WO2024036225A1 - Epicatechin for inhibiting glutamate toxicity - Google Patents

Abstract

The present invention provide methods of treating or preventing a condition related to acute or chronic excessive glutamate exposure using epicatechin or pharmaceutical compositions thereof. Methods of decreasing reactive oxygen species (ROS) and lowering intracellular Ca2+ levels using epticatechin or pharmaceutical compositions thereof are also provided.

Description

EPICATECHIN FOR INHIBITING GLUTAMATE TOXICITY

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Application Serial No.

63/371,056, filed August 10, 2022, which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates in some aspects to treating or preventing a condition related to acute or chronic excessive glutamate exposure using epicatechin.

BACKGROUND

[0003] Glutamate is one of the most important excitatory neurotransmitters, and binds to a family of glutamate receptors. When glutamate levels in the extracellular fluid are too high, a subset of glutamate receptors, known as the N-methyl-D-aspartate (NMD A) receptors, become activated as calcium (Ca²⁺) channels that let in excess quantities of Ca²⁺. NMD A receptors permit the influx of Ca²⁺ to the extent that it is toxic to mitochondria if glutamate activation of NMDA receptors is excessive. High Ca²⁺ impairs mitochondrial membrane potential, reducing the production of ATP and increasing hydrolysis of ATP by ATP synthase, thereby reducing availability of ATP required for cellular functioning. If the neuron is unable to produce an adequate amount of ATP, it cannot subsequently remove the excess Ca²⁺. Acute exposure Ca²⁺ may cause several downstream effects, such as: (i) sustained activation of Ca²⁺ activated enzymes such as calpain, caspases, and phospholipases, (ii) mitochondrial damage, via opening of the mitochondrial permeability transition pore (mPTP), and (iii) bioenergetic depletion of the central nervous system (CNS), ultimately resulting in loss of postsynaptic structures neuronal cell death and brain dysfunction.

[0004] Glutamate toxicity is implicated in several different acute CNS syndromes and in progressive neurodegeneration. In particular, acute glutamate exposure is associated with both calcium (Ca²⁺) overload of the neuron and accompanying mitochondrial injury and depletion. Acute release of toxic levels of glutamate has been implicated in the neurological impairment associated with traumatic injury to the head and spinal cord, epilepsy, and stroke, among other acute neurological syndromes. The release of glutamate into the extracellular space by damaged or dying neurons has been implicated as a progression factor in chronic neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and ALS.

1 [0005] Palliating glutamate toxicity has been a drug target for several decades, with the main strategy to antagonize the NMDA receptor. While such strategies may be effective at reducing glutamate toxicity, NMDA receptor antagonists can have harmful side effects, as signaling through the NMDA receptor has many important roles in normal neuronal physiology which can be also be affected by currently available glutamate receptor blockers. The present disclosure addresses this and other unmet needs.

BRIEF SUMMARY

[0006] Provided herein are methods of treating or preventing diseases or disorders resulting from acute or chronic excessive glutamate exposure, excess calcium (Ca²⁺) levels, and/or reactive oxygen species, using epicatechin. Such methods are useful for the treatment of a variety of diseases or disorders, such as central nervous system (CNS) diseases or disorders and/or diseases or disorders associated with intracellular Ca²⁺ buildup.

[0007] One aspect of the present application provides a method of treating or preventing a condition related to acute or chronic excessive glutamate exposure in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of epicatechin or a pharmaceutically acceptable salt thereof.

[0008] In some embodiments, the glutamate exposure induces intracellular calcium (Ca²⁺) buildup.

[0009] In some embodiments, the condition is selected from the group consisting of status epilepticus, neuroinflammatory disorders, pediatric seizure disorders, neuronal exocitoxicity, over activation of the N-methyl-D-aspartate (NMDA) receptor, post-operative syndromes of cognition loss, and loss of synaptic density.

[0010] In other aspects, provided herein is a method of decreasing reactive oxygen species (ROS) in a subject need thereof, comprising administering to the subject a therapeutically effective amount of epicatechin or a pharmaceutically acceptable salt thereof.

[0011] In further aspects, provided herein is a method of treating a condition related to acute or chronic exposure to intracellular calcium (Ca²⁺) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of epicatechin or a pharmaceutically acceptable salt thereof.

[0012] In some embodiments, the condition is neuronal exci totoxi city.

[0013] In additional aspects, provided herein is a method of lowering intracellular Ca²⁺ levels in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of epicatechin or a pharmaceutically acceptable salt thereof. [0014] In some embodiments, the subject has a disease or disorder selected from the group consisting of spinal cord injury or abnormality, liver disease, kidney disease, impaired cognition, neurodegenerative disease, dystonia, sarcopenia, cardiomyopathy of aging or other diseases associated with mitochondrial dysfunction, cardiomyopathy, ischemic vascular disease, immunodeficiency states, ataxia, pulmonary inflammation and fibrosis, infantile encephalomyopathy, epilepsy, Charcot-Marie-Tooth disease, exocrine pancreatic insufficiency, impaired wound healing, and growth of cancer cells. In some embodiments, the subject has a neurodegenerative disease selected from the group consisting of Alzheimer’s disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), Leigh syndrome, Duchenne muscular dystrophy, Becker muscular dystrophy, and peripheral and central neuropathies.

[0015] In some embodiments, the epicatechin is administered in the form of a pharmaceutical composition comprising the epicatechin or the pharmaceutically acceptable salt, solvate, or co-crystal thereof, and a pharmaceutically acceptable excipient. In some embodiments, the epicatechin is in the form of a co-crystal.

[0016] In some embodiments, the epicatechin is administered orally, sublingually, subcutaneously, parenterally, intravenously, intranasally, topically, transdermally, intraperitoneally, intramuscularly, intrapulmonarily, vaginally, rectally, or intraocularly. In some embodiments, the epicatechin is administered orally or intravenously.

[0017] In some embodiments, the epicatechin is administered in the form of a tablet, a capsule, a powder, a liquid, a suspension, a suppository, or an aerosol.

[0018] In some embodiments, the epicatechin is administered at a daily dosage of about 5 to about 500 mg. In some embodiments, the daily dosage is administered in a single dose or in 2, 3, or 4 divided doses. In some embodiments, the epicatechin is administered each day, every other day, weekly, every two weeks, every three weeks, or every four weeks.

[0019] In some embodiments, the epicatechin is (+)-epicatechin. In some embodiments, the epicatechin is (-)-epicatechin. In some embodiments, the epicatechin comprises at least 75% (+)-epicatechin. In some embodiments, the epicatechin comprises at least 75% (-)- epicatechin.

[0020] In some embodiments, the epicatechin is administered in combination with one or more additional therapeutic agents. In some embodiments, the epicatechin and the one or more additional therapeutic agents are administered concurrently. In some embodiments, the epicatechin and the one or more additional therapeutic agents are administered sequentially. [0021] Also provided are kits and articles of manufacture comprising any one of the compositions described above and instructions for any one of the methods described above.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The drawings illustrate certain embodiments of the features and advantages of this disclosure. These embodiments are not intended to limit the scope of the appended claims in any manner.

[0023] FIGS. 1A-1E show the effects of EPM-01 (i.e., (+)-epicatechin; FIG. 1A), EPM-03 (z.e., (-)-epicatechin; FIG. IB), EPM-06 i.e., 11 -hydroxypregnenolone; FIG. 1C), EPM-07 i.e., 11- hydroxyprogesterone; FIG. ID), or catechin (FIG. IE) on NMDA-induced neurotoxicity. Values are expressed as percentage of control values and are from 4 independent experiments, each with 4-10 replicates per condition. **p< 0.01, ***p<0.001, ****p<0.0001

[0024] FIG. 2A shows an exemplary scheme of rat hippocampal circuitry.

[0025] FIG. 2B shows a representative transverse section of a rat hippocampal slice. The closed circle shows the stimulating electrode, the large rectangle shows the recording electrodes for fESPS, and the small rectangle shows the recording electrodes for population spikes.

[0026] FIGS. 2C-2D show that EPM-01 i.e., (+)-epicatechin) improved fESPS recovery following NMDA activation (FIG. 2C) or oxygen-glucose deprivation (OGD) (FIG. 2D). The statistics were performed using a two-way Anova with a Dunett’s multiple comparisons test. P -values were marked in the data presentations using the following notation: ns not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

[0027] FIGS. 3 A-3B show bodyweight (FIG. 3 A) and survival (FIG. 3B) of mice treated with EPM-01 (i.e., (+)-epicatechin).

[0028] FIGS. 4A-4C show weekly assessment of body condition score (FIG. 4A), clinical score (FIG. 4B), and neurological score (FIG. 4C) of R6/2 mice treated with EPM-01 (i.e., (+)-epicatechin). ***indicates p<0.001, ** indicates p<0.01 comparison of non-carrier vehicle vs R6/2 hemi-vehicle (Tukey’s multiple comparisons following two-way repeated measures ANOVA).

[0029] FIGS. 5A-5D show activity chamber evaluations, including distance moved (FIG.

5 A), vertical count (FIG. 5B), vertical time (FIG. 5C), and duration in the center of the chamber (FIG. 5D), of R6/2 mice treated with EPM-01 (i.e., (+)-epicatechin). *p< 05, ***p< 001, comparison of non-carrier-vehicle vs R6/2 hemi-vehicle Tukey’s post hoc comparison of means.

[0030] FIG. 6 shows rotarod evaluations of R6/2 mice treated with EPM-01 (z.e., (+)- epicatechin). Data represent average latency to fall across 3 trials performed on each of weeks 7, 9 and 11. **p<0.01, ***p<0.001, comparison of non-carrier-vehicle vs R6/2 hemi- vehicle Tukey’s post hoc comparison of means.

[0031] FIGS. 7A-7H show distance (FIG. 7A), velocity (FIG. 7B), active behavior duration (FIG. 7C), climbing (FIG. 7D), rearing (FIG. 7E), grooming (FIG. 7F), digging (FIG. 7G), and seizure duration (FIG 7H), of R6/2 mice treated with EPM-01 (z.e., (+)-epicatechin) during the novel cage observation test across weeks 6, 9, 12 and 13. *p<0.05, **p<0.01, ***p<0.001, comparison of non-carrier-vehicle vs R6/2 hemi-vehicle, Tukey’s post hoc comparison of means.

[0032] FIGS. 8A-8B show fear conditioning analysis of R6/2 mice treated with EPM-01 (z.e., (+)-epicatechin). Line graphs indicate % Freezing during Day 1 training and Day 2 context for each group in FIG. 8A and 8B. *p< 05; posthoc comparisons of group means, Tukey’s multiple comparisons following two-way ANOVA. *p<0.05, **p<0.01, comparison of non-carrier-vehicle vs R6/2 hemi-vehicle, Tukey’s post hoc comparison of means.

[0033] FIGS. 9A-9C show log2-fold change bar graphs (mean ± SEM, n=5) indicating changes in immune related proteins in the hippocampus of R6/2 mice treated with EPM-01 (z.e., (+)-epicatechin). Bars represent mean Log2 of fold-change between R6/2-vehicle (FIG.

9 A) relative to non-carrier-vehicle, R6/2-EPM-01 (FIG. 9B) relative to R6/2-vehicle, and R6/2-EPM-01 (FIG. 9C) relative to non-carrier-vehicle. *p < 0.05, **p < 0.01, ***p < 0.001; t-test between group means for relevant group comparisons within each protein.

DETAILED DESCRIPTION

[0034] Described herein are methods of treating or preventing diseases or disorders resulting from acute or chronic excessive glutamate exposure, excess calcium (Ca²⁺) levels, and/or reactive oxygen species, using epicatechin. Such methods are useful for the treatment of a variety of diseases or disorders, such as central nervous system (CNS) diseases or disorders and/or diseases or disorders associated with intracellular Ca²⁺ buildup.

[0035] The present application is based at least in part on the inventor’s finding decreasing Ca²⁺ levels via treatment with epicatechin, can be used to boost mitochondrial function rather than blocking the glutamate receptor outright. Through normalization of Ca²⁺ levels, toxic enzyme activation and cell death is prevented while preserving the normal and essential role of glutamate neurotransmission. The novel mechanism of epicatechin preserves neuronal function in the setting of glutamate neurotoxicity, with clear therapeutic implications for a spectrum of central nervous system (CNS) disorders. CNS diseases including seizure disorders, spinal cord injury, and neurodegeneration, may be amenable to epicatechin mitochondrial-targeted therapies, because of a shared mechanism of action unique to epicatechin.

[0036] Thus, the present application in one aspect provides a method of treating or preventing a condition related to acute or chronic excessive glutamate exposure in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of epicatechin or a pharmaceutically acceptable salt thereof.

I. Definitions

[0037] As used herein and in the appended claims, the singular forms “a”, “an” and “the” include plural forms, unless the context clearly dictates otherwise.

[0038] As used herein, and unless otherwise specified, the terms “about” and “approximately,” when used in connection with doses, amounts, molar percent, or weight percent of ingredients of a composition or a dosage form, mean a dose, amount, molar percent, or weight percent that is recognized by those of ordinary skill in the art to provide a pharmacological effect equivalent to that obtained from the specified dose, amount, molar percent, or weight percent. Specifically, the terms “about” and “approximately,” when used in this context, contemplate a dose, amount, molar percent, or weight percent within 15%, within 10%, within 5%, within 4%, within 3%, within 2%, within 1%, or within 0.5% of the specified dose, amount, molar percent, or weight percent.

[0039] As used herein, “therapeutically effective amount” indicates an amount that results in a desired pharmacological and/or physiological effect for the condition. The effect may be prophylactic in terms of completely or partially preventing a condition or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for the condition and/or adverse effect attributable to the condition.

[0040] As used herein, the term “pharmaceutically acceptable excipient,” and cognates thereof, refers to adjuvants, binders, diluents, etc. known to the skilled artisan that are suitable for administration to an individual (e.g., a mammal or non-mammal). Combinations of two or more excipients are also contemplated. The pharmaceutically acceptable excipient(s) and any additional components, as described herein, should be compatible for use in the intended route of administration (e.g., oral, parenteral) for a particular dosage form, as would be recognized by the skilled artisan.

[0041] As used herein, the term "co-crystal" denotes crystalline molecular complexes, encompassing hydrates and solvates. "Co-crystals" are composed of multi-component, stoichiometric and neutral molecular species, each existing as a solid under ambient conditions. Co-crystals exhibit properties different from free drugs or salts. The solid form influences relevant physico-chemical parameters such as solubility, dissolution rate of the drug, chemical stability, melting point, and hygroscopicity, which can result in solids with superior properties.

[0042] The terms “treat,” “treating,” and “treatment” are meant to include alleviating or abrogating a disorder, disease, or condition, or one or more of the symptoms associated with the disorder, disease, or condition; or to slowing the progression, spread or worsening of a disease, disorder or condition or of one or more symptoms thereof. Often, the beneficial effects that a subject derives from a therapeutic agent do not result in a complete cure of the disease, disorder or condition.

[0043] The term “subject” refers to an animal, including, but not limited to, a primate (e.g., human), monkey, cow, pig, sheep, goat, horse, dog, cat, rabbit, rat, or mouse. The terms “subject” and “patient” are used interchangeably herein in reference, for example, to a mammalian subject, such as a human.

[0044] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

[0045] It is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. [0046] The following statements provide a summary of some aspects of the inventive nucleic acids and methods described herein. II. Methods of use

[0047] The present application provides methods of using epicatechin. Epicatechin may boost mitochondrial function by increasing the availability of ATP, which can be used to pump excess calcium (Ca²⁺) out of the cell or into internal storage compartments. Through normalization of Ca²⁺ levels, toxic enzyme activation, and cell death can be prevented while preserving the normal and essential role of glutamate neurotransmission. As described in the working examples of the present disclosure, in live cells and tissues, epicatechin preserves neuronal function in the setting of glutamate neurotoxicity, with clear therapeutic implications for a spectrum of CNS disorders.

A. Epicatechin

[0048] Epicatechin is flavonoid that is a more active isomer of catechin. Catechin is a flavan-3-ol, a type of natural phenol and antioxidant. Epicatechin and catechin are abundant flavanols in fruits, chocolate, red wine, and tea. In some aspects, provided herein is a method comprising use of a therapeutically effective amount of epicatechin or a pharmaceutically acceptable salt thereof. The term “epicatechin” as used herein refers to epicatechin in its various forms, including but not limited to epicatechin, a co-crystal of epicatechin, a therapeutically effective amount of epicatechin, a pharmaceutically acceptable salt of epicatechin, a pharmaceutical composition comprising epicatechin, or any combination thereof.

[0049] The epicatechin of the present disclosure may promote neuronal survival. In some embodiments, the epicatechin is neuroprotective. In some embodiments, the epicatechin increases neuronal survival at concentrations greater than about 0.1 pM, such greater than any of about 10 pM, 0.1 nM, 1 nM, 2 nM, 5 nM, 10 nM, or greater. In some embodiments, the epicatechin increases neuronal survival at concentrations less than about 10 nM, such as less than any of about 5 nM, 2 nM, 1 nM, 0.1 nM, 10 pM, 0.1 pM, or less. In some embodiments, the epicatechin is neuroprotective against CNS disease, metabolic disease, mitochondrial disease, or any combination thereof. In some embodiments, the epicatechin is neuroprotective against stroke, Parkinson’s disease, Huntington's disease, oxygen deprivation (e.g., cardiac ischemia), ischemic penumbra, seizure conditions, and spinal cord injury.

[0050] In some embodiments, the epicatechin reduces the expression of anti-inflammatory markers. In some embodiments, the anti-inflammatory marker is IL-10, ILlb, or MCP.l [0051] In some embodiments, the epicatechin reduces seizures associated with disease. [0052] In some embodiments, the epicatechin reduces mortality in early phases of disease progression.

[0053] In some embodiments, the epicatechin increases neuronal survival against NMDA toxicity (e.g., neuronal excitotoxicity). In some embodiments, the epicatechin promotes sustained neuronal recovery. In some embodiments, the epicatechin accelerates recovery of neuronal membrane potential during neuronal excitotoxicity. In some embodiments, the epicatechin increases ATP availability during neuronal excitotoxicity. In some embodiments, the increased ATP availability is used to pump Ca²⁺ out of the neuron. In some embodiments, the increased ATP availability is used to pump Ca²⁺ into intracellular stores (e.g., the endoplasmic reticulum). In some embodiments, the pumping of Ca²⁺ prevents the activation of caspase. In some embodiments, the pumping of Ca²⁺ prevents the opening of the mitochondrial permeability transition pore (mPTP).

[0054] In some embodiments, the epicatechin stimulates an adaptive stress response. In some embodiments, the epicatechin promotes tissue regeneration.

[0055] In some embodiments, epicatechin is enantiomerically pure or enantiomerically enriched. In some embodiments, the epicatechin is enantiomerically pure or enantiomerically enriched (+) epicatechin. In other embodiments, the epicatechin is enatiomerically pure or enantiomerically enriched (-) epicatechin. The purity of the enantiomerically pure or enantiomerically enriched (+)/(-) epicatechin is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, or 100%. In some embodiments, the epicatechin comprises at least about 75% (+)-epicatechin, such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, or greater, (+)-epicatechin. In some embodiments, the epicatechin comprises at least about 75% (-)-epicatechin, such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, or greater, (-)-epicatechin.

[0056] In some embodiments, the epicatechin is in the form of a co-crystal. Co-crystals of epicatechin have been previously described, for example, in US2021/0380535, which is hereby incorporated by reference in its entirety. Co-crystals described herein can have a purity of at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 99.9%. In some embodiments, the co-crystal is a co-crystal of (+) epicatechin : trigonelline. In some embodiments, the co-crystal is a co-crystal of (-) epicatechin : trigonelline. In some embodiments, the co-crystal is a co-crystal of (+) epicatechin : D-proline. In some embodiments, the co-crystal is a co-crystal of (+) epicatechin : L-proline. B. Methods

[0057] In some aspects, provided herein is a method of treating or preventing a condition related to acute or chronic excessive glutamate exposure in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of epicatechin or a pharmaceutically acceptable salt thereof.

[0058] As used herein the phrase “excessive glutamate exposure” refers to an amount of glutamate that induces excessive stimulation of glutamate receptors. In some embodiments, the excessive glutamate exposure results from increased extracellular glutamate concentrations. In some embodiments, the excessive glutamate exposure results from excessive glutamate release from the presynaptic membrane. In some embodiments, the excessive glutamate exposure results from impaired glutamate reuptake function. In some embodiments, the excessive glutamate refers to glutamate levels that are increased by greater than any of about 0.05-fold, 0.1-fold, 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, or more, compared to the glutamate levels prior to exci totoxi city. In some embodiments, the excessive glutamate refers to glutamate levels that are increased by less than any of about 5-fold, 4- fold, 3-fold, 2-fold, 1-fold, 0.5-fold, 0.1-fold, 0.05-fold, or less, compared to the glutamate levels prior to excitotoxicity.

[0059] In some embodiments, the excessive glutamate exposure causes glutamate excitotoxicity. Glutamate excitotoxicity is the excessive stimulation of glutamate receptors, such as N-methyl-D-aspartate (NMDA) receptors, a-amino-3-hydroxy-5-methyl-4- isoxazol epropionic acid (AMP A) receptors, and/or kainate receptors. In some embodiments, glutamate excitotoxicity may induce neuronal degeneration, such as degeneration of dopaminergic neurons, thereby resulting in motor dysfunction. In some embodiments, the excessive glutamate exposure causes excessive stimulation of glutamate receptors.

[0060] In some embodiments, the excessive glutamate exposure increases intracellular sodium (Na²⁺) buildup. In some embodiments, the excessive glutamate exposure induces intracellular calcium (Ca²⁺) buildup. In some embodiments, the intracellular Ca²⁺ and/or Na²⁺ is increased by greater than any of about 0.05-fold, 0.1-fold, 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, or more, compared to the intracellular Ca²⁺ and/or Na²⁺ prior to the glutamate exposure. In some embodiments, the intracellular Ca²⁺ and/or Na²⁺ is increased less than about 5-fold, 4-fold, 3-fold, 2-fold, 1-fold, 0.5-fold, 0.1-fold, 0.05-fold, or less, compared to the intracellular Ca²⁺ and/or Na²⁺ prior to the glutamate exposure. In some embodiments, the intracellular Ca²⁺ and/or Na²⁺ buildup may cause neuronal damage. In some embodiments, the intracellular Ca²⁺ and/or Na²⁺ buildup may cause cell death.

[0061] The conditions for treatment or prevention related to acute or chronic excessive glutamate exposure may be any condition for which epicatechin is indicated, including, without limitation, any of the disease or conditions described in US 11,154,546, US 9,187,448, US 11,273,144, US2018/0193306, US 9,975,869, US 10,898,465, US2019/0262347, and US2021/0380535, each of which is hereby incorporated by reference in its entirety. In some embodiments, the condition is related to acute excessive glutamate exposure. In some embodiments, the condition is related to chronic excessive glutamate exposure.

[0062] In some embodiments, the condition is selected from the group consisting of status epilepticus, neuroinflammatory disorders, pediatric seizure disorders, neuronal exocitoxicity, over activation of the NMDA receptor, post-operative syndromes of cognition loss, and loss of synaptic density.

[0063] In other aspects, provided herein is a method of decreasing reactive oxygen species (ROS) in a subject need thereof, comprising administering to the subject a therapeutically effective amount of epicatechin or a pharmaceutically acceptable salt thereof. The ROS may be any ROS known in the art. In some embodiments, the ROS comprises alpha-oxygen (a- O).In some embodiments, the ROS comprises superoxide (O’ U). In some embodiments, the ROS comprises a hydroxyl radical (OH’). In some embodiments, the ROS comprises a hydrogen peroxide (H2O2). In some embodiments, the ROS comprises singlet oxygen (¹O2). In some embodiments, the ROS comprises a combination of superoxide, hydroxyl radical, hydrogen peroxide, singlet oxygen, and/or alpha-oxygen. In some embodiments, the ROS is a result of acute or chronic excessive glutamate exposure. In some embodiments, the ROS is a result of acute or chronic exposure to intracellular Ca²⁺.

[0064] In some embodiments, the subject in need of a method of decreasing ROS has a disease or disorder. The disease or disorder may be any disease or disorder for which epicatechin is indicated, including, without limitation, any of the disease or conditions described in US 11,154,546, US 9,187,448, US 11,273,144, US2018/0193306, US 9,975,869, US 10,898,465, US2019/0262347, and US2021/0380535, each of which is hereby incorporated by reference in its entirety. In some embodiments, the subject has a disease or disorder selected from the group consisting of spinal cord injury or abnormality, liver disease, kidney disease, impaired cognition, neurodegenerative disease, dystonia, sarcopenia, cardiomyopathy of aging or other diseases associated with mitochondrial dysfunction, cardiomyopathy, ischemic vascular disease, immunodeficiency states, ataxia, pulmonary inflammation and fibrosis, infantile encephalomyopathy, epilepsy, Charcot-Marie-Tooth disease, exocrine pancreatic insufficiency, impaired wound healing, and growth of cancer cells. In some embodiments, the subject has a neurodegenerative disease selected from the group consisting of Alzheimer’s disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), Leigh syndrome, Duchenne muscular dystrophy, Becker muscular dystrophy, and peripheral and central neuropathies.

[0065] In additional aspects, provided herein is a method of treating a condition related to acute or chronic exposure to intracellular Ca²⁺ in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of epicatechin or a pharmaceutically acceptable salt thereof. In some embodiments, the exposure to intracellular Ca²⁺ exposure results from excessive glutamate exposure. In some embodiments, the exposure to intracellular Ca²⁺ exposure results from excessive glutamate release from the presynaptic membrane. In some embodiments, the exposure to intracellular Ca²⁺ exposure results from impaired glutamate reuptake function. In some embodiments, the condition is related to acute exposure to intracellular Ca²⁺. In some embodiments, the condition is related to chronic exposure to intracellular Ca²⁺. In some embodiments, the condition may be any condition related to acute or chronic exposure to intracellular Ca²⁺. In some embodiments, the acute or chronic exposure to intracellular Ca²⁺is caused by excessive glutamate exposure. In some embodiments, the excessive glutamate exposure is acute excessive glutamate exposure. In some embodiments, the excessive glutamate exposure is chronic excessive glutamate exposure. In some embodiments, the condition is neuronal excitotoxicity. In some embodiments, the condition is glutamate excitotoxicity.

[0066] In other aspects, provided herein is a method of lowering intracellular Ca²⁺ levels in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of epicatechin or a pharmaceutically acceptable salt thereof. In some embodiments, the Ca²⁺ levels are reduced by greater than any of about 0.05-fold, 0.1-fold, 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, or more, compared to the intracellular Ca²⁺ prior to performing a method described herein. In some embodiments, the Ca²⁺ levels are reduced by less than any of about 5-fold, 4-fold, 3-fold, 2-fold, 1-fold, 0.5-fold, 0.1-fold, 0.05-fold, or less, compared to the intracellular Ca²⁺ prior to performing a method described herein.

[0067] In some embodiments, the subject in need of a method of lowering intracellular Ca²⁺ levels has a disease or disorder. The disease or disorder may be any disease or disorder for which epicatechin is indicated, including, without limitation, any of the disease or conditions described in US 11,154,546, US 9,187,448, US 11,273,144, US2018/0193306, US 9,975,869, US 10,898,465, US2019/0262347, and US2021/0380535, each of which is hereby incorporated by reference in its entirety. In some embodiments, the subject has a disease or disorder selected from the group consisting of spinal cord injury or abnormality, liver disease, kidney disease, impaired cognition, neurodegenerative disease, dystonia, sarcopenia, cardiomyopathy of aging or other diseases associated with mitochondrial dysfunction, cardiomyopathy, ischemic vascular disease, immunodeficiency states, ataxia, pulmonary inflammation and fibrosis, infantile encephalomyopathy, epilepsy, Charcot-Marie-Tooth disease, exocrine pancreatic insufficiency, impaired wound healing, and growth of cancer cells. In some embodiments, the subject has a neurodegenerative disease selected from the group consisting of Alzheimer’s disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), Leigh syndrome, Duchenne muscular dystrophy, Becker muscular dystrophy, and peripheral and central neuropathies.

III. Pharmaceutical compositions and dosage

[0068] According to the methods described herein, the epicatechin or pharmaceutical composition thereof may be administered at a therapeutically effective dosage, e.g., a dosage sufficient to provide treatment for the disease state. While human dosage levels have yet to be optimized for the chemical entities described herein (e.g., epicatechin, co-crystals of epicatechin, etc.), generally, a daily dose ranges from about 0.01 to 100 mg/kg of body weight; in some embodiments, from about 0.05 to 10.0 mg/kg of body weight, and in some embodiments, from about 0.10 to 1.4 mg/kg of body weight. Thus, for administration to a 70 kg person, in some embodiments, the dosage range would be about from 0.7 to 7000 mg per day; in some embodiments, about from 3.5 to 700.0 mg per day, and in some embodiments, about from 7 to 100.0 mg per day.

[0069] In some embodiments, the epicatechin is administered each day, every other day, weekly, every two weeks, every three weeks, or every four weeks. The amount of the chemical entity administered will be dependent, for example, on the subject and disease state being treated, the severity of the affliction, the manner and schedule of administration and the judgment of the prescribing physician. For example, an exemplary dosage range for oral administration is from about 5 mg to about 500 mg per day, and an exemplary intravenous administration dosage is from about 5 mg to about 500 mg per day, each depending upon the pharmacokinetics. In some embodiments, the epicatechin is administered at a daily dosage of between about 5 mg to about 500 mg, such as between about 5 mg and about 100 mg, about 50 mg and about 200 mg, about 100 mg and about 300 mg, about 200 mg and about 400 mg, or about 300 mg and about 500 mg. In some embodiments, the epicatechin is administered at a daily dosage of less than about 500 mg, such as less than any of about 450 mg, 400 mg, 350 mg, 300 mg, 250 mg, 200 mg, 150 mg, 100 mg, 50 mg, 40 mg, 30 mg, 20 mg, 10 mg, 5 mg, or less. In some embodiments, the epicatechin is administered at a daily dosage of greater than about 5 mg, such as greater than any of about 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, or greater, [0070] A daily dose is the total amount administered in a day. A daily dose may be, but is not limited to be, administered each day, every other day, each week, every 2 weeks, every month, or at a varied interval. In some embodiments, the daily dose is administered for a period ranging from a single day to the life of the subject. In some embodiments, the daily dose is administered once a day. In some embodiments, the daily dose is administered in multiple divided doses, such as in 2, 3, or 4 divided doses. In some embodiments, the daily dose is administered in 2 divided doses.

[0071] Administration of the epicatechin (e.g., epicatechin, a pharmaceutical composition comprising the epicatechin, and/or a co-crystal of the epicatechin) can be via any accepted mode of administration for therapeutic agents including, but not limited to, oral, sublingual, subcutaneous, parenteral, intravenous, intranasal, topical, transdermal, intraperitoneal, intramuscular, intrapulmonary, vaginal, rectal, or intraocular administration. In some embodiments, the epicatechin or pharmaceutical composition thereof is administered orally, sublingually, subcutaneously, parenterally, intravenously, intranasally, topically, transdermally, intraperitoneally, intramuscularly, intrapulmonarily, vaginally, rectally, or intraocularly. In some embodiments, the epicatechin or pharmaceutical composition thereof is administered orally or intravenously. In some embodiments, the epicatechin or pharmaceutical composition thereof is administered orally.

[0072] Pharmaceutically acceptable compositions include solid, semi-solid, liquid and aerosol dosage forms, such as tablet, capsule, powder, liquid, suspension, suppository, and aerosol forms. In some embodiments, the epicatechin is administered in the form of a tablet, a capsule, a powder, a liquid, a suspension, a suppository, or an aerosol. The epicatechin or pharmaceutical composition thereof can also be administered in sustained or controlled release dosage forms (e.g., controlled/sustained release pill, depot injection, osmotic pump, or transdermal (including electrotransport) patch forms) for prolonged timed, and/or pulsed administration at a predetermined rate. In some embodiments, the epicatechin or pharmaceutical composition thereof are provided in unit dosage forms suitable for single administration of a precise dose.

[0073] The epicatechin or pharmaceutical composition thereof can be administered either alone or in combination with one or more conventional pharmaceutical carriers or excipients (e.g., mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, sodium crosscarmellose, glucose, gelatin, sucrose, magnesium carbonate). If desired, the pharmaceutical composition can also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifying agents, solubilizing agents, pH buffering agents and the like (e.g., sodium acetate, sodium citrate, cyclodextrine derivatives, sorbitan monolaurate, triethanolamine acetate, triethanolamine oleate). Generally, depending on the intended mode of administration, the pharmaceutical composition will contain about 0.005% to 95%, or about 0.5% to 50%, by weight of a compound disclosed and/or described herein. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania.

[0074] In some embodiments, the epicatechin or pharmaceutical composition thereof will take the form of a pill or tablet and thus the composition may contain, along with epicatechin (e.g., an epicatechin co-crystal), one or more of a diluent (e.g., lactose, sucrose, dicalcium phosphate), a lubricant (e.g., magnesium stearate), and/or a binder (e.g., starch, gum acacia, polyvinylpyrrolidine, gelatin, cellulose, cellulose derivatives). Other solid dosage forms include a powder, marume, solution or suspension (e.g., in propylene carbonate, vegetable oils or triglycerides) encapsulated in a gelatin capsule.

[0075] Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing or suspending etc. a co-crystal disclosed and/or described herein and optional pharmaceutical additives in a carrier (e.g., water, saline, aqueous dextrose, glycerol, glycols, ethanol or the like) to form a solution or suspension. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, as emulsions, or in solid forms suitable for dissolution or suspension in liquid prior to injection. The percentage of the epicatechin contained in such parenteral compositions depends, for example, on the physical nature of the co-crystal, the activity of the epicatechin, and the needs of the subject. However, percentages of active ingredient of 0.01% to 10% in solution are employable, and may be higher if the composition is a solid which will be subsequently diluted to another concentration. In some embodiments, the composition will comprise from about 0.2 to 2% of epicatechin in solution. [0076] Pharmaceutical compositions of epicatechin may also be administered to the respiratory tract as an aerosol or solution for a nebulizer, or as a microfine powder for insufflation, alone or in combination with an inert carrier such as lactose. In such a case, the particles of the pharmaceutical composition may have diameters of less than 50 microns, or in some embodiments, less than 10 microns.

[0077] In addition, pharmaceutical compositions can include epicatechin, epicatechin cocrystals, and one or more additional medicinal agents, pharmaceutical agents, adjuvants, and the like. In some embodiments, the epicatechin is administered in the form of a pharmaceutical composition comprising the epicatechin or the pharmaceutically acceptable salt, solvate, or co-crystal thereof, and a pharmaceutically acceptable excipient.

[0078] The epicatechin may be administered in combination with one or more additional therapeutic agents. In some embodiments, the epicatechin and the one or more additional therapeutic agents are administered concurrently. In some embodiments, the epicatechin and the one or more additional therapeutic agents are administered sequentially.

IV. Kits

[0079] Also provided are articles of manufacture and kits containing epicatechin, such as epicatechin, a co-crystal of epicatechin, or pharmaceutical compositions thereof, provided herein. The article of manufacture may comprise a container with a label. Suitable containers include, for example, bottles, vials, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. The container may hold a pharmaceutical composition provided herein. The label on the container may indicate that the pharmaceutical composition is used for preventing, treating or suppressing a condition described herein, and may also indicate directions for either in vivo or in vitro use.

[0080] In one aspect, provided herein are kits containing epicatechin, such as epicatechin, a co-crystal of epicatechin, or pharmaceutical compositions thereof, described herein and instructions for use. The kits may contain instructions for use in the treatment of any disorder, disease, or condition, provided herein in a subject in need thereof. A kit may additionally contain any materials or equipment that may be used in the administration of the epicatechin, such as epicatechin, a co-crystal of epicatechin, or pharmaceutical compositions thereof, such as vials, syringes, or IV bags. A kit may also contain sterile packaging. EXAMPLES

[0081] The invention will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the invention. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

Example 1: Epicatechin blocks NMDA induced neuronal toxicity in primary cortical neurons

[0082] This Example shows that (+)-epicatechin (z.e., EPM-01) and (-)-epicatechin (z.e., EPM-03) block N-methyl-D-aspartate (NMDA) induced neuronal toxicity in vitro in primary cultures of rat cortical neurons.

[0083] An in vitro model of glutamate exci totoxi city associated with prolonged Ca²⁺ elevation and cell death was used. Primary cortical neurons were prepared from El 7 Sprague Dawley rat embryos. The brain cortices were dissected into Brooks Logan Solution containing IxPBS, sucrose, glucose and HEPES buffer. Neurons were dissociated with lx trypsin and plated in PDL-coated 96-well plates at a density of 50k per well. Three days later, 5-fluoro-2'-deoxyuridine (30 pM) was added to prevent glial cell proliferation. The cells were maintained in Neurobasal medium supplemented with B27, 2 mM glutamine, 25 mM glucose, and antibiotic solution consisting of penicillin (100 U/mL) and streptomycin (100 mg/mL), in a humidified atmosphere at 37 °C, with 5% CO2. Half-media changes were performed every 4-5 days, or as required. All experiments were conducted at 12-14 DIV.

[0084] Neurons were pretreated on DIV 12 to 14 with either EPM-01 (z.e., (+)- epicatechin), EPM-03 (z.e., (-)-epicatechin), EPM-06 (z.e., 11 -hydroxypregnenolone), EPM- 07 (z.e., 11- hydroxyprogesterone), or catechin, at various concentrations for 30 minutes. Neurons were then exposed to 20 or 25 uM of NMDA, determined by a dose response curve, for 30 minutes. After NMDA exposure, media with test compounds was replaced and the cells were allowed to recover for 18-24 hours. To assess cell death, CellTiter-Glo Luminescent Cell Viability Assay (Promega) was used. Luminescence was measured according to manufacturer (BMG LABTECH CLARIOstar) instructions.

[0085] As shown in FIGS. 1 A-1E, the addition of NMDA caused a decrease in neuronal survival. Primary cortical neurons treated with EPM-01 at 10 pM, 1 nM, and 10 nM showed increased survival, compared to vehicle treated neurons (FIG. 1 A). Primary cortical neurons treated with EPM-03 at 0.1 pM, 10 pM, and 0.1 nM showed increased survival, compared to vehicle treated neurons (FIG. IB). EPM-06, EPI-07, and catechin treatment did not increase survival in primary cortical neurons exposed to NMD A (FIGS. 1C-1E).

Example 2: Epicatechin improves fESPS recovery following NMDA activation or oxygen-glucose deprivation (OGD) ex vivo

[0086] This Example shows that epicatechin improves field excitatory postsynaptic potential (fESPS) in a translational model exci totoxi city using brain slices from rats. [0087] Brain slices (FIGS. 2A-2B) were exposed to NMDA or oxygen-glucose deprivation (OGD), either in the presence or absence of EPM-01 (z.e., (+)-epicatechin), according to standard protocols. The OGD model leads to lack of ATP production and opening of the glutamate receptors and channels, resulting in neuronal cell death. Field excitatory postsynaptic potential (fESPS) was measured to determine how epicatechin influences ion transport required to reset the neuron back to its resting membrane potential.

[0088] The fEPSP amplitude was measured as the difference between the baseline (before stimulation) and the maximal peak amplitude. The population spike was measured as peak to peak amplitude. To be validated, fEPSPs and population spikes needed to be stable (less than 15% fluctuation) and larger than 100 pV during the baseline. Data from each electrode in a given slice were averaged and the slice was considered as an individual sample (n). The distribution of raw amplitudes between groups was plotted as a scatter plot. The normalized amplitude values (± SEM) were plotted as a function of time. The percentage of change over the last 2 minutes of each period of vehicle or compound application (in regards to the baseline period) were plotted in a scatter plot with each slice considered as an individual sample (n).

[0089] As shown in FIGS. 2C-2D, while both NMDA and OGD caused neurons to remain depolarized the presence of 0.01, 0.1, and 1 pM epicatechin enabled neurons to return closer to resting membrane potential. Strikingly, 0.1 pM epicatechin almost returned fESPS back to normal after 20-30 mins.

[0090] Taken together, these data demonstrate that epicatechin can accelerate acute recovery of neuronal membrane potential during glutamate-induced exci totoxi city probably by promoting mitochondrial health and bioenergetic function. The increased ATP availability during glutamate-induced excitotoxicity can be used to pump Ca²⁺ out of the neuron or into intracellular stores, such as the endoplasmic reticulum. Such activity may prevent activation of intracellular proteases, including caspase, and opening of the mitochondrial permeability transition pore (mPTP).

Example 3: Epicatechin protects against neuroinflammatory disorders in vivo in a mouse model

[0091] This Example shows the effects of epicatechin (EPM-01) treatment on mice with neuroinflammatory disorders. In particular, R6/2 mice dosed with EPM-01 had lower mortality than vehicle-dosed R6/2 mice. EPM-01 had limited effects on behavior of R6/2 mice, but seemed to reduce seizures in R6/2 mice and to reduce freezing in non-carrier controls. EPM-01 displayed anti-inflammatory properties in R6/2 mice brains compared to the vehicle treated controls suggesting its utility and anti-inflammatory mode of action for the treatment of central nervous system (CNS) disorders.

[0092] The effects of epicatechin (EPM-01) treatment on disease progression and behavior in the Activity Chamber, Rotarod, Novel Cage Observation, and Fear Conditioning in the R6/2 transgenic mouse model of Huntington's disease, compared to vehicle treated R6/2 and wild type (WT) littermates were evaluated.

[0093] Briefly, four to five weeks old male, R6/2 mice from Jackson Laboratory (Stock# 006494; B6CBA-Tg(HDexonl)62Gpb/3J) and wild-type (WT) littermates were used in the study. The experimental groups are described in Table 1. A baseline Activity Chamber and body weight were conducted at 5 weeks old to randomize the mice into n=15 x 4 different groups. Mice received daily treatment of EPM-01 (z.e., (+)-epicatechin) (3 mg/kg, Per Oral) or vehicle (0.5% Carboxymethylcellulose sodium salt in water) starting at 5 weeks old until 16 weeks old for tissue collection. Behavioral tests included Activity Chamber (AC), Rotarod (RR), Novel Cage Observation (NCO) and Fear Conditioning (FC). In addition, weekly Body Weight, Clinical Score, Body Condition Score, Neurological Score and Survival follow up were performed throughout the study. Tissue samples were collected at the end of study for ex vivo analysis. Brain, plasma, heart, liver, lungs, spleen, gastrocnemius muscle, kidneys, and testes were collected. Table 1, below outlines the experimental design.

Table 1. Experimental design.

A. Assays

(i) Body weight and survival

[0094] Body weight was assessed weekly during the study.

[0095] Chronic dosing with EPM-01 had no impact on body weight loss across disease progression in R6/2 mice (FIG. 3 A). The Survival graph indicates that while EPM-01 delayed mortality, with a survival curve approaching significance (p=.O7) at 70 days, by day 76, EPM-01 -dosed R6/2 and vehicle-dosed R6/2 groups had similar mortality rates (FIG. 3B).

(ii) Body condition, clinical score, and neurological score

[0096] Body condition, clinical score, and neurological were assessed weekly during the study.

[0097] Body Condition Score was assessed according to the following scale: 1 = emaciated mice (skeletal structure extremely prominent, vertebrae distinctly segmented); 2 = underconditioned mice (segmentation of the vertebral column is evident and the dorsal pelvic bones are easily palpable); 3 = well -conditioned mice (the vertebrae and dorsal pelvis are not prominent and are palpable with slight pressure); 4 = over-conditioned mice (spine is a continuous column and the vertebrae are palpable only with firm pressure); 5 = obese mice. [0098] Clinical Score was assessed according to the following scale: 0 = Normal Gait; 0.5 = slight dragging of knuckles (at least 2x during circling of arena); 1 = dragging feet/knuckles; 1.5 = single leg extremely weak/ limp (little to no use for walking); 2 = weakness/limpness in 2 hind limbs; 3 = single leg paralysis; 4 = 2 legs paralysis; 5 = advanced paralysis or cannot up-right in 20 sec.

[0099] Neurological Score was assessed according to the following scale: 0 = Full extension (WT picture) of hind legs away from lateral midline when suspended by tail, hold for 2 sec., suspended 2-3 times; 0.5 = Shaking; 1 = Collapse or partial collapse of leg extension (R6/2 picture) toward midline (weakness).

[0100] Disease progression in R6/2 mice was detected by neurological score as early as 9- 10 weeks of age (FIG. 4C). Body condition score (FIG. 4A) and clinical score (FIG. 4B) were not as sensitive to disease progression.

(Hi) Activity chamber assay

[0101] The Activity Chamber is used to determine general activity levels, gross locomotor activity, and exploration habits in rodents. The assessment took place in an Open Field Activity Arena (Med Associates Inc., St. Albans, VT. Model ENV-515) mounted with three planes of infrared detectors, within a specially designed sound-attenuating chamber (Med Associates Inc., St. Albans, VT. MED-017M-027). The arena is 43cm (L) x 43cm (W) x 30cm (H) and the sound-attenuating chamber is 74cm (L) x 60cm (W) x 60cm (H). The mice were placed in the comer of the testing arena and allowed to explore the arena for 10 minutes while being tracked by an automated tracking system. Parameters including Distance moved, velocity, rearing, and times spent in periphery and center of the arena were analyzed. The periphery was defined as the zone 5cm away from the arena wall. The arena was cleaned with a 1% Virkon solution at the end of each trial. AC was conducted at 5, 8 and 11 weeks postdosing.

[0102] R6/2 mice have deficits in Distance Moved, Vertical Count, Vertical Time, and Duration in the Center of the Arena as detected in the Activity Chamber (FIGS. 5A-5D).

(iv) Rotarod

[0103] The Rotarod test is designed to evaluate the motor coordination and balance of mice by allowing the mice to locomote on a rotating rod (Med Associate Inc, Model ENV-575M) elevated 16.5cm above the testing floor. The experiment consisted of one day of training and one day of testing. On the training day, mice were placed on the rod at a fixed speed of 4rpm for 1 minute. They were returned to the home cage after training. On the testing days, the mice were tested with an accelerating speed of 4-40rpm for maximum duration of 5 minutes. They were tested for 3 trials per day with 15-20 minutes inter-trial-intervals (ITIs). A 4th trial was tested on the mice that hold onto the rod for 2 consecutive revolutions or falls within 5 seconds of the start of a trial. Latencies to fall from the rod from 3 highest trials during the testing days were recorded. The apparatus was cleaned with 1% Virkon between trials. Mice were tested in Rotarod at 7, 9 and 11 weeks postdose. [0104] Reduced latency to fall in R6/2 mice across the Rotarod behavioral testing sessions (FIG. 6).

(v) Novel cage observation

[0105] The Novel Cage Observation (NCO) is an assessment used to determine general activity levels, locomotion activity and exploration habits in a novel cage environment. NCO was conducted at 6, 9, 12, and 13 weeks post-dose. Each mouse was placed in the center of a clean cage and allowed to freely move while tracked by an automated tracking system Ethovision XT software. The distance moved and average velocity were acquired over the 5 minutes trial. An experimenter blind to the treatment groups assessed other parameters such as digging, grooming, rearing, climbing, and seizure duration. All parameters were assessed as duration of bouts (start-stop behaviors) and are defined as follows: Digging: disturbance of cage bedding with either nose of front paws; Grooming: licking of any body part (paws, head, body, legs, tail and genitals); Rearing: standing on hind legs either in a protected (wallleaning) or unprotected (front paws in the air) manner; Climbing: attempts by subject to climb onto rim of cage by jumping from the reared position and time spent walking along rim of cage if jumping attempt is successful; and, Seizure Duration: cumulative time spent during seizure.

[0106] A behavioral phenotype for R6/2 mice in novel cage observation was observed across multiple individual measures (FIGS. 7A-7G). Comparison of R6/2 mice dosed with EPM-01 relative to R6/2 mice dosed with vehicle was not significant for any of the measures. [0107] Regarding seizure duration (FIG. 7H), at 13 weeks average seizure duration was elevated in R6/2 vehicle-dosed mice but not R6/2 EPM-01 -dosed mice relative to non-carrier vehicle dosed mice. This effect is likely due to a greater percentage of mice in the R6/2 vehicle-dosed group (5 out of 12; 41.7%) having seizures than in the R6/2 EPM-01 dosed group (2 out of 13; 15.4%). No mice had seizures in the non-carrier groups.

(vi) Fear conditioning assay

[0108] The Fear Conditioning (FC) assay is an associative learning task in which mice learn to associate a particular neutral Conditional Stimulus (CS; a context) with an aversive Unconditional Stimulus (US; a mild electrical foot shock) and show a Conditional Response (CR; freezing). The experiment consisted of 1 day of Training and 1 day of Contextual testing. The chamber walls were made of aluminum; the floor was a gray metal grid through which the US (shock) was delivered. The conditioning was conducted under yellow light and the chamber was scented with mint extract to produce a unique smell. The chambers were cleaned with a 10% simple green solution (Sunshine Makers, Inc.) between each mouse. Chambers were mounted on the wall and includes an exhaust fan and camera. On Day 1 Training, mice were placed individually into a training chamber for 90 seconds. Three electrical shocks (intensity 0.5mA, 2 seconds) were presented 90s apart and mouse was removed from the chamber 90s after the last shock. On Day 2, the mice were placed back into the training chamber without any shock for contextual memory testing for 5 minutes.

Coulbourn Instruments (Holliston, MA, USA) fear conditioning system and FreezeFrame software were used for analysis. The FC test was conducted at 8 weeks post-dose.

[0109] In the fear conditioning test (FIGS. 8A-8B), R6/2 mice show reduced freezing to context on Day 2 (FIG. 8B). EPM-01 reduced freezing to context in non-carrier mice, with no further effect in R6/2 mice.

(vii) Neuroinflammation - Luminex cytokine assay

[0110] Tissue cytokines were analyzed in brain homogenate from hippocampal-containing dissections from frozen coronal sections spanning the rostrocaudal extent of the hippocampus, using a Luminex 48-plex mouse cytokine assay. The Luminex assay was performed in the Human Immune Monitoring Center at Stanford University, following manufacturer instructions. Briefly, hippocampal tissue was homogenized in lysis buffer containing proteinase inhibitor by pulling tissue through a 23 g needle (15x) and then sonicated for 3x3 second pulses. Homogenate was spun at 14,000 g for 10 minutes, and protein concentrations were determined by Pierce BCA assay. Samples were diluted to a common concentration of 6 ug/uL. Brain homogenate samples were run in singlets on a 96 well plate alongside standard curve and quality control calibration samples.

[0111] As shown in FIGS. 9A-9C, EPM-01 blunted the induction of MCP1/CCL3 seen in the R6/2 mice and blunted the reduction in IL- 10 seen in the R6/2 mice. EPM-01 also reduced IL-ip in R6/2 mice relative to vehicle-treated R6/2.

B. Conclusions

[0112] R6/2 mice dosed with EPM-01 had lower mortality than vehicle-dosed R6/2 mice, with an effect that approached significance at 70 days (p = 0.07), but not at study termination at 76 days. R6/2 mice had impaired behavioral phenotypes in Activity Chamber, Rotarod and Novel Cage Observation and Fear Conditioning. EPM-01 had limited effects on behavior, including a tendency to reduce seizures in R6/2 mice and to reduce freezing in non-carrier controls. Several markers of neuroinflammation in R6/2 mice were attenuated with EPM-01 treatment. Example 4: Epicatechin mitigates disease phenotypes in CNS and neurodegenerative diseases of acute glutamate toxicity

[0113] Phase II proof-of-concept trials are conducted with epicatechin in the clinic. These trials validate the preclinical findings that epicatechin is effective in promoting neurological recovery. The efficacy of epicatechin in one or more central nervous system (CNS) diseases of acute glutamate toxicity, examples of which include head trauma, spinal cord injury, pediatric seizure disorders, and post-operative syndromes of cognition loss, is determined.

[0114] Neurodegenerative diseases in which chronic glutamate toxicity has been implicated in disease progression, examples of which include Parkinson’s disease, ALS, and Alzheimer’s disease, may also be evaluated in the clinic.

Claims

1. A method of treating or preventing a condition related to acute or chronic excessive glutamate exposure in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of epicatechin or a pharmaceutically acceptable salt thereof.

2. The method of claim 1, wherein the glutamate exposure induces intracellular calcium (Ca²⁺) buildup.

3. The method of claim 1 or 2, wherein the condition is selected from the group consisting of status epilepticus, neuroinflammatory disorders, pediatric seizure disorders, neuronal exocitoxicity, over activation of the N-methyl-D-aspartate (NMD A) receptor, postoperative syndromes of cognition loss, and loss of synaptic density.

4. A method of decreasing reactive oxygen species (ROS) in a subject need thereof, comprising administering to the subject a therapeutically effective amount of epicatechin or a pharmaceutically acceptable salt thereof.

5. A method of treating a condition related to acute or chronic exposure to intracellular calcium (Ca²⁺) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of epicatechin or a pharmaceutically acceptable salt thereof.

6. The method of claim 5, wherein the condition is neuronal exci totoxi city.

7. A method of lowering intracellular Ca²⁺ levels in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of epicatechin or a pharmaceutically acceptable salt thereof.

8. The method of claim 7, wherein the subject has a disease or disorder selected from the group consisting of spinal cord injury or abnormality, liver disease, kidney disease, impaired cognition, neurodegenerative disease, dystonia, sarcopenia, cardiomyopathy of aging or other diseases associated with mitochondrial dysfunction, cardiomyopathy, ischemic vascular disease, immunodeficiency states, ataxia, pulmonary inflammation and fibrosis, infantile encephalomyopathy, epilepsy, Charcot-Marie-Tooth disease, exocrine pancreatic insufficiency, impaired wound healing, and growth of cancer cells.

9. The method of claim 8, wherein the subject has a neurodegenerative disease selected from the group consisting of Alzheimer’s disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), Leigh syndrome, Duchenne muscular dystrophy, Becker muscular dystrophy, and peripheral and central neuropathies.

10. The method of any one of claims 1-9, wherein the epicatechin is administered in the form of a pharmaceutical composition comprising the epicatechin or the pharmaceutically acceptable salt, solvate, or co-crystal thereof, and a pharmaceutically acceptable excipient.

11. The method of any one of claims 1-10, wherein the epicatechin is in the form of a cocrystal.

12. The method of any one of claims 1-11, wherein the epicatechin is administered orally, sublingually, subcutaneously, parenterally, intravenously, intranasally, topically, transdermally, intraperitoneally, intramuscularly, intrapulmonarily, vaginally, rectally, or intraocularly.

13. The method of any one of claims 1-11, wherein the epicatechin is administered orally or intravenously.

14. The method of any one of claims 1-13, wherein the epicatechin is administered in the form of a tablet, a capsule, a powder, a liquid, a suspension, a suppository, or an aerosol.

15. The method of any one of claims 1-14 wherein the epicatechin is administered at a daily dosage of about 5 to about 500 mg.

16. The method of claim 15, wherein the daily dosage is administered in a single dose or in 2, 3, or 4 divided doses.

17. The method of any one of claims 1-16, wherein the epicatechin is administered each day, every other day, weekly, every two weeks, every three weeks, or every four weeks.

18. The method of any one of claims 1-17, wherein the epicatechin is (+)-epicatechin.

19. The method of any one of claims 1-17, wherein the epicatechin is (-)-epicatechin.

20. The method of any one of claims 1-17, wherein the epicatechin comprises at least 75% (+)-epicatechin.

21. The method of any one of claims 1-17, wherein the epicatechin comprises at least 75% (-)-epicatechin.

22. The method of any one of claims 1-21, wherein the epicatechin is administered in combination with one or more additional therapeutic agents.

23. The method of claim 22, wherein the epicatechin and the one or more additional therapeutic agents are administered concurrently.

24. The method of claim 22, wherein the epicatechin and the one or more additional therapeutic agents are administered sequentially.