Compositions for intravenous administration

Abstract

The present invention provides compounds, compositions, and methods of use thereof for treatment of certain disorders and conditions, for example brain injuries such as stroke or traumatic brain injuries.

Description

COMPOSITIONS FOR INTRAVENOUS ADMINISTRATION CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of United States Provisional Application Nos. 63 / 521,523, filed June 16, 2023; and 63 / 422,292, filed November 3, 2022; the entirety of each of which is incorporated herein by reference. STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under Grant No. 5R44NS093756-04 awarded by the National Institutes of Health. The government has certain rights in the invention. FIELD OF THE INVENTION

[0003] The present invention relates to compounds, compositions, and methods of use thereof for treating, ameliorating, or promoting recovery from certain conditions of the brain, central nervous system (CNS), or cardiovascular system such as a brain injury, a neurodegenerative condition, or cardiac ischemia. BACKGROUND OF THE INVENTION

[0004] Brain injuries are a distressingly common medical condition and one of the leading causes of morbidity and mortality worldwide. The brain is particularly susceptible to injury as neurons have a limited capacity to repair. When an individual is born, the brain already has essentially all the neurons it will have in life. Unlike other cells in the body, neurons stop reproducing shortly after birth. If these cells are injured or die, they are not replaced, often culminating in the disabling and largely irreversible degradation of a person’s cognitive and sensorimotor capacity. Conditions that result in nerve cell death and damage range from ischemic episodes (e.g., stroke) and trauma, to degenerative disorders (e.g., Alzheimer’s disease).

[0005] Injury to the Central Nervous System (CNS) is a substantial cause of death and disability worldwide. For example, according to the CDC approximately 1.7 million people sustain a Traumatic Brain Injury (TBI) annually, costing the U.S. economy in excess of $60 billion per year in terms of medical costs and lost productivity (Finkelstein, E; Corso, P; Miller, T, The Incidence and Economic Burden of Injuries in the United States, Oxford University 1 BUSINESS.30655163.1 393522-004WO (204863)Press: New York, 2006). Additionally, stroke is the third leading cause of death in the U.S. with an estimated incidence of 795,000 cases annually, a major cause of disability, and costing the U.S. economy over $34 billion per year (NINDS, 2014; stroke.nih.gov; and Mozaffarian D, Benjamin EJ, Go AS, et al. “Heart disease and stroke statistics-2015 update: a report from the American Heart Association,” Circulation.2015 ;e29-322).

[0006] In the acute setting, there is an opportunity to treat patients within 24 hours that can limit the extent of the damage. Immediately after an ischemic or hemorrhagic stroke, the site of insult in the brain typically contains a core of tissue that is irreversibly damaged, and then also an area of viable but at-risk tissue called the penumbra. During this period, the insufficient oxygen and glucose supply to brain cells results in further secondary injury to the penumbra. The lack of oxygen and glucose decreases energy production by cell mitochondria. An immediate effect of this energy depletion is failure of the ion pumps, which by elevating extracellular potassium (K+) ions, results in waves of recurrent spreading depolarizations in brain tissue. At the same time, influx of sodium (Na+) ions into cells, followed by chloride (Cl-) ions, results in the swelling of cells due to osmotic pressure elevation, pressuring nearby neurons and their processes, ultimately leading to lysis (cell rupture) and inflammatory responses. In general, this disruption of ion homeostasis leads to excitotoxicity, cell swelling and cell death that extends damage to adjacent tissue and expands lesions by secondary mechanisms. There is a need for effective treatments during the initial 24 hours to protect the stressed brain cells. The propagation of brain damage in stroke is similar to that observed in other forms of brain injury such as trauma and concussions.

[0007] Beyond acute treatment, effective astrocyte function plays a key role in broader neurorestoration – in the period 24-96 hours following brain insult, in the period months-years in patients with neurodegeneration such as Alzheimer’s, or most generally in aged individuals. The inability of brain cells to regenerate requires the remaining intact brain tissue to reorganize in an attempt to recover any loss of function. This potential for neural reorganization is diminished in older individuals.

[0008] GPCR receptors have been suggested to mediate cardioprotective effects. Therefore, there is potential to treat heart and cardiovascular conditions by similar mechanisms of action via modulation of these receptors.

[0009] There is urgent and compelling unmet medical need for more effective treatments for brain injuries, CNS injuries, heart and cardiovascular diseases, and related conditions, as well as promoting neurorestoration in patients having a neurodegenerative condition such as Alzheimer’s. 2 BUSINESS.30655163.1 393522-004WO (204863)SUMMARY OF THE INVENTION

[0010] In one aspect, the present disclosure provides an injectable liquid pharmaceutical composition, comprising: (a) compound A:at a concentration of about 5 mg / mL to about 75 mg / mL; (b) 30-60% v / v glycol solvent in water; and (c) a buffer.

[0011] Various embodiments and additional aspects of the present disclosure are described below. DETAILED DESCRIPTION OF THE INVENTION General Description of Certain Aspects of the Invention

[0012] The present invention provides pharmaceutical compositions for injectable, e.g., intravenous, administration. As is well understood in the art, many active pharmaceutical agents (APIs) are not orally bioavailable. Even for APIs that are orally bioavailable, the amount of the API that is ultimately absorbed and distributed to the site(s) of action in the body is greatly influenced by the pharmaceutical formulation used. Discovering an optimal injectable formulation presents various challenges including solubilizing and stabilizing the API in a sufficiently small volume so that injection is possible. A formulation that has been successful for one API will not necessarily be successful for a different API, even a structurally- similar one. In large part, formulation studies depend on trial and error for success and are not predictable in advance.

[0013] The present invention provides liquid oral pharmaceutical compositions comprising an agonist of the adenosine receptor 1 (A1R) and / or adenosine receptor 3 (A3R). United States Patent Nos. 9,789,131, the entirety of which is hereby incorporated herein by reference, describe certain therapeutically beneficial compounds. Such compounds include compound A: 3 BUSINESS.30655163.1 393522-004WO (204863)A.

[0014] Compound A is designated as MRS4322 in the ’131 patent and the synthesis of compound A is described in detail at Example 9 of the ’131 patent, which is hereby incorporated by reference in its entirety. Compound A may also be prepared by the convergent synthesis described in PCT / US2022 / 071971, which is hereby incorporated by reference.

[0015] Compound A has shown effectiveness in treatment of diseases such as TBI and stroke. See, e.g.: 1. Liston TE, Hama A, Boltze J, et al. Adenosine A1R / A3R (Adenosine A1 and A3 Receptor) Agonist AST-004 Reduces Brain Infarction in a Nonhuman Primate Model of Stroke. Stroke. 2022;53(1):238-248. Epub 2021 / 11 / 23. doi: 10.1161 / strokeaha.121.036396. PubMed PMID: 34802248. 2. Fisher ES, Chen Y, Sifuentes MM, et al. Adenosine A1R / A3R Agonist AST-004 Reduces Brain Infarction in Mouse and Rat Models of Acute Ischemic Stroke. Frontiers in Stroke (In Press).2022. 3. Bozdemir E, Vigil FA, Chun SH, et al. Neuroprotective Roles of the Adenosine A(3) Receptor Agonist AST-004 in Mouse Model of Traumatic Brain Injury. Neurotherapeutics. 2021;18(4):2707-2721. Epub 2021 / 10 / 06. doi: 10.1007 / s13311-021-01113-7. PubMed PMID: 34608616; PMCID: PMC8804149. 4. Liston TE, Hinz S, Müller CE, et al. Nucleotide P2Y(1) receptor agonists are in vitro and in vivo prodrugs of A(1) / A(3) adenosine receptor agonists: implications for roles of P2Y(1) and A(1) / A(3) receptors in physiology and pathology. Purinergic Signal. 2020. Epub 2020 / 11 / 01. doi: 10.1007 / s11302-020-09732-z. PubMed PMID: 33129204. 5. Suresh RR, Gao ZG, Salmaso V, et al. Selective A(3) Adenosine Receptor Antagonist Radioligand for Human and Rodent Species. ACS Med Chem Lett.2022;13(4):623-631. Epub 2022 / 04 / 23. doi: 10.1021 / acsmedchemlett.1c00685. PubMed PMID: 35450351; PMCID: PMC9014498; Suresh RR, Poe RB, Lin B, et al. Convergent synthesis of 2-thioether- substituted (N)-methanocarba-adenosines as purine receptor agonists. RSC Adv. 2021;11(44):27369-27380. Epub 2022 / 04 / 29. doi: 10.1039 / d1ra05096f. PubMed PMID: 35480676; PMCID: PMC9037833. 4 BUSINESS.30655163.1 393522-004WO (204863)

[0016] In some embodiments, Form A or Form B solid form of compound A is used in the manufacture of a disclosed pharmaceutical composition. Form A and Form B are described in PCT Publication WO 2020 / 069068, which is hereby incorporated by reference.

[0017] It would be desirable to have compositions for parenteral, e.g., intravenous (IV), administration of compound A. Accordingly, the present invention provides compositions for intravenous (IV) administration of compound A. In some embodiments, the composition is administered via a subcutaneous (SC / SQ), intraperitoneal (IP), intravenous (IV), intradermal (ID), or intramuscular (IM) route.

[0018] In some embodiments, the composition is administered parenterally. In some embodiments, the compound or pharmaceutically acceptable salt thereof is administered intravenously. In some embodiments, the compound or pharmaceutically acceptable salt thereof is administered as a continuous intravenous (IV) infusion. In some embodiments, the compound or pharmaceutically acceptable salt thereof is administered initially as an IV bolus, followed by continuous IV infusion, e.g., to maintain a desired plasma concentration. In some embodiments, the compound or pharmaceutically acceptable salt thereof is administered as a slow bolus / rapid infusion. In some embodiments, the slow bolus / rapid infusion comprises IV administration over an about 5-60 minute period, e.g., a 5-30, 5-20, 10-20, or about 10 minute period. In some embodiments, the slow bolus / rapid infusion is then followed by continuous IV infusion. In some embodiments, the slow bolus / rapid infusion followed by continuous IV infusion is for treating TBI or stroke in a subject.

[0019] In some embodiments, the slow bolus / rapid infusion (whether or not followed by a continuous IV infusion) consists of about 5 mg to about 500 mg of Compound A. In certain embodiments, the slow bolus / rapid infusion consists of about 15 mg to about 375 mg of Compound A. In certain embodiments, the slow bolus / rapid infusion consists of about 25 mg to about 125 mg of Compound A. In certain embodiments, the slow bolus / rapid infusion consists of about 75 mg to about 180 mg of Compound A. In certain embodiments, the slow bolus / rapid infusion consists of about 100 mg to about 150 mg of Compound A. In some embodiments, the slow bolus / rapid infusion consists of about 5 mg to about 130 mg of Compound A.

[0020] In certain embodiments, the slow bolus / rapid infusion consists of about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, or about 200 mg of Compound A. In certain embodiments, the slow bolus / rapid infusion consists of about 150 mg of Compound A. 5 BUSINESS.30655163.1 393522-004WO (204863)

[0021] In some embodiments, the compound or composition is administered over a 6-hour period. In some embodiments, the compound or composition is administered over an 8-hour period. In some embodiments, the compound or composition is administered over a 4-hour period. In some embodiments, the compound or composition is administered over a 2-hour period. In some embodiments, the compound or composition is administered over a 1-hour period.

[0022] In some embodiments, the compound or composition is administered at a dose, wherein the dose achieves a plasma concentration level of between 1000 ng / mL and 6000 ng / mL. In some embodiments, the compound or composition is administered at a dose, wherein the dose achieves a plasma concentration level of between 1000 ng / mL and 5000 ng / mL. In some embodiments, the compound or composition is administered at a dose, wherein the dose achieves a plasma concentration level of between 1000 ng / mL and 4000 ng / mL. In some embodiments, the compound or composition is administered at a dose, wherein the dose achieves a plasma concentration level of between 1000 ng / mL and 3000 ng / mL. In some embodiments, the compound or composition is administered at a dose, wherein the dose achieves a plasma concentration level of between 1000 ng / mL and 2000 ng / mL.

[0023] In some embodiments, the compound or composition is administered at a dose, wherein the dose achieves a plasma concentration level of between 2000 ng / mL and 6000 ng / mL. In some embodiments, the compound or composition is administered at a dose, wherein the dose achieves a plasma concentration level of between 3000 ng / mL and 6000 ng / mL. In some embodiments, the compound or composition is administered at a dose, wherein the dose achieves a plasma concentration level of between 4000 ng / mL and 6000 ng / mL. In some embodiments, the compound or composition is administered at a dose, wherein the dose achieves a plasma concentration level of between 5000 ng / mL and 6000 ng / mL.

[0024] In some embodiments, the compound or composition is administered at a dose, wherein the dose achieves a plasma concentration level of between 2000 ng / mL and 6000 ng / mL. In some embodiments, the compound or composition is administered at a dose, wherein the dose achieves a plasma concentration level of between 3000 ng / mL and 6000 ng / mL. In some embodiments, the compound or composition is administered at a dose, wherein the dose achieves a plasma concentration level of between 4000 ng / mL and 6000 ng / mL. In some embodiments, the compound or composition is administered at a dose, wherein the dose achieves a plasma concentration level of between 5000 ng / mL and 6000 ng / mL.

[0025] In certain embodiments, the method comprises the steps: A) administering a single bolus comprising Compound A; and B) administering an infusion of a pharmaceutical 6 BUSINESS.30655163.1 393522-004WO (204863)composition comprising Compound A over a period of 1 to 6 hours. In certain embodiments, the method comprises the steps: A) administering a single bolus comprising Compound A; and B) administering an infusion of a pharmaceutical composition comprising Compound A over a period of 1 to 5 hours. In certain embodiments, the method comprises the steps: A) administering a single bolus comprising Compound A; and B) administering an infusion of a pharmaceutical composition comprising Compound A over a period of 1 to 4 hours. In certain embodiments, the method comprises the steps: A) administering a single bolus comprising Compound A; and B) administering an infusion of a pharmaceutical composition comprising Compound A over a period of 1 to 3 hours. In certain embodiments, the method comprises the steps: A) administering a single bolus comprising Compound A; and B) administering an infusion of a pharmaceutical composition comprising Compound A over a period of 1 to 2 hours.

[0026] In certain embodiments, the single bolus comprises about 5 mg to about 500 mg of Compound A. In certain embodiments, the single bolus comprises about 15 mg to about 375 mg of Compound A. In certain embodiments, the single bolus comprises about 25 mg to about 125 mg of Compound A. In certain embodiments, the single bolus comprises about 75 mg to about 180 mg of Compound A. In certain embodiments, the single bolus comprises about 100 mg to about 150 mg of Compound A. In some embodiments, the single bolus comprises about 5 mg to about 130 mg of Compound A.

[0027] In certain embodiments, the single bolus comprises about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, or about 200 mg of Compound A. In certain embodiments, the single bolus comprises about 150 mg of Compound A.

[0028] In certain embodiments, the infusion comprises a dose of about 600 mg to about 2900 mg of Compound A. In certain embodiments, the infusion comprises a dose of about 700 mg to about 2800 mg of Compound A. In certain embodiments, the infusion comprises a dose of about 800 mg to about 2700 mg of Compound A. In certain embodiments, the infusion comprises a dose of about 900 mg to about 2600 mg of Compound A. In certain embodiments, the infusion comprises a dose of about 1000 mg to about 2500 mg of Compound A. In certain embodiments, the infusion comprises a dose of about 1100 mg to about 2400 mg of Compound A. In certain embodiments, the infusion comprises a dose of about 500 mg to about 1800 mg of Compound A. In certain embodiments, the infusion comprises a dose of about 600 mg to about 1700 mg of Compound A. In certain embodiments, the infusion comprises a dose of about 700 mg to about 1600 mg of Compound A. In certain embodiments, the infusion 7 BUSINESS.30655163.1 393522-004WO (204863)comprises a dose of about 800 mg to about 1500 mg of Compound A. In certain embodiments, the infusion comprises a dose of about 900 mg to about 1400 mg of Compound A. In certain embodiments, the infusion comprises a dose of about 1000 mg to about 1300 mg of Compound A.

[0029] In certain embodiments, the infusion comprises a dose of about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, about 1600 mg, about 1700 mg, about 1800 mg, about 1900 mg, about 2000 mg, about 2100 mg, about 2200 mg, about 2300 mg, about 2400 mg, about 2500 mg, about 2600 mg, about 2700 mg, about 2800 mg, about 2900 mg, or about 3000 mg of Compound A.

[0030] In certain embodiments, the total dose of Compound A administered is about 750 mg to about 3000 mg. In certain embodiments, the total dose of Compound A administered is about 1000 mg to about 2500 mg. In certain embodiments, the total dose of Compound A administered is about 1250 mg to about 2000 mg. In certain embodiments, the total dose of Compound A administered is about 1300 mg to about 1800 mg. In certain embodiments, the total dose of Compound A administered is about 1000 mg to about 1500 mg.

[0031] In some embodiments, the total dose of Compound A administered is about 500 mg, about 600 mg, about 700 mg, about 750 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, about 1600 mg, about 1700 mg, about 1800 mg, about 1900 mg, about 2000 mg, about 2100 mg, about 2200 mg, about 2300 mg, about 2400 mg, about 2500 mg, about 2600 mg, about 2700 mg, about 2800 mg, about 2900 mg, or about 3000 mg of Compound A.

[0032] In some embodiments, Compound A is administered at a higher (loading) dose sufficient to achieve a desired plasma concentration that is then followed by a continuous infusion (maintenance dose) over a period of time such as about 6 hours or 1-6 hours.

[0033] In some embodiments, the loading dose is administered using an IV bag at a higher concentration and the maintenance dose is administered using a second IV bag in which Compound A is at a lower concentration. In some embodiments, the loading dose is administered using a faster rate of infusion and the maintenance dose is administered using the same IV bag at a slower rate of infusion. Brief Description of Formulations of Compound A for IV Administration

[0034] In one aspect, the present disclosure provides an injectable liquid pharmaceutical composition, comprising: 8 BUSINESS.30655163.1 393522-004WO (204863)(a) compound A:at a concentration of about 5 (b) 30-60% v / v glycol solvent in water; and (c) a buffer.

[0035] In some embodiments, the concentration of compound A is about 15 mg / mL to about 50 mg / mL. In some embodiments, the concentration of compound A is about 25 mg / mL.

[0036] In some embodiments, the composition comprises 30-50% v / v glycol solvent in water.

[0037] In some embodiments, the composition does not comprise a glycol solvent.

[0038] In some embodiments, the concentration of compound A is about 15 mg / mL to about 50 mg / mL, about 16 mg / mL to about 45 mg / mL, about 17 mg / mL to about 40 mg / mL, about 16 mg / mL to about 35 mg / mL, about 15 mg / mL to about 35 mg / mL, or about 20 mg / mL to about 30 mg / mL.

[0039] In some embodiments, the concentration of compound A is about 15 mg / mL, about 20 mg / mL, about 21 mg / mL, about 22 mg / mL, about 23 mg / mL, about 24 mg / mL, about 25 mg / mL, about 26 mg / mL, about 27 mg / mL, about 28 mg / mL, about 29 mg / mL or about 30 mg / mL. In some embodiments, the concentration of compound A is about 25 mg / mL.

[0040] A glycol solvent is any solvent comprising repeating polyether units, e.g., - ((CH2)nO)-, where n is an integer from 2 to 50,000. Glycol ethers are either “e-series” or “p- series” glycol ethers, depending on whether they are made from ethylene oxide or propylene oxide, respectively. Typically, e-series glycol ethers are found in pharmaceuticals, sunscreens, cosmetics, inks, dyes and water-based paints, while p-series glycol ethers are used in degreasers, cleaners, aerosol paints and adhesives. Both E-series glycol ethers and P-series glycol ethers can be used as intermediates that undergo further chemical reactions, producing glycol diethers and glycol ether acetates. P-series glycol ethers are marketed as having lower toxicity than the E-series. Most glycol ethers are water-soluble, biodegradable, and only a few are considered toxic. 9 BUSINESS.30655163.1 393522-004WO (204863)

[0041] In some embodiments, the glycol solvent comprises a dialkylene glycol monoalkyl ether, such as, e.g., diethylene glycol monoethyl ether.

[0042] In certain embodiments, a solvent is an alkylene glycol solvent. For instance, in some embodiments, the alkylene glycol solvent is ethylene glycol, propylene glycol, butylene glycol, or the like. In certain embodiments, the glycol solvent is propylene glycol.

[0043] In some embodiments, a solvent is a glycol ether. For instance, in some embodiments, a solvent is a dialkylene glycol monoalkyl ether, such as, e.g., diethylene glycol monoethyl ether. Other such glycol ethers are known in the chemical and pharmaceutical arts and are contemplated by the present invention. In some embodiments, the glycol solvent comprises a polyethylene glycol. In some embodiments, the glycol solvent comprises propylene glycol.

[0044] In some embodiments, the glycol solvent is ethylene glycol monomethyl ether (2- methoxyethanol, CH3OCH2CH2OH), ethylene glycol monoethyl ether (2-ethoxyethanol, CH3CH2OCH2CH2OH), ethylene glycol monopropyl ether (2-propoxyethanol, CH3CH2CH2OCH2CH2OH), ethylene glycol monoisopropyl ether (2-isopropoxyethanol, (CH3)2CHOCH2CH2OH), ethylene glycol monobutyl ether (2-butoxyethanol, CH3CH2CH2CH2OCH2CH2OH), ethylene glycol monophenyl ether (2-phenoxyethanol, C6H5OCH2CH2OH), ethylene glycol monobenzyl ether (2-benzyloxyethanol, C6H5CH2OCH2CH2OH), propylene glycol methyl ether, (1-methoxy-2-propanol, CH3OCH2CH(OH)CH3), diethylene glycol monomethyl ether (2-(2-methoxyethoxy)ethanol, methyl carbitol, CH3OCH2CH2OCH2CH2OH), diethylene glycol monoethyl ether (2-(2- ethoxyethoxy)ethanol, carbitol cellosolve, CH3CH2OCH2CH2OCH2CH2OH), diethylene glycol mono-n-butyl ether (2-(2-butoxyethoxy)ethanol, butyl carbitol, CH3CH2CH2CH2OCH2CH2OCH2CH2OH), dipropyleneglycol methyl ether, ethylene glycol dimethyl ether (dimethoxyethane, CH3OCH2CH2OCH3), ethylene glycol diethyl ether (diethoxyethane, CH3CH2OCH2CH2OCH2CH3), or ethylene glycol dibutyl ether (dibutoxyethane, CH3CH2CH2CH2OCH2CH2OCH2CH2CH2CH3).

[0045] In some embodiments, the polyethylene glycol is selected from PEG 200, PEG 300, PEG 400, SR PEG 400, PEG 540, PEG 600, PEG 900, or a mixture of any of the foregoing.

[0046] In some embodiments, the polyethylene glycol is PEG 400.

[0047] In certain embodiments, a composition of the present invention comprises more than one solvent, wherein at least one of the more than one solvents is an alcohol solvent and one of the more than one solvents is a glycol solvent. In certain embodiments, an alcohol solvent and a glycol solvent are each present in an amount of about 15 wt% to about 35 wt%. 10 BUSINESS.30655163.1 393522-004WO (204863)In certain embodiments, an alcohol solvent and a glycol solvent are each present in an amount of about 15 wt% to about 30 wt%. In certain embodiments, an alcohol solvent and a glycol solvent are each present in an amount of about 15 wt% to about 25 wt%. In certain embodiments, an alcohol solvent and a glycol solvent are each present in an amount of about 18 wt% to about 21 wt%. In some embodiments, the alcohol solvent is an alkyl alcohol (e.g., ethanol) present in an amount of about 1-25 wt% and the glycol solvent is an alkylene glycol (e.g., propylene glycol) present in an amount of about 5-35 wt%. In some embodiments, the alcohol solvent is ethanol and is present in an amount of about 1-10 wt% and the glycol solvent is propylene glycol and is present in an amount of about 10-30 wt%.

[0048] Glycol solvents are vulnerable to oxidation and can generate peroxides, such as H2O2, resulting in discoloration of the IV solution. A number of measures can be taken to prevent undesired oxidation of the glycol solvent, such as pH adjustment, and the use of antioxidants and / or a nitrogen purge.

[0049] In some embodiments, the polyethylene glycol is substantially free of peroxides and other degradants.

[0050] In other embodiments, the polyethylene glycol contains ≤0.04% peroxides, ≤0.03% peroxides, ≤0.02% peroxides, or ≤0.01% peroxides.

[0051] In some embodiments, the polyethylene glycol contains about 1.0 meqO2 / kg (milliequivalents / kg) or less of peroxide content. In some embodiments, the polyethylene glycol contains about 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 meqO2 / kg or less of peroxide content. In some embodiments, the polyethylene glycol contains 100 ppm meqO2 / kg or less of peroxide content.

[0052] In some embodiments, the glycol solvent comprises 35-50% v / v of a polyethylene glycol. In some embodiments, the glycol solvent comprises 35-45% v / v, or 35-40% v / v of a polyethylene glycol.

[0053] In some embodiments, the glycol solvent comprises about 40% v / v of a polyethylene glycol. In some embodiments, the glycol solvent comprises about 30% v / v, 35% v / v, 45% v / v ¸50% v / v¸ 55% v / v ¸or 65% v / v of a polyethylene glycol.

[0054] In some embodiments, the polyethylene glycol has an average molecular weight (MW) of 200-600. In some embodiments, the polyethylene glycol has an average molecular weight (MW) of 200-600. In some embodiments, the polyethylene glycol has an average molecular weight (MW) of 250-350. In some embodiments, the polyethylene glycol has an average molecular weight (MW) of 350-450. In some embodiments, the polyethylene glycol has an average molecular weight (MW) of 450-550. In some embodiments, the polyethylene 11 BUSINESS.30655163.1 393522-004WO (204863)glycol has an average molecular weight (MW) of 550-650. In some embodiments, the polyethylene glycol has an average molecular weight (MW) of 650-750. In some embodiments, the polyethylene glycol has an average molecular weight (MW) of 750-850. In some embodiments, the polyethylene glycol has an average molecular weight (MW) of 850-950. In some embodiments, the polyethylene glycol has an average molecular weight (MW) of 380- 420.

[0055] In some embodiments, a solvent other than a glycol solvent is used. Any pharmacueutically acceptable solvent that enhances the solubility of Compound A in an aqueous solution may be used. In some embodiments, the solvent is selected from a cyclodextrin, a mannitol solution, benzylbenzoate, an alcohol such as ethanol or benzyl alcohol, or n-methyl pyrrolidine (NMP).

[0056] As mentioned above, PEG is prone to oxidation if stored for periods of time before use or if not carefully refined. It has been found that failing to control levels of peroxides in the PEG solvents described herein led to undesired color changes, pH changes, and decomposition of Compound A when the PEG solvents were used for preparing IV compositions. It was found that controlling the pH of the Compound A IV formulation provided certain advantages for product stability.

[0057] In some embodiments, the buffer is a citrate, phosphate, borate, or acetate buffer.

[0058] In some embodiments, the buffer is a phosphate buffer.

[0059] In some embodiments, the composition has a pH of about 6.0 to about 9.0. the composition has a pH of about 7.0 to about 8.5. In some embodiments, the composition has a pH of about 7.5 to about 8.3. In some embodiments, the composition has a pH of about 7.8.

[0060] In some embodiments, the composition further comprises an antioxidant. In some embodiments, the antioxidant is sodium metabisulfite.

[0061] In some embodiments, the antioxidant is selected from BHT and BHA.

[0062] In some embodiments, after storage at 25 °C and 60% relative humidity (RH) for a period of 6 months, the composition contains an amount of compound A within 0.5% of the amount of compound A of initial manufacture of the composition. In some embodiments, the composition contains less than 0.1% impurities after storage at 25 °C and 60% RH for a period of 6 months.

[0063] In some embodiments, the pH of the composition after storage at 25 °C and 60% RH for a period of 6 months remains within 0.2 units of the pH measured at initial manufacture of the composition. 12 BUSINESS.30655163.1 393522-004WO (204863)

[0064] In some embodiments, after storage at 40 °C and 75% relative humidity (RH) for a period of 1 month, the composition contains an amount of compound A within 0.5% of the amount of compound A of initial manufacture of the composition.

[0065] In some embodiments, the composition contains less than 0.1% impurities after storage at 40 °C and 75% RH for a period of 1 month.

[0066] In some embodiments, the pH of the composition after storage at 40 °C and 75% RH for a period of 1 month remains within 0.2 units of the pH measured at initial manufacture of the composition.

[0067] In some cases, impurities from manufacture of Compound A may oxidize. In some embodiments, the composition contains no more than 0.8% by HPLC, as compared to the AUC (area under the curve) of compound A, of the following compound:

[0068] In anotherprovides an injectable liquid pharmaceutical composition, comprising: (a) compound A:at a concentration of about 5 mg / mL to about 75 mg / mL; (b) a buffer; and (c) optionally, a pharmaceutically acceptable solvent.

[0069] In another aspect, the present disclosure provides a unit dosage form comprising a pharmaceutical composition disclosed herein. In some embodiments, the unit dosage form contains about 15 mg to about 500 mg of compound A. In some embodiments, the unit dosage form contains about 25 mg to about 375 mg of compound A. In some embodiments, the unit dosage form contains about 25 mg, about 125, or about 375 mg of compound A. In some 13 BUSINESS.30655163.1 393522-004WO (204863)embodiments, the unit dosage form contains about 125 mg of compound A in a fill volume of about 5 mL.

[0070] In some embodiments, the unit dosage form is packaged in a glass or plastic vial. In some embodiments, the unit dosage form is packaged in an IV bag. In some embodiments, the unit dosage form is packaged in a pre-filled syringe. In some embodiments, the unit dosage form is packaged as a dry powder for reconstitution.

[0071] In some embodiments, the present invention provides a method of treating an injury, disease, or condition selected from traumatic brain injury (TBI), concussion, stroke, polytrauma, cardiac arrest, near drowning, altitude sickness, brain injuries from directed energy or Havanna Syndrome, partial or total spinal cord transection, malnutrition, toxic neuropathies, meningoencephalopathies, neurodegeneration caused by a genetic disorder, age-related neurodegeneration, vascular disease, Alzheimer’s Disease (AD), Parkinson’s Disease (PD), Huntington’s Disease (HD), Multiple Sclerosis (MS), amyotrophic lateral sclerosis (ALS), chronic traumatic encephalopathy (CTE), cardiovascular disease, autoimmune diseases, allergic diseases, transplant rejection, graft-versus-host disease, intraocular hypertension, glaucoma, odor sensitivity, an olfactory disorder, type 2 diabetes, pain control, respiratory diseases, deficits in CNS function, deficits in learning, deficits in cognition, otic disorders, Meniere’s disease, endolymphatic hydrops, progressive hearing loss, noise-induced hearing loss, dizziness, vertigo, tinnitus, collateral brain damage associated with radiation cancer therapy, migraine treatment, sleep disorders in the elderly, epilepsy, schizophrenia, symptoms experienced by recovering alcoholics, damage to neurons or nerves of the peripheral nervous system during surgery, gastrointestinal conditions, pain mediated by the CNS, migraine, collateral brain damage associated with radiation cancer therapy, depression, mood or behavioral changes, dementia, erratic behavior, suicidality, tremors, Huntington’s chorea, loss of coordination of movement, deafness, impaired speech, dry eyes, hypomimia, attention deficit, memory loss, cognitive difficulties, vertigo, dysarthria, dysphagia, ocular abnormalities or disorientation, and addiction; comprising administering to a patient in need thereof an effective amount of a composition described herein.

[0072] In some embodiments, the injury, disease, or condition is selected from acute pain, chronic pain, nociceptive pain, neuropathic pain, inflammatory pain, and acute pain.

[0073] In some embodiments, the pain is selected from musculoskeletal pain, fibromyalgia, myofascial pain, pain during menstruation, pain during osteoarthritis, pain during rheumatoid arthritis, pain during gastrointestinal inflammation, pain during inflammation of the heart muscle, pain during multiple sclerosis, pain during neuritis, pain during AIDS, pain during 14 BUSINESS.30655163.1 393522-004WO (204863)chemotherapy, tumor pain, headache, chronic pain syndrome (CPS), central pain, trigeminal neuralgia, shingles, stamp pain, phantom limb pain, temporomandibular joint disorder, nerve injury, migraine, post-herpetic neuralgia, neuropathic pain encountered as a consequence of injuries, amputation infections, metabolic disorders or degenerative diseases of the nervous system, neuropathic pain associated with diabetes, pseudesthesia, hypothyroidism, uremia, vitamin deficiencies or alcoholism, acute pain after injuries, postoperative pain, pain during acute gout, and pain from an operation.

[0074] In some embodiments, the injury, disease, or condition is selected from traumatic brain injury (TBI), stroke, a neurodegenerative condition, and a heart and cardiovascular disease.

[0075] In some embodiments, the injury, disease, or condition is TBI selected from concussion, blast injury, combat-related injury, a mild, moderate or severe blow to the head, whiplash, sports-related injury, or a head injury sustained from a fall or other accident.

[0076] In some embodiments, the injury, disease, or condition is a stroke selected from ischemic stroke, hemorrhagic stroke, subarachnoid hemorrhage, cerebral vasospasm, and transient ischemic attacks (TIA).

[0077] In some embodiments, the compound or composition is administered within 24 hours of the TBI or stroke.

[0078] In some embodiments, the compound or composition is administered within 8 hours of the TBI or stroke.

[0079] In some embodiments, the compound or composition is administered at least during the first 8-48 hours following the TBI or stroke.

[0080] In some embodiments, neuroprotection or neurorestoration is increased in the patient as compared with an untreated patient.

[0081] In some embodiments, the injury, disease, or condition is a neurodegenerative disease selected from Alzheimer’s Disease (AD), Parkinson’s Disease (PD), Huntington’s Disease (HD), Multiple Sclerosis (MS), amyotrophic lateral sclerosis (ALS), chronic traumatic encephalopathy (CTE), and a neurodegenerative condition caused by a virus, alcoholism, tumor, toxin, or repetitive brain injuries.

[0082] In some embodiments, the injury, disease, or condition is heart or cardiovascular disease selected from cardiac ischemia, myocardial infarction, a cardiomyopathy, coronary artery disease, arrhythmia, myocarditis, pericarditis, angina, hypertensive heart disease, endocarditis, rheumatic heart disease, congenital heart disease, and atherosclerosis. 15 BUSINESS.30655163.1 393522-004WO (204863)

[0083] In some embodiments, the present invention provides a method of increasing neuroprotection or neurorestoration in a patient in need thereof who has suffered a TBI or stroke, comprising administering to the patient an effective amount of a composition described herein.

[0084] In some embodiments, the present invention provides a method of treating an injury, disease, disorder, or condition selected from: (i) brain damage caused by radiation or collateral brain damage associated with radiation cancer therapy or migraine treatment; (ii) migraine headache; (iii) a condition associated with a brain injury or a neurodegenerative condition; and (iv) an autoimmune disease or condition, glaucoma, an otic disorder, progressive hearing loss, tinnitus, epilepsy, pain control, pain mediated by the CNS, neuropathic pain, inflammatory pain, or acute pain; comprising administering to a patient in need thereof an effective amount of a composition described herein.

[0085] In some embodiments, the composition increases neuroprotection or neurorestoration in the patient as compared with an untreated patient.

[0086] In some embodiments, the condition associated with a brain injury or a neurodegenerative condition is selected from epilepsy, migraine, collateral brain damage associated with radiation cancer therapy, depression, mood or behavioral changes, dementia, erratic behavior, suicidality, tremors, Huntington’s chorea, loss of coordination of movement, deafness, impaired speech, dry eyes, hypomimia, attention deficit, memory loss, cognitive difficulties or deficit in cognition, deficit in CNS function, deficit in learning, vertigo, dysarthria, dysphagia, ocular abnormalities, and disorientation.

[0087] In some embodiments, the injury, disease, or condition is migraine.

[0088] In some embodiments, the injury, disease, or condition is pain selected from central pain syndrome, peripheral neuropathy, corneal neuropathic pain, post stroke pain, and pain caused by multiple sclerosis.

[0089] In some embodiments, the present invention provides a method of increasing cardioprotection or regeneration of damaged heart tissue in a patient in need thereof who has suffered a cardiac ischemia or myocardial infarction, comprising administering to the patient an effective amount of a composition described herein.

[0090] In some embodiments, the present invention provides a method of treating an addiction, addictive behavior, behavioral addiction, brain reward system disorder, or a 16 BUSINESS.30655163.1 393522-004WO (204863)compulsive disorder, comprising administering to a patient in need thereof an effective amount of a composition described herein.

[0091] In some embodiments, the present invention provides a method of treating a disease, disorder, or condition selected from deficit in cognition, deficit in CNS function, deficit in learning, and memory loss, comprising administering to a subject in need thereof an effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable composition thereof.

[0092] In some embodiments, the disease, disorder, or condition is deficit in cognition.

[0093] In some embodiments, the disease, disorder, or condition is deficit in CNS function.

[0094] In some embodiments, the disease, disorder, or condition is deficit in learning.

[0095] In some embodiments, the disease, disorder, or condition is memory loss.

[0096] In some embodiments, the subject has suffered one or more traumatic brain injuries (TBI or TBIs) and the disease, disorder, or condition is associated with the TBI or TBIs.

[0097] In some embodiments, the subject has suffered one or more strokes and the disease, disorder, or condition is associated with the one or more strokes.

[0098] In some embodiments, the subject has suffered one or more ischemic strokes, hemorrhagic strokes, subarachnoid hemorrhages, cerebral vasospasms, or transient ischemic attacks (TIA).

[0099] In some embodiments, the subject has Alzheimer’s disease and the disease, disorder, or condition is associated with the Alzheimer’s disease.

[0100] In some embodiments, a method provided herein improves cognitive or neurological function as measured by a score increase between about 1% and 40% in the delayed verbal recall task of the revised Wechsler Memory Scale.

[0101] In some embodiments, a method provided herein improves the score between about 5-10%, 10-20%, 15-30%, 20-30%, 30-40%, or 5-30% in the delayed verbal recall task of the revised Wechsler Memory Scale.

[0102] In some embodiments, the method increases synaptic plasticity, improves hippocampal long-term potentiation, improves cognitive function, decreases cognitive impairment, and / or improves or restores memory or learning.

[0103] In some embodiments, the method increases synaptic plasticity, improves hippocampal long-term potentiation, improves cognitive function, decreases cognitive impairment, prevents or delays cognitive decline, decreases plaque burden, enhances beta amyloid clearance, and / or improves or restores memory or learning. 17 BUSINESS.30655163.1 393522-004WO (204863)

[0104] In some embodiments, the method improves or enhances cognition or neurological function by enhancing synaptogenesis.

[0105] In one aspect, the present invention provides a method of improving cognitive or neurological function in a subject having Alzheimer’s disease, comprising administering to a subject in need thereof an effective amount of Compound A, or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable composition thereof. In some embodiments, the improvement in cognitive or neurological function as measured by a score increase between about 1% and 40%, or about 5-10%, 10-20%, 15-30%, 20-30%, 30-40%, or 5-30%, in the delayed verbal recall task of the revised Wechsler Memory Scale. Uses of Compounds and Pharmaceutically Acceptable Compositions Thereof

[0106] As used herein, the terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease or disorder, or one or more symptoms thereof, as described herein. In some embodiments, treatment is administered after one or more symptoms have developed. In other embodiments, treatment is administered in the absence of symptoms. For example, treatment is administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and / or in light of genetic or other susceptibility factors). Brain, CNS, Cardiovascular, and Other Injuries and Conditions

[0107] In one aspect, the present invention provides a method of preventing and / or treating brain damage associated with acute brain trauma as well as longer term diseases of the brain and CNS and heart and cardiovascular diseases and conditions. In one aspect, the present invention provides methods of treating such injuries, diseases, and conditions by utilizing neuroprotective and neurorestorative effects mediated by astrocytes, which are now understood as the key natural caretaker cell of neurons, as well as the astrocyte mitochondria, which supply a significant portion of the brain’s energy. In another aspect, the present invention provides methods of treating such injuries, diseases, and conditions by cardioprotective and regenerative effects mediated by A3R receptors. Regarding neuroprotective and neurorestorative effects, without wishing to be bound by theory, it is believed that selective enhancement of astrocyte energy metabolism mediated by A3R and / or P2Y1 receptors promotes astrocyte caretaker functions, such as their neuroprotective and neurorestorative functions, in turn enhancing the resistance of neurons and other cells to both acute injury and long-term stress. In some cases, it may be advantageous to achieve biased, i.e., selective or preferential, of one or more 18 BUSINESS.30655163.1 393522-004WO (204863)pathways mediated by A3R and / or P2Y1and / or A1R receptors wherein one or more undesired pathways are not activated or activated to a lesser degree. In addition to or as an alternative to astrocytes, neuroprotective or neurorestorative function of glia, microglia, neurons, endothelium cells and other brain and / or CNS cell types may be activated. Accordingly, in one aspect, the present invention provides compounds and methods of use thereof for treating, ameliorating, or promoting recovery from certain conditions of the brain or central nervous system (CNS) such as brain injuries, for example by increasing neuroprotection and / or neurorestorative effects mediated by astrocytes, glia, microglia, neurons, endothelium cells or other cells of the brain and / or CNS, comprising administering to a patient in need thereof an effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof or composition comprising the same.

[0108] Astrocytes play key roles in supporting and protecting neurons and they critically affect the outcome of brain injuries that cause brain damage, such as ischemic injuries. The central role astrocyte mitochondria themselves play in these brain functions is less well appreciated. For example, inhibition of astrocyte mitochondria increases swelling and leads to necrotic cell death. Neurons are permanently injured by recurrent spreading depolarizations only if astrocyte mitochondrial function fails, and astrocyte mitochondria are required for reduction of pathophysiological elevations of extracellular K+, which initiate spreading depolarizations. Activation of purinergic receptors on astrocytes results in increased mitochondrial Ca2+that enhances mitochondrial citric acid cycle function and increases respiration and ATP production. Accordingly, in one aspect, the present invention relates to the discovery that activation of astrocyte purinergic receptors enhances brain cell survival signalling pathways, enabling both astrocyte and neuronal viability during oxidative stress. Furthermore, activated astrocytes generate and supply reduced glutathione, a key antioxidant that aids in the resistance of both astrocytes and neurons to oxidative stress. Thus, in one aspect, the present invention provides a method of modulating astrocyte purinergic receptors to promote survival and viability of one or more cell types in the brain of a patient after oxidative stress, such as oxidative stress caused by a brain injury, ischemia-reperfusion or a neurodegenerative condition, comprising administering to a patient in need thereof a compound described herein, or a pharmaceutically acceptable salt thereof or composition comprising the same.

[0109] In some embodiments, activation of astrocytes is achieved through contacting with a disclosed compound one or more purinergic receptors such as adenosine receptors (ARs), for example those associated with or expressed by astrocytes, thus modulating the activity of the 19 BUSINESS.30655163.1 393522-004WO (204863)one or more receptors. In some embodiments, through effects on adenosine receptors such as A1, A2A, A2Band A3on astrocytes, the compound activates astrocytes to treat one or more disclosed diseases or conditions. In some embodiments, after administration to a patient in need thereof, a disclosed compound influences one or more astrocyte functions. In some embodiments, the astrocyte function is selected from glutamate uptake, reactive gliosis, swelling, or release of neurotrophic and neurotoxic factors that act to ameliorate metabolic stress and its consequences. In some embodiments, the compound is an AR agonist. In some embodiments, the purinergic receptor is an A3adenosine receptor (A3R). In some embodiments, the compound is an A3R agonist. In some embodiments, the compound is a partial agonist or biased agonist or biased partial agonist, at an A3receptor (A3R), such as a human A3 receptor (hA3R). In some embodiments, the compound acts as an agonist of an A1 adenosine receptor (A1R). In some embodiments, the compound is a biased agonist at an A1 and / or A3 receptor. In some embodiments, the compound acts by dual agonism at an A3R and an A1R.

[0110] In another aspect, the present invention provides a method of treating or ameliorating a brain injury, disease, or condition, such as a brain injury resulting from a TBI or progressive neurodegenerative disorder, in a patient in need thereof, comprising administering to the patient an effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof or composition comprising the same. In some embodiments, the subject has suffered a TBI, concussion, stroke, partial or total spinal cord transection, or malnutrition. In other embodiments, the subject has suffered toxic neuropathies, meningoencephalopathies, neurodegeneration caused by a genetic disorder, age-related neurodegeneration, or a vascular disease; or another disease disclosed in US 8,691,775, which is hereby incorporated by reference. In some embodiments, the present invention provides a method of treating or ameliorating a brain injury, disease, or condition, such as a brain injury resulting from a TBI or progressive neurodegenerative disorder, in a patient in need thereof, comprising administering to the patient an effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof or composition comprising the same, wherein the compound is an A1R and / or A3R agonist. In some embodiments, the compound is a biased agonist, partial agonist, or biased partial agonist at an A1receptor. In some embodiments, the compound is a biased agonist, partial agonist, or biased partial agonist at an A3 receptor. In some embodiments, the compound acts by dual agonism at an A3R and an A1R.

[0111] In another aspect, the present invention provides a method of promoting or increasing neuroprotection, neurorestoration, or neuroregeneration in a patient suffering from 20 BUSINESS.30655163.1 393522-004WO (204863)a disease or condition, comprising administering to the patient an effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof or composition comprising the same. In some embodiments, the patient is suffering from a neurodegenerative disease or condition. In some embodiments, the patient has suffered a TBI or stroke. Traumatic Brain Injuries

[0112] Traumatic brain injuries (TBI) are a distressingly common medical condition. There are no approved treatments for TBI, and most TBI patients are discharged from the hospital with no pharmacological treatment. Repetitive TBI such as concussions can trigger age-associated neurodegeneration that results in a range of symptoms and disabilities over decades. TBIs can happen through sports-related injuries, motor vehicle accidents, falls, explosive impacts, physical assaults, etc. Injuries range widely in their complexity and severity, from “mild” concussions with brief alterations in mental status, cognitive difficulties, or loss of consciousness to “severe” with prolonged periods of unconsciousness and / or amnesia after the injury. In the U.S., approximately 1.7 million people have an injury resulting in a TBI annually and seek medical intervention (USCSF and CDC), and the CDC estimates that 1.6 to 3.8 million additional concussion incidents occur in sports and other recreational pursuits annually that do not present to hospital or emergency departments. Approximately 5-10% of athletes will receive a concussion each sport season. Football is the sport with the highest concussion risk for males (75% chance for concussion), while soccer has the highest concussion risk for females (50% chance for concussion). TBI is the leading cause of death and disability in children and young adults (CDC) and the most commonly received military- related injury; approximately 20% of U.S. Service Members deployed since 2003 have sustained at least one TBI. Total TBI-related indirect and direct medical costs are estimated at $77 billion annually. At least 5 million Americans require ongoing daily support in performing activities as a result of TBI.

[0113] Provided herein in one aspect is a method of treating TBI or promoting recovery from TBI, comprising administering to a patient in need thereof an effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof or composition comprising the same. In some embodiments, the TBI is selected from traumatic injuries to the brain (such as concussion, blast injury, combat-related injury) or spinal cord (such as partial or total spinal cord transection). In some embodiments, the TBI results from a mild, moderate, or severe blow to the head, comprises an open or closed head wound, or results from a penetrating or non-penetrating blow to the head. In some embodiments, the TBI is selected from 21 BUSINESS.30655163.1 393522-004WO (204863)concussion, blast injury, combat-related injury, a mild, moderate or severe blow to the head, whiplash, sports-related injury, or a head injury sustained from a fall or other accident. Stroke

[0114] A stroke occurs when a blood vessel that transports oxygen and nutrients to the brain is disrupted due to an ischemic blockage or from the hemorrhagic rupture of a blood vessel in the brain, causing neurons, glia and endothelial cells in the disrupted region of the brain to die. The outcome of the stroke depends upon the location and breadth of damage, and the impacts of that damage are observed in the body functions regulated by the damaged brain region. Strokes can cause unilateral or bilateral paralysis, speech and language disabilities, memory loss, behavioral changes, and even death. Stroke is the fourth leading cause of death in the United States and is a major cause of adult disability. Each year, ~800,000 people experience a new or recurrent stroke. Each day, over 2,000 Americans will have a stroke, resulting in death in over 400 of these incidents. Stroke accounted for ~1 of every 19 deaths in the United States in 2010. An estimated 6.8 million Americans ≥20 years of age have had a stroke. As of 2010, the annual direct and indirect cost of stroke was estimated at $36.5 billion. Within minutes of a stroke, the lack of blood flow will permanently damage a core of brain tissue. Between this damaged core and normal brain tissue is a region of tissue known as the penumbra – tissue that is under gradated stress from lessened blood flow and some disruption of energy metabolism. Over the first 24-48 hours following a stroke incident, the stress on neuronal and glia cells in the penumbra resolves either with some recovery or further cell death.

[0115] In one aspect, the present invention provides a method of neuroprotective therapy in a stroke patient, comprising administering to a patient in need thereof an effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof or composition comprising the same. In some embodiments, such therapy salvages as much of the penumbra as possible, and / or limits further acute tissue damage, and / or promotes neuron recovery. In another aspect is provided a method of treating stroke or promoting recovery from stroke, comprising administering to a patient in need thereof an effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof or composition comprising the same. In another aspect is provided a method of promoting or increasing neuroprotection, neuroregeneration, or neurorestoration in a patient who has suffered a stroke, comprising administering to the patient an effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof or composition comprising the same. 22 BUSINESS.30655163.1 393522-004WO (204863)

[0116] In some embodiments, the stroke is selected from ischemic stroke, hemorrhagic stroke, subarachnoid hemorrhage, cerebral vasospasm, and transient ischemic attacks (TIA). In some embodiments, the stroke is ischemic, e.g., an acute ischemic stroke (AIS). In some embodiments, the stroke is hemorrhagic. In some embodiments, the compound is administered within 48 hours of the stroke. In some embodiments, the compound is administered within 24 hours of the stroke. In some embodiments, the compound is administered within 16 hours of the stroke. In some embodiments, the compound is administered within 8, 4, 2, or 1 hours of the stroke. In some embodiments, the compound is administered for at least the first 1-72 hours following the stroke. In some embodiments, the compound is administered for at least the first 8-52 hours following the stroke. In some embodiments, the compound is administered for at least the first 8-48 hours following the stroke. In some embodiments, the compound is administered for at least the first 24-48 hours following the stroke. In some embodiments, the compound is administered chronically to treat the stroke as it occurs. In some embodiments, the compound is administered chronically to treat Transient Ischemic Attacks (TIA).

[0117] In some embodiments, the compound is administered chronically to treat ischemic stroke, hemorrhagic stroke, a subarachnoid hemorrhage, cerebral vasospasm, transient ischemic attacks (TIA), or treat a patient who is at an increased risk for a stroke, such as a patient who has had a stroke in the past and is at risk for a further stroke, such as a patient over the age of 40, 45, 50, 55, 60, 65, 70, 75, or 80 years of age.

[0118] In some embodiments, the compound treats an ischemia-reperfusion injury caused by the stroke.

[0119] In certain embodiments, for treatment of stroke and related conditions, a recanalization procedure such as thrombolysis by recombinant tissue plasminogen activator (r- tPA) or mechanical thrombectomy is used in combination with a presently disclosed method of treating stroke or the related condition. Neurodegenerative Diseases

[0120] Neurodegenerative diseases are incurable, progressive, and ultimately debilitating syndromes resulting from the progressive degeneration and / or death of neurons in the brain and spinal cord. Neurodegeneration results in movement (ataxias) and / or cognitive function (dementias) disorders, and includes a spectrum of diseases such as Alzheimer’s Disease (AD), Parkinson’s Disease (PD), Huntington’s Disease (HD), Multiple Sclerosis (MS), amyotrophic lateral sclerosis (ALS), and chronic traumatic encephalopathy (CTE). While many 23 BUSINESS.30655163.1 393522-004WO (204863)neurodegenerative diseases are principally genetic in origin, other causes can include viruses, alcoholism, tumors or toxins, and as is now clear, repetitive brain injuries.

[0121] Neurons accumulate cellular damage over time due to the foregoing factors, which is generally considered the reason why many neurodegenerative diseases associated with prolonged cellular stress, such as Alzheimer’s disease and Parkinson’s disease, occur in aged individuals. Dementias represent the predominant outcome of neurodegenerative diseases with AD representing approximately 60-70% of cases. As discussed above, activation of neuroprotective and neurorestorative mechanisms can ameliorate the progression of one or more neurodegenerative diseases. Accordingly, in one aspect the present invention provides a method of treating a neurodegenerative disease or promoting recovery from a neurodegenerative disease, comprising administering to a patient in need thereof an effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof or composition comprising the same.

[0122] In one aspect, the present invention provides a method of promoting neuroprotection or neurorestoration in a patient suffering from a neurodegenerative disease, comprising administering to the patient an effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof or composition comprising the same. Alzheimer’s Disease (AD)

[0123] An estimated 5.2 million Americans of all ages had AD in 2014; 11% of the population age 65 and older have AD. By 2050, the number of people age 65 and older with AD is projected to nearly triple to a projected 13.8 million. In the U.S., the cost of providing care for AD patients is about $214 billion per year; 70% of this cost is covered by Medicare and Medicaid. The current trends would project these costs to grow to $1.2 trillion per year by 2050.

[0124] Activation of astrocytes and promoting neuroprotection and neurorestoration according to the present invention represents a new treatment option for AD. Accordingly, provided herein in one aspect is a method of treating AD or promoting neuroprotection or neurorestoration in a patient suffering from AD, comprising administering to the patient an effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof or composition comprising the same.

[0125] In some embodiments, beneficial effects resulting from a method of treating AD provided herein include, but are not limited to, one or more of: improving cognitive function, 24 BUSINESS.30655163.1 393522-004WO (204863)decreasing cognitive impairment, decreasing plaque burden, enhancing beta amyloid clearance, increasing synaptogenesis, and improving memory. Parkinson’s Disease (PD)

[0126] As many as one million Americans live with PD, and each year approximately 60,000 Americans are newly diagnosed not including the thousands of cases that go undetected. The total combined direct and indirect cost of PD, including medical treatment, social security payments and lost income, is estimated to be nearly $25 billion per year in the United States.

[0127] Activation of neuroprotection and neurorestoration according to the present invention represents a new treatment option for PD. Accordingly, provided herein in one aspect is a method of treating PD or promoting neuroprotection or neurorestoration in a patient suffering from PD, comprising administering to the patient an effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof or composition comprising the same. Multiple Sclerosis (MS)

[0128] More than 400,000 people in the United States have MS. In young adults, MS represents the most prevalent disease of the central nervous system. There is potential for astrocytes to reverse the destruction of nerve cell myelin coatings that is caused by MS by their neurorestorative effects and promotion of healing in the damaged CNS of MS patients.

[0129] Activation of neuroprotection and neurorestoration in the CNS according to the present invention thus represents a new treatment option for MS. Accordingly, provided herein in one aspect is a method of treating MS or promoting neuroprotection or neurorestoration in a patient suffering from MS, comprising administering to the patient an effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof or composition comprising the same. Amyotrophic Lateral Sclerosis (ALS) / Lou Gehrig’s Disease

[0130] Approximately 5,600 people in the U.S. are diagnosed with ALS each year; as many as 30,000 Americans may have the disease concurrently. Activation of astrocytes can provide stimulation of recovery and repair of the neurons and their connections in an ALS patient.

[0131] Accordingly, provided herein in one aspect is a method of treating ALS or promoting neuroprotection or neurorestoration in a patient suffering from ALS, comprising administering to the patient an effective amount of a compound described herein, or a 25 BUSINESS.30655163.1 393522-004WO (204863)pharmaceutically acceptable salt thereof or composition comprising the same. Also provided in other embodiments is a method of stimulating recovery and repair of the neurons and their connections in an ALS patient, comprising administering to the patient an effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof or composition comprising the same. Chronic Traumatic Encephalopathy (CTE)

[0132] CTE (a form of tauopathy) is a progressive neurodegenerative disease found in individuals who have suffered one or more (often multiple or repeated over the course of time) severe blows to the head. CTE is most often diagnosed in professional athletes in American football, soccer, hockey, professional wrestling, stunt performing, bull riding and rodeo performing, motocross, and other contact sports who have experienced brain trauma and / or repeated concussions. A subset of CTE sufferers have chronic traumatic encephalomyopathy (CTEM), which is characterized by motor neuron disease symptoms that mimic ALS. Progressive muscle weakness and motor and gait abnormalities are believed to be early signs of CTEM. First stage symptoms of CTE include progressive attention deficit, disorientation, dizziness, and headaches. Second stage symptoms comprise memory loss, social instability, erratic behavior, and poor judgment. In third and fourth stages, patients suffer progressive dementia, slowed movements, tremors, hypomimia, vertigo, speech impediments, hearing loss, and suicidality, and may further include dysarthria, dysphagia, and ocular abnormalities, e.g., ptosis.

[0133] Accordingly, provided herein in one aspect is a method of treating or preventing CTE or promoting neuroprotection or neurorestoration in a patient suffering from CTE, comprising administering to the patient an effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof or composition comprising the same. Also provided in other embodiments is a method of stimulating recovery and repair of the neurons and their connections in a CTE patient, comprising administering to the patient an effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof or composition comprising the same. In some embodiments, the compound treats one or more symptoms of first stage, second stage, third stage, or fourth stage CTE.

[0134] On a microscopic scale the pathology includes neuronal death, tau deposition, TAR DNA-binding Protein 43 (TDP 43) beta-amyloid deposition, white matter changes, and other abnormalities. Tau deposition includes the increasing presence of dense neurofibrillary tangles (NFT), neurites, and glial tangles, which are made up of astrocytes and other glial cells. Thus, 26 BUSINESS.30655163.1 393522-004WO (204863)in some embodiments, the method treats, enhances clearance or prevents neuronal death, tau deposition, TAR DNA-binding Protein 43 (TDP 43) beta-amyloid deposition, white matter changes, and other abnormalities associated with CTE. Cardiovascular Diseases

[0135] Disclosed compounds are also useful in treating a variety of cardiovascular diseases and conditions. In some embodiments, the present invention provides a method of treating a heart (cardiac) or cardiovascular disease, such as cardiac ischemia, myocardial infarction, a cardiomyopathy, chest pain, stress, coronary artery disease, arrhythmia, myocarditis, pericarditis, angina, hypertensive heart disease, endocarditis, rheumatic heart disease, congenital heart disease, or atherosclerosis, comprising administering an effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof or composition comprising the same.

[0136] In some embodiments, the heart or cardiovascular disease is cardiac ischemia or myocardial infarction.

[0137] In some embodiments, the present invention provides a method of promoting or increasing cardioprotection, cardiorestoration, or cardioregeneration in a patient suffering from a heart (cardiac) or cardiovascular disease or condition, comprising administering to the patient an effective amount of a disclosed compound, or a pharmaceutically acceptable salt thereof or a composition comprising the same.

[0138] In some embodiments, the heart (cardiac) or cardiovascular disease from which the patient is suffering is cardiac ischemia, myocardial infarction, a cardiomyopathy, coronary artery disease, arrhythmia, myocarditis, pericarditis, angina, hypertensive heart disease, endocarditis, rheumatic heart disease, congenital heart disease, or atherosclerosis. Other Diseases

[0139] Compounds that modulate beneficial effects such as neuroprotection, for example by increasing astrocyte mitochondrial activity, also have the potential to treat a variety of other diseases. For example, due to the role of astrocytes in neuroprotection disclosed in the present invention, activation of astrocytes, for example via modulation of A3R and / or A1R, is useful in treating various diseases and conditions discussed below.

[0140] Accordingly, in some embodiments, the present invention provides a method of treating neurodegeneration in a patient suffering from a disease or condition, comprising 27 BUSINESS.30655163.1 393522-004WO (204863)administering to the patient an effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof or composition comprising the same.

[0141] In some embodiments, the present invention provides a method of promoting or increasing neuroprotection, neurorestoration, or neuroregeneration in a patient suffering from a disease or condition, comprising administering to the patient an effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof or composition comprising the same.

[0142] In some embodiments, the disease or condition is selected from autoimmune diseases, allergic diseases, and / or transplant rejection and graft-versus-host disease (for the use of certain nucleoside and nucleotide compounds in treating these conditions, see, for example, WO 2007 / 20018, hereby incorporated by reference). In other embodiments, the disease or condition is selected from intraocular hypertension and / or glaucoma (for the use of certain nucleoside and nucleotide compounds in treating these conditions, see, for example, WO 2011 / 77435, hereby incorporated by reference). In other embodiments, the disease or condition is selected from odor sensitivity and / or an olfactory disorder (for the use of certain nucleoside and nucleotide compounds in treating these conditions, see, for example, EP1624753, hereby incorporated by reference). In other embodiments, the disease or condition is type 2 diabetes (for the use of certain nucleoside and nucleotide compounds in treating these conditions, see, for example, US 2010 / 0256086, hereby incorporated by reference).

[0143] In other embodiments, the disease or condition is selected from respiratory diseases and / or cardiovascular (CV) diseases (for the use of certain nucleoside and nucleotide compounds in treating these conditions, see, for example, FASEB J. (2013) 27:1118.4 (abstract of meeting), hereby incorporated by reference). In other embodiments, the disease or condition is selected from deficits in CNS function, deficits in learning and / or deficits in cognition (for the use of certain nucleoside and nucleotide compounds in treating these conditions, see, for example, Neuropsychopharmacology 2015 Jan;40(2):305-14. doi: 10.1038 / npp.2014.173. Epub 2014 Jul 15. “Impaired cognition after stimulation of a P2Y1 receptor in the rat medial prefrontal cortex,” Koch, H. et al. PMID: 25027332, hereby incorporated by reference). In other embodiments, the disease or condition is selected from a neurodegenerative disease such as Alzheimer's disease, Parkinson’s disease, Huntington’s disease, prion disease, and / or amyotrophic lateral sclerosis (for the use of certain nucleoside and nucleotide compounds in treating these conditions, see, for example, US 8,691,775, hereby incorporated by reference). In other embodiments, the disease or condition is selected from otic disorders, Meniere’s disease, endolymphatic hydrops, progressive hearing loss, noise-induced hearing loss, 28 BUSINESS.30655163.1 393522-004WO (204863)dizziness, vertigo, tinnitus, collateral brain damage associated with radiation cancer therapy, and / or migraine treatment (for the use of certain nucleoside and nucleotide compounds in treating these conditions, see, for example, US 2009 / 0306225; UY31779; and US 8,399,018, each of which is hereby incorporated by reference). In other embodiments, the disease or condition is selected from pathological sleep perturbations, depression, sleep disorders in the elderly, Parkinson’s disease, Alzheimer’s disease, epilepsy, schizophrenia, and / or symptoms experienced by recovering alcoholics (for the use of certain nucleoside and nucleotide compounds in treating these conditions, see, for example, US 2014 / 0241990, hereby incorporated by reference). In other embodiments, the disease or condition is selected from damage to neurons or nerves of the peripheral nervous system during surgery (for the use of certain nucleoside and nucleotide compounds in treating these conditions, see, for example, US 8,685,372, hereby incorporated by reference). In other embodiments, the disease or condition is a cancer such as prostate cancer (for the use of certain nucleoside and nucleotide compounds in treating these conditions, see, for example, Biochem Pharmacol. 2011 August 15; 82(4): 418–425. doi:10.1016 / j.bcp.2011.05.013. “Activation of the P2Y1 Receptor Induces Apoptosis and Inhibits Proliferation of Prostate Cancer Cells,” Qiang Wei et al., hereby incorporated by reference). In other embodiments, the disease or condition is selected from one or more gastrointestinal conditions such as constipation and / or diarrhea (for the use of certain nucleoside and nucleotide compounds in treating these conditions, see, for example, Acta Physiol (Oxf). 2014 Dec;212(4):293-305. doi: 10.1111 / apha.12408. “Differential functional role of purinergic and nitrergic inhibitory cotransmitters in human colonic relaxation,” Mañé N1, Gil V, Martínez-Cutillas M, Clavé P, Gallego D, Jiménez M.; and Neurogastroenterol. Motil. 2014 Jan;26(1):115-23. doi: 10.1111 / nmo.12240. Epub 2013 Oct 8. “Calcium responses in subserosal interstitial cells of the guinea-pig proximal colon,” Tamada H., Hashitani H. PMID: 24329947, hereby incorporated by reference).

[0144] In other embodiments, the disease or condition is selected from cancer of the brain, such as glioblastoma (for the use of certain nucleoside and nucleotide compounds in treating these conditions, see, for example, Purinergic Signal. 2015 Sep;11(3):331-46. doi: 10.1007 / s11302-015-9454-7. Epub 2015 May 15. “Potentiation of temozolomide antitumor effect by purine receptor ligands able to restrain the in vitro growth of human glioblastoma stem cells.” D’Alimonte, I. et al. PMID: 25976165, hereby incorporated by reference). In other embodiments, the disease or condition is selected from a gastrointestinal disorder such as diarrhea (for the use of certain nucleoside and nucleotide compounds in treating these conditions, see, for example, Acta Physiol (Oxf). 2014 Dec;212(4):293-305. doi: 29 BUSINESS.30655163.1 393522-004WO (204863)10.1111 / apha.12408. “Differential functional role of purinergic and nitrergic inhibitory cotransmitters in human colonic relaxation,” Mañé N., Gil V, Martínez-Cutillas M, Clavé P, Gallego D, Jiménez M., hereby incorporated by reference). In other embodiments, the disease or condition is impaired cognition (for the use of certain nucleoside and nucleotide compounds in treating this condition, see, for example, Neuropsychopharmacology. 2015 Jan;40(2):305- 14. doi: 10.1038 / npp.2014.173. Epub 2014 Jul 15. “Impaired cognition after stimulation of P2Y1 receptors in the rat medial prefrontal cortex,” Koch H, Bespalov A, Drescher K, Franke H, Krügel U. PMID: 25027332, hereby incorporated by reference).

[0145] In some embodiments, the present invention provides a method of treating a disease or condition associated with brain injury or a neurodegenerative condition, such as epilepsy, migraine, collateral brain damage associated with radiation cancer therapy, depression, mood or behavioral changes, dementia, erratic behavior, suicidality, tremors, Huntington’s chorea, loss of coordination of movement, deafness, impaired speech, dry eyes, hypomimia, attention deficit, memory loss, cognitive difficulties, vertigo, dysarthria, dysphagia, ocular abnormalities, or disorientation, comprising administering to a patient in need thereof an effective amount of a disclosed compound.

[0146] In some embodiments, the improvement in cognitive or neurological function is measured as a score increase between about 1% and 20% in the delayed verbal recall task of the revised Wechsler Memory Scale. For example, the improvement in cognitive function may be measured as a score increase between about 10% and 20%, or between about 10% and 15%, or between about 5% and 15%.

[0147] In some embodiments, the present invention provides a method of treating an alcohol-related disorder such as ethanol toxicity, hangover, and neurological and other effects of excessive alcohol consumption. In some embodiments, the alcohol-related disorder is selected from Wernicke-Korsakoff Syndrome, alcoholic neuropathy, alcohol withdrawal syndrome, alcoholic cerebellar degeneration, and alcoholic myopathy. In a large epidemiological survey of headache in Danish 25- to 64-year-olds, the lifetime prevalence of hangover headache was 72 percent, making it the most common type of headache reported. Alcohol intoxication results in vasodilatation, which may induce headaches. Alcohol has effects on several neurotransmitters and hormones that are implicated in the pathogenesis of headaches, including histamine, serotonin, and prostaglandins. The alcohol withdrawal syndrome following the cessation of excessive drinking results from compensatory changes in the central nervous system that take place in response to chronically administered depressant substances (in this case, alcohol, or more specifically, ethanol). These changes include 30 BUSINESS.30655163.1 393522-004WO (204863)alterations in two types of receptors embedded in nerve cell membranes. One receptor type binds with an important chemical messenger (i.e., neurotransmitter) called gamma- aminobutyric acid (GABA), and the other type binds with another neurotransmitter, glutamate. Both GABA and glutamate are critical in regulating nerve cell activity: GABA is the body’s primary means of inhibiting nerve cell activity, and glutamate is the primary means of exciting it. Following chronic alcohol exposure, the body decreases (i.e., downregulates) the number or sensitivity of GABA receptors and increases (i.e., upregulates) the number or sensitivity of glutamate receptors in an effort to counterbalance alcohol’s sedative effects. When alcohol is removed from the body, however, the central nervous system and the portion of the nervous system that coordinates response to stress (i.e., the sympathetic nervous system) remain in an unbalanced “overdrive” state. Addictive Disorders

[0148] Disclosed compounds are also useful in treating addictions, addictive behaviors, behavioral addictions, compulsive disorders and behaviors, and related conditions.

[0149] The use of certain compounds in treating such addictions, behaviors, and disorders is described in WO / 2019 / 157317, the contents of which are hereby incorporated by reference.

[0150] Cocaine self-administering mice exhibit significantly higher glutamate levels in the VTA (ventral tegmental area) of the brain. The VTA, in particular the VTA dopamine neurons, serve several functions in the reward system, motivation, cognition, and drug addiction, and may be the focus of several psychiatric disorders. The elevated glutamate levels appear to be due, at least in part, to loss of glutamate uptake into astrocytes. Without wishing to be bound by theory, it is believed that reduced availability of glutamate has negative effects on astrocyte function and this loss of function affects neuronal activity and drug-seeking behavior. It has now been found that the compounds disclosed herein treat or prevent relapse in addicted individuals, for example by reversing such loss of astrocyte function. Such loss of astrocyte function may be partly due to reduced expression of the glutamate transporter (GLT-1) in astrocytes. Since astrocytes metabolize glutamate to produce ATP, this likely impairs glutamate uptake, weakens astrocyte oxidative metabolism and downstream ATP-dependent processes and thereby weakens their ability to maintain an optimal environment for VTA neuronal activity.

[0151] Accordingly, in one aspect, the present invention provides a method of preventing, ameliorating, treating, or promoting recovery from an addiction, addictive behavior, behavioral addiction, brain reward system disorder, compulsive disorder, or related condition, comprising 31 BUSINESS.30655163.1 393522-004WO (204863)administering to a subject in need thereof an effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof or composition comprising the same.

[0152] In some embodiments, the addiction is to an addictive substance. In some embodiments, the addictive substance is a prescription or recreational drug.

[0153] In some embodiments, the addictive substance is selected from alcohol, nicotine, a stimulant, a cannabinoid agonist, or an opioid agonist. In some embodiments, the addictive substance is selected from heroin, cocaine, alcohol, an inhalant, an opioid, nicotine, an amphetamine, or a synthetic analog, salt, composition, or combination thereof.

[0154] In some embodiments, the amphetamine is selected from bupropion, cathinone, MDMA, or methamphetamine.

[0155] In some embodiments, the prescription or recreational drug is selected from a cannabinoid agonist or opioid agonist.

[0156] In some embodiments, the addiction is an alcohol or nicotine addiction.

[0157] In some embodiments, the subject is a polydrug abuser.

[0158] In some embodiments, the prescription or recreational drug is selected from cocaine, heroin, bupropion, cathinone, MDMA, or methamphetamine morphine, oxycodone, hydromorphone, fentanyl, or a combination thereof.

[0159] In some embodiments, a disclosed compound increases energy metabolism mediated by astrocytes, such as astrocyte mitochondria. In some embodiments, the compound reverses loss of glutamate uptake into astrocytes caused by a substance with abuse potential. In some embodiments, the compound at least partially reverses the remodeling of the brain reward system caused by the addiction. In some embodiments, such effects are mediated by brain or CNS adenosine A3receptors, such as astrocyte A3R in the VTA; or microglia A3R.

[0160] In another aspect, the present invention provides a method of preventing, ameliorating, treating, or promoting recovery from an addiction, addictive behavior, behavioral addiction brain reward system disorder, compulsive disorder, or related condition by increasing energy metabolism mediated by astrocytes, glia, microglia, neurons, endothelium cells, or other cells of the brain and / or CNS, comprising administering to a subject in need thereof an effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof or composition comprising the same.

[0161] In some embodiments, the method treats or prevents a relapse of an addiction or addictive behavior in the subject. In some embodiments, the subject is addicted to one or more addictive substances such as addictive drugs (drugs having abuse potential). As described 32 BUSINESS.30655163.1 393522-004WO (204863)below, such drugs include prescription drugs and recreational drugs such as heroin, cocaine, nicotine, or an opioid agonist.

[0162] In another aspect, the present invention provides a method of treating or preventing withdrawal caused by addiction to one or more addictive substances or drugs, comprising administering to a subject in need thereof an effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof or composition comprising the same. In some embodiments, the compound decreases withdrawal symptoms in an addicted individual in withdrawal. In some embodiments, the compound treats withdrawal in an addicted individual in withdrawal. In some embodiments, the method further comprises co-administering another drug for treating withdrawal and, optionally, counseling such as psychotherapy. In some embodiments, the method further comprises a cognitive behavioral therapy. In some embodiments, the method further comprises a digital therapeutic. Digital therapeutics include, for example, reSET or reSET-O (Pear Therapeutics).

[0163] In some embodiments, the present invention provides a method of treating or preventing a relapse of a compulsive disorder or compulsive behavior, comprising administering to a subject in need thereof an effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof or composition comprising the same.

[0164] In some embodiments, the compulsive disorder is obsessive-compulsive disorder (OCD), Tourette syndrome, trichotillomania, anorexia, bulimia, anxiety disorder, psychosis, or post-traumatic stress disorder.

[0165] According to another aspect, the present invention provides a method for treating one or more behavioral addictions and addictive behaviors or disorders comprising administering to a subject in need thereof a compound described herein, or a pharmaceutically acceptable salt thereof or composition comprising the same. Behavioral addictions and addictive disorders result from the intoxication one senses from the release of brain chemicals (e.g., serotonin, adrenaline, epinephrine, etc.) during certain activities. Such disorders are known in the art and include gambling, sex addiction, pornography addiction, eating disorders, spending addiction, rage / anger, workaholism, exercise addiction, risk taking addictions (e.g. kleptomania and pyromania), perfectionism, internet or video game addiction, and compulsive use of electronic devices such as texting and checking social media, to name a few.

[0166] As used herein, the term “addiction” includes, unless otherwise specified, physical or psychological dependence on a substance. Addiction may involve withdrawal symptoms or mental or physical distress if the substance is withdrawn. Addiction includes drug liking, drug dependence, habit-formation, neurological and / or synaptic changes, development of brain 33 BUSINESS.30655163.1 393522-004WO (204863)reward system disorders, behavioral changes, or other signs or symptoms of addiction in a subject.

[0167] As used herein, the term “addictive drug” or “drug having abuse potential” includes drugs and other substances such as nicotine, whether approved by a regulatory body for treatment of a disease or not, that are known to result in clinical, behavioral, or neurological manifestations of addiction or compulsive behavior. In some embodiments, the addictive drug includes nicotine, a cannabinoid agonist, a stimulant, or an opioid agonist. “Addictive substance” refers to addictive drugs as well as other substances of abuse such as alcohol. Examples of addictive substances thus include heroin, cocaine, alcohol, opiates, nicotine, inhalants, amphetamines, and their synthetic analogs. Pain Conditions and Disorders

[0168] Disclosed compounds are also useful in treating pain, pain disorders, and related conditions. Accordingly, in one aspect, the present invention provides a method of treating, preventing, promoting recovery from, or ameliorating a pain condition or disorder, comprising administering to a subject in need thereof an effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof or pharmaceutical composition thereof.

[0169] In some embodiments, the pain condition or disorder is pain control (pain management, e.g., management of chronic pain). For the use of certain nucleoside and nucleotide compounds in treating this and related conditions, see, for example, US 2010 / 0256086, hereby incorporated by reference.

[0170] In other embodiments, the pain condition or disorder is selected from pain mediated by the CNS, such as neuropathic pain, inflammatory pain, or acute pain. For the use of certain nucleoside and nucleotide compounds in treating these conditions, see, for example, Br J Pharmacol. 2010 Mar;159(5):1106-17. doi: 10.1111 / j.1476-5381.2009.00596.x. Epub 2010 Feb 5. “A comparative analysis of the activity of ligands acting at P2X and P2Y receptor subtypes in models of neuropathic, acute and inflammatory pain.” Andó RD1, Méhész B, Gyires K, Illes P, Sperlágh B. PMID: 20136836, hereby incorporated by reference.

[0171] In some embodiments, the pain condition or disorder is migraine.

[0172] In some embodiments, the pain condition or disorder is neuropathic pain, inflammatory pain, or acute pain. See, e.g., Tosh, D.K.; Padia, J.; Salvemini, D.; Jacobson, K.A. Efficient, large-scale synthesis and preclinical studies of MRS5698, a highly selective A3adenosine receptor agonist that protects against chronic neuropathic pain. Purinergic Signalling 2015, 11, 371–387. 34 BUSINESS.30655163.1 393522-004WO (204863)

[0173] In some embodiments, the pain condition or disorder is central pain syndrome, peripheral neuropathy, corneal neuropathic pain, post stroke pain, or pain caused by multiple sclerosis.

[0174] In another aspect, the present invention provides a method of treating pain, comprising administering to a subject in need thereof an effective amount of a disclosed compound, or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable composition thereof.

[0175] In some embodiments, the pain is neuropathic pain. In some embodiments, the pain is inflammatory pain. In some embodiments, the pain is acute pain. In some embodiments, the pain is chronic pain. In some embodiments, the pain is nociceptive pain. In some embodiments, the pain is non-inflammatory musculoskeletal pain, fibromyalgia syndrome (FMS), or myofascial pain syndrome (MPS).

[0176] In some embodiments, the pain is selected from musculoskeletal pain, fibromyalgia, myofascial pain, pain during menstruation, pain during osteoarthritis, pain during rheumatoid arthritis, pain during gastrointestinal inflammation, pain during inflammation of the heart muscle, pain during multiple sclerosis, pain during neuritis, pain during AIDS, pain during chemotherapy, tumor pain, headache, chronic pain syndrome (CPS), central pain, trigeminal neuralgia, shingles, stamp pain, phantom limb pain, temporomandibular joint disorder, nerve injury, migraine, post-herpetic neuralgia, neuropathic pain encountered as a consequence of injuries, amputation infections, metabolic disorders or degenerative diseases of the nervous system, neuropathic pain associated with diabetes, pseudesthesia, hypothyroidism, uremia, vitamin deficiencies or alcoholism, acute pain after injuries, postoperative pain, pain during acute gout, and pain from an operation.

[0177] In some embodiments, the musculoskeletal pain is neck and shoulder pain and / or spasms, back pain, sciatica, chest ache, or thigh muscle ache.

[0178] In some embodiments, the pain is, or is associated with, otitis externa (OE), otitis media (OM), mastoiditis, bullous myringitis, eustachian tubal catarrh, labyrinthitis, facial nerve neuritis, temporal bone osteoradionecrosis, mal de debarquement, temporal bone fracture, or temporomandibular joint disease. Combinations with Other Therapeutic Agents

[0179] Depending upon the particular condition, or disease, to be treated, additional therapeutic agents that are normally administered to treat that condition, may also be present in the compositions of this invention. As used herein, additional therapeutic agents that are 35 BUSINESS.30655163.1 393522-004WO (204863)normally administered to treat a particular disease, or condition, are known as “appropriate for the disease, or condition, being treated.”

[0180] In another aspect, the present disclosure provides combination treatments comprising 1) an injectable liquid pharmaceutical composition, comprising: (a) compound A:at a concentration of about 5 mg / mL to about 75 mg / mL; and 2) a second agent to be administered together or separately with the injectable liquid pharmaceutical composition.

[0181] In some embodiments, the second agent is a thromobolytic agent.

[0182] In some embodiments, the thrombolytic agent is alteplase or tenecteplase.

[0183] In some embodiments, the second agent is effective for stroke or traumatic brain injury.

[0184] Depending upon the particular condition, or disease, to be treated, additional therapeutic agents that are normally administered to treat that condition, may also be present in the compositions of this invention. As used herein, additional therapeutic agents that are normally administered to treat a particular disease, or condition, are known as “appropriate for the disease, or condition, being treated.”

[0185] In certain embodiments, a provided compound, or composition thereof, is administered in combination with other therapeutic agents, such as tissue plasminogen activators and other thrombolytics, blood thinners, statins, ACE inhibitors, angiotensin II receptor blockers (ARBs), beta blockers, calcium channel blockers or diuretics, to a patient in need thereof.

[0186] In certain embodiments, the tissue plasminogen activator used in combination with compounds or compositions of the invention include, but are not limited to, alteplase, desmoteplase, reteplase, streptokinase, tenecteplase, urokinase, or combinations of any of the above. 36 BUSINESS.30655163.1 393522-004WO (204863)

[0187] In certain embodiments, the blood thinners used in combination with compounds or compositions of the invention include, but are not limited to, warfarin, heparin, apixabam, clopidogrel, aspirin, rivaroxaban, dabigatran, or combinations of any of the above.

[0188] In certain embodiments, the statins used in combination with compounds or compositions of the invention include, but are not limited to, atorvastatin, rosuvastatin, fluvastatin, lovastatin, pravastatin, simvastatin and pitavastatin, cerivastatin, mevastatin, or combinations of any of the above.

[0189] In certain embodiments, the ACE inhibitors used in combination with compounds or compositions of the invention include, but are not limited to, captopril, enalapril, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril, trandolapril benazepril, or combinations of any of the above.

[0190] In certain embodiments, the angiotensin II receptor blockers (ARBs) used in combination with compounds or compositions of the invention include, but are not limited to, azilsartan, candesartan, eprosartan, irbesartan, losartan, olmesartan, telmisartan, valsartan, fimasartan, or combinations of any of the above.

[0191] In certain embodiments, the beta blockers used in combination with compounds or compositions of the invention include, but are not limited to, atenolol, bisoprolol, betaxolol, carteolol, carvedilol, labetalol, metoprolol, nadolol, nebivolol, oxprenolol, penbutolol, pindolol, propranolol, timolol, or combinations of any of the above.

[0192] In certain embodiments, the calcium channel blockers used in combination with compounds or compositions of the invention include, but are not limited to, dihydropyridines: amlodipine, cilnidipine, clevidipine, felodipine, isradipine, lercanidipine, levamlodipine, nicardipine, nifedipine, nimodipine, nisoldipine, nitrendipine, diltiazem, verapamil, or combinations of any of the above.

[0193] In certain embodiments, the diuretics used in combination with compounds or compositions of the invention include, but are not limited to, loop diuretics, thiazide diuretics, thiazide-like diuretics and potassium-sparing diuretics, or combinations of any of the above.

[0194] In certain embodiments, the loop diuretics used in combination with compounds or compositions of the invention include, but are not limited to, bumetanide, ethacrynic acid, furosemide, torsemide, or combinations of any of the above.

[0195] In certain embodiments, the thiazide diuretics used in combination with compounds or compositions of the invention include, but are not limited to, epitizide, hydrochlorothiazide and chlorothiazide, bendroflumethiazide, methyclothiazide, polythiazide, or combinations of any of the above. 37 BUSINESS.30655163.1 393522-004WO (204863)

[0196] In certain embodiments, the thiazide-like diuretics used in combination with compounds or compositions of the invention include, but are not limited to, indapamide, chlorthalidone, metolazone, or combinations of any of the above.

[0197] In certain embodiments, the potassium-sparing diuretics used in combination with compounds or compositions of the invention include, but are not limited to, amiloride, triamterene, spironolactone, eplerenone, or combinations of any of the above.

[0198] In certain embodiments, a provided compound, or composition thereof, is administered in combination with a second agent selected from nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids, muscle relaxants, anti-anxiety drugs, serotonin receptor agonists, antidepressants, anticonvulsants, and corticosteroids. In some embodiments, the second agent is selected from opioids, triptans, and COX-2 inhibitors. In some embodiments, the second agent is ibuprofen, acetaminophen, aspirin, naproxen, diclofenac, diflunisal, etodolac, fenoprofen, flurbiprofen, indomethacin, ketorolac, mefenamic acid, meloxicam, nabumetone, oxaprozin, piroxicam, sulindac, tolmetin, celecoxib, a triptan, oxycodone, morphine, codeine, hydromorphone, oxymorphone, fentanyl, or sufentanil. In some embodiments, the second agent is acetaminophen with codeine (Tylenol #2, #3, #4), buprenorphine, fentanyl transdermal patches, hydrocodone with acetaminophen, hydrocodone with ibuprofen, hydrocodone, hydromorphone, meperidine, methadone, morphine, morphine sustained-release (e.g., MS-Contin®, Avinza®, Kadian®) oxycodone sustained-release (e.g., OxyContin®), Oxycodone with acetaminophen (e.g., Percocet®) oxycodone with aspirin (e.g., Percodan®), oxycodone with ibuprofen (e.g., Combunox®), oxymorphone (e.g., Opana®, Opana ER®), pentazocine, propoxyphene with aspirin, propoxyphene with acetaminophen, tapentadol (e.g., Nucynta®, Nucynta ER®), tramadol, tramadol with acetaminophen (Ultram, Ultracet), a mixed opioid agonist / antagonist such as pentazocine / naloxone (e.g., Talwin NX®) butorphanol, or nalbuphine (e.g., Nubain®), an antidepressant such as amitriptyline (e.g., Elavil®), bupropion, desipramine (e.g., Norpramin®) duloxetine (e.g., Cymbalta®), imipramine (e.g., Tofranil®), venlafaxine (e.g., Effexor®), an anticonvulsant such as carbamazepine (e.g., Tegretol®), clonazepam (e.g., Klonopin®), gabapentin (e.g., Neurontin®), lamotrigine (e.g., Lamictal), pregabalin (e.g., Lyrica®), tiagabine (e.g., Gabitril®), or topiramate (e.g., Topamax®), a fibromyalgia medication such as milnacipran (e.g., Savella®), an anxiolytic such as alprazolam (e.g., Xanax®), diazepam (e.g., Valium®), lorazepam (e.g., Ativan®), triazolam (e.g., Halcion®), a muscle relaxant such as baclofen (e.g., Lioresal®), carisoprodol (e.g., Soma®), chlorzoxazone (e.g., Parafon Forte®, DSC), cyclobenzaprine (e.g., Flexeril®), dantrolene (e.g., Dantrium®), metaxalone (e.g., Skelaxin®), 38 BUSINESS.30655163.1 393522-004WO (204863)methocarbamol (e.g., Robaxin®), orphenadrine (e.g., Norflex®), or tizanidine (e.g., Zanaflex®), a corticosteroid such as cortisone, prednisone, prednisolone, dexamethasone, methylprednisolone (e.g., Medrol®, A-Methapred®, Depo Medrol®, Solu Medrol®), or triamcinolone. In some embodiments, the second agent is a triptan (serotonin receptor agonist) or other migraine treatment, such as almotriptan (e.g., Axert®), eletriptan (e.g., Relpax®), frovatriptan (e.g., Frova®), naratriptan ((e.g., Amerge®), rizatriptan (e.g., Maxalt®), sumatriptan (e.g., Imitrex®), sumatriptan / naproxen sodium (e.g., Treximet®), zolmitriptan (e.g., Zomig), erenumab (e.g., Aimovig®), or fremanezumab (e.g., Ajovy®), meclizine, dimenhydrinate, ubrogepant (e.g., Ubrelvy®), or botulinum toxin type A (e.g., Botox®).

[0199] In certain embodiments, a provided compound, or composition thereof, is administered in combination with a mechanical thrombectomy device, to a patient in need thereof. In certain embodiments, the mechanical thrombectomy device is a stroke thrombectomy device or a coil embolization device for cerebral aneurysm. In certain embodiments, such a device includes, but is not limited to, a coil retriever, an aspiration device or a stent retriever.

[0200] In certain embodiments, a combination of 2 or more therapeutic agents may be administered together with compounds or compositions of the invention. In certain embodiments, a combination of 3 or more therapeutic agents may be administered together with compounds or compositions of the invention.

[0201] Those additional agents may be administered separately from an inventive compound-containing composition, as part of a multiple dosage regimen. Alternatively, those agents may be part of a single dosage form, mixed together with a compound of this invention in a single composition. If administered as part of a multiple dosage regime, the two active agents may be submitted simultaneously, sequentially or within a period of time from one another, normally within five hours from one another.

[0202] As used herein, the term “combination,” “combined,” and related terms refers to the simultaneous or sequential administration of therapeutic agents in accordance with this invention. For example, a compound of the present invention may be administered with another therapeutic agent simultaneously or sequentially in separate unit dosage forms or together in a single unit dosage form. Accordingly, the present invention provides a single unit dosage form comprising a compound of the present invention, an additional therapeutic agent, and a pharmaceutically acceptable carrier, adjuvant, or vehicle.

[0203] The amount of both, a provided compound and additional therapeutic agent (in those compositions which comprise an additional therapeutic agent as described above) that 39 BUSINESS.30655163.1 393522-004WO (204863)may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. Preferably, compositions of this invention should be formulated so that a dosage of between 0.01 - 100 mg / kg body weight / day of an inventive compound can be administered.

[0204] In those compositions which comprise an additional therapeutic agent, that additional therapeutic agent and the compound of this invention may act synergistically. Therefore, the amount of additional therapeutic agent in such compositions will be less than that required in a monotherapy utilizing only that therapeutic agent. In such compositions a dosage of between about 0.001 - 100 mg / kg body weight / day of the additional therapeutic agent can be administered, or about 0.001 mg / kg to about 500 µg / kg, or about 0.005 mg / kg to about 250 µg / kg, or about 0.01 mg / kg to about 100 µg / kg body weight / day of the additional therapeutic agent can be administered.

[0205] The amount of additional therapeutic agent present in the compositions of this invention will be no more than the amount that would normally be administered in a composition comprising that therapeutic agent as the only active agent. Preferably the amount of additional therapeutic agent in the presently disclosed compositions will range from about 50% to 100% of the amount normally present in a composition comprising that agent as the only therapeutically active agent.

[0206] In one embodiment, the present invention provides a composition comprising a compound of the present invention and one or more additional therapeutic agents. The therapeutic agent may be administered together with a compound of the present invention or may be administered prior to or following administration of a compound of the present invention. Suitable therapeutic agents are described in further detail below. In certain embodiments, a compound of the present invention may be administered up to 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5, hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, or 18 hours before the therapeutic agent. In other embodiments, a compound of the present invention may be administered up to 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5, hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, or 18 hours following the therapeutic agent.

[0207] In some embodiments, the present invention provides a medicament comprising at least one compound of the present invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. 40 BUSINESS.30655163.1 393522-004WO (204863)

[0208] All features of each of the aspects of the invention apply to all other aspects mutatis mutandis.

[0209] In order that the invention described herein may be more fully understood, the following examples are set forth. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting this invention in any manner. EXEMPLIFICATION

[0210] As depicted in the Examples below, in certain exemplary embodiments, compounds are prepared according to the following general procedures. It will be appreciated that, although the general methods depict the synthesis of certain compounds of the present invention, the following general methods, and other methods known to one of ordinary skill in the art, can be applied to all compounds and subclasses and species of each of these compounds, as described herein. Example 1 – Synthesis of Compound A NH NH NBoc NBoc NN20% NaSMe aq.NNBoc O, DMAP, 2-Me-THFNN10% NH H O, 2-Me-THFNN

[0211] The formation of the methylthioether (AST-004-01) used 6 equivalents of sodium thiol methane (NaSMe) under aqueous conditions, at 90-105°C for approximately 72 hours. Upon reaction completion by IPC, the reaction mixture was cooled to 15-30°C and the pH was adjusted to 8-9 with hydrochloric acid. The mixture was heated to 70-80°C for 30-60 minutes 41 BUSINESS.30655163.1 393522-004WO (204863)and then cooled to 15-25 °C for 2-3 hours. The mixture was filtered, washed with acetonitrile, then dried. Steps 2 and 3

[0212] Prior to the reaction, the Step 1 product (AST-004-01) was azeotropically dried to remove residual water in 2-methyl tetrahydrofuran (2-MeTHF). The formation of the tri-Boc protected intermediate (AST-004-02) used 5 equivalents of di-tert-butyl dicarbonate (Boc2O) and 0.3 equivalents of N,N-dimethylpyridin-4-amine (DMAP) at 25℃ for 16 hours in 2-methyl tetrahydrofuran (2-MeTHF) to form the tri-Boc protected intermediate (AST-004-02) in situ. Upon reaction completion by IPC, the reaction mixture was cooled to 0-10 C, water was added, the mixture was filtered, and the organic layer was separated, washed with water and separated again. The Boc group in the 9 position was removed by reacting with 10% ammonium hydroxide at 0-5 C to form the di-Boc protected intermediate, AST-004-03. Upon reaction completion by IPC, the pH was adjusted to 7-8 hydrochloric acid and the organic portion is separated and then concentrated. N-heptane is added, heated to 60 ~70℃ for 30 to 60 minutes, cooled to 15 ~25℃ for ~2 to 3 hours and the product was filtered, washed with n-heptane, then dried. The Step 3 product (AST-004-03) can be recrystallized a second time from 2-MeTHF and n-heptane to increase purity of the intermediate if needed. Steps 4 and 4P

[0213] Step 4 utilized the Mitsunobu reaction to couple the methanocarba moiety to the di- Boc protected intermediate (AST-004-03) from Step 3. Equal equivalents of di-Boc protected intermediate (AST-004-03) and methanocarba moiety were charged in tetrahydrofuran (THF) to azeotropically dried to remove residual water. Triphenylphosphine (TPP) and diisopropyl azodicarboxylate (DIAD) were added at 1.5 equivalents each at 15-30℃ for approximately 7 hours. A solvent exchange was performed to remove THF and replace it with methyl tert-butyl ether (MTBE). The mixture was then cooled to 0 -10℃ for 1 to 2 hours and filtered. The filtrate was washed with MTBE and is collected and concentrated and a solvent exchange was performed to replace MTBE with DCM. The concentrated DCM mixture was used for the Step 4P purification step.

[0214] The purification step (Step 4P) used preparative chromatography to purify the Step 4 crude product. Step 4P started by concentrating the crude Step 4 product in dichloromethane. Once concentrated, the solution was diluted with n-heptane for loading on a silica column. Prior to the preparative chromatography, two use-tests were performed to confirm elution time of the desired product (approximately 37 to 55 minutes). Small preparative chromatography 42 BUSINESS.30655163.1 393522-004WO (204863)batches were prepared and each batch was purified by column chromatography with a SiO2sorbant and n-heptane / isopropyl alcohol in a mobile phase gradient over 141 minutes. All fractions of the peak of interest were tested, pooled, concentrated, and solvent exchanged with isopropyl alcohol. Step 5 Crude

[0215] The purified product was charged and was concentrated under reduced pressure and temperature. An excess of trifluroacetic acid (TFA) was added for the global deprotection and heated to 60℃ for 17 hours. The reaction was cooled, and MTBE was added. The mixture was further cooled between 0℃ and10℃ for 2 to 3 hours and the solids were filtered and washed with MTBE. The crude TFA salt was stirred for 1 to 2 hours at 15℃-30℃ and was cooled between 0℃ and10℃ for 2 to 3 hours and the solids filtered and washed with MTBE for a second time. Methanol and water for injection (WFI) were added and the pH is adjusted to pH 8-9 with aqueous sodium hydroxide. The solvent was concentrated and water for injection was added. This solvent mixture was concentrated until methanol is less than 3%. The mixture was heated to 80-90℃ for 1 to 2 hours then cooled to 10 to 20℃ and crystalized for 1 to 3 hours. The solids were filtered, and the filter cake washed with WFI and dried with nitrogen. Step 5R Recrystallization of AST-004 Drug Substance

[0216] The crude API was dissolved in 16 volumes of 50 / 50 methanol and WFI and heated to 50℃ to 60℃ for 1 to 2 hours. The mixture was cooled to 20℃ to 40℃ and passed through a bed of diatomacious earth and a capsule filter. The solvent mixture was concentrated under reduced pressure and temperature ≤60℃. The solvent was exchanged with isopropanol until water was less than 3%. The mixture was cooled to 0℃ to 10℃ for 2 to 3 hours to crystalize the AST-004. The solids were filtered and the filter cake was washed with isopropanol. The AST-004 solids were dried on the filter for 31 hours with nitrogen. The material was sieved through 20 mesh screen and packaged. Step 5M Recovery of API from Mother Liquor

[0217] Additional API may be recovered from the mother liquor following a similar process as Step 5 Recrystallization. The mother liquor from the Step 5 Recrystallization was exchanged with isopropanol until water content was ≤3% and concentrated to 5 to 10 solvent volumes under reduced pressure and temperature ≤60℃. The mixture was cooled to 0℃ to 5℃ for ~13 hours to crystalize additional AST-004. The AST-004 solids were filtered and the 43 BUSINESS.30655163.1 393522-004WO (204863)cake was washed with isopropanol. The solids were dried on the filter for 24 hours with nitrogen. The material was sieved through 20 mesh screen and packaged.

[0218] Powder x-ray powder diffraction (XRPD) shows a crystalline material of Form B. Two crystal forms (Form A and Form B) have been observed. Form B was selected for the clinical development of AST-004. All released lots of AST-004 have been Form B. Form A is a hemihydrate. It was used in in early development and exploratory toxicology. The manufacturing process as described yields only Form B, the anhydrous form. A bridging study was conducted and concluded that Form B was the most stable form when water activity is ≤ 63% and Form A is the most stable form when the water activity is ≥ 72%. Example 2 - Preparation of an Injectable Composition of Compound A

[0219] Compound A Injection, for intravenous use, contains 125 mg of Compound A active pharmaceutical ingredient in a 5 mL fill volume resulting in a 25 mg mg / mL clear and colorless solution. A 40% PEG 400 / water for injection (WFI) (v / v) was added as a vehicle, with a 10 mM phosphate buffer, q.s to apparent pH of 7.8 with either sodium hydroxide or ortho-phosphoric acid. Each vial contained a 0.3 mL overfill to allow for adequate withdrawal of the label volume. Each 6R vial is a clear borosilicate glass with a grey West stopper and a white flip off cap. Compound A Injection is intended to be delivered in a 50 mL admixture of Normal Saline (0.9% NaCl) over a 10-minute infusion. Table 1 contains the composition of Compound A Injection. Table 1: Composition of Compound A Injection Component Function Grade Molecular Molar Ratio Weight per44 BUSINESS.30655163.1 393522-004WO (204863)Component Function Grade Molecular Molar Ratio Weight per Weight (%) mL (mg) lc = QS to 1 mL d = Final density is 1.079 g / mL. Example 3 – Solubility of Compound A

[0220] The solubility of Compound A was determined in common pharmaceutical excipients used as solubility enhancers and determined by HPLC. Table 2: Compound A Solubility in Solubility Enhancers Solvent Solubility (mg / mL)45 BUSINESS.30655163.1 393522-004WO (204863)Example 4 – Stability of Compound A

[0221] In order to determine the need for antioxidants in a formulation of Compound A, four prototype lots were manufactured in a development lab with Compound A compounded in a 40% PEG 400 (v / v) vehicle with antioxidants and without antioxidants. These lots were manufactured in a 2 x 2 full factorial design where one configuration (two prototypes) was compounded with 0.01% BHT (w / w) and 0.004% BHA (w / w) antioxidants and the other configuration (two prototypes) was vialed with an inert nitrogen overlay headspace. Air in the Vial Headspace

[0222] To address the concern of instability caused by oxygen present in air the headspace, two prototypes used air in the headspace while the other two contained an inert nitrogen overlay headspace. All test between the two configurations were indistinguishable except for BHA quantity which indicted that air being present in the headspace consumed more BHA antioxidant. Since no other differences between air and a nitrogen overlay were observed, air and nitrogen headspaces appear equivocal for Compound A drug product formulations.

[0223] The following solutions A-D were prepared: Table 3: Overview of Prototype Batches Test Article Antioxidants N2 overlay46 BUSINESS.30655163.1 393522-004WO (204863)

[0224] The following stability testing protocol was followed: Table 4: Stability Protocol for Prototype Batches (Solution A, Solution B, Solution C and Solution D) Storage Period Condition Initial 2W 1M 2M 3MMost Sensitive Stability Indicating Tests

[0225] The appearance and pH were the most sensitive stability indicating outcomes. These values changed as a function of time and storage condition. The initial appearance was an essentially clear and colorless solution but the solution changed to slightly to light brown solution as a function of time and temperature. Additionally, 3 out of 4 of the vials stored at - 20℃ had white precipitates attributed to precipitation of Compound A and therefore, freezing is contraindicated. In regard to pH, the composition of the prototypes used in this stability study did not contain a buffer and thus the compositions are sensitive to slight changes that cause a corresponding shift in pH. The most prominent shift in pH was for the 2 week accelerated stability sample at 40℃ where the pH shifted more acidic by 0.6 to 1.0 pH units. However, at 1 month, the pH was essentially unchanged from the 2 week, 40℃ samples. To a lesser extent, there was a slight change in pH towards more acidic for the 25℃ samples up to 3 months. HPLC Degradants

[0226] The ingoing Compound A used had no reportable API-related substances reported above the 0.05% LOQ level. Therefore, all impurities in the drug product were a result of degradation during manufacturing or while on stability. The manufacture of formulation was primarily responsible for all related substances in the drug product with total impurities between 0.44% and 0.46% for the four prototypes. The total impurities appear to decline slightly over time. The largest and primary degradation products (AST-004-020 and AST-004- 021) are the two sulfoxide impurities form during manufacture. Typically, the two sulfoxides 47 BUSINESS.30655163.1 393522-004WO (204863)appeared at approximately the same concentration and remained constant over time or storage conditions.

[0227] Polyethylene glycols are known to contain peroxides and other oxidizing species (Wu et al 2011, Santos et al 2009, Wasylaschuke et al 2006). Peroxide content in PEG increases during storage with high temperatures and exposure to light (Santos et al 2009). Additionally, the oxidative degradation of PEG ultimately leads to the formation of small reactive molecules such as formaldehyde, acetylaldehyde, formic acid and acetic acid (Hemenway et al 2012). Due of the formation of small organic acids, the pH of the PEG solutions becomes more acidic and is correlated with the degradation of the PEG (Hemenway et al 2012).

[0228] In a forced degradation study with hydrogen peroxide, Compound A was reactive with 1% hydrogen peroxide and formed the purported sulfoxides as its primary oxidative degradation products. The prototype formulations had (AST-004-020 and AST-004-021) the two primary degradants, which likely resulted from the presence of residual peroxide found in PEG-400. In consideration of the impact of residual peroxides, the two sulfoxides (AST-004- 020 and AST-004-021) are specified impurities in the drug product. Antioxidants

[0229] 0.01% BHT (w / w) and 0.004% BHA (w / w) antioxidants were investigated to see if beneficial to the stability of the formulated product. When included, BHT was essentially completely consumed from the initial testing and BHA was partially consumed to approximately 10% to 80% of initial value for the stability program with a trend in consumption with temperature and time. Antioxidants where unable to prevent the appearance of the solution turning brown with time and temperature and did not prevent the change in pH over time and temperature. Furthermore, the incorporation of antioxidants did not change qualitatively or quantitatively change the related substances profile; either for individual degradants or total impurities. Therefore, the conclusion is that antioxidants are not beneficial to the formulation and are not used in the injectable composition.

[0230] Results of the appearance of the solutions are reported in Tables 5-9. Table 5: Appearance Stability of Compound A Injection with and Without Antioxidants and With and Without Nitrogen 48 BUSINESS.30655163.1 393522-004WO (204863)Storage Results Test TestArticleCondition T0 2W 1M 2M 3M s w / ne s n te n te s w / ne s n te n te49 BUSINESS.30655163.1 393522-004WO (204863)Storag Results Test Teste ArticleCondition T0 2W 1M 2M 3M s n te n te n te s n te ne es n te50 BUSINESS.30655163.1 393522-004WO (204863)estSt Results TTestorage ArticleCondition T0 2W 1M 2M 3M n teAdditional test results are shown in Tables 6- 9: Table 6: Assay (% of Label Claim) Stability of Compound A Injection with and Without Antioxidants and With and Without Nitrogen TeStorage StorageResults Tes stTest Test tArticle ConditionResults29351 BUSINESS.30655163.1 393522-004WO (204863)and without 40°C / 98.0 9NT NT Nitrogen)75%RH8.7Table 7: Total Impurities (%) Stability of Compound A Injection with and Without Antioxidants and With and Without Nitrogen Test Test Test TestStorage StorageResultsArticleConditionArticle ConditionResultsTable 8: Assay BHA (µg / mL) Stability of Compound A Injection with and Without Antioxidants and With and Without Nitrogen Tt T t TestStorage Stora eR l R lts52 BUSINESS.30655163.1 393522-004WO (204863)antioxidants 25°C / NT ND ND ND and 60%RH i53 BUSINESS.30655163.1 393522-004WO (204863)Table 9: pH Stability of Compound A Injection With and Without Antioxidants and With and Without Nitrogen StorTestStorage Test TestageResults TestArticle Article ConditionResultsConditionxa p e – o pa y o jec a e o u a o o po e s

[0231] In a follow-up in-use stability and compatibility study of the admixture of Compound A, Injection with 0.9% NaCl normal saline, samples were tested for Visible Particles (Ph. Eur.2.9.20) with the acceptance criteria “Essentially free from visible particles”. The sample preparation conditions were changed to use a laminar flow hood and aseptic transfers to an empty 100 mL sterile vial to eliminate cross contamination from extraneous environmental particulates from the analytical laboratory. The plumb set compatibility was a single pass over 20 minutes though the apparatus to match the clinical protocol. The low dose was changed to 5 mg and the high dose was changed to 125 mg to match the updated range of 54 BUSINESS.30655163.1 393522-004WO (204863)doses contemplated in the current clinical protocol. The protocol for the testing is described below. All admixtures are diluted to 50 mL with 0.9% NaCl, normal saline. Compatibility Protocols Table 10: Admixture Stability Protocol for Compound A Injection Time Point (Hours) Storage Conditions T0 2hr 8hr 24hr ted, , . Table 11: Follow-up Admixture Stability Protocol for Compound A Injection Storage Time Point (Hours) Conditions55 BUSINESS.30655163.1 393522-004WO (204863)Table 12: Admixture Stability and Infusion Set Compatibility Acceptance Criteria Attribute Acceptance Criteria Assay (mg / mL) Report resultsCompatibility Data Table 13: Low Dose 25mg Admixture Stability and Infusion Set Compatibility of Compound A Injection Results g: H56 BUSINESS.30655163.1 393522-004WO (204863)Results In Plumb 50 mL 50 mL 50 mL g: H 9) at e L LTable 14: High Dose 375mg Admixture Stability and Infusion Set Compatibility of Compound A Injection Results g: H57 BUSINESS.30655163.1 393522-004WO (204863)Results In Plumb 50 mL 50 mL 50 mL g: H 9) at e d r L LTable 15: Visible Particles (Ph. Eur.2.9.20) Test Results for Compound A Doses in Saline Time Points m s m s m s58 BUSINESS.30655163.1 393522-004WO (204863)High Free from Free from D Visible Visible NT NTCompatibility Summary and Conclusions

[0232] The admixture stability and preparation for use of Compound A Injection was validated for stability in saline and compatibility with the syringe and infusion set. A low dose of 25 mg (1 mL of Compound A Injection) and a high dose 375 mg (15 mL of Compound A Injection) was diluted in NaCl 0.9% IV and qs to 50 mL and prepared for use. Preparation for use was performed by transfer of the Compound A Injection to a syringe and then transferred into the saline. After transfer of the Compound A Injection to a 50-mL NaCl 0.9% IV, samples were taken at baseline (T0), 8 and 24 hours at 5°C, and at 8 hours at 25°C. In addition, stability was verified at 2 hours at 25°C for the syringe and infusion set where the admixture was flushed through the infusion set for 5 cycles during the 2 hour period. Testing included visual appearance, color, clarity, visible particles, particulate matter in injections, assay, and purity. For visual appearance, color, and clarity, all results met the predefined acceptance criteria and were identical to the T0 samples. For particulate matter for injection, the acceptance criteria were “report results”; however, all results also met the Ph. Eur. 2.9.19 criteria and were not essentially different to the T0 samples. For visible particles, there were some small white particles in all samples, including the T0 samples and occasionally a fiber. The samples were prepared open to the atmosphere in a non-particle free normal analytical laboratory and as such, some visible particles or fiber were introduced to the samples. For pH, the ingoing saline had a pH value of 5.0, the low dose had a pH between 5.9 and 6.0 and the high dose had a pH of 6.3. For assay, all results met the predefined acceptance criteria and were essentially identical to the T0 samples. For purity, all results met the predefined acceptance criteria and were essentially identical to the T0 samples. The only reported impurity was at RRT 1.06 and ranged between 0.14% to 0.15%. This same impurity was seen at 0.14% at the time of release.

[0233] In the follow-up in-use stability and compatibility study of the admixture of Compound A Injection with 0.9% NaCl, normal saline to demonstrate absence of visible particles according to Ph. Eur. 2.9.20. All samples in the protocol were “free from visible particles” demonstrating that the visible particle observed in the previous compatibility study 59 BUSINESS.30655163.1 393522-004WO (204863)are attributed to the sample preparation technique specific to the previous study which used open containers in an analytical laboratory and are not present when prepared using aseptic technique. These acceptable results support the storage conditions for the admixtures up to 24 hours at 5°C or up to 8 hours at 25°C as well as the compatibility with the syringe and infusion set. Example 6 – Preparation of Large Batch of Compound A Injectable Formulation

[0234] The batch formula for a 14 liter, 2,642 vial batch of Compound A Injection is provided in Table 16. The first 500 mL is discarded as the flush volume. Table 16: Batch Formula Table Component Function GradeAmount perBatch

[0235] Compound A Drug Substance was dispensed into a 20L glass vessel and an aqueous buffer solution of disodium phosphate dihydrate and PEG400 was added. The mixture was stirred to mix and dissolve. The pH was checked and 1N sodium hydroxide or ortho- phosphoric acid, 85% was added to adjust to a target pH of 7.8 as necessary.

[0236] The solution was transferred by peristaltic pump from the first 20L glass vessel to a second 20L glass vessel and pre-filtered using a 0.45 µm filter.

[0237] Using a peristaltic pump, the solution was transferred from the second glass vessel through a 0.22 µm filter in the Grade C environment and passed through into the Grade A environment and filtered through redundant 0.22 µm filters. The filter integrity test of the two, in series, sterile filters in the Grade A area were performed prior and after the sterile filtration step by the bubble point test. Washed and depyrogentated vials were filled to a target volume of 5.3 mL. 60 BUSINESS.30655163.1 393522-004WO (204863)

[0238] The filled vials were stoppered and sealed by an automatic crimping machine. The vials were inspected manually inspect for defects (unsealed crimp, missing seal, missing flip- off, damaged flip-off, damaged seal, missing stopper, or cracked vial). Example 7 – Stability testing Stability Data Table 17: Stability of Compound A Injection, Lot P992-01 at 5ºC Lot: P992-01 Storage Conditions: 5°C Site of Manufacture: Patheon Date of Manufacture: 15-Apr- M61 BUSINESS.30655163.1 393522-004WO (204863)Table 18: Stability of Compound A Injection, Lot P992-01 at 25ºC / 60% RH Lot: P992-01 Storage Conditions: 25ºC / 60% RH Site of Manufacture: Patheon Date of Manufacture: 15- UP t tTable 19: Stability of Compound A Injection, Lot P992-01 at 40ºC / 75% RH Lot: P992-01 Storage Conditions: 40ºC / 75% RH Sit f Mn f t r Pth Dt f Mn f t r 1562 BUSINESS.30655163.1 393522-004WO (204863)Table 20: Stability of Compound A Injection, Lot P99202A at 25ºC / 60% RH Lot: P99202AP Storage Conditions: 25ºC / 60% RH Site of Manufacture: Patheon Date of Use: ClinicalTable 21: Stability of Compound A Injection, Lot P99202A at 30ºC / 65% RH Lot: P99202AP Storage Conditions: 30ºC / 65% RH63 BUSINESS.30655163.1 393522-004WO (204863)Lot: P99202AP Storage Conditions: 30ºC / 65% RH Site of Manufacture: Patheon Date of Manufacture: 1-Sep- Use: ClinicalTable 22: Stability of Compound A Injection, Lot P99202A at 40ºC / 75% RH Lot: P99202AP Storage Conditions: 40ºC / 75% RH Site of Manufacture: Patheon Date of Manufacture: 1-Sep- Use: Clinical

[0239] Example 8 - Evaluation of the pharmacokinetics (PK) and bioavailability of Compound A when administered as a single intravenous (IV) injection 64 BUSINESS.30655163.1 393522-004WO (204863)

[0240] The purpose of this study was to evaluate the bioavailability of AST-004 following administration of an ODT (oral disintegrating tablet) formulation in comparison with IV administration. Part I – Procedure

[0241] Administration was once per phase. IV bolus via the cephalic vein. Dosing occurred at 4.598 mg / kg, with a dose concentration of 0.9196 mg / mL, and a dose volume of 5 mL / kg. Following IV administration, blood samples were collected from an alternate vein through 1- hour postdose. Information related to the test articles used and their preparation can be found in Tables 23 and 24, below. Table 23. Test Article Information Identification Compound ATest Article n O. d65 BUSINESS.30655163.1 393522-004WO (204863)

[0242] Whole blood samples were collected from all animals as noted in the following table for determination of test article exposure. Sample collection requirements are outlined in Table 25. Table 25. Sample Collection Requirements for Pharmacokinetic Analysis Targeted Sample Collection Timepoints Collection Device Volume SiteMethod: LC–MS / MS Column: Restek Raptor ARC-182.7 µm (30 x 2.1 mm) Mobile Phase A: 0.1% Formic Acid in Water Mobile Phase B: 0.1% Formic Acid in Acetonitrile Ionization: (+) ESI with Ion Spray Analyte: Compound A MRM: m / z 324.200 / 182.200 amu Internal Standard: Labetalol MRM: m / z 329.200 / 294.200 amu Assay Range (LLOQ and ULOQ): 1-5000 ng / mL

[0244] Pharmacokinetic Evaluation:

[0245] Pharmacokinetic concentration-time data for Compound A was analyzed using non- compartmental methods (Phoenix®WinNonlin®Version 8.1) with nominal sampling times and dosages. The following parameters were calculated whenever possible and as data allowed: Volume of distribution, Clearance, t1 / 2, C0 / Cmax, Tmax, %F, and AUC. Additional parameters may have been calculated as appropriate. Descriptive statistics were generated using Phoenix WinNonlin. Part II – Results

[0246] Following bioanalysis, pharmacokinetic calculations were conducted.

[0247] Individual animal and group mean plasma concentration versus time data are shown in Table 26. 66 BUSINESS.30655163.1 393522-004WO (204863)Table 26. Individual and Mean Compound A Plasma Concentration (ng / mL) versus Time Data Following a Single IV Bolus Injection of Compound A to Male Beagle Dogs Animal Time (hr) ID 6 7 0 7 8 5 3 a. PhaseNote: NA=Not applicable.

[0248] Individual animal and group mean pharmacokinetic parameters following a single IV bolus dose of Compound A are shown in Table 27. Table 27. Individual and Mean Pharmacokinetic Parameters Following a Single IV Bolus Injection of Compound A to Male Beagle Dogs C0 Tmax Cmax AUC0-24 AUCinf t1 / 2 Vz Cl Animal ID g)67 BUSINESS.30655163.1 393522-004WO (204863)C0 Tmax Cmax AUC0-24 AUCinf t1 / 2 Vz Cl Animal ID (ng / mL) (hr) (ng / mL) (hr*ng / mL) (hr*ng / mL) (hr) (L / kg) (mL / min / kg) anest mate va ue w c s ca cu ate rom nom na ose concentrat ons an est mate AUCinf values. Note: Clearance following IV dosing is CL; Volume following IV dosing is Vz ; NA=Not applicable; NC=Not calculated

[0249] Summary of mean pharmacokinetic parameters data are shown Table 28. Table 28. Summary of Mean Pharmacokinetic Parameters Following a Single Oral Disintegrating Tablet Dose of Compound A to Male Beagle Dogs C0CmaxAUC0-24AUCinft1 / 2Vz Cl )b%F is an estimated value which is calculated from nominal IV dose concentrations and estimated AUCinf values. Note: Clearance following IV dosing is CL; Volume following IV is Vz; NA=Not applicable

[0250] Following a single IV bolus injection of Compound A to male Beagle dogs, Compound A was quantifiable in plasma throughout the sampling period post injection (from 0.083 to 24 hours postdose). Mean back-extrapolated concentration at time 0 hour (C0), peak (Cmax) and total AUC0-24 exposures to Compound A, half-life (t1 / 2), volume of distribution (Vz), and clearance (Cl) were 5220 ng / mL, 4500 ng / mL, and 11200 hr*ng / mL, 4.64 hours, 2.67 L / kg, and 6.81 mL / min / kg, respectively. Example 9 – Noncompartmental Analysis of Plasma, Urine, and Cerebrospinal Fluid for Compound A for First-In-Human Phase 1 Study in Healthy Subjects

[0251] The primary study objective was to evaluate the safety and tolerability profile of single ascending IV doses of Compound A in healthy subjects. The secondary study objective 68 BUSINESS.30655163.1 393522-004WO (204863)was to characterize the single dose pharmacokinetic (PK) profile of Compound A in plasma, CSF, and urine. Part I - Procedures

[0252] This study was an adaptive-design, subject-blinded, first-in-human (FIH) Phase 1 safety, tolerability, and pharmacokinetic (PK) study, conducted in 2 study parts:

[0253] Part 1: single ascending dose (SAD); and

[0254] Part 2: single dose with cerebrospinal fluid collection (SD CSF).

[0255] A total of 40 subjects for Part 1 (SAD) and 12 subjects for Part 2 (SD CSF) were enrolled in the study. The subjects were healthy male and female subjects 18 to 55 years of age at the time of signing the informed consent form.

[0256] For Part 1 (SAD), 40 subjects in 5 independent, ascending-dose cohorts were enrolled. The doses explored in these 5 cohorts were 5, 25, 50, 75, and 100 mg, which were administered as single 10-minute intravenous (IV) infusions. Each cohort consisted of 8 subjects randomized to Compound A or placebo, whereby 6 subjects received a single dose of Compound A and 2 subjects received placebo.

[0257] For Part 2 (SD CSF), approximately 12 subjects in 1 dose cohort were enrolled who each received a single dose of 100 mg Compound A as a single 10-minute IV infusion (no subjects in SD CSF received a placebo).Sentinel dosing (1 subject to receive Compound A and 1 subject to receive placebo amongst the first 2 subjects) was used in each cohort to ensure adequate safety and tolerability evaluation prior to dosing the remainder of subjects within the cohort.

[0258] Sampling: Plasma samples for Part 1 SAD were taken at predose; 10 and 20 minutes; and 0.5, 1, 2, 4, 8, 12, 24, 36, 48, 72, 96, and 120 hours post start of the dose; and optional extended PK timepoints of 144, 168, and 192 hours postdose. Plasma samples for Part 2 SD CSF were taken at predose and 10, 30, 50, and 70 minutes postdose.

[0259] The complete urine output was collected from each subject during the following intervals: 0 to 6, 6 to 12, 12 to 24, 24 to 36, 36 to 48, 48 to 72, 72 to 96, and 96 to 120 hours postdose. If optional extended plasma PK collections on Day 6, Day 7, and Day 8 through discharge on Day 8 were decided by the safety review committee (SRC), 24-hour urine collections also occurred on Day 6, Day 7, and Day 8. No urine samples were collected in Part 2 SD CSF.

[0260] The CSF samples were collected from subjects enrolled in Part 2 SD CSF only. The CSF samples were collected at 20, 40, and 60 minutes postdose. All 12 volunteers in this cohort 69 BUSINESS.30655163.1 393522-004WO (204863)were sampled for CSF. Only one sample was taken from each volunteer in a subject-staggered manner with 4 subjects per timepoint.

[0261] Bioanalytical Analysis: The primary analysis variables were the plasma, urine, and CSF concentrations of Compound A, as determined using validated ultra performance liquid chromatography coupled to tandem mass spectrometry (UPLC / MS / MS) bioanalytical assays. The determination of Compound A concentration ranged from 5 to 5000 ng / mL in plasma, 30 to 15,000 ng / mL in urine, and 1 to 500 ng / mL in CSF. The lower limit of quantitation (LLOQ) for the assay used for the quantitation of plasma, urine, and CSF was 5, 30, and 1 ng / mL, respectively.

[0262] Pharmacokinetic Analysis: All exploratory data analyses and presentations of data were performed using RVersion 4.1.3. Noncompartmental analysis modeling was performed using the computer program PKanalix Version 2021R1.

[0263] The following is a list of Plasma Pharmacokinetic Parameters Calculated, and how they are defined and abbreviated herein. Cmax Maximum concentration Tmax Time to Cmax Clast Last quantifiable concentrationTlastTime of ClastAUC0-24Area under the plasma concentration-time curve from 0 to 24 hours AUC0-t Area under the plasma concentration-time curve from 0 to the last measurable plasma concentration AUC0-∞ Area under the plasma concentration-time curve from 0 to infinity fextPercent of AUC0-∞extrapolated from the last plasma drug concentration to infinity AUMC0-∞ Area under the first moment curve from 0 to infinity CL Total plasma clearance, calculated as: Dose / AUC0-∞MRT0-∞ Mean residence time from 0 to infinity, calculated as: (AUMC0-∞ / AUC0-∞) / (D / 2), where D is infusion duration Vss Volume of distribution at steady-state, calculated as: MRT 0-∞ ×CL VzVolume of distribution based on terminal phase, calculated as: Dose / (AUC0-∞ ×kel) kelFirst-order terminal elimination rate constant t1 / 2 Terminal elimination phase half-life, calculated as: ln(2) / kel. 70 BUSINESS.30655163.1 393522-004WO (204863)

[0264] Dose Proportionality Assessment: Dose proportionality of plasma concentration data was assessed using the power model approach. Dose proportionality was assessed over the dose range of 5 to 100 mg Compound A administered intravenously. The key assumption of this method was that the logarithm of exposure (maximum concentration [Cmax],area under the plasma concentration-time curve from 0 to the last measurable plasma concentration [AUC0-t],and area under the plasma concentration-time curve from 0 to infinity [AUC0-∞]) was linearly related to the logarithm of the dose. To meet the dose proportionality criteria, the calculated point estimate and associated upper and lower confidence interval (CI) must be contained wholly within the calculated CI criteria limits.

[0265] Data Accounting, Inclusion, and Exclusion: There were a total of 30 healthy subjects included in the Part 1 SAD analysis, with 6 subjects in each of the dosing cohorts (the 10 placebo subjects were not included in the analyses). One subject randomized to the 5 mg dosing cohort failed to meet the inclusion criteria of negative Hepatitis B surface antigen and was excluded from the PK analyses for plasma and urine, bringing the total number of subjects to 29 for the Part 1 SAD PK analyses. Of the subjects remaining, 1 subject had an outlier observation at 2 hours postdose and another subject had 3 samples collected outside the predetermined sampling windows (deemed as protocol deviations). These samples were excluded from the concentration summary statistics and mean plots. These values were also removed from the calculation of the PK parameters and were not included in the individual concentration-time listings and figures. There were no samples obtained from optional extended PK assessments for either plasma or urine analyses.

[0266] Data from all 12 subjects in Part 2 included in the Part 2 SD CSF analysis. No deletions or omissions were performed.

[0267] Pharmacokinetics of Compound A in Plasma:

[0268] In general, the following procedures were used for computation of area under the plasma concentration-time curve from 0 to 24 hours (AUC0-24), area under the plasma concentration-time curve from 0 to the last measurable plasma concentration (AUC0-t) and area under the plasma concentration-time curve from 0 to infinity (AUC0-∞): ^ AUC0-24, AUC0-t, and AUC0-∞were computed using the linear trapezoidal rule between concentration-time points that either increased in value or remained at the same value and using the logarithmic trapezoidal rule between concentration-time points that decreased in value (linear up / log down). 71 BUSINESS.30655163.1 393522-004WO (204863)^ At least 4 quantifiable concentration-time values must have been available to compute AUC0-24, AUC0-tand AUC0-∞.^ AUC0-24 was computed as the cumulative AUC from dose administration to 24 hours after dose. ^ AUC0-twas computed as the cumulative AUC from dose administration to the last observable concentration. ^ AUC0-∞was computed as the cumulative AUC to time to last quantifiable concentration (Tlast) of last quantifiable concentration (Clast), plus the extrapolated ^ AUC from Tlast to time infinity. The area under the plasma concentration-time curve from the last quantifiable observation to infinity (AUCtlast-∞) is estimated by dividing the Clast by the first-order elimination rate constant (kel). AUC0-∞ was not computed or reported in the following circumstances: ^ Data were not amenable to determination of kel; and ^ Extrapolated component of AUC0-∞ was > 25% of AUC0-∞. ^ The percentage of AUC0-∞that was due to extrapolation from Tlastto infinity was computed as: percent of AUC0-∞extrapolated from the last plasma drug concentration to infinity (fext) = (1-(Tlast / AUC0-∞)) × 100.

[0269] In general, the following procedures were used for the estimation of kel: ^ Terminal elimination rate constant was computed by linear regression of the terminal linear portion of the log concentration-time data. ^ The terminal elimination phase half-life (t1 / 2) was calculated as: Ln(2)=kel (1).

[0270] Terminal elimination t1 / 2 was initially estimated using the default method in PKanalix. The plots were analyzed by visual inspection to assess the quality of the estimation, and the following constraints were placed on the computation of kel: ^ There must have been at least 3 quantifiable concentration-time values included in the regression analysis of the terminal linear phase. ^ Adjusted R2 > 0.8 was used. ^ Maximum concentration was not used in the determination of kel.^ If the estimated t1 / 2 was greater than the duration of the PK sampling interval, then any parameter dependent upon kelfor computation (for example, t1 / 2and AUC0-∞) and kel were not reported (due to inherent inaccuracy in such an estimation).

[0271] Statistical Methods: 72 BUSINESS.30655163.1 393522-004WO (204863)

[0272] For all PK parameters, appropriate descriptive statistics were calculated based on dose amounts and dose time. Descriptive statistics included sample size, arithmetic mean, standard deviation (SD), percent coefficient of variation (%CV), minimum, median, maximum, geometric mean, and geometric mean %CV.

[0273] Dose proportionality of plasma concentration data was assessed using the power model approach. Part II - Results

[0274] Pharmacokinetics of Compound A in Plasma: The study design provided sufficient plasma concentration sampling times to characterize the terminal slope in all the concentration- time profiles after administering the single dose of Compound A intravenously, even though the majority of the plasma concentrations were below the lower limit of quantitation (BLQ) by 8 hours postdose for the lower dosing cohorts (5 and 25 mg), and 12 hours postdose for 50 mg and above cohorts. Summary statistics for parameters for Compound A plasma concentrations were calculated for each dose group.

[0275] A total of 434 plasma concentration obtained from 29 subjects enrolled in the Part 1 SAD. No plasma samples were available from optional extended PK assessments (144, 168, 192 h postdose) for analysis. The majority of the plasma concentrations were BLQ by 8 hours postdose for the lower dosing cohorts (5 mg and 25 mg). For cohorts 50 mg and above, the majority of the concentrations were BLQ by 12 hours postdose. For all 5 dosing cohorts, predose sample concentrations were BLQ. As expected, the plasma concentrations increased with increase in dose. The greatest variability between individual profiles was observed in the lowest dose group (5 mg). Noncompartmental analysis of Compound A plasma concentrations was performed for all subject concentration-time profiles. The clearance and volume of distribution parameters were weight adjusted (assuming 70 kg body weight).

[0276] Summary statistics for Cmax,Tmax, AUC0-t, and Half-life for all dose groups can be found in Tables 29, 30, 31, and 32 below. Table 29. Summary Statistics for Cmax (ng / mL) Dose (mg) N Mean CV (%) SD Min Median Max Geo. Mean Geo. CV (%)73 BUSINESS.30655163.1 393522-004WO (204863)Dose (mg) N Mean CV (%) SD Min Median Max Geo. Mean Geo. CV (%)Dose (mg) N Median Min Max T. u y 0-t g Dose (mg) N Mean CV (%) SD Min Median Max Geo. Mean Geo. CV (%)Table 32. Summary Statistics for Half-Life (h) Dose (mg) N Mean CV (%) SD Min Median Max Geo. Mean Geo. CV (%)74 BUSINESS.30655163.1 393522-004WO (204863)

[0277] Dose-normalized Cmax, AUC0-24, AUC0-t, and AUC0-∞, were also calculated. The geometric mean t1 / 2of Compound A ranged from 1.1 to 1.36 hours, and CL ranged from 11.41 to 19.17 mL / min / kg after administering a single dose of Compound A. The highest variability in CL was observed for the lowest dose group with geometric %CV as 34.39%. The geometric mean values for CL for the higher dose groups (50 mg or above) were similar, ranging from 15.95 to 17.47 mL / min / kg. The geometric mean values for Vz ranged from 1131.62 to 2056.12 mL / kg. The Vz parameter was observed to be lower for 50 mg dose group when compared to the 25 mg dose group. Additionally, the highest variability was observed in the 5-mg dose group (geometric %CV as 27.88%) followed by 50-mg dose group (geometric %CV as 27.41%) for the Vz parameter.

[0278] Boxplots comparing dose-normalized parameters for Cmax,AUC0-24,AUC0-t,and AUC0-∞ indicate there was an overlapping of the percentiles between the doses for each dose level, suggesting dose linearity.

[0279] Pharmacokinetics of Compound A in Urine: A total of 224 urine concentrations were obtained from 29 subjects enrolled in the Part 1 SAD. No urine concentrations were available from optional extended PK collections (Day 6, Day 7 and Day 8 postdose) for analysis. As expected, the amount recovered in the urine increased with an increase in dose. Summary statistics for urine parameters for Compound A were calculated for each dose group. The geometric mean of percent of unchanged dose recovered in urine ranged from 9.93% to 35.3%.

[0280] Noncompartmental analysis of cumulative Compound A urine concentrations was performed for all subjects. The renal clearance (CLr) parameter was weight adjusted (assuming 70 kg body weight). The geometric mean of percent of unchanged dose recovered in urine ranged from 9.93% to 35.3%. The geometric mean of CLr ranged from 1.9 to 5.74 mL / min / kg. The percent of unchanged dose recovered and CLrwere lowest for 25-mg dose group. This group also had the largest variability compared to the other dose groups for all the computed parameters.

[0281] The mean (SD) percent of unchanged dose recovered at this collection interval for this particular dosing cohort (25 mg) was 0.003% (0.0055) of the total dose. As the value recovered in urine was <1%, and did not seem to impact the overall results, the sample was therefore included in the analysis.

[0282] Pharmacokinetics of Compound A in Cerebrospinal Fluid: A total of 12 observations each for plasma and CSF were obtained from 12 subjects enrolled in the Part 2 75 BUSINESS.30655163.1 393522-004WO (204863)SD CSF. Mean CSF and plasma Compound A concentrations at each nominal time are summarized in Table 33 and Table 34, respectively. Table 33. Descriptive Statistics for AST-004 Cerebrospinal Fluid Concentration-Time Data After Single Intravenous Administration of 100 mg for Part 2 SD CSF Dose Nominal Mean SD Min Median Max CV Geo. Mean G (mg)e (h)Neo. CV Tim(ng / mL) / L (ng / mL) (ng / mL) (ng / mL) (%) (ng / mL) (%). p Single Intravenous Administration of 100 mg for Part 2 SD CSF Dose Nominal N Mean SD Min Median Max CV Geo. Mean Geo. CV Ti h

[0283] Summary statistics for CSF / plasma ratio were calculated. The CSF / plasma ratio was calculated for matched samples obtained from subjects enrolled in Part 2 SD CSF. As the plasma sampling times did not match the exact CSF sampling times, the following samples were paired and evaluated: 20-minute (0.333-hour) CSF sample with 10-minute (0.166-hour) plasma sample, 40-minute (0.666-hour) CSF sample with 30-minute (0.500-hour) plasma sample, and 60-minute (1.00-hour) CSF sample with 50-minute (0.833-hour) plasma sample.

[0284] As expected, the ratio of CSF / plasma increased over time, indicating an increase in CSF concentration with time. Summary statistics for CSF / plasma ratio is summarized in Table 35, below. The high variability was observed for the CSF / plasma ratio at 40 minutes (0.666 h) postdose with a geometric %CV of 62.5%. 76 BUSINESS.30655163.1 393522-004WO (204863)Table 35. Summary Statistics for CSF / Plasma Ratio Dose Nominal NMean SD MinCV Geo. Geo. CV (mg)Time (h)Median Max(%) Mean (%) 9 4 2parameters included, Compound A appeared to be dose-proportional. All exposure metric exponents and the 90% CI were within the critical region.

[0286] While we have described a number of embodiments of this invention, it is apparent that our basic examples may be altered to provide other embodiments that utilize the compounds and methods of this invention. Therefore, it will be appreciated that the scope of this invention is to be defined by the appended claims rather than by the specific embodiments that have been represented by way of example. 77 BUSINESS.30655163.1 393522-004WO (204863)

Claims

CLAIMS We claim:

1. An injectable liquid pharmaceutical composition, comprising: (a) compound A:at a concentration of about 5 mg / mL to about 75 mg / mL; (b) 30-60% v / v glycol solvent in water; and (c) a buffer.

2. The composition of claim 1, wherein the concentration of compound A is about 15 mg / mL to about 50 mg / mL.

3. The composition of claim 1, wherein the concentration of compound A is about 25 mg / mL.

4. The composition of any one of claims 1-3, wherein the glycol solvent comprises a dialkylene glycol monoalkyl ether or polyethylene glycol.

5. The composition of any one of claims 1-4, wherein the glycol solvent comprises a polyethylene glycol.

6. The composition of claim 4, wherein the polyethylene glycol is substantially free of peroxides and other degradants.

7. The composition of any one of claims 1-6, wherein the glycol solvent comprises 35- 50% v / v of a polyethylene glycol.

8. The composition of any one of claims 1-6, wherein the glycol solvent comprises about 40% v / v of a polyethylene glycol.393522-004WO (204863)9. The composition of any one of claims 5-8, wherein the polyethylene glycol has an average molecular weight (MW) of 200-600.

10. The composition of any one of claims 5-9, wherein the polyethylene glycol is selected from PEG 200, PEG 300, PEG 400, SR PEG 400, PEG 540, PEG 600, PEG 900, or a mixture of any of the foregoing.

11. The composition of any one of claims 5-9, wherein the polyethylene glycol is PEG 400.

12. The composition of any one of claims 1-11, wherein the buffer is a citrate, phosphate, borate, or acetate buffer.

13. The composition of any one of claims 1-11, wherein the buffer is a phosphate buffer.

14. The composition of any one of claims 1-13, wherein the composition has a pH of about 6.0 to about 9.

0.

15. The composition of any one of claims 1-13, wherein the composition has a pH of about 7.0 to about 8.

5.

16. The composition of any one of claims 1-13, wherein the composition has a pH of about 7.5 to about 8.

3.

17. The composition of any one of claims 1-13, wherein the composition has a pH of about 7.

8.

18. The composition of any one of claims 1-17, wherein the composition further comprises an antioxidant.

19. The composition of claim 18, wherein the antioxidant is selected from BHT and BHA. 79 BUSINESS.30655163.1 393522-004WO (204863)20. The composition of any one of claims 1-19, wherein, after storage at 25 °C and 60% relative humidity (RH) for a period of 6 months, the composition contains an amount of compound A within 0.5% of the amount of compound A of initial manufacture of the composition.

21. The composition of claim 20, wherein the composition contains less than 0.1% impurities after storage at 25 °C and 60% RH for a period of 6 months.

22. The composition of claim 20 or 21, wherein the pH of the composition after storage at 25 °C and 60% RH for a period of 6 months remains within 0.2 units of the pH measured at initial manufacture of the composition.

23. The composition of any one of claims 1-22, wherein, after storage at 40 °C and 75% relative humidity (RH) for a period of 1 month, the composition contains an amount of compound A within 0.5% of the amount of compound A of initial manufacture of the composition.

24. The composition of claim 23, wherein the composition contains less than 0.1% impurities after storage at 40 °C and 75% RH for a period of 1 month.

25. The composition of claim 23 or 24, wherein the pH of the composition after storage at 40 °C and 75% RH for a period of 1 month remains within 0.2 units of the pH measured at initial manufacture of the composition.

26. The composition of any one of claims 1-25, wherein the composition contains no more than 0.8% by HPLC, as compared to the AUC (area under the curve) of compound A, of the following compound: BUSINESS.30655163.1393522-004WO (204863)27. A unit dosage form comprising the composition of any one of claims 1-26, wherein the unit dosage form contains about 15 mg to about 500 mg of compound A.

28. The unit dosage form of claim 27, containing about 25 mg to about 375 mg of compound A.

29. The unit dosage form of claim 27, containing about 25 mg, about 125, or about 375 mg of compound A.

30. The unit dosage form of claim 27, wherein the unit dosage form contains about 125 mg of compound A in a fill volume of about 5 mL.

31. A method of treating an injury, disease, or condition selected from traumatic brain injury (TBI), concussion, stroke, polytrauma, cardiac arrest, near drowning, altitude sickness, brain injuries from directed energy or Havanna Syndrome, partial or total spinal cord transection, malnutrition, toxic neuropathies, meningoencephalopathies, neurodegeneration caused by a genetic disorder, age-related neurodegeneration, vascular disease, Alzheimer’s Disease (AD), Parkinson’s Disease (PD), Huntington’s Disease (HD), Multiple Sclerosis (MS), amyotrophic lateral sclerosis (ALS), chronic traumatic encephalopathy (CTE), cardiovascular disease, autoimmune diseases, allergic diseases, transplant rejection, graft- versus-host disease, intraocular hypertension, glaucoma, odor sensitivity, an olfactory disorder, type 2 diabetes, pain control, respiratory diseases, deficits in CNS function, deficits in learning, deficits in cognition, otic disorders, Meniere’s disease, endolymphatic hydrops, progressive hearing loss, noise-induced hearing loss, dizziness, vertigo, tinnitus, collateral brain damage associated with radiation cancer therapy, migraine treatment, sleep disorders in the elderly, epilepsy, schizophrenia, symptoms experienced by recovering alcoholics, damage to neurons or nerves of the peripheral nervous system during surgery, gastrointestinal conditions, pain mediated by the CNS, migraine, collateral brain damage associated with radiation cancer therapy, depression, mood or behavioral changes, dementia, erratic behavior, suicidality, tremors, Huntington’s chorea, loss of coordination of movement, deafness, impaired speech, dry eyes, hypomimia, attention deficit, memory loss, cognitive difficulties, vertigo, dysarthria, dysphagia, ocular abnormalities or disorientation, and addiction; comprising administering to a patient in need thereof an effective amount of a composition of any one of claims 1-26. 81 BUSINESS.30655163.1 393522-004WO (204863)32. The method according to claim 31, wherein the injury, disease, or condition is selected from acute pain, chronic pain, nociceptive pain, neuropathic pain, inflammatory pain, and acute pain.

33. The method of claim 31, wherein the compound or pharmaceutically acceptable salt thereof is co-administered to the patient with a tissue plasminogen activator, a blood thinner, a statin, an ACE inhibitor, an angiotensin II receptor blocker (ARB), a beta blocker, a calcium channel blocker, or a diuretic.

34. The method of claim 31 or 33, wherein the injury, disease, or condition is selected from traumatic brain injury (TBI), stroke, a neurodegenerative condition, and a heart or cardiovascular disease.

35. The method of claim 34, wherein the injury, disease, or condition is TBI selected from concussion, blast injury, combat-related injury, a mild, moderate or severe blow to the head, whiplash, sports-related injury, or a head injury sustained from a fall or other accident.

36. The method of claim 31, wherein the injury, disease, or condition is a stroke selected from ischemic stroke, hemorrhagic stroke, subarachnoid hemorrhage, cerebral vasospasm, and transient ischemic attacks (TIA).

37. The method of claim 31, wherein neuroprotection or neurorestoration is increased in the patient as compared with an untreated patient.

38. The method of claim 31, wherein the injury, disease, or condition is a neurodegenerative disease selected from Alzheimer’s Disease (AD), Parkinson’s Disease (PD), Huntington’s Disease (HD), Multiple Sclerosis (MS), amyotrophic lateral sclerosis (ALS), chronic traumatic encephalopathy (CTE), or a neurodegenerative condition caused by a virus, alcoholism, tumor, toxin, and repetitive brain injuries.

39. The method of claim 31, wherein the injury, disease, or condition is heart or cardiovascular disease selected from cardiac ischemia, myocardial infarction, a cardiomyopathy, coronary artery disease, arrhythmia, myocarditis, pericarditis, angina, 82 BUSINESS.30655163.1 393522-004WO (204863)hypertensive heart disease, endocarditis, rheumatic heart disease, congenital heart disease, or atherosclerosis.

40. A method of increasing neuroprotection or neurorestoration in a patient in need thereof who has suffered a TBI or stroke, comprising administering to the patient an effective amount of the composition of any one of claims 1-26.

41. A method of treating an injury, disease, disorder, or condition selected from: (i) brain damage caused by radiation or collateral brain damage associated with radiation cancer therapy or migraine treatment; (ii) migraine headache; (iii) a condition associated with a brain injury or a neurodegenerative condition; and (iv) an autoimmune disease or condition, glaucoma, an otic disorder, progressive hearing loss, tinnitus, epilepsy, pain control, pain mediated by the CNS, neuropathic pain, inflammatory pain, or acute pain; comprising administering to a patient in need thereof an effective amount of the composition of any one of claims 1-26.

42. The method of claim 41, wherein the compound increases neuroprotection or neurorestoration in the patient as compared with an untreated patient.

43. The method of claim 41, wherein the condition associated with a brain injury or a neurodegenerative condition is selected from epilepsy, migraine, collateral brain damage associated with radiation cancer therapy, depression, mood or behavioral changes, dementia, erratic behavior, suicidality, tremors, Huntington’s chorea, loss of coordination of movement, deafness, impaired speech, dry eyes, hypomimia, attention deficit, memory loss, cognitive difficulties or deficit in cognition, deficit in CNS function, deficit in learning, vertigo, dysarthria, dysphagia, ocular abnormalities, or disorientation.

44. A method of increasing cardioprotection or regeneration of damaged heart tissue in a patient in need thereof who has suffered a cardiac ischemia or myocardial infarction, comprising administering to the patient an effective amount of the composition of any one of claims 1-26. 83 BUSINESS.30655163.1 393522-004WO (204863)45. A method of treating an addiction, addictive behavior, behavioral addiction, brain reward system disorder, or a compulsive disorder, comprising administering to a patient in need thereof an effective amount of the composition of any one of claims 1-26.

46. The method according to claim 41, wherein the injury, disease, or condition is migraine.

47. The method according to claim 41, wherein the injury, disease, or condition is pain selected from central pain syndrome, peripheral neuropathy, corneal neuropathic pain, post stroke pain, and pain caused by multiple sclerosis.

48. A method of treating stroke, comprising administering to a subject in need thereof an effective amount of the composition of any one of claims 1-26 in combination with a thrombolytic agent.

49. The method of claim 48, wherein the thrombolytic agent is selected from alteplase, desmoteplase, reteplase, streptokinase, tenecteplase, urokinase, or a combination thereof.

50. The combination treatment of claim 48, wherein the thrombolytic agent is alteplase or tenecteplase.

51. An injectable liquid pharmaceutical composition, comprising: (a) compound A:at a concentration of about 5 mg / mL to about 75 mg / mL; (b) a buffer; and (c) optionally, a glycol solvent. 84 BUSINESS.30655163.1 393522-004WO (204863)52. The composition of claim 51, wherein the concentration of compound A is about 15 mg / mL to about 50 mg / mL.

53. The composition of claim 51 or 52, wherein the composition comprises 30-60% v / v glycol solvent in water. 85 BUSINESS.30655163.1 393522-004WO (204863)

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