embodiments, the present invention provides a method for increasing neuronal protein expression in a subject, the method comprising the step of administering to the subject compound I in the form of a lithium chloride salt or eutectic or a pharmaceutically acceptable composition thereof. In some embodiments, the increase in neuronal protein expression occurs in postsynaptic neurons. In some embodiments, the increase in neuronal protein expression includes an increase in brain-derived neurotrophic factor (BDNF) expression. In some embodiments, the increase in neuronal protein expression includes an increase in glutamate receptor 1 (GluR1) expression. In some embodiments, the increase in neuronal protein expression includes an increase in synapsin expression. In some embodiments, the increase in neuronal protein expression includes an increase in PSD95 expression.

[0083] In some embodiments, the present invention provides a method for increasing synaptogenesis in an object, the method comprising the step of administering to the object a compound I in the form of a lithium chloride salt or a eutectic, or a pharmaceutically acceptable composition thereof. In some embodiments, the increase in synaptogenesis involves synaptic remodeling. In some embodiments, the increase in synaptogenesis involves the induction of dendritic spines. In some embodiments, the induction of dendritic spines results in an increase in the density of dendritic spines. In some embodiments, the dendritic spines are thin spines. In some embodiments, the dendritic spines are mushroom spines.

[0084] In some embodiments, the present invention provides a method for enhancing the weather function in an object, the method comprising the step of administering to the object a compound I in the form of a lithium chloride salt or a eutectic, or a pharmaceutically acceptable composition thereof. In some embodiments, the enhanced weather function in the object involves an increase in excitatory postsynaptic current (EPSC).

[0085] In some embodiments, the present invention provides a method for treating a disorder of the central nervous system (CNS), the method comprising the step of administering to the patient a compound I in the form of a lithium chloride salt or a eutectic, or a pharmaceutically acceptable composition thereof. In some embodiments, the CNS disorder is bipolar disorder, depression, or schizophrenia.

[0086] In some embodiments, the present invention provides a method for treating a dietary dysfunction, the method comprising the step of administering to the patient a compound I in the form of a lithium chloride salt or a eutectic, or a pharmaceutically acceptable composition thereof. In some embodiments, the dietary dysfunction is anorexia or bulimia.

[0087] In some embodiments, the present invention provides a method for treating a blood disorder, the method comprising the step of administering to the patient a compound I in the form of a lithium chloride salt or a eutectic, or a pharmaceutically acceptable composition thereof. In some embodiments, the blood disorder is anemia and low white blood cell count (neutropenia).

[0088] In some embodiments, the present invention provides a method for treating headache, alcoholism, post-traumatic stress disorder (PTSD), epilepsy, diabetes, liver disease, kidney disease, arthritis, skin conditions such as seborrhea, hyperthyroidism, asthma, Huntington's disease, Graves' disease, herpes simplex, movement disorders such as tardive dyskinesia, Tourette syndrome, cyclic vomiting, Meniere's disease, skin prickling or "crawling" sensations (paresthesia), and aggressive behavior in attention deficit hyperactivity disorder (ADHD), the method comprising the step of administering to the patient a compound I in the form of a lithium chloride salt or a eutectic, or a pharmaceutically acceptable composition thereof.

[0089] The pharmaceutically acceptable compositions of the present invention can be administered to humans and other animals orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (such as by powder, ointment or drops), buccally, in the form of an oral spray or nasal spray, etc., depending on the severity of the infection being treated. In certain embodiments, Compound I in the form of a lithium chloride salt or eutectic can be administered orally or parenterally once or more times a day at a dose level of about 0.01 mg / kg of subject body weight / day to about 200 mg / kg of subject body weight / day, or about 0.01 mg / kg of subject body weight / day to about 50 mg / kg of subject body weight / day and preferably about 1 mg / kg of subject body weight / day to about 25 mg / kg of subject body weight / day to obtain the desired therapeutic effect.

[0090] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compound, the liquid dosage forms may also contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifying agents, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 - butanediol, dimethylformamide, oils (specifically, cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan and mixtures thereof. In addition to the inert diluent, the oral compositions may also include adjuvants such as wetting agents, emulsifying agents and suspending agents, sweetening agents, flavoring agents and fragrances.

[0091] Injectable preparations, such as sterile injectable aqueous or oily suspensions, can be formulated according to known techniques using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparations may also be sterile injectable solutions, suspensions or emulsions in a parenterally acceptable non - toxic diluent or solvent, such as a solution in 1,3 - butanediol. Acceptable vehicles and solvents that can be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, a sterile non - volatile oil is commonly used as a solvent or suspending medium. For this purpose, any mild non - volatile oil can be employed, including synthetic mono - or di - glycerides of fatty acids. In addition, fatty acids such as oleic acid are used to prepare injectables.

[0092] The injectable formulations can be sterilized, for example, by filtration through a bacteria - retaining filter, or by incorporating a sterilizing agent in the form of a sterile solid composition which can be dissolved or dispersed in sterile water or other sterile injectable medium before use.

[0093] To prolong the effect of Compound I in the form of lithium chloride salt or eutectic, it is generally desirable to slow the absorption of the compound from subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of a poorly water-soluble crystalline or amorphous material. Then, the absorption rate of the compound depends on its dissolution rate, which in turn can depend on crystal size and crystal form. Alternatively, delayed absorption of the parenterally administered form of the compound is achieved by dissolving or suspending the compound in an oily vehicle. Injectable depot forms are prepared by forming a microcapsule matrix of the compound in a biodegradable polymer such as poly(lactide-co-glycolide). The release rate of the compound can be controlled according to the ratio of the compound to the polymer and the nature of the specific polymer employed. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the compound in liposomes or microemulsions compatible with human tissue.

[0094] Compositions for rectal or vaginal administration are preferably suppositories, which can be prepared by mixing Compound I in the form of lithium chloride salt or eutectic with a suitable non-irritating excipient or carrier such as cocoa butter, polyethylene glycol or suppository wax, which is solid at room temperature but liquid at body temperature and thus melts in the rectal or vaginal cavity and releases the active compound.

[0095] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is admixed with: at least one inert pharmaceutically acceptable excipient or carrier such as sodium citrate or dibasic calcium phosphate and / or a) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol, and silicic acid; b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia; 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 promoters such as quaternary ammonium compounds; g) wetting agents such as cetyl alcohol and glycerol monostearate; h) adsorbents such as kaolin and bentonite; and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also contain buffering agents.

[0096] Solid compositions of a similar type can also be used as fillers in soft - filled and hard - filled gelatin capsules which use excipients such as lactose (lactose or milk sugar) and high - molecular - weight polyethylene glycol. 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 art of pharmaceutical formulation. The dosage forms can optionally contain emulsifiers and their composition can also be such that the dosage form releases the active ingredient only or preferentially in a particular part of the intestine, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type can also be used as fillers in soft - filled and hard - filled gelatin capsules which use excipients such as lactose and high - molecular - weight polyethylene glycol.

[0097] The active compound can also be in micro - encapsulated form together with one or more of the 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 - control coatings, and other coatings well - known in the art of pharmaceutical formulation. In such solid dosage forms, the active compound can be mixed with at least one inert diluent such as sucrose, lactose, or starch. Under normal circumstances, in addition to the inert diluent, such dosage forms can also contain additional substances, 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 form can also contain buffering agents. The dosage forms can optionally contain emulsifiers and their composition can also be such that the dosage form releases the active ingredient only or preferentially in a particular part of the intestine, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

[0098] Dosage forms for topical or transdermal administration of Compound I in the form of lithium chloride salt or eutectic include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches. The active ingredient is mixed with a pharmaceutically acceptable carrier and any required preservatives or buffer solutions that may be needed under sterile conditions. Ophthalmic formulations, ear drops, and eye drops are also contemplated within the scope of the present invention. Additionally, the present invention contemplates the use of transdermal patches which have the additional advantage of delivering the compound to the body in a controlled manner. Such dosage forms can be prepared by dissolving or dispersing the compound in a suitable medium. Penetration 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 polymeric matrix or gel.

[0099] According to one embodiment, the present invention relates to a method of modulating mTORC1 activity in a biological sample, the method comprising the step of contacting the biological sample with Compound I in the form of lithium chloride salt or eutectic or a composition comprising the compound.

[0100] According to one embodiment, the present invention relates to a method for selectively modulating mTORC1 activity in a biological sample, the method comprising the step of contacting the biological sample with Compound I in the form of a lithium chloride salt or a co-crystal, or a composition comprising the compound.

[0101] According to one embodiment, the present invention relates to a method for activating mTORC1 in a biological sample, the method comprising the step of contacting the biological sample with Compound I in the form of a lithium chloride salt or a co-crystal, or a composition comprising the compound.

[0102] As used herein, the term "biological sample" includes, but is not limited to, cell cultures or extracts thereof; biopsy material obtained from a mammal or an extract thereof; and blood, saliva, urine, feces, semen, tears, or other body fluids or extracts thereof.

[0103] Another embodiment of the present invention relates to a method for modulating mTORC1 activity in a patient, the method comprising the step of administering to the patient Compound I in the form of a lithium chloride salt or a co-crystal, or a composition comprising the compound.

[0104] Another embodiment of the present invention relates to a method for selectively modulating mTORC1 activity in a patient, the method comprising the step of administering to the patient Compound I in the form of a lithium chloride salt or a co-crystal, or a composition comprising the compound.

[0105] Another embodiment of the present invention relates to a method for activating mTORC1 in a patient, the method comprising the step of administering to the patient Compound I in the form of a lithium chloride salt or a co-crystal, or a composition comprising the compound.

[0106] In other embodiments, the present invention provides a method for treating an mTORC1-mediated disorder in a patient in need thereof, the method comprising the step of administering to the patient Compound I in the form of a lithium chloride salt or a pharmaceutically acceptable composition thereof. Such disorders are described in detail herein.

[0107] Depending on the particular condition or disease to be treated, additional therapeutic agents that are typically administered for the treatment of the condition may also be present in the compositions of the present invention. As used herein, additional therapeutic agents that are typically administered for the treatment of a particular disease or condition are referred to as "suitable for the disease or condition being treated".

[0108] In some embodiments, Compound I in the form of a lithium chloride salt or co-crystal is administered in combination with an antidepressant therapeutic agent. Antidepressant therapeutic agents are well known to those of ordinary skill in the art and include selective serotonin reuptake inhibitors (“SSRI”, such as sertraline, escitalopram, citalopram, fluvoxamine, fluoxetine, paroxetine), antidepressants (such as bupropion, venlafaxine, mirtazapine, duloxetine, amitriptyline, imipramine, selegiline, nortriptyline, trazodone, desvenlafaxine, and aripiprazole).

[0109] In some embodiments, Compound I in the form of a lithium chloride salt or co-crystal is administered in combination with an additional therapeutic agent or method useful for treating one or more LSDs. In some embodiments, Compound I in the form of a lithium chloride salt or co-crystal is administered in combination with enzyme replacement therapy, chemical chaperone therapy, bone marrow transplantation, substrate reduction therapy, α-L-iduronidase, recombinant human N-acetylgalactosamine-4-sulfatase (arylsulfatase B), glycosphingolipid biosynthesis inhibitors, N-butyl-deoxynojirimycin (Miglustat), hydrophobic iminosugars, or an inhibitor of α-galactosidase A (e.g., 1-deoxygalactonojirimycin).

[0110] Those additional agents can be administered separately from the composition containing the compound of the invention as part of a multi-dose regimen. Alternatively, those agents can be part of a single dosage form that is mixed together with Compound I in the form of a lithium chloride salt or co-crystal in a single composition. If administered as part of a multi-dose regimen, the two active agents can be delivered simultaneously, sequentially, or at intervals (usually five hours apart) from each other.

[0111] As used herein, the terms “combination”, “combined” and related terms refer to the administration of therapeutic agents simultaneously or sequentially according to the present invention. For example, Compound I in the form of a lithium chloride salt or co-crystal can be administered simultaneously or sequentially with another therapeutic agent in separate unit dosage forms or together in a single unit dosage form. Accordingly, the present invention provides a single unit dosage form comprising Compound I in the form of a lithium chloride salt or co-crystal, an additional therapeutic agent, and a pharmaceutically acceptable carrier, adjuvant, or vehicle.

[0112] The amounts of Compound I in the form of lithium chloride salt or eutectic and the additional therapeutic agent (in those compositions that contain an additional therapeutic agent as described above), which can be combined with a carrier material to produce a single dosage form, will vary depending on the subject being treated and the particular mode of administration. Preferably, the compositions of the present invention should be formulated such that a dose of 0.01 - 100 mg / kg body weight / day of Compound I in the form of lithium chloride salt or eutectic can be administered.

[0113] In those compositions that contain an additional therapeutic agent, the additional therapeutic agent and Compound I in the form of lithium chloride salt or eutectic can act synergistically. Thus, the amount of the additional therapeutic agent in such compositions will be less than the amount required in a single therapy using only the therapeutic agent. In such compositions, an additional therapeutic agent can be administered at a dose between 0.01 - 1,000 μg / kg body weight / day.

[0114] The amount of the additional therapeutic agent present in the compositions of the present invention will not exceed the amount typically administered in compositions that contain the therapeutic agent as the sole active agent. Preferably, the amount of the additional therapeutic agent in the compositions disclosed in the present invention will be in the range of about 50% to 100% of the amount typically present in compositions that contain the agent as the sole therapeutic active agent.

[0115] Compound I in the form of lithium chloride salt or eutectic or its pharmaceutical composition can also be incorporated into compositions for coating implantable medical devices such as prostheses, artificial valves, vascular grafts, stents, and catheters. For example, vascular stents have been used to overcome restenosis (the restenosis of the blood vessel wall after injury). However, patients using stents or other implantable devices are at risk of clot formation or platelet activation. These undesirable effects can 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 in the form of lithium chloride salt or eutectic is another embodiment of the present invention.

[0116] All features of each aspect in the aspects of the present invention apply to all other aspects, with necessary modifications.

[0117] In order to more fully understand the present invention described herein, the following examples are set forth. It should be understood that these examples are for illustrative purposes only and should not be construed as limiting the present invention in any way.

[0118] Illustration

[0119] As depicted in the following examples, in certain exemplary embodiments, Compound I in the form of a lithium chloride salt or eutectic is prepared according to the following general procedure. It should be understood that although the general method depicts the synthesis of a certain compound of the present invention, the following general method and other methods known to those of ordinary skill in the art can be applied as described herein.

[0120] General Procedure for the Preparation of Compounds

[0121] Compound I is prepared according to the method described in U.S. Patent No. 10,100,066, the entire content of which is incorporated herein by reference.

[0122] The preparation of Compound I in the form of a lithium chloride eutectic is described below.

[0123] Example 1: Compound I in the Form of a Lithium Chloride Salt or Eutectic

[0124] Two (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 obtain a colorless solution. The resulting solution was concentrated to 10 vol at atmospheric pressure over approximately 5 hours. The solution was then cooled to room temperature over a 1.5-hour period. A precipitate was observed during the cooling process. The solid material was separated by filtration and dried under vacuum overnight to obtain a white solid (950 mg; 38.5% yield). The solid, as Compound I in the form of a lithium chloride eutectic, was characterized using different techniques, and the results are summarized in Table 1 below.

[0125] Table 1. Characterization Techniques

[0126]

[0127] Based on HPLC determination, ICP, and the stoichiometry of chloride, the ratio between Compound I and LiCl was revealed to be 1:1, as summarized in Table 2 below.

[0128] Table 2. Stoichiometry Data

[0129] Molar weight (g / mol) w / w% Mole Eutectic 223.577 89.94 0.402 Compound I 181.183 0.402 <![CDATA[Li + > 6.941 2.84 0.409 <![CDATA[Cl - > 35.453 14.17 0.4

[0130] General Procedure for In Vivo Testing

[0131] Animal use: Male Sprague Dawley rats (Charles River Laboratories, Wilmington, MA) weighing 175 - 200 g will be group-housed upon arrival (Yale University, New Haven, CT) and allowed to acclimate for 5 days prior to the start of experimental studies. Rats will have free access to food and water except during the fasting periods specified by the protocol. Clinical signs of the animals will be monitored daily. Qualified veterinarians will supervise all rodent procedures. All personnel will be trained by the Yale animal care and use committee (IACUC). All animal procedures will be conducted at Yale University in strict accordance with the National Institutes of Health IACUC and will be approved by the Yale animal care and use committee.

[0132] Behavioral analysis using the female urine sniff test (FUST): FUST will be performed 24 hours after dosing according to published procedures (Malkesman, O. et al., Biol Psychiatry 67(9):864 - 71 (2010)). Briefly, rats will be habituated to a cotton swab dipped in tap water in their home cage for 60 minutes. Next, the rats will be exposed to a second cotton swab dipped in tap water and, 45 minutes later, to a third cotton swab soaked in fresh rat urine from 11 - to 14 - week - old female rats in estrus. The total time (seconds) spent sniffing the cotton - tipped applicator will be quantified for each animal over 5 minutes.

[0133] Behavioral analysis using locomotor activity assessment (LMA): LMA will be evaluated on an open field equipped with an automated activity meter consisting of parallel rows of infrared beams according to published procedures (Warner - Schmidt, J. L. and Duman, R. S., Proc Natl Acad Sci USA 104(11):4647 - 52 (2007)). The number of beam breaks will be recorded for each animal at 30 - minute intervals.

[0134] Behavioral analysis using the novelty-suppressed feeding test (NSFT): The NSFT will be conducted as previously described (Warner-Schmidt, J. L., and Duman, R. S. Proceedings of the National Academy of Sciences of the United States of America 104(11):4647-52 (2007)). Rats will be fasted in their home cages for 20 hours and then placed in a Plexiglas open field (76.5 cm x 76.5 cm x 40 cm) with a small amount of food in the center. Animals will be allowed to explore the open field for 8 minutes, and the latency to feed (in seconds) will be recorded.

[0135] Behavioral analysis using the sucrose preference test (SPT): Rats will be habituated to a palatable 1% sucrose solution for 48 hours to avoid neophobia. At the end of day 0, rats will be treated with compound I or Veh in the form of lithium chloride salt or eutectic, and the SPT will be performed 24 hours after administration on day 1. For the SPT, rats will be water-deprived for 6 hours and exposed to two bottles with equal volumes of 1% sucrose or water for 60 minutes. The ratio of the volume of sucrose water consumed to the total volume of water consumed during the 1-hour test period will be defined as sucrose preference (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).

[0136] Chronic unpredictable stress (CUS) condition: Rats will be exposed to a variable sequence of 12 unpredictable stressors to prevent habituation, as described in the literature (Li, N. et al., Biological Psychiatry 69(8):754-61 (2011)). The following twelve stressors will be applied (2 per day for 25 days): cage rotation, light on, light off, cold stress, isolation, swim stress, food and water deprivation, wet bedding, strobe light, cage tilt, odor exposure, and group housing. Animals in the non-stress (NS) group will be normally housed without the application of external stressors. NS and CUS rats will be handled and weighed weekly.

[0137] Marmoset human threat test (HTT): Marmosets will be challenged by the presence of a human observer at regular intervals over a long period. This chronic stimulation is known to increase plasma cortisol, and subsequent increases in hypothalamic-pituitary-adrenal function contribute to the pathophysiology of depressive illness.

[0138] Example A: Behavioral changes in the novelty-suppressed feeding test and the female urine sniffing test after a single dose of compound I or ketamine in the form of lithium chloride salt or eutectic.

[0139] Study Design: Male Sprague-Dawley rats weighing between 175 and 200 g will be randomly divided into four (4) study groups after a 5-day acclimation period. On study day 0, rats in groups 1 and 2 will receive a single dose of saline (Sal) or ketamine (Ket) by intraperitoneal injection (i.p.), respectively. Rats in groups 3 and 4 will receive a single dose of vehicle (Veh, 0.5% methylcellulose / 0.1% Tween-80) or Compound I in the form of lithium chloride salt or eutectic (160 mg / kg) by oral gavage, respectively. All rats will undergo FUST on day 1 (i.e., 24 hours after dosing). On day 2, i.e., 48 hours after dosing, LMA will be measured for all rats in an open field. Then, the rats will be fasted for 20 hours and NSFT will be performed 72 hours after dosing.

[0140] Preparation of Test Articles: Ket (Sigma, catalog #K1884) will be dissolved in Sal at a concentration of 10 mg / mL. For groups 1 and 2, a volume of 1 mL / kg of Sal or Ket will be injected intraperitoneally, respectively. Compound I in the form of lithium chloride salt or eutectic will be prepared by dissolving it at a concentration of 50 mg / mL in Veh (0.5% methylcellulose / 0.1% Tween-80). A dosing volume (3.2 mL / kg) of Veh or Compound I in the form of lithium chloride salt or eutectic will be administered to the study animals in groups 3 and 4 by oral gavage, respectively. The test articles will be prepared on the day of dosing.

[0141] Example B: Comparison of the Effects of Single Doses of Compound I in the Form of Lithium Chloride Salt or Eutectic and Ketamine Administration on the mTORC1 Signaling Pathway and Synaptic Protein Expression in Synaptosomal Preparations Derived from the Rat Prefrontal Cortex

[0142] Study Design: Male Sprague-Dawley rats weighing between 175 and 200 g will be randomly divided into eight (8) study groups after a 5-day acclimation period. On study day 0, rats in groups 3 and 7 will receive a single dose of Sal, while groups 4 and 8 will receive a single dose of Ket (10 mg / kg), each by intraperitoneal injection. Rats in groups 1 and 5 will receive a single dose of Veh, while groups 2 and 6 will receive a single dose of Compound I in the form of lithium chloride salt or eutectic (160 mg / kg), each by oral gavage. One hour after dosing, rats in groups 1-4 will be sacrificed by conscious decapitation and then the PFC will be collected. Crude synaptosomes will be prepared from the PFC and three mTORC1 substrates pmTOR, pp70S6K, and p4E-BP1, and the corresponding total protein loading controls (mTOR, p70S6K, and GAPDH) will be quantified by Western blot. Twenty-four hours after dosing, rats in groups 5-8 will be sacrificed by conscious decapitation and the PFC will be collected. Crude synaptosomes will be prepared from the PFC and synaptic proteins (GluR1 and PSD95), and the total protein loading control (GAPDH) will be quantified by Western blot.

[0143] Formulations of Ket and Compound I in the form of lithium chloride salt or eutectic for administration: Ket (Sigma, catalog #K1884) will be dissolved in Sal at a concentration of 10 mg / mL. A volume of 1 mL / kg will be injected intraperitoneally. Compound I in the form of lithium chloride salt or eutectic will be prepared by dissolving it in Veh at a concentration of 50 mg / mL. The dosing volume based on animal weight (3.2 mL / kg) will be administered by oral gavage. The test article will be prepared on the day of dosing.

[0144] Prefrontal Cortex Synaptosomal Preparation: Brains will be dissected from rats in all groups and rinsed in PBS. The PFC will be collected and homogenized 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)) at 4°C. The homogenate will be centrifuged at 2,800 rpm for 10 minutes at 4°C, then the supernatant will be removed, and it will be centrifuged again at 12,000 rpm for 10 minutes at 4°C. The resulting pellet containing crude synaptosomes will be 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 NaVO3, 5 mM NaF, and protease inhibitor cocktail) and sonicated at 50% amplitude for 20 seconds on ice. Protein concentration will be determined by Bradford assay, and all samples will be 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.

[0145] Western blot analysis: Western blot analysis of GluR1, PSD95, and GAPDH was performed as previously described. Briefly, synaptosomal preparations (15 μg total protein) were loaded onto 10 - 15% SDS PAGE gels for electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes in transfer buffer (10X premixed electrophoresis buffer containing 25 mM Tris, 192 mM glycine, pH 8.3; Bio-Rad). The PVDF membranes were blocked with blocking buffer (PBS-T containing 2% BSA (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 in blocking buffer with primary antibodies 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; #13185) at 1:1000, rabbit anti-synapsin 1 (Cell Signaling; #5297) at 1:1000, rabbit anti-PSD95 (Cell Signaling; #9644) at 1:1000, and rabbit anti-GAPDH (Cell Signaling; #5174) at 1:1000. The next day, the membranes were washed three times in PBS-T buffer and incubated for 1 hour with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (Vector Laboratories Inc) at 1:5000 to 1:10000. After the last three washes with PBS-T buffer, bands were detected using enhanced chemiluminescence. The blots were then incubated for 30 minutes at 50 - 55°C in stripping buffer (2% SDS, 100 mM β-mercaptoethanol, 50 mM Tris-HCl pH 6.8), then washed three times with PBS-T buffer. The stripped blots were kept in blocking solution for 1 hour and incubated with primary antibodies against the corresponding protein for total levels or GAPDH for loading control. Densitometric analysis of the phospho- and total immunoreactivity of each protein was performed using NIH Image J software. The resulting density readings were used to generate the ratio of phosphoprotein to its corresponding total protein level or GAPDH as indicated.For each protein, the resulting ratio was further normalized relative to the control group treated with Sal or Veh.

[0146] Example C: Effect of a single oral dose of Compound I in lithium chloride salt or eutectic form on the mTORC1 signaling pathway in multiple regions of the rat brain

[0147] Study design: Male rats weighing between 175 and 200 g were randomly divided into two study groups after a 5-day acclimation period. Group 1 would receive a single administration of Veh by oral gavage, and Group 2 would receive a single administration of Compound I (160 mg / kg, prepared in Veh) in lithium chloride salt or eutectic form by oral gavage. One hour after administration, the rats were sacrificed by conscious decapitation. In addition to separating the PFC, hippocampus, striatum, neocortex, and cerebellum by microdissection, plasma was collected to analyze the exposure of Compound I in lithium chloride salt or eutectic form. Total protein extracts were prepared from the harvested tissues and subjected to WB analysis, followed by quantitative analysis of selected mTORC1 substrates.

[0148] Formulation of Compound I in lithium chloride salt or eutectic form (160 mg / ml): Compound I in lithium chloride salt or eutectic form was prepared by dissolving it at a concentration of 160 mg / mL in Veh. The dosing volume based on animal weight (10 mL / kg) was administered to the study animals in Group 2 by oral gavage. The test article was prepared on the day of dosing.

[0149] Western blot analysis: Synaptosomal preparations (15 μg total protein) were loaded and separated on NuPAGE 4-12% Bis-Tris gels and transferred to PVDF membranes (Immobilon-FL PVDF membranes, Millipore) using CAPS buffer (10 mM 3-(cyclohexylamino)-1-propanesulfonic acid, 12.5% ethanol, pH = 10). After transfer, the membranes were incubated in Odyssey blocking buffer (Licor) for 1 hour at room temperature. After blocking, the membranes were incubated overnight at 4 °C with primary antibodies. The primary antibodies used were rabbit anti- at 1:1000 in Odyssey blocking buffer S400 / 440pS6 (Cell Signaling; #5364) and mouse anti-α-tubulin at 1:10,000 (Sigma; #T5168). 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 for 30 minutes at 1:20,000 in Odyssey blocking buffer with dye-conjugated secondary antibodies (goat anti-mouse IRdye680 and goat anti-rabbit IRdye800 from LI-COR), then washed three times in 1X TBS-Tween. Signals were quantified using an Odyssey infrared imaging system (LI-COR Bioscience). The resulting density readings were used to generate the ratio of phosphoprotein to α-tubulin. The resulting ratios were further normalized relative to vehicle-treated controls.

[0150] Prefrontal cortex synaptosome preparation: One hour after dosing, rats were sacrificed by decapitation under conscious conditions, and plasma and brains were collected. Brains were dissected for each group and rinsed in PBS. The PFC, striatum, hippocampus, neocortex, and cerebellum were collected and homogenized in homogenization buffer (0.32 M sucrose, 20 mM HEPES at pH 7.4, 1 mM EDTA, 5 mM NaF, 1 mM NaVO3, and protease inhibitor mixture (Roche; #19543200)) at 4°C. The homogenate was centrifuged at 2,800 rpm for 10 minutes at 4°C, then the supernatant was removed and centrifuged again at 12,000 rpm for 10 minutes at 4°C. The resulting pellet 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 NaVO3, 5 mM NaF, and protease inhibitor mixture) and sonicated at 50% amplitude for 20 seconds on ice. 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.

[0151] Compound analysis: To determine the compound levels in plasma, proteins were precipitated from 50 μL of the resulting tissue homogenate in 150 μL of acetonitrile containing an internal standard (tolbutamide), then centrifuged at 3,000 rpm for 10 minutes. One hundred microliters of the resulting supernatant was added to 100 μL of water, mixed well, and injected onto an LC-MS / MS system using the following program to assess compound levels:

[0152] ● Phenomenex LUX cellulose column (4.6 x 150 mm, 5 μm)

[0153] ● Mobile phase A – water containing 0.1% formic acid

[0154] ● Mobile phase B – acetonitrile containing 0.1% formic acid

[0155] ● Gradient:

[0156] ○ Initial – 40% A

[0157] ○ 2 minutes – 40% A

[0158] ○ 2.1 minutes – 2% A

[0159] ○ 3 minutes – 2% A

[0160] ○ 3.1 minutes – 40% A

[0161] ○ 4 minutes – 40% A

[0162] ● Flow rate 0.8 mL / min

[0163] ● Column temperature 40 °C

[0164] ● Sciex 5500 triple quadrupole mass spectrometer

[0165] Example D: Effect of a single oral dose of Compound I in the form of its lithium chloride salt or co-crystal or leucine on the mTORC1 signaling pathway in the rat brain and selected peripheral organs

[0166] Study design: Male rats weighing between 175 and 200 g were randomly divided into three (3) study groups after a 5-day acclimation period. The test articles were administered by oral gavage. One hour after dosing, the rats were sacrificed by conscious decapitation, and plasma, brain, and selected peripheral tissues were harvested for compound level and Western blot analysis. Tissues were prepared for Western blot to quantify the mTORC1 substrate pS6 as a measure of mTORC1 activity.

[0167] Preparation of test articles: Compound I in the form of its lithium chloride salt or co-crystal and leucine (Leu, Sigma; #L8912) were prepared by dissolving them at concentrations of 16 mg / mL and 100 mg / mL, respectively, in Veh (0.5% methylcellulose / 0.1% Tween-80). The dosing volume based on animal weight (10 mL / kg) was administered by oral gavage. The test articles were prepared on the day of dosing.

[0168] Tissue preparation: One hour after administration, rats will be sacrificed by decapitation under anesthesia. Plasma, brain, and peripheral tissues will be harvested and immediately frozen in liquid nitrogen. Tissues will be thawed and homogenized twice for 1 minute each in Lysis buffer (Cell lysis buffer: 1% Triton X-100, 50 mM HEPES pH 7.4, 100 mM NaCl, 2 mM EDTA, 10 mM β-glycerophosphate, 10 mM sodium pyrophosphate, and 1 protease inhibitor tablet per 50 mL of fresh buffer) using an MP homogenizer at 4°C. The lysates will then be sonicated for 20 seconds at 50% amplitude on ice. Protein concentration will be determined by Bradford assay, and all samples will be 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.

[0169] Western blot (WB) analysis: Equal amounts of each sample (15 μg total protein) will be 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 will be incubated for 1 hour at room temperature in Odyssey blocking buffer (LI-COR). After blocking, the membrane will be incubated overnight at 4°C with primary antibodies. The primary antibodies used will be 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 1:10,000 in Odyssey blocking buffer. The next day, the membrane will be 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 for 30 minutes at room temperature in Odyssey blocking buffer with dye-conjugated secondary antibodies (goat anti-mouse IRdye680 and goat anti-rabbit IRdye800 from LI-COR) at 1:20,000, followed by three washes in 1X TBS-Tween. Signals will be quantified using an Odyssey infrared imaging system (LI-COR Biosciences). The resulting densitometry readings will be used to generate the ratio of phosphoprotein to α-tubulin or GAPDH. The resulting ratios will be further normalized relative to vehicle-treated control groups.

[0170] Compound analysis: To determine the compound levels in tissue preparations, 70% isopropanol was added to tissue samples at a ratio of 3:1 v:w (μL:mg), and then homogenized using a bead mill (Biospec). Proteins were precipitated from 50 μL of the resulting tissue homogenate in 150 μL of acetonitrile containing an internal standard (tolbutamide), and then centrifuged at 3000 rpm for 10 minutes. One hundred microliters of the resulting supernatant was added to 100 μL of water, mixed well, and injected onto an LC-MS / MS system using the following program to assess compound levels:

[0171] ● Phenomenex LUX cellulose column (4.6 x 150 mm, 5 μm)

[0172] ● Mobile phase A – water containing 0.1% formic acid

[0173] ● Mobile phase B – acetonitrile containing 0.1% formic acid

[0174] ● Gradient:

[0175] ○ Initial – 40% A

[0176] ○ 2 minutes – 40% A

[0177] ○ 2.1 minutes – 2% A

[0178] ○ 3 minutes – 2% A

[0179] ○ 3.1 minutes – 40% A

[0180] ○ 4 minutes – 40% A

[0181] ● Flow rate 0.8 mL / min

[0182] ● Column temperature 40 °C

[0183] ● Sciex 5500 triple quadrupole mass spectrometer

[0184] Example E: Effects of a single dose of Compound I in lithium chloride salt or eutectic form on sucrose preference and novelty suppressed feeding tests and synaptic protein expression

[0185] Study Design: Male rats weighing between 175 and 200 g were randomly divided into four (4) study groups after a 5-day acclimation period. On study day -20, two (2) groups of rats were subjected to CUS for 25 days, and two (2) groups of rats were normally housed as the NS group. On day 21 of the CUS protocol, rats received a single dose of Veh or Compound I in the form of lithium chloride eutectic (160 mg / kg) by oral gavage (day 0). SPT and NSFT were performed at 24 hours and 48 hours (day 1 and day 2) after administration, respectively. After completion of the behavioral tests, 25 days after the CUS protocol, a second dose of Compound I or Veh in the form of lithium chloride salt or eutectic was administered on day 5, and the rats were sacrificed by decapitation under anesthesia 24 hours later. Crude synaptosomes were prepared from the PFC, and synaptic proteins GluR1 and PSD95 were quantified by WB.

[0186] Formulation of Compound I in the form of lithium chloride salt or eutectic (50 mg / mL): Compound I in the form of lithium chloride salt or eutectic was prepared by dissolving it in Veh to a concentration of 50 mg / mL. The solution was administered to the rats in groups 2 and 4 by oral gavage at a volume of 10 mL / kg, with a final dose of 160 mg / kg. An equal volume of Veh was administered to groups 1 and 3.

[0187] Prefrontal cortex synaptosome preparation: The brain was dissected from the rats and rinsed in PBS. The PFC was collected and homogenized in homogenization buffer (0.32 M sucrose, 20 mM HEPES at pH 7.4, 1 mM EDTA, 5 mM NaF, 1 mM NaVO3, and protease inhibitor mixture (Roche; #19543200)) at 4°C. The homogenate was centrifuged at 2,800 rpm for 10 minutes at 4°C, then the supernatant was removed, and it was centrifuged again at 12,000 rpm for 10 minutes at 4°C. The resulting pellet 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 NaVO3, 5 mM NaF, and protease inhibitor mixture) and sonicated at 50% amplitude for 20 seconds on ice. 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.

[0188] Western blot analysis: Western blot analysis of GluR1, PSD95, and GAPDH was performed as previously described (Li, N. et al., Science 329(5994):959 - 964(2010)). Briefly, synaptosomes (15 μg 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 (PBS - T containing 2% BSA (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 in blocking buffer 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. The next day, the membrane was washed 3 times in PBS - T buffer and incubated for 1 hour with horseradish peroxidase - conjugated anti - mouse or anti - rabbit secondary antibody (Vector Laboratories) at 1:5000 to 1:10000. After the last three washes with PBS - T buffer, the bands were detected using enhanced chemiluminescence. The blot was then incubated for 30 minutes at 50 - 55°C in stripping buffer (2% SDS, 100 mM β - mercaptoethanol, 50 mM Tris pH 6.8), then washed three times with PBS - T buffer. The stripped blot was kept in blocking solution for 1 hour and incubated with the primary antibody against GAPDH used as a loading control. The total immunoreactivity of each protein was densitometrically analyzed using NIH Image J software. The resulting density readings were used to generate the ratio of total protein to GAPDH. For each protein, the resulting ratio was further normalized relative to the NS - Veh group.

[0189] Example F: Dependence of the pharmacological activity of Compound I in the lithium chloride salt or eutectic form on mTORC1 activation in forced swimming and novelty - suppressed feeding tests after single oral administration in rats

[0190] Study Design: Male rats weighing between 175 and 200 g will be randomly divided into three (3) study groups after a 5-day acclimation period. All rats will have bilateral IT cannulas implanted in the PFC surgically 2 weeks before drug administration. On the day of drug administration, all treatment groups will receive bilateral IT infusions (0.5 μL / side) containing rapamycin (R) vehicle (Veh-R, 10% DMSO) or rapamycin (R, 0.01 nmol / μL), which has been previously shown to completely inhibit mTORC1 activity. Thirty minutes after intrathecal infusion, compound I vehicle (Veh-NV, 0.5% methylcellulose / 0.1% Tween-80) or compound I in the form of lithium chloride salt or eutectic (160 mg / kg) will be administered by oral gavage. Each treatment group will be evaluated at specified times after oral administration (FST 24 hours (day 1), LMA 48 hours (day 2), and NSFT 72 hours (day 3 after a 20-hour fasting period)). LMA will be measured to exclude overall changes in general locomotor activity.

[0191] Preparation of Test Articles: Rapamycin (Cell Signaling; #9904) will be prepared in a solution of 10% DMSO (Veh-R) to a final concentration of 10 μM. Thirty minutes before treatment with compound I in the form of lithium chloride salt or eutectic or Veh-NV by oral gavage, R or Veh-R will be administered bilaterally (0.005 nmol / 0.5 μL per side) to the medial PFC by IT infusion. Compound I in the form of lithium chloride salt or eutectic will be prepared by dissolving it at a concentration of 50 mg / mL in Veh (0.5% methylcellulose / 0.1% Tween-80). The dosing volume based on animal weight (3.2 mL / kg) will be administered by oral gavage to the study animals in groups 2 and 3.

[0192] Surgical Procedure and Administration of Rapamycin: Guide cannulas (22GA) will be stereotaxically implanted into the medial PFC of rats (coordinates relative to bregma: +3.2 AP, ±1.0 ML, -3.5 DV relative to dura mater). The surgical procedure will be performed under pentobarbital sodium anesthesia (intraperitoneal 55 mg / kg). Postoperative care will consist of perioperative administration of carprofen (5 mg / kg) and topical triple antibiotic. After a 2-week recovery period, 30 minutes before oral administration of compound I in the form of lithium chloride salt or eutectic or Veh-NV, R (0.01 nmol in 1 μL for PFC infusion) or Veh-R will be delivered at a rate of 0.25 μL / minute using an injection cannula (26GA) extending 0.5 mm from the guide cannula. The dose of rapamycin will be selected based on previous reports demonstrating efficacy and selective inhibition of mTORC1 activity.

[0193] Example G: Duration of Behavioral Changes in the Forced Swim Test and Novelty Suppressed Feeding Test after a Single Dose of Compound I in Lithium Chloride Salt or Eutectic Form or Ketamine

[0194] Study Design: Male rats weighing between 175 and 200 g were randomly divided into six (6) study groups after a 5-day acclimation period. A single dose of all test articles was administered on Day 0, and behavioral tests were performed 3, 7, and 10 days later. Rats in Groups 1 and 2 were dosed with Compound I in lithium chloride salt or eutectic form (160 mg / kg, by oral gavage) and Ket (10 mg / kg, by intraperitoneal injection) on Day 0, respectively, and the FST was performed on Day 3. Rats in Groups 3 and 4 received a single dose of Compound I vehicle (Veh) or Compound I in lithium chloride salt or eutectic form (160 mg / kg) by oral gavage on Day 0, respectively. Rats in Groups 5 and 6 received a single dose of Ket vehicle (Sal) or Ket (10 mg / kg) by intraperitoneal injection on Day 0, respectively. Rats in Groups 3-6 were subjected to the FST on Day 7 and the NSFT on Day 10. All rats in Groups 3-6 were fasted for 20 hours the night before the NSFT.

[0195] Example H: Physiological Changes in Layer V Pyramidal Neurons after a Single Dose of Compound I in Lithium Chloride Salt or Eutectic Form

[0196] Study Design: Male rats weighing between 175 and 200 g were randomly divided into two (2) study groups after a 5-day acclimation period. On Study Day 0, rats received a single dose of NV vehicle (Veh, 0.5% methylcellulose / 0.1% Tween-80) or Compound I in lithium chloride salt or eutectic form (160 mg / kg) by oral gavage. On Day 1, rats were sacrificed 24 hours after dosing, and brain slices were prepared and whole-cell patch-clamp recordings were made on layer V pyramidal neurons in the PFC.

[0197] Preparation of Test Articles: Compound I in lithium chloride salt or eutectic form was prepared by dissolving it at a concentration of 50 mg / mL in Veh (0.5% methylcellulose / 0.1% Tween-80). The dosing volume based on animal weight (3.2 mL / kg) was administered to study animals by oral gavage. Test articles were prepared on the day of dosing.

[0198] Brain slice preparation: Brain slices were prepared according to a published procedure (Liu, R. J., et al., J. Neurosci. 22(21):9453 - 9464 (2002)). Briefly, rats were anesthetized with chloral hydrate (400 mg / kg, intraperitoneally) according to a protocol approved by the Yale Animal Care and Use Committee. After decapitation, the brain was quickly removed and placed into ice - cold (4°C) artificial cerebrospinal fluid (ACSF) in which sucrose (252 mM) replaced NaCl (sucrose - ACSF) to prevent cell swelling. A tissue block containing the PFC was dissected and cut into coronal slices (400 μm) in sucrose - ACSF using a vibrating blade microtome (Leica VT1000S). After placing the slices into an immersion recording chamber, the bath temperature was raised to 32°C. Known concentrations of drugs dissolved in ACSF and applied via a stopcock arrangement at a fast flow rate (∼4 ml / min) reached the slices within 7 - 10 s. Standard ACSF (pH = 7.35) was equilibrated with 95% O2 / 5% CO2 and contained 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 ∼1 - 2 h recovery period was allowed before starting the recordings.

[0199] Electrophysiological recordings: Pyramidal neurons in layer V will be visualized by video microscopy using an Olympus BX50WI microscope (×60IR lens) and infrared differential interference contrast (IR / DIC) video microscopy (Olympus) according to published procedures (Lambe, E.K. and Aghajanian, G.K. Neuron 40(1):139 - 150 (2003)). Low-resistance patch microelectrodes (3 - 5 MΩ) will be pulled from patch-clamp glass pipettes (Warner Instruments) using a Flaming-Brown horizontal puller (model P-97; Sutter Instruments). The microelectrodes will be filled with the following solution: 115 mM potassium gluconate, 5 mM KCl, 2 mM MgCl2, 2 mM Mg-ATP, 2 mM Na2ATP, 10 mM Na2-phosphocreatine, 0.4 mM Na2GTP, and 10 mM HEPES, pH 7.33. Neurobiotin (0.3%) will be added to the microelectrode solution to label cells for later imaging. Whole-cell recordings will be performed using an Axoclamp-2B amplifier (Axon Instruments). The output signal will be low-pass filtered at 3 KHz, amplified ×100 by a Cyberamp, digitized at 15 kHz, and acquired using pClamp 9.2 / Digidata 1320 software (Axon Instruments). The series resistance, monitored throughout the experiment, will typically be between 4 and 8 MΩ. To minimize series resistance errors, cells will be discarded if the series resistance rises above 10 MΩ. Postsynaptic currents will be studied in the continuous single-electrode voltage-clamp mode (3000 Hz low-pass filter), which is clamped near the resting potential (75 mV ± 5 mV) to minimize the holding current. After recording, the slices will be transferred to 0.1 M phosphate buffer containing 4% paraformaldehyde and stored overnight at 4°C. Then, the slices will be treated with streptavidin conjugated to Alexa 594 (1:1000; Invitrogen) to visualize neurobiotin in the labeled cells.

[0200] Dendritic spine density analysis: Labeled neurons within layer V of the cingulate anterior (Cg1) and medial prefrontal cortex anterior (Cg3) will be imaged using a two-photon Ti:sapphire laser scanning system (810 nm; Mai Tai, Spectra-Physics, Mountain View, California) for analysis of dendritic spine density and morphology, which is 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. This will include the total number of dendritic spines on proximal and distal clusters of layer V neurons, as well as dendritic spine head diameter and an indication of dendritic spine maturation. The length of apical cluster branch segments will be determined within the 3D matrix of each Z-stack using Neurolucida 10.2 (MicroBrightField). Dendritic spine density and dendritic spine head diameter analysis are performed on the raw image stacks (2 - 5 optical sections, 1 μm apart) using the Autospine module of Neurolucida Explorer (version 10.2). Dendritic spine density and segmentation (beaded) will be sampled in three regions: the tip of the cluster branch as it approaches the pia mater; the intermediate dendrite approximately midway between the pia mater and the apical trunk bifurcation; and the proximal cluster dendrite located immediately distal to the bifurcation. Results will be expressed as total dendritic length, dendritic spine density, and segmentation density. Results will be expressed as dendritic spine density per 10 μm.

[0201] Example I: Behavioral changes in the forced swim and novelty suppressed feeding tests following daily administration of Compound I in the form of the lithium chloride salt or eutectic, or ketamine administered every other day

[0202] Study Design: Male Sprague-Dawley rats weighing between 175 and 200 g will be randomly divided into four (4) study groups after a 5-day acclimation period. On study day -1, the rats will be pre-treated before swimming. Starting from study day 0, the rats in Group 2 will receive a certain dose of Ket (10 mg / kg) every other day (days 0, 2, 4, and 6), each time by intraperitoneal injection. The rats in Group 1 will receive a daily dose of Veh by oral gavage for 7 days (days 0 to 6). The rats in Groups 3 and 4 will receive a daily dose of Compound I in the form of lithium chloride salt or eutectic (40 or 80 mg / kg) for 7 days (days 0 to 6), each time by oral gavage. All rats will undergo the FST on day 7 (i.e., 24 hours after the last administration). On day 8, i.e., 48 hours after the last administration, the LMA of all rats will be measured in an open field. Then, the rats will be fasted for 20 hours and undergo the NSFT on day 9 (72 hours after the last administration).

[0203] Preparation of Test Articles: Ket (Sigma, catalog #K1884) will be dissolved in Sal at a concentration of 10 mg / mL. An injection volume of 1 mL / kg of Ket (intraperitoneal) will be administered to Group 2. Compound I in the form of lithium chloride salt or eutectic will be prepared by dissolving it in Veh (0.5% methylcellulose / 0.1% Tween-80) at a concentration of 50 mg / mL. A dosing volume of Veh or Compound I in the form of lithium chloride salt or eutectic based on the animal weight (3.2 mL / kg) will be administered daily by oral gavage to the study animals in Groups 1, 3, and 4, respectively. The test articles will be prepared on the day of administration.

[0204] Example J: Marmoset Human Threat Test

[0205] Study Design: Marmosets (Callithrix jacchus) will be paired and randomly divided into treatment groups. Twenty-four hours before the Human Threat Test (HTT), the animals will be treated with vehicle, ketamine (0.3 mg / kg; intramuscular (i.m.)) or Compound I in the form of lithium chloride salt or eutectic (160 mg / kg; oral (p.o.)). The next day, the same animals will be treated with vehicle (subcutaneous (s.c.)) or chlordiazepoxide (1 mg / kg; subcutaneous). Then, in the presence of a human observer, the number of threat postures of the animals will be monitored within two (2) minutes. The locomotor activity will be monitored during the same time period, as measured by the number of jumps observed.

[0206] Although many embodiments of the present invention have been described, it will be apparent that the basic examples can be varied to provide other embodiments that utilize the compounds and methods of the present invention. Accordingly, it is to be understood that the scope of the present invention will be defined by the appended claims and not by the specific embodiments that have been illustrated by way of example.

Claims

1. A compound having the formula LiCl⋅Compound I, or a solvate or hydrate thereof, wherein Compound I has the following structure:

2. The compound according to claim 1, wherein the LiCl⋅Compound I is a eutectic.

3. The compound according to claim 1, wherein the stoichiometric ratio of lithium to Compound I is about 1 to about 1.

4. A pharmaceutical composition comprising the compound according to claim 1 and a pharmaceutically acceptable carrier, adjuvant, or vehicle.

5. The pharmaceutical composition according to claim 4, wherein the composition is formulated for oral administration.

6. The pharmaceutical composition according to claim 4, which further comprises an additional therapeutic agent.

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

8. The method according to 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 disease, 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, skin prickling or crawling sensations (paresthesia), or aggressive behavior in attention deficit hyperactivity disorder (ADHD).

9. A method of treating refractory depression in a patient in need, said method comprising administering to said patient an effective amount of the compound according to claim 1 or the pharmaceutical composition according to claim 4.

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

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