WO2022214964A2 - Selective connexin hemichannel blockers as therapeutic compounds and methods for treating inflammatory diseases - Google Patents

Abstract

Provided herein are hemichannel-selective inhibitors and stabilized compositions thereof. Also provided are methods of stabilizing hemichannel-selective inhibitor compounds. Also provided are methods of making, methods of using, and methods of dosing the foregoing. Methods of screening for hemichannel-selective inhibitors using engineered cells are also described herein.

Description

SELECTIVE CONNEXIN HEMICHANNEL BLOCKERS AS THERAPEUTIC COMPOUNDS AND METHODS FOR TREATING INFLAMMATORY DISEASES

SPECIFICATION

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority benefit from U.S. Provisional Application No. 63/171,507 filed April 6, 2021, entitled “SELECTIVE CONNEXIN HEMICHANNEL BLOCKERS AS THERAPEUTIC COMPOUNDS AND METHODS FOR TREATING INFLAMMATORY DISEASES,” the contents of which are incorporated herein by reference in their entireties for all purposes.

BACKGROUND OF THE INVENTION

[0002] Connexin-based channels represent the hallmark of intercellular communication in eukaryotic systems. The connexin gene family comprises several connexin proteins (Cx proteins) in chordate animals, with about 20 connexin isoforms in humans. Connexin proteins assemble into hexameric transmembrane structures called connexons, which can be homohexameric (comprised of six identical connexin proteins) or hetero-hexameric (comprised of a mixture of different connexin proteins). These connexons, or hemichannels, provide channels between the cytosol and the extracellular space that allow passage of ions and small molecules, with permeability and selectivity that are dependent on the hemichannel connexin composition. Alternatively, a hemichannel can dock to a second hemichannel embedded in a nearby cell membrane, forming a gap junction between adjacent cells and allowing transfer of substances between the adjoined cells.

[0003] In addition to a well-documented role for the maintenance of homeostasis between cells forming tissues and organs, connexin-based channels play an important role as mediators of the cellular inflammation process. In general, hemichannels tend to rest in a closed state to prevent loss of critical analytes to the extracellular space, opening selectively in response to specific signals such as membrane depolarization or divalent cation concentration. Dysregulation of hemichannel activity, particularly overactivation (an increase in function of hemichannels, or an increase in the expression of functional hemichannels) can have a variety of detrimental effects to the cell, such as Ca²⁺ overload, increased permeability to ATP, and dysregulation of signaling and neurotransmission. Hence, connexins have gained importance as a therapeutic target for inflammation-related diseases, and inhibitors or modulators of hemichannels may have therapeutic utility in the treatment of these diseases.

[0004] Cellular inflammation is the hallmark of a plethora of both acute and chronic conditions such as muscle wasting diseases (muscular atrophies and dystrophies), neuromuscular disorders (amyotrophic lateral sclerosis, spinal muscular atrophy), neurodegenerative disorders (Alzheimer, Parkinson, Epilepsy), metabolic disorders (obesity, type II diabetes, metabolic syndrome), and complications derived from central nervous system trauma, stroke or ischemia, among others. These inflammatory diseases are characterized by the overexpression and/or overactivation of connexin hemichannels as a common molecular denominator. Since hemichannels can passively transport small and charged molecules when they become active, an ion imbalance such as cation imbalance may occur, causing inflammation and promoting the activation of apoptotic or necrotic pathways, leading to cell death.

[0005] As one example, muscular dystrophies (MDs) are degenerative genetic diseases caused by mutations in the dystrophin gene. Among these, Duchenne MD (DMD) is the most serious, causing death around the age of 30. Being a rare disease (15.9-19.8 per 100,000 male births worldwide), it has limited treatment options, mainly palliative. Despite new treatments improve the course of the disease they do not reduce its morbidity, and, in the case of oligo-based therapies, its efficacy is limited to patients harboring specific mutations. As the dystrophin gene is one of the longest human genes known in humans and more than 500 different mutations have been described on it to date, a mutation-based treatment as unfeasible. In the case of DMD, a prolonged inflammatory state of the muscle cell is a key factor to the development of the progressive muscular atrophy.

[0006] Targeting hemichannels while avoiding concomitant blockage of gap junctions is desirable to minimize disruption of physiological signaling through gap junctions. Few hemichannel inhibitors demonstrate selectivity for hemichannels over gap junctions. One such inhibitor, 2-(4- chlorophenyl)-2-oxo-l-phenylethyl quinoline-2-carboxylate, has been previously identified as a blocker of Cx43 and Cx45 hemichannels, and has shown potential for selective inhibition of hemichannels in cellular studies (for instance, as shown in US2017/0115276 A1 and US 2018/0050029 Al), with the goal of preventing the activation of inflammatory pathways, cell death and therefore muscle dysfunction. However, it was not previously known that 2-(4-chlorophenyl)-2-oxo-l-phenylethyl quinoline-2- carboxylate exhibits significant chemical instability in aqueous solutions, making it impractical for prolonged dosing and hampering its utility as a research tool or therapeutic. Therefore, there is a need in the field for stable compositions 2-(4-chlorophenyl)-2-oxo-l-phenylethyl quinoline -2-carboxylate, and for other chemically stable hemichannel-selective inhibitors and compositions thereof.

[0007] Furthermore, classical methods for assessing the activity of a blocker of connexin hemichannels depend on imaging live mammalian cells that produce said hemichannels. This method is slow, requires a high level of technical proficiency, and is subject to variations and inconsistent results from one experiment to the next. Therefore, this method is not suitable for high throughput screening, and there is a need in the field for new methods to identify hemichannel-selective inhibitors. BRIEF SUMMARY OF THE INVENTION

[0008] Compositions and compounds that address the unmet need for chemically stable hemichannel-selective inhibitors have been identified. Such compositions, compounds, methods of making or dosing the foregoing, and related assays, are described herein. [0009] Provided herein are compositions comprising a compound of Formula (I), which is 2-

(4-chlorophenyl)-2-oxo-l-phenylethyl quinoline -2-carboxylate, or a salt thereof, and a hydrophilic polymer, a nonionic surfactant, and a solvent. The compositions provided herein are chemically stable compared to known formulations of a compound of Formula (I) or a salt thereof, which is particularly useful in therapeutic methods or in in vitro assay methods. Further provided are methods of stabilizing the compound of Formula (I) or a salt thereof and methods of preparing the compositions comprising a compound of Formula (I) or a salt thereof. Also provided are dosage forms comprising the compound of Formula (I) or a salt thereof and methods of treatment comprising administering compositions comprising a compound of Formula (I) or a salt thereof and a hydrophilic polymer, a nonionic surfactant, and a solvent. As an added benefit, the same composition can be used to deliver the compound orally, intravenously, or by intraperitoneal injection.

[0010] Also provided herein are compositions comprising quinaldic acid (also referred to herein as compound 2) or a salt thereof, such as a composition comprising quinaldic acid or a salt thereof, a hydrophilic polymer, a nonionic surfactant, and a solvent, based upon the discovery of quinaldic acid as a degradation product of compound 1 in cellular assays which retains its biological activity. Further provided are methods of preparing compositions comprising quinaldic acid or a salt thereof and dosage forms comprising quinaldic acid or a salt thereof. Methods of treatment comprising administering quinaldic acid or a salt thereof are also provided.

[0011] The compound of Formula (I) has a stereocenter as indicated by the carbon bearing * in the structure of Formula (I) below. All stereoisomeric forms of Formula (I) are described herein, including stereoisomeric forms where the carbon bearing * is in the R configuration, S configuration, or mixtures comprising the R and S configurations in any ratio, including racemic mixtures. In some embodiments, the compound of Formula (I) is a stereoisomer of 2-(4-chlorophenyl)-2-oxo-l-phenylethyl quinoline-2-carboxylate or a salt thereof. In some embodiments, the compound is (R)-2-(4-chlorophenyl)- 2-oxo- 1-phenylethyl quinoline-2-carboxylate (compound 1) or a salt thereof. In some embodiments, a composition comprises a mixture of enantiomers of 2-(4-chlorophenyl)-2-oxo- 1-phenylethyl quinoline-2- carboxylate, such as a racemic mixture.

[0012] Also provided herein are methods of screening and identifying compounds that modulate connexin hemichannel activity. In some embodiments, the methods are cell-based assays

Compound 2

Formula (I) Compound 1 (quinaldic acid) appropriate for high-throughput screening. Further provided are engineered cells for use in these methods.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The drawings illustrate embodiments with certain features and advantages as described in this disclosure, and are not intended to limit the claims.

[0014] FIG. 1A is a schematic depicting the state-of-the-art HeLa-based screening assay for identifying compounds that modulate connexin hemichannel activity, and the degree of modulation.

[0015] FIG. IB shows the connexin hemichannel blocking activity of compound 1 relative to (S)-2-(4-chlorophenyl)-2-oxo-l-phenylethyl quinoline-2-carboxylate.

[0016] FIG. 2A shows the chemical stability of compound 1 over the course of two hours in

DMSO.

[0017] FIG. 2B shows the chemical stability of compound 1 over the course of two hours in ethanol.

[0018] FIG. 2C shows the chemical stability of compound 1 over the course of two hours in phosphate buffered saline (“PBS”). [001 ] FIG. 2D shows the chemical stability of compound 1 over thirty minutes in phosphate buffer.

[0020] FIG. 2E shows the chemical stability of 1 mM compound 1 in a stabilized composition of 30% PEG 400, 20% polyethoxylated castor oil, and 50% PBS by volume, as described herein.

[0021] FIG. 3 demonstrates the variation in fluorescence intensity that can occur with the state-of-the-art HeLa-based screening assay.

[0022] FIG. 4A is a schematic depicting a yeast-based screening assay for identifying compounds that modulate connexin hemichannel activity, and the degree of modulation. [0023] FIG. 4B shows the change in connexin hemichannel activity upon treatment with

IOmM compound 1 or compound 2, as measured by the yeast assay depicted in FIG. 4A.

[0024] FIG. 5A shows the concentration of compound 2 present over time in the serum of mice treated orally with stabilized composition of compound 1.

[0025] FIG. 5B shows the concentration of compound 2 present over time in the serum of mice treated orally with a composition of compound 2.

[0026] FIG. 5C shows the concentration of compound 2 present over time in the serum of mice treated intravenously with stabilized composition of compound 1.

[0027] FIG. 5D shows the concentration of compound 2 present over time in the serum of mice treated with stabilized composition of compound 1 by intraperitoneal injection. [0028] FIG. 6A demonstrates the lack of toxicity to mice upon daily oral dosing with 50 mg/kg compound 2 as compared to vehicle.

[0029] FIG. 6B illustrates the conditions of a hanging test to assess improvement in muscle performance (hanging time) in mdx mice upon treatment with compound 1 or compound 2.

[0030] FIGS. 6C-F show the results of the hanging test over time for treated mdx mice relative to control untreated wild type mice and control untreated mdx mice upon daily oral dosing. FIG 6C illustrates results from the last 5 weeks of treatment with compound 1 of an 8 week treatment period. FIG 6D illustrates results with compound 2 over an 8 week treatment period. FIG 6E illustrates the statistical significance of the hanging time results obtained for compound 1. FIG 6F illustrates the statistical significance of the hanging time results obtained for compound 2. [0031] FIG. 7 A and FIG. 7B show the effect of daily oral dosing of compound 1 on necrotic markers (creatine kinase activity, atrogen levels).

[0032] FIG. 8 shows the effect of daily oral dosing with compound 1 on muscle fiber histology in mdx mice (images bl-b3), relative to vehicle -treated wild-type mice (al-a3) and vehicle- treated mdx mice (cl-c3).

DETAILED DESCRIPTION OF THE INVENTION

[0033] This description and the accompanying drawings should be understood as non limiting.

[0034] As used herein, “treatment” or “treating” is an approach for obtaining beneficial or desired results. For example, beneficial or desired results include, but are not limited to, one or more of the following: decreasing symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, delaying the progression of the disease, and/or prolonging survival of individuals.

[0035] As used herein, an “effective amount” of compound or salt thereof or pharmaceutical composition is an amount sufficient to effect beneficial or desired results.

[0036] The word “about” in the context of a given value or range of values refers to a value or range that is within the usual error known to a person skilled in the technical field, for example, within 2%, within 5%, or within 10% of said value or range. It is understood that reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”.

[0037] A compound or composition that is “substantially free” of a given impurity intends a composition that contains no more than 35% impurity. In some embodiments, a composition of substantially pure compound or a salt thereof is provided wherein the composition contains no more than 25%, 20%, 15%, 10%, or 5% impurity. In some embodiments, a composition of substantially pure compound or a salt thereof is provided wherein the composition contains or no more than 3%, 2%, 1% or 0.5% impurity.

[0038] It is understood that aspects and variations described herein also include “consisting” and/or “consisting essentially of’ aspects and variations. [0039] An “individual” as described herein encompasses, but is not limited to, mammals, such as mice, rats, dogs, primates and humans. In some embodiments, the individual is human.

[0040] The term “ambient conditions” refers to a temperature of between about 18 °C and about 25 °C.

Compositions

[0041] Although the compound of Formula (I) is stable in organic solvents such as DMSO and ethanol, these solvents are toxic to humans and animals, making compositions in these solvents problematic for repeated dosing and therapeutic use. The compositions provided herein are chemically stable compared to known formulations of a compound of Formula (I) or a salt thereof, and are therefore suitable for therapeutic use or in in vitro assay methods. Provided herein are compositions comprising a compound of Formula (I), which is 2-(4-chlorophenyl)-2-oxo-l-phenylethyl quinoline-2-carboxylate, or a stereoisomer thereof, or a salt of any of the foregoing; a hydrophilic polymer, a nonionic surfactant, and a solvent. Compositions comprising quinaldic acid or a salt thereof are also provided, as are compositions comprising a combination of a compound of Formula (I) and quinaldic acid or a salt of any of the foregoing.

[0042] Provided herein are compositions comprising a compound of Formula (I), which is 2- (4-chlorophenyl)-2-oxo-l-phenylethyl quinoline-2-carboxylate, or a stereoisomer thereof, or a salt of any of the foregoing; a hydrophilic polymer, a nonionic surfactant, and a solvent. In some embodiments, the composition comprises (R)-2-(4-chlorophenyl)-2-oxo-l-phenylethyl quinoline-2-carboxylate (compound 1) or a salt thereof. In some embodiments, the composition comprises a racemic mixture of enantiomers of 2-(4-chlorophenyl)-2-oxo-l-phenylethyl quinoline -2-carboxylate. In some embodiments, the composition is substantially free of (S)-2-(4-chlorophenyl)-2-oxo-l-phenylethyl quinoline-2-carboxylate or a salt thereof. Also provided are compositions comprising quinaldic acid (compound 2) or a salt thereof, a hydrophilic polymer, a nonionic surfactant, and a solvent. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the salt of compound 2 or the salt of a compound of Formula (I) is a pharmaceutically acceptable salt.

[0043] In some embodiments, the composition comprises compound 2 or the compound of Formula (I) or a salt of the foregoing at a purity of at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, or at least 99%. In some such embodiments, the compound of Formula (I) is compound 1 or a salt thereof. In some embodiments, a composition comprising compound 2 or the compound of Formula (I) or a salt of the foregoing is substantially free of impurities such as agents other than the active pharmaceutical ingredient (compound 2 or the compound of Formula (I)) or a salt thereof and pharmaceutically acceptable excipients, such as a hydrophilic polymer, a nonionic surfactant and a

7 solvent. In some embodiments, such a composition contains no more than 25%, 20%, 15%, 10%, 5%,

3%, 2%, 1% or 0.5% impurity. In some embodiments, the active pharmaceutical ingredient is a compound of formula 1 and the composition is substantially free of (S)-2-(4-chlorophenyl)-2-oxo-l- phenylethyl quinoline-2-carboxylate or a salt thereof .

[0044] In some embodiments, the composition comprises a compound of Formula (I) or a salt thereof at a concentration between about 0.0001 mg/rnL and about 10 mg/mL. In some embodiments, the composition comprises the compound of Formula (I) or a salt thereof at a concentration between about 0.0001 mg/mL and about 0.001 mg/mL, between about 0.001 mg/mL and about 0.01 mg/mL, between about 0.01 mg/mL and about 0.1 mg/mL, between about 0.1 mg/mL and about 1 mg/mL, or between about 1 mg/mL and about 10 mg/mL. In some embodiments, the composition comprises the compound of Formula (I) or a salt thereof at a concentration of about 0.0004 mg/mL, about 1 mg/mL, about 5 mg/mL, or about 10 mg/mL.

[0045] In some embodiments, the composition comprises compound 2 or a salt thereof at a concentration between about 0.0001 mg/mL and about 5 mg/mL. In some embodiments, the composition comprises the compound 2 or a salt thereof at a concentration between about 0.0001 mg/mL and about 0.001 mg/mL, between about 0.001 mg/mL and about 0.01 mg/mL, between about 0.01 mg/mL and about 0.1 mg/mL, between about 0.1 mg/mL and about 1 mg/mL, or between about 1 mg/mL and about 5 mg/mL. In some embodiments, the composition comprises the compound of compound 2 or a salt thereof at a concentration of about 5 mg/mL.

[0046] In some embodiments is a composition comprising compound 2 or a salt thereof and/or the compound of Formula (I) or a salt thereof. In some embodiments, the composition comprises compound 2 or a salt thereof. In some embodiments, the composition comprises the compound of Formula (I), such as compound 1, or a salt thereof. In some embodiments, the composition comprising compound 2 or a salt thereof and/or the compound of Formula (I) or a salt thereof further comprises a hydrophilic polymer. In some embodiments, the hydrophilic polymer is polyacrylamide, polyvinyl pyrrolidone, polyvinyl alcohol, polyacrylic acid, polyethylene glycol, or polypropylene glycol. In some embodiments, the hydrophilic polymer comprises polyethylene glycol or polypropylene glycol. In some embodiments, the hydrophilic polymer comprises polyethylene glycol. In some embodiments, the hydrophilic polymer has an average molecular weight of between about 100 Da and 2000 Da. In some embodiments, the hydrophilic polymer has an average molecular weight between about 200 Da and about 600 Da. In some embodiments, the hydrophilic polymer has an average molecular weight between about 380 Da and 420 Da. In some embodiments, the hydrophilic polymer has an average molecular weight of about 100 Da, about 200 Da, about 300 Da, about 400 Da, about 500 Da, about 600 Da, about 700 Da, about 800 Da, about 900 Da, about 1000 Da, about 1200 Da, about 1400 Da, about 1600 Da, about 1800 Da, or about 2000 Da. In some embodiments, the hydrophilic polymer is PEG400. In some embodiments, the hydrophilic polymer comprises between about 1% and about 50% of the composition by volume. In some embodiments, the hydrophilic polymer comprises between about 10% and about 50% of the composition by volume. In some embodiments, the hydrophilic polymer comprises between about 20% and about 40% of the composition by volume. In some embodiments, the hydrophilic polymer comprises about 1%, about 5%, about 10%, about 20%, about 25%, about 30%, about 40%, or about 50% of the composition by volume.

[0047] In some embodiments, the composition comprising compound 2 or a salt thereof and/or the compound of Formula (I) or a salt thereof further comprises a nonionic surfactant. In some embodiments, the nonionic surfactant is a poloxamer, a polysorbate, or an ethoxylated fatty acid. In some embodiments, the nonionic surfactant is polyethoxylated castor oil or Kolliphor® EL. In some embodiments, the poloxamer is polyethoxylated castor oil or Kolliphor® EL. In some embodiments, the nonionic surfactant comprises between about 1% and about 50% of the composition by volume. In some embodiments, the nonionic surfactant comprises between about 5% and about 40% of the composition by volume. In some embodiments, the nonionic surfactant comprises between about 10% and about 30% of the composition by volume. In some embodiments, the nonionic surfactant comprises about 1%, about 5%, about 10%, about 20%, about 25%, about 30%, about 40%, or about 50% of the composition by volume.

[0048] In some embodiments, the composition comprising compound 2 or a salt thereof and/or the compound of Formula (I) or a salt thereof further comprises a solvent. In some embodiments, the solvent is a pharmaceutically acceptable solvent. In some embodiments, the solvent comprises water. In some embodiments, the solvent comprises saline. In some embodiments, the solvent comprises a buffer solution. In some embodiments, the solvent is phosphate buffered saline. In some embodiments, the solvent has a pH between about 6.5 and about 8.5. In some embodiments, the solvent has a pH of between about 6.5 and 7, between about 7 and 7.5, between about 7.5 and 8.0, or between about 8.0 and 8.5. In some embodiments, the solvent has a pH of about 7.4. In some embodiments, the solvent is at physiological pH. In some embodiments, the solvent comprises between about 50% and about 98% of the composition by volume. In some embodiments, the solvent comprises between about 50% and about 60%, between about 60% and about 70%, between about 70% and about 80%, between about 80% and about 90%, or between about 90% and about 98% of the composition by volume. In some embodiments, the solvent comprises up to or about 50%, about 60%, about 70%, about 80%, about 90%, or about 98% of the composition by volume.

[0049] In some embodiments, the composition has a pH between about 6.5 and about 8.5. In some embodiments, the composition has a pH of between about 6.5 and 7, between about 7 and 7.5, between about 7.5 and 8.0, or between about 8.0 and 8.5. In some embodiments, the composition has a pH of about 7.4. In some embodiments, the composition is at physiological pH. [0050] In some embodiments, the composition comprising compound 2 or a salt thereof and/or the compound of Formula (I) or a salt thereof further comprises any one or more of a hydrophilic polymer, a nonionic surfactant and a solvent. For example, in some embodiments is a composition comprising compound 2 or a salt thereof and/or the compound of Formula (I) or a salt thereof and further comprising both a hydrophilic polymer and a nonionic surfactant, and optionally comprises a solvent.

Also provided is a composition comprising compound 2 or a salt thereof and/or the compound of Formula (I) or a salt thereof and further comprising each of a hydrophilic polymer, a nonionic surfactant and a pharmaceutically acceptable solvent. It is understood that each description of compositions further comprising a hydrophilic polymer may be combined with descriptions of compositions further comprising a nonionic surfactant and/or solvent the same as if each and every combination were specifically and individually listed for such a composition. Likewise, it is understood that each description of compositions further comprising a nonionic surfactant may be combined with descriptions of compositions further comprising a hydrophilic polymer and/or solvent the same as if each and every combination were specifically and individually listed for such a composition.

[0051] In some embodiments, the composition stabilizes the compound of Formula (I) or a salt thereof compared to currently known compositions of the compound of Formula (I). In some embodiments, the compositions are pharmaceutically acceptable. In some embodiments, the compositions are substantially free of polymers, surfactants, solvents or other components that are not generally recognized as safe, as determined by the U.S. Food and Drug Administration. In some embodiments of the compositions herein, the compound of Formula (I) is chemically stable for up to about 1 hour under ambient conditions.

Methods of Stabilizing Compounds of Formula (I)

[0052] Provided herein are methods of stabilizing a compound of Formula (I) or a salt thereof in a composition. In some embodiments, the method comprises mixing the compound of Formula (I) with a hydrophilic polymer. In some embodiments, the method comprises mixing the compound of Formula (I) with a nonionic surfactant. In some embodiments, the method comprises mixing the compound of Formula (I) with a solvent. In some embodiments, the method comprises sonication of the compound of Formula (I) with a hydrophilic polymer, a nonionic surfactant, and a solvent, or any combination of the same. In some embodiments, the method comprises vortexing the compound of Formula (I) with a hydrophilic polymer, a nonionic surfactant, and a solvent, or any combination of the same. In some embodiments, the method comprises a combination of sonication and vortexing of the compound of Formula (I) with the hydrophilic polymer, the nonionic surfactant, the solvent, or any combination of the same. In some embodiments, the method comprises mixing a compound of Formula (I) with a hydrophilic polymer, followed by mixing with a nonionic surfactant, followed by mixing with a solvent. In some embodiments, the method comprises mixing a compound of Formula (I) with a nonionic surfactant, followed by mixing with a hydrophilic polymer, followed by mixing with a solvent. In some embodiments, the method comprises mixing a compound of Formula (I) with a hydrophilic polymer, followed by mixing with a solvent, followed by mixing with a nonionic surfactant.

[0053] In some embodiments, the method comprises mixing a compound of Formula (I) or a salt thereof at a concentration between about 0.0001 mg/mL and about 10 mg/mL with a hydrophilic polymer, a nonionic surfactant, a solvent, or a combination of the same. In some embodiments, the method comprises mixing the compound of Formula (I) or a salt thereof at a concentration between about 0.0001 mg/mL and about 0.001 mg/mL, between about 0.001 mg/mL and about 0.01 mg/mL, between about 0.01 mg/mL and about 0.1 mg/mL, between about 0.1 mg/mL and about 1 mg/mL, or between about 1 mg/mL and about 10 mg/mL with a hydrophilic polymer, a nonionic surfactant, a solvent, or a combination of the same.

[0054] In some embodiments, the hydrophilic polymer is polyacrylamide, polyvinyl pyrrolidone, polyvinyl alcohol, polyacrylic acid, polyethylene glycol, or polypropylene glycol. In some embodiments, the hydrophilic polymer comprises polyethylene glycol or polypropylene glycol. In some embodiments, the hydrophilic polymer comprises polyethylene glycol. In some embodiments, the hydrophilic polymer has an average molecular weight of between about 100 and 2000. In some embodiments, the hydrophilic polymer has an average molecular weight between about 200 and about 600. In some embodiments, the hydrophilic polymer has an average molecular weight between about 380 and 420. In some embodiments, the hydrophilic polymer has an average molecular weight of about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 1200, about 1400, about 1600, about 1800, or about 2000. In some embodiments, the method comprises mixing the compound of Formula (I) with an amount of the hydrophilic polymer that comprises between about 1% and about 50% of the composition by volume after mixing all components. In some embodiments, the method comprises mixing the compound of Formula (I) with an amount of the hydrophilic polymer that comprises between about 10% and about 50% of the composition by volume after mixing all components. In some embodiments, the method comprises mixing the compound of Formula (I) with an amount of the hydrophilic polymer that comprises between about 20% and about 40% of the composition by volume after mixing all components. In some embodiments, the method comprises mixing the compound of Formula (I) with an amount of the hydrophilic polymer that comprises between about 1%, about 5%, about 10%, about 20%, about 25%, about 30%, about 40%, or about 50% of the composition by volume after mixing all components. [0055] In some embodiments, the nonionic surfactant is a poloxamer, a polysorbate, or an ethoxylated fatty acid. In some embodiments, the nonionic surfactant is polyethoxylated castor oil or Kolliphor® EL. In some embodiments, the poloxamer is polyethoxylated castor oil or Kolliphor® EL. In some embodiments, the method comprises mixing the compound of Formula (I) with an amount of the nonionic surfactant that comprises between about 1% and about 50% of the composition by volume after mixing all components. In some embodiments, the method comprises mixing the compound of Formula (I) with an amount of the nonionic surfactant that comprises between about 5% and about 40% of the composition by volume after mixing all components. In some embodiments, the method comprises mixing the compound of Formula (I) with an amount of the nonionic surfactant that comprises between about 10% and about 30% of the composition by volume after mixing all components. In some embodiments, the method comprises mixing the compound of Formula (I) with an amount of the nonionic surfactant that comprises about 1%, about 5%, about 10%, about 20%, about 25%, about 30%, about 40%, or about 50% of the composition by volume after mixing all components.

[0056] In some embodiments, the solvent is a pharmaceutically acceptable solvent. In some embodiments, the solvent comprises water. In some embodiments, the solvent comprises saline. In some embodiments, the solvent comprises a buffer solution. In some embodiments, the solvent is phosphate buffered saline. In some embodiments, the solvent has a pH between about 6.5 and about 8.5. In some embodiments, the solvent has a pH of between about 6.5 and 7, between about 7 and 7.5, between about 7.5 and 8.0, or between about 8.0 and 8.5. In some embodiments, the solvent has a pH of about 7.4. In some embodiments, the solvent is at physiological pH. In some embodiments, the method comprises mixing the compound of Formula (I) with an amount of the solvent that comprises between about 50% and about 98% of the composition by volume after mixing all components. In some embodiments, the method comprises mixing the compound of Formula (I) with an amount of the solvent that comprises between about 50% and about 60%, between about 60% and about 70%, between about 70% and about 80%, between about 80% and about 90%, or between about 90% and about 98% of the composition by volume after mixing all components. In some embodiments, the method comprises mixing the compound of Formula (I) with an amount of the solvent that comprises up to or about 50%, about 60%, about 70%, about 80%, about 90%, or about 98% of the composition by volume after mixing all components.

[0057] In some embodiments, the composition has a pH between about 6.5 and about 8.5. In some embodiments, the composition has a pH of between about 6.5 and 7, between about 7 and 7.5, between about 7.5 and 8.0, or between about 8.0 and 8.5. In some embodiments, the composition has a pH of about 7.4. In some embodiments, the composition is at physiological pH.

[0058] In some embodiments, the method stabilizes the compound of Formula (I) or a salt thereof. In some embodiments, the compound of Formula (I) is chemically stable for up to, greater than, or about 1 hour under ambient conditions. Dosage Forms

[0059] Provided herein are unit dosage forms configured for administration to an individual, comprising a composition including a compound of Formula (I) or a salt thereof as disclosed herein. Also provided herein are unit dosage forms configured for administration to an individual, comprising a composition including compound 2 or a salt thereof as disclosed herein. In some embodiments, the unit dosage form is configured for oral administration. In some embodiments, the unit dosage form is configured for daily oral administration. In some embodiments, the unit dosage form is configured for intravenous administration. In some embodiments, the unit dosage form is configured for intraperitoneal administration.

[0060] In some embodiments, the unit dosage form comprises between about 1 mg and about 200 mg of a compound of Formula (I) per kilogram of the individual. In some embodiments, the unit dosage form comprises between about 1 mg and about 10 mg, between about 10 mg and about 50 mg, between about 50 mg and about 100 mg, between about 100 mg and about 150 mg, or between about 150 mg and about 200 mg of a compound of Formula (I) per kilogram of the individual. In some embodiments, the unit dosage form comprises about 2 mg, about 50 mg, about 100 mg, or about 200 mg of a compound of Formula (I) per kilogram of the individual.

[0061] In some embodiments, the unit dosage form comprises between about 1 mg and about 100 mg of compound 2 or a salt thereof per kilogram of the individual. In some embodiments, the unit dosage form comprises between about 1 mg and about 10 mg, between about 10 mg and about 50 mg, or between about 50 mg and about 100 mg of compound 2 or a salt thereof per kilogram of the individual. In some embodiments, the unit dosage form comprises about 2 mg, about 50 mg, or about 100 mg of compound 2 per kilogram of the individual.

Methods of Modulating Connexin Hemichannel Activity

[0062] Provided herein are methods of modulating connexin hemichannel activity in an individual. In some embodiments, the method comprises administering to the individual an effective amount of a compound of Formula (I) or a salt thereof, compound 2 or a salt thereof, or a composition or dosage form of the foregoing. In some embodiments, the individual has a disease associated with the overactivation of connexin hemichannels. In some embodiments, the disease or condition comprises inflammatory disease, neurodegenerative disorder, vascular disorder, arrhythmia, chronic injury, retinal neuroprotection, treatment of pain, skeletal muscle denervation, scarred tissue, muscular dystrophy, post ischemia reperfusion injury, damage to the spinal cord, or a genetic disease characterized by an increased activity of connexin hemichannels, or at least one symptom of any of the foregoing. In some embodiments, the disease or condition comprises an inflammatory disease or a disease characterized by inflammation, such as epilepsy, type II diabetes, shock, or inflammatory ocular disorder, or at least one symptom of any one of the foregoing. In some embodiments, the disease or condition comprises treating a neurodegene rati ve disorder, such as Alzheimer’s disease or Parkinson’s disease, or at least one symptom of any of the foregoing. In some embodiments, the disease or condition is muscular dystrophy or at least one symptom thereof. In some embodiments, the disease or condition is Duchenne muscular dystrophy. In some embodiments, the connexin hemichannels are hemichannels of connexin 43. In some embodiments, the connexin hemichannels are hemichannels of connexin 26.

Methods of Treatment and Diseases

[0063] Provided herein are methods of treating a disease or condition associated with overactivation of connexin hemichannels in an individual in need thereof, or at least one symptom of said disease or condition, comprising administering to the individual a composition including a compound of Formula (I) or a salt thereof as disclosed herein. Also provided are methods of treating a disease or condition associated with overactivation of connexin hemichannels in an individual in need thereof, or at least one symptom of said disease or condition, comprising administering to the individual a dosage form including a compound of Formula (I) or a salt thereof as disclosed herein.

[0064] In some embodiments, the disease or condition comprises an acquired or inherited inflammatory disease. In some embodiments, the disease or condition comprises inflammatory disease, neurodegenerative disorder, vascular disorder, arrhythmia, chronic injury, retinal neuroprotection, treatment of pain, skeletal muscle denervation, scarred tissue, muscular dystrophy, post-ischemia reperfusion injury, damage to the spinal cord, or a genetic disease characterized by an increased activity of connexin hemichannels, or at least one symptom of any of the foregoing. In some embodiments, the disease or condition comprises an inflammatory disease or a disease characterized by inflammation, such as epilepsy, type II diabetes, shock, or inflammatory ocular disorder, or at least one symptom of any one of the foregoing. In some embodiments, the disease or condition comprises treating a neurodegenerative disorder, such as Alzheimer’s disease or Parkinson’s disease, or at least one symptom of any of the foregoing. In some embodiments, the disease or condition is muscular dystrophy or at least one symptom thereof. In some embodiments, the disease or condition is Duchenne muscular dystrophy.

[0065] Further provided are methods of treating a disease or condition associated with overactivation of connexin hemichannels in an individual in need thereof, or at least one symptom of said disease or condition, comprising administering to the individual compound 2 or a salt thereof as disclosed herein. Also provided are methods of treating a disease or condition associated with overactivation of connexin hemichannels in an individual in need thereof, or at least one symptom of said disease or condition, comprising administering to the individual a dosage form including a compound 2 or a salt thereof as disclosed herein. [0066] In some embodiments, the disease or condition comprises inflammatory disease, neurodegenerative disorder, vascular disorder, arrhythmia, chronic injury, retinal neuroprotection, treatment of pain, skeletal muscle denervation, scarred tissue, muscular dystrophy, post-ischemia reperfusion injury, damage to the spinal cord, or a genetic disease characterized by an increased activity of connexin hemichannels, or at least one symptom of any of the foregoing. In some embodiments, the disease or condition comprises an inflammatory disease or a disease characterized by inflammation, such as epilepsy, type II diabetes, shock, or inflammatory ocular disorder, or at least one symptom of any one of the foregoing. In some embodiments, the disease or condition comprises treating a neurodegenerative disorder, such as Alzheimer’s disease or Parkinson’s disease, or at least one symptom of any of the foregoing. In some embodiments, the disease or condition is muscular dystrophy or at least one symptom thereof. In some embodiments, the disease or condition is Duchenne muscular dystrophy.

[0067] In some embodiments, overactivation of connexin hemichannels is characterized by elevated hemichannel activity in at least one tissue, compared to a healthy state of that tissue. In some embodiments, overactivation of connexin hemichannels is characterized by elevated serum creatine kinase activity, for instance, between about 10- to about 100-fold increase in activity relative to normal levels for the individual. In some embodiments, overactivation of connexin hemichannels is characterized by elevated atrogen protein levels in at least one tissue of the individual, relative to a healthy tissue of the individual.

[0068] In some embodiments, the method of treating a disease or condition associated with overactivation of connexin hemichannels (or at least one symptom of said disease or condition) in an individual in need thereof comprising administering to the individual a compound, composition, salt, or dosage form as disclosed herein further comprises determining whether the individual has an elevated level of connexin hemichannel activity in at least one tissue compared to a healthy state of that tissue. In some embodiments, determining whether the individual has an elevated level of connexin hemichannel activity occurs prior to administering a compound, salt, or dosage form disclosed herein. In some embodiments, determining whether the individual has an elevated level of connexin hemichannel activity comprises detecting elevated serum kinase activity in the individual, for instance, detecting between about 10- to about 100-fold increase in activity relative to normal levels for the individual. In some embodiments, determining whether the individual has an elevated level of connexin hemichannel activity comprises detecting elevated atrogen protein levels in at least one tissue of the individual, relative to a healthy tissue of the individual, for instance, by isolating muscle tissue from the individual and measuring atrogen protein levels by Western blot.

Methods of Screening Compounds [0069] Further provided herein are methods of screening compounds to identify compounds that modulate connexin hemichannel activity. In some embodiments, the methods comprise conducting a cell assay with the following steps (l)-(4):

(1) providing to a cell that expresses connexin hemichannels a condition that favors an open state of the connexin hemichannel;

(2) applying to the cell a reporter that is cell-impermeable or poorly permeable;

(3) applying to the cell a compound of interest; and

(4) quantifying the accumulation of the reporter in the cell.

[0070] In some embodiments, the method further comprises conducting at least one control assay. In some embodiments, the control assay is a positive control comprising the following steps (1)- (3):

(1) providing to a cell that expresses connexin hemichannels a condition that favors an open state of the connexin hemichannel;

(2) applying to the cell a reporter that is cell-impermeable or poorly permeable; and (3) quantifying the accumulation of the reporter in the cell.

[0071 ] In some embodiments, the control assay is a negative control comprising the following steps (l)-(3):

(1) providing to a cell that expresses connexin hemichannels a condition that disfavors an open state of the connexin hemichannel; (2) applying to the cell a reporter that is cell-impermeable or poorly permeable; and

(3) quantifying the accumulation of the reporter in the cell.

[0072] In some embodiments, the condition which favors the open state of the connexin hemichannel comprises a solution that is free or substantially free of Ca²⁺ and Mg²⁺. In some embodiments, the condition which favors the open state of the connexin hemichannel comprises a divalent cation-free solution, including divalent cation-free buffer such as Locke solution or Krebs solution, or divalent cation-free growth medium. In some embodiments, the condition which favors the open state of the connexin hemichannel comprises a solution, buffer, or growth medium which is substantially free of divalent cations. In some embodiments, the condition which favors the open state of the connexin hemichannel comprises application of positive voltage to the cellular membrane.

[0073] In some embodiments, the condition which disfavors the open state of the connexin hemichannel comprises a divalent cation solution, such as a buffer or growth medium including divalent cations. In some embodiments, the divalent cation solution is a buffer such as Krebs solution or a growth medium such as YPD. In some embodiments, the divalent cation solution is supplemented with potassium at a concentration of between about 5 mM and about 100 mM K⁺, between about 10 mM and about 100 mM K⁺, between about 25 mM and about 75 mM K⁺, or about 50 mM K⁺. In some embodiments, the divalent cation solution is YPD (yeast extract -peptone-dextrose) medium supplemented with K⁺. In some embodiments, the source of K⁺ is an organic or inorganic potassium salt. In some embodiments, the source of K⁺ is KC1.

[0074] In some embodiments, the reporter comprises a fluorescent or colorimetric dye. In some embodiments, the reporter comprises a DNA intercalating molecule such as propidium iodide, ethidium bromide, or DAPI (4’,6-diamidino-2-phenylindole). In some embodiments, quantifying the accumulation of the reporter in the cell comprises measuring absorption or fluorescence intensity. In some embodiments, the method comprises comparing the accumulation of the reporter in the cell assay to the accumulation of the reporter in the positive control. In some embodiments, the method comprises comparing the accumulation of the reporter in the cell assay to the accumulation of the reporter in the negative control.

[0075] In some embodiments, the assay is completed in less than about 5 minutes, less than about 10 minutes, or less than about 15 minutes. In some embodiments, the assay is suitable for simultaneously screening more than one compound. In some embodiments, the assay is configured in a multi-well plate or array. In some embodiments, at least one step of the assay is configured in a multi well plate or array. In some embodiments, at least one step of the assay and at least one step of a control assay are conducted simultaneously. In some embodiments, the assay and at least one control assay are configured in the same multi-well plate or array. In some embodiments, the assay is suitable for high- throughput screening.

[0076] In some embodiments, the methods comprise conducting a cell assay using yeast cells expressing connexin hemichannels, as described herein. In some embodiments, the gene encoding a connexin hemichannel is the GJA1 gene. In some embodiments, the yeast cells are further engineered to lack functional potassium channels. In some embodiments, the yeast cells are engineered to knock out functional potassium channels. In some embodiments, the yeast cells are engineered to delete the genes for functional potassium channels. In some embodiments, the yeast are engineered to replace potassium channels with resistance genes. In some embodiments, the yeast are engineered to replace trkl or trk2 with one or more genes. In some embodiments, the yeast are engineered to replace trkl and trlc2 with one or more genes. In some embodiments, the yeast are engineered to replace the trkl gene with a cassette comprising GJA1 and hphMX. In some embodiments, the yeast are engineered to replace the trk2 gene with a cassette comprising natMX. In some embodiments, the gene replacement is achieved by homologous recombination.

Yeast Expressing Connexin Hemichannels

[0077] Further provided herein is an engineered strain of yeast capable of expressing human connexin 43, and methods of producing said yeast strain. In some embodiments, the yeast is appropriate for use in the methods described herein for screening compounds to identify molecules capable of modulating connexin hemichannel activity.

[0078] In some embodiments, the engineered strain of yeast comprises the human GJA1 gene. In some embodiments, the engineered strain of yeast comprises resistance gene(s). In some embodiments, the engineered strain of yeast comprises hphMX or natMX. In some embodiments, the engineered strain of yeast comprises hphMX and natMX. In some embodiments, the engineered strain of yeast comprises hygromycin or nourseothricin resistance. In some embodiments, the engineered strain of yeast comprises hygromycin and nourseothricin resistance. In some embodiments, the engineered strain comprises a knockout of an ion channel. In some embodiments, the ion channel is a potassium channel. In some embodiments, the ion channel is trkl or trk2. In some embodiments, the engineered strain comprises a knockout of both trkl and trk2.

[0079] In some embodiments, the engineered yeast are maintained in growth medium supplemented with ions. In some embodiments, the engineered yeast are maintained in growth medium supplemented with cations. In some embodiments, the engineered yeast are maintained in a growth medium supplemented with potassium at a concentration of between about 5 mM and about 100 mM K⁺, between about 10 mM and about 100 mM K⁺, between about 25 mM and about 75 mM K⁺, or about 50 mM K⁺. In some embodiments, the growth medium is YPD (yeast extract-peptone-dextrose) medium. In some embodiments, the source of K⁺ is an organic or inorganic potassium salt. In some embodiments, the source of K⁺ is KC1.

[0080] All references and publications listed herein are hereby incorporated herein by reference in their entireties. EXAMPLES

[0081] The following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention.

Example 1. Identification of compound 1 as the active isomer

[0082] This example describes the state of the art assay for analyzing inhibition of connexin hemichannels, and the identification of (R)-2-(4-chlorophenyl)-2-oxo-l-phenylethyl quinoline -2- carboxylate (compound 1) as the active isomer.

[0083] Preparation: HeLa cells were transfected with human connexin 43 to produce a stably transfected cell line. 10³ cells per well were added to a 90-well plate, 24 hours prior to the start of each experiment. The wells were then divided into four groups (A, Bl, B2, and B3).

[0084] To initiate the assay, the wells in Group A were washed and incubated with a Locke solution containing normal levels of divalent cations (Ca²⁺ and Mg²⁺) and 5 mM of ethidium bromide. The wells in Groups Bl-3 were washed and incubated with a divalent cation-free Locke solution containing 5 u M of ethidium bromide. The wells of groups B2 and B3 were further treated with compound 1 or (S)-2- (4-chlorophenyl)-2-oxo-l-phenylethyl quinoline -2-carboxylate, respectively. After at least 5 minutes, ethidium bromide fluorescence was evaluated for all cells, and fluorescence intensity were averaged for each group.

[0085] The presence of divalent cations favors the closed state of connexin hemichannels, whereas solutions that are free of divalent cations favor the open state of connexin hemichannels. The wells of Group A (“baseline” in FIG. IB) are expected to contain HeLa cells with closed connexin hemichannels, which block the entry of ethidium bromide (“EtBr” in FIG. 1 A) and result in negligible fluorescence response. On the other hand, the wells of Group Bl (“DCFS” in FIG. IB) are expected to contain HeLa cells with open connexin hemichannels, which allow the entry of ethidium bromide, resulting in a fluorescent readout. Therefore, the fluorescence intensity serves as a proxy for hemichannel activity. In Groups B2 and B3, a fluorescence response similar to Group Bl indicates that the compound does not affect the connexin hemichannels, whereas a decrease in fluorescence relative to Group B 1 indicates that the compound blocks the connexin hemichannels (see FIG. 1A).

[0086] Surprisingly, the wells of Group B2 (“R” in FIG. IB) yielded a significant decrease in fluorescence intensity relative to Group B 1 “DCFS”, approaching the intensity of Group A “Baseline”, indicating significant inhibition of the connexin hemichannels. On the other hand, the wells of Group B3 (“S” in FIG. IB) demonstrated a negligible effect on fluorescence intensity relative to Group Bl “DCFS”, indicating poor efficacy. Therefore, compound 1 was pursued for further studies. Example 2. Chemical synthesis of compound 1

[0087] Efforts to obtain pure or substantially pure enantiomers of a compound of Formula (I) by chemical synthesis of a racemate followed by chiral purification were challenging due to the low stability of the compounds of Formula (I). As are result, synthesis of compound 1 from a single enantiomer of starting material was preferred, as described herein.

Trimethyl aluminium

SOCI₂ OH N,O-Dimethylhydroxylamine

OH hydrochloride

Reflux, 16 h

Step-1 Step-2

[0088] Step 1: Synthesis of (R)-methyl 2-hydroxy-2-phenylacetate.

To a solution of (R)-2-hydroxy-2-phenylacetic acid (60.0 g, 0.395 moles) in methanol (300 ml) at 0 °C was added thionyl chloride (9.3 ml, 0.0788 moles) dropwise. The resultant reaction mixture was then refluxed for 4h. The TLC (20 % ethyl acetate in hexane) showed that starting material was consumed completely. The reaction mixture was then concentrated under reduced pressure and then diluted using demineralized water (400 ml). The reaction mixture was then extracted using ethyl acetate (300 ml x 2). The organic layer was then dried over sodium sulfate and concentrated to get crude product. The crude product was then purified using silica gel chromatography; the eluent was 0-30 % ethyl acetate in hexane to get 64.0 g (R)- methyl 2-hydroxy-2-phenylacetate (97.6 %) as white solid. ¹H NMR: (400 MHz, Chloroform-cf) d 7.46 - 7.25 (m, 5H), 5.18 (d, /= 5.4 Hz, 1H), 3.77 (s, 3H), 3.42 (d, /= 5.6 Hz, 1H).

[0089] Step 2: Synthesis of (R)-2-hydroxy-N-methoxy-N-methyl-2- phenylacetamide.

In two neck RBF (2.0 liter) equipped with over stirrer, N, O-dimethyl hydroxylamine hydrochloride (75.12 g, 0.77 moles) was dissolved in DCM (800 ml) and cooled to 0 °C. To this reaction mixture was added trimethyl aluminum solution (385 ml, 0.77 moles) dropwise over the period of 2 h. To this reaction mixture was added solution of (R)-methyl 2-hydroxy-2-phenylacetate (64.0 g, 0.385 moles) in DCM (200 ml) dropwise. The resultant reaction mixture was then refluxed for 16 h. The TLC (30 % ethyl acetate in hexane) showed that starting material consumed completely. The reaction mixture was cooled to room temperature and quenched by the addition of 1 % HC1 solution (500 ml) and diluted using DCM (500 ml). The reaction mixture was then partitioned in separating funnel and separated both layers. The aqueous layer was extracted by DCM (500 ml). The combined organic layer was dried and concentrated to get crude product. The crude product was then purified using silica gel chromatography; the eluent was 0-40 % ethyl acetate in hexane to get (R)-2-hydroxy-N-methoxy-N-methyl-2-phenylacetamide (46.0 g, 61.1 %). ¾ NMR (400 MHz, DMSO-d6) d 7.39 - 7.23 (m, 5H), 5.58 - 5.52 (m, 1H), 5.38 (d, J = 6.9 Hz, 1H), 3.48 (s, 3H), 3.09 (s, 3H).

[0090] Step 3: Synthesis of (R)-l-(4-chlorophenyl)-2-hydroxy-2-phenylethanone:

To the stirred solution of (R)-2-hydroxy-N-methoxy-N-methyl-2-phenylacetamide (20.0 g, 0.10 moles ) in dry THF (150 ml) at 0 °C was added chlorophenyl magnesium bromide (300 ml, 0.30 moles) dropwise using dropping funnel over the period of 2 h. The reaction mixture was stirred for 2h at 0 °C. The TLC (10 % ethyl acetate in hexane) showed complete consumption of starting material. The reaction mixture was then quenched by ice-cold water (150 ml), during addition white precipitation formed which was filtered through Buchner funnel. The filtrate was then extracted using ethyl acetate (400 ml). The organic layer was then concentrated to afford crude product. The crude product was purified using flash chromatography; the eluent was 0-10 % ethyl acetate in hexane to get (R)-l-(4-chlorophenyl)-2-hydroxy- 2-phenylethanone as (13.5 g, 53.4 %) white solid. Ή NMR (400 MHz, DMSO-de) d 8.04 - 7.97 (m, 2H), 7.57 - 7.49 (m, 2H), 7.42 - 7.39 (m, 2H), 7.32 - 7.30 (m, 2H), 7.28 - 7.20 (m, 1H), 6.14 (d, /= 5.7 Hz, 1H), 6.04 (d, J= 5.7 Hz, 1H).

[0091] Step 4: Synthesis of (R)-2-(4-chlorophenyl)-2-oxo-l-phenylethyl quinoline-2- carboxylate (compound 1).

To a stirred solution of quinoline-2-carboxylic acid (5.27 g, 0.030 moles) in DCM (100 ml) was added EDC-HC1 (5.84 g, 0.030 moles) and DMAP (0.037 g, 0.0030 moles) at 0 °C. The reaction mixture was stirred for 20 min at 0 °C. To this reaction mixture was added (R)-l-(4-chlorophenyl)-2-hydroxy-2- phenylethanone (5.0 g, 0.020 moles) at 0 °C. The reaction mixture was continued stirred for 2 h at 0 °C. The TLC (10 % ethyl acetate in hexane) showed formation of new spot along with starting material (~ 30 %), providing a first crude batch of product. A second batch was prepared in the same way. After 2 h of reaction, both batches were mixed, silica gel (100 -200 mesh size) was added to reaction mixture and concentrated to get slurry to purify using flash chromatography. The eluent was 0-45 % ethyl acetate in hexane to get 4.5 g of (R)-2-(4-chlorophenyl)-2-oxo-l-phenylethyl quinoline-2-carboxylate (compound 1) as white solid. *H NMR (400 MHz, DMSO-d6) d 8.63 (d, /= 8.6 Hz, 1H), 8.22 - 8.10 (m, 5H), 7.89 - 7.80 (m, 1H), 7.78 (t, J = 7.5 Hz, 1H), 7.74 - 7.65 (m, 2H), 7.65 - 7.60 (m, 2H), 7.53 - 7.39 (m, 4H). The product was further purified by HPLC and SFC (supercritical fluid chromatography) and specific optical rotation was determined [a] = -209.1° (±0.43°)

Example 3. Chemical stability of compound 1 in solution.

[0092] This example demonstrates the chemical stability of compound 1 in stabilized compositions as described herein, in contrast to the poor chemical stability of compound 1 in unstabilized aqueous solutions.

[0093] Compound 1 was dissolved in DMSO, ethanol, PBS, phosphate buffer, or in a mixture comprising 30% PEG400, 20% polyethoxylated castor oil (obtained as Kolliphor® EL, BASF Corp.), and 50% PBS by volume (the “stabilized composition”). Phosphate-buffered saline (PBS) contains 137 mM NaCl, 2.7 mM KC1, 10 mM NaiHPCL and 1.8 mM KH2PO4, adjusted to pH 7.4 with HC1. Phosphate buffer contains KH2PO4100 mM and then pH is adjusted to 7.4 using K2HPO4. For the purposes of these stability experiments, compound 1 was dissolved at a concentration of 1 mM. To prepare the stabilized composition, the compound was added to the PEG400 component of the mixture, vortexed for 5 seconds, and sonicated for a total of 15 minutes in 5-minute increments. The mixture was then diluted with the polyethoxylated castor oil component and vortexed for 5 seconds. The mixture was finally diluted with PBS, vortexed for 5 seconds, and sonicated for 5 minutes to provide the stabilized composition.

[0094] Each solution was incubated under ambient conditions, and the chemical stability of compound 1 was monitored for up to two hours, as measured by removing a small portion of the solution and quantifying the remaining compound 1 using LCMS/MS. While the compound showed stability in organic solvents (DMSO or ethanol, FIGS. 2A-B, with approximately 100% of the compound remaining after 2 hours), rapid decomposition occurred in aqueous solvents, with less than 40% of compound 1 remaining after 2 h in PBS (FIG. 2C) and less than 40% of compound 1 remaining after 20 minutes in phosphate buffer (FIG. 2D). Strikingly, in the stabilized composition, approximately 100% of compound 1 was still remaining after 1 hour. This stabilization in a nontoxic aqueous solution is necessary to enable dosing of compound 1 into humans or other animals, as organic solvents such as DMSO and ethanol are toxic and are therefore not appropriate for dosing to humans, especially for long-term or repeated dosing schedules.

Example 4: Inconsistency of HeLa-based assay identifying connexin hemichannel inhibitors or modulators

[0095] This example establishes a need in the field for an improved assay for identifying compounds that inhibit or modulate connexin hemichannels. [0096] As described in Example 1 , the HeLa-based assay requires long incubation times of up to 24 hours, making it an inefficient assay for identifying connexin hemichannel inhibitors or modulators. In addition, the use of HeLa cells was shown to yield quantitatively inconsistent results from one experiment to the next. For instance, a single experimental group yielded drastically different fluorescence intensity values across four separate replicates of said experiment (see FIG. 3, experiments N1-N4).

[0097] In conclusion, although the HeLa assay can provide a reliable results between groups in a single experiment, greater consistency is desirable across different experiments such that data between experiments can be reasonably compared.

Example 5: Yeast-based screening assay for connexin hemichannel inhibitors or modulators

[0098] This example demonstrates a yeast-based assay for analyzing inhibition of connexin hemichannels as an improvement over the state of the art HeLa cell assay described in Examples 1 and 4.

Genetically engineered yeast strain

[0099] Genes trkl and trk2 encode potassium ion transporters in various yeast strains. Starting from Saccharomyces cerevisiae BY4741, trkl was replaced using a cassette containing GJA1 (encoding connexin 43) and hphMX (encoding hygromycin resistance) genes, and trkl was replaced with a cassette containing natMX (encoding nourseothricin resistance), using the yeast’s endogenous homologous recombination machinery. Primers with a 30 bp homology sequence were used to amplify each of the cassettes used in the recombination process. The resulting modified strain was maintained in YPD culture medium supplemented with 50 mM KC1.

Yeast-based assay

[0100] An experiment similar to that described in Example 1 was conducted using the genetically engineered yeast cells.

[0101] Engineered yeast cells were grown overnight in YPD medium (10 g yeast extract, 20 g peptone, and 20 g dextrose in 1 L distilled water) supplemented with 50 mM KC1. The culture was then diluted to an optical density of 1.0 at 600nm. After diluting, two portions of a desired volume of cells was centrifuged for 10 seconds at 15000 x g. One portion of cells was resuspended in the same volume of Krebs solution; the second portion was resuspended in the same volume of divalent cation free Krebs solution (DCFS). 50 m L of suspension was added per well of a 96-well plate with optical bottom (6 replicates for each condition tested). Finally, 50 m L of a DCFS solution containing 10 m M ethidium bromide, or containing 10 mM ethidium bromide and 20 mM of the drug, was added per well. The plate was incubated for 1 to 5 minutes, and the hemichannel activity was determined, measured as fluorescence intensity in a plate reader using a 285 nm excitation and 605 nm emission configuration. Hemichannel activity was measured as the percentage of fluorescence intensity relative to a DCFS solution without any inhibitor (see FIG. 4A).

[0102] This yeast-based assay significantly reduced the time required (relative to the HeLa based assay), while simultaneously providing more consistently reliable quantitative results (data not shown). Moreover, using this yeast-based assay, compound 1 decreased the connexin hemichannel activity by about 30%, and compound 2 decreased the connexin hemichannel activity by about 50% (see FIG. 4B).

Example 6. Pharmacokinetic properties of compound 2 after dosing with a stabilized composition of compound 1 or compound 2.

[0103] C57BL6 mice were orally dosed with a stabilized composition comprising compound 1 (10 mg compound 1 per mL of 30% PEG400/20% polyethoxylated castor oil/50% PBS, dosed at 20 mg compound 1 per kg of mouse) or compound 2 (5 mg compound 2 per mL of 30% PEG400/20% polyethoxylated castor oil/50% PBS, dosed at 10 mg compound 2 per kg of mouse). Since compound 1 decomposes into active compound 2 in the animal, the concentration of active compound 2 remaining in the mouse was measured over the course of 8 hours in each case by LCMS/MS (see FIG. 5A-B). The half-life of compound 2, after dosing compound 2, was 69 minutes. Dosing the animal with compound 1 increased the half-life of compound 2 to 81 minutes.

[0104] To determine if the stabilized composition of compound 1 was suitable for intravenous (IV) or intraperitoneal (IP) administration, C57BL6 mice (Mus musculus) were dosed with 2 mg/kg compound 1 (IV) or 10 mg/kg compound 1 (IP). The concentration of active compound 2 remaining in the mouse was measured over time (see FIG. 5C-D).

Example 7. Orally dosing connexin hemichannel inhibitors in an mdx model of muscular dystrophy

[0105] This example demonstrates the effect of administering compositions of compound 1 or compound 2 on connexin hemichannel activity in vivo, using a mouse model of muscular dystrophy as a model system.

[0106] The mdx mouse is a genetically engineered dystrophin -negative mouse which is a common model for muscular dystrophy, particularly Duchenne muscular dystrophy. While normal skeletal myofibers do not present connexin hemichannel activity, recent research has shown that mdx mice express elevated levels of connexins and connexin hemichannels, which is correlated with an increase in necrotic features (such as cellular size and increase in creatine kinase activity), causing poor muscle performance (such as poor performance in a hanging test) relative to mdx mice that are deficient in connexins (see Cea et. al., Cell. Mol. Life. Sci. 2016. 73, 2583-2599). Thus, the mdx mouse is an ideal model system for evaluating potential hemichannel inhibitors.

[0107] In order to determine the activity of D4R with the new formulation, mice (Mus musculus) strain B6Ros.Cg-DMD^{mdx 4Cv} /J ( mdx mouse) were used to assess its potential as a treatment for Duchenne Muscular Distrophy. Transgenic mice were separated in two groups of nine each and were dosed either compound 1 prepared in the stabilized composition, or dosed with the vehicle. As a control, five C57BL6 mice were dosed with the vehicle.

[0108] The solution was prepared daily and mice were orally dosed with a stabilized composition comprising compound 1 (10 mg compound 1 per mL of 30% PEG400/20% polyethoxylated castor oil/50% PBS, dosed at 100 mg compound 1 per kg of mouse) daily from week 5 until week 13 of age. Mice were monitored regularly and showed no behavioral changes and no signs of toxicity through the course of the experiment (as determined by monitoring weight of each animal, see FIG. 6A). Once per week, a muscular functionality test known as “hanging test” was performed, as described below. Performance of the treated mdx mice was compared to performance of wild-type mice (normal muscle function) dosed with vehicle and mdx mice (impaired muscle function) dosed with vehicle.

[0109] A similar experiment was conducted with mdx mice orally dosed with a composition comprising compound 2 (5 mg compound 2 per mL of 30% PEG400/20% polyethoxylated castor oil/50% PBS, dosed at 50 mg compound 2 per kg of mouse), every day for eight weeks. At the start of the experiment, and at the end of each subsequent week, each mouse was subjected to a hanging test, as described below.

[0110] Hanging Test: Each mouse was placed on a wire, and the amount of time the mouse was able to hang from the wire without falling (up to 20 minutes total) was measured (see FIG. 6B). Each mouse was provided with three attempts, with a one minute rest between attempts. Attempts from the mice of each category (treated mdx mice dosed with a composition of compound 1 or compound 2, wild- type dosed with vehicle only, and mdx dosed with vehicle only) were averaged and plotted over time.

[0111] Treatment with either compound 1 compound 2 significantly improved performance in the hanging test, relative to untreated mdx mice dosed with vehicle (see FIG. 6C and FIG. 6E, respectively). For instance, after eight weeks of daily treatment, compound 1 provided more than a 3-fold improvement in hanging time relative to untreated mdx mice dosed with vehicle (see FIG. 6C-D), and compound 2 provided more than a 4-fold improvement in hanging time (see FIG. 6F).

[0112] After completion of the hanging test experiment, the serum creatine kinase activity and atrogen levels in muscle tissue were also measured, as markers of tissue necrosis. Creatine kinase was measured using a commercially available kit. Atrogen levels were determined by isolating and homogenizing muscular tissue samples and detecting atrogen by western blot. As demonstrated in FIGS. 7A-B, untreated mdx mice showed more necrotic features than treated mdx mice or wild type mice.

[0113] Histological analysis of the muscle fibers of treated mice also showed significant improvements in morphology. FIG. 8 illustrates muscle tissue slices obtained from three wild-type mice treated with vehicle (al-a3), three mdx mice treated with stabilized compositions of compound 1 (bl-b3), and three mdx mice treated with vehicle (cl-c3). This improvement in muscle cell morphology reflects mitigation of the increased cell necrosis caused by active connexin hemichannels in the muscular tissue.

Claims

CLAIMS What is claimed as new and desired to be protected by Letters Patent of the United States is:

1. A stabilized composition comprising a compound of Formula (I) or a stereoisomer thereof

a hydrophilic polymer, a nonionic surfactant, and a solvent.

2. The composition of claim 1, wherein the compound of Formula (I) is (R)-2-(4-chlorophenyl)-2- oxo- 1 -phenylethyl quinoline-2-carboxylate

3. The composition of claim 1, wherein the composition is substantially free of (S)-2-(4- chlorophenyl)-2-oxo- 1 -phenylethyl quinoline-2-carboxylate.

4. The composition of any of the preceding claims, wherein the hydrophilic polymer is selected from the group consisting of polyacrylamide, polyvinyl pyrrolidone, polyvinyl alcohol, polyacrylic acid, polyethylene glycol, and polypropylene glycol.

5. The composition of any of the preceding claims, wherein the hydrophilic polymer comprises polyethylene glycol.

6. The composition of any of the preceding claims, wherein the hydrophilic polymer has an average molecular weight of between about 100 and about 2000.

7. The composition of any of the preceding claims, wherein the hydrophilic polymer has an average molecular weight of between about 200 and about 600.

8. The composition of any of the preceding claims, wherein the hydrophilic polymer has an average molecular weight of between about 380 and about 420.

9. The composition of any of the preceding claims, wherein the hydrophilic polymer comprises between about 1 % and about 50% of the composition by volume.

10. The composition of any of the preceding claims, wherein the hydrophilic polymer comprises between about 10% and about 50% of the composition by volume.

11. The composition of any of the preceding claims, wherein the hydrophilic polymer comprises between about 20% and about 40% of the composition by volume.

12. The composition of any of the preceding claims, wherein the hydrophilic polymer comprises about 30% of the composition by volume.

13. The composition of any of the preceding claims, wherein the nonionic surfactant is a poloxamer, a polysorbate, or an ethoxylated fatty acid.

14. The composition of claim 13, wherein the poloxamer is polyethoxylated castor oil.

15. The composition of any of the preceding claims, wherein the nonionic surfactant comprises between about 1 % and about 50% of the composition by volume.

16. The composition of any of the preceding claims, wherein the nonionic surfactant comprises between about 5% and about 40% of the composition by volume.

17. The composition of any of the preceding claims, wherein the nonionic surfactant comprises between about 10% and about 30% of the composition by volume.

18. The composition of any of the preceding claims, wherein the nonionic surfactant comprises about 20% of the composition by volume.

19. The composition of any of the preceding claims, wherein the solvent comprises water, saline, or a buffer solution.

20. The composition of any of the preceding claims, wherein the solvent comprises phosphate buffered saline.

21. The composition of any of the preceding claims, wherein the solvent comprises between about 50% and about 98% of the composition by volume.

22. The composition of any of the preceding claims, wherein the solvent comprises about 50% of the composition by volume.

23. The composition of any of the preceding claims, wherein the compound of Formula (I) is present at a concentration between about 0.0004 mg/mL and about 10 mg/mL.

24. The composition of any of the preceding claims, wherein 2-(4-chlorophenyl)-2-oxo-l- phenylethyl quinoline-2-carboxylate is chemically stable for about 1 hour under ambient conditions.

25. A method of stabilizing 2-(4-chlorophenyl)-2-oxo-l-phenylethyl quinoline-2-carboxylate or a stereoisomer thereof in a composition, the method comprising mixing 2-(4-chlorophenyl)-2-oxo-l- phenylethyl quinoline-2-carboxylate or a stereoisomer thereof with a hydrophilic polymer and a nonionic surfactant in a solvent.

26. The method of claim 25, wherein the hydrophilic polymer is selected from the group consisting of polyacrylamide, polyvinyl pyrrolidone, polyvinyl alcohol, polyacrylic acid, polyethylene glycol, and polypropylene glycol.

27. The method of claim 25 or 26, wherein the hydrophilic polymer comprises polyethylene glycol.

28. The method of any of claims 25-27, wherein the hydrophilic polymer has an average molecular weight of between about 100 and about 2000.

29. The method of any of claims 25-28, wherein the hydrophilic polymer has an average molecular weight of between about 200 and about 600.

30. The method of any of claims 25-29, wherein the hydrophilic polymer has an average molecular weight of between about 380 and about 420.

31. The method of any of claims 25-30, wherein the hydrophilic polymer comprises between 1% and 50% of the composition by volume.

32. The method of any of claims 25-31, wherein the hydrophilic polymer comprises between 10% and 50% of the composition by volume.

33. The method of any of claims 25-32, wherein the hydrophilic polymer comprises between 20% and 40% of the composition by volume.

34. The method of any of any of claims 25-33, wherein the hydrophilic polymer comprises about 30% of the composition by volume.

35. The method of any of claims 25-34, wherein the nonionic surfactant is a poloxamer, a polysorbate, or an ethoxylated fatty acid.

36. The method of claim 35, wherein the poloxamer is polyethoxylated castor oil.

37. The method of any of claims 25-35, wherein the nonionic surfactant comprises between about 1% and about 50% of the composition by volume.

38. The method of any of any of claims 25-37, wherein the nonionic surfactant comprises between about 5% and about 40% of the composition by volume.

39. The method of any of claims 25-38, wherein the nonionic surfactant comprises between about 10% and about 30% of the composition by volume.

40. The method of any of claims 25-39, wherein the nonionic surfactant comprises about 20% of the composition by volume.

41. The method of any of claims 25-40, wherein the solvent comprises water, saline, or a buffer solution.

42. The method of any of claims 25-41, wherein the solvent comprises phosphate buffered saline.

43. The method of any of claims 25-42, wherein the solvent comprises between about 50% and 98% of the composition by volume.

44. The method of any of claims 25-43, wherein the solvent comprises about 50% of the composition by volume.

45. The method of any of claims 25-44, wherein 2-(4-chlorophenyl)-2-oxo-l-phenylethyl quinoline- 2-carboxylate is present at a concentration between about 0.0004 mg/mL and about 10 mg/mL.

46. The method of any of claims 25-45, wherein the method comprises mixing 2-(4-chlorophenyl)-2- oxo-l-phenylethyl quinoline-2-carboxylate or a stereoisomer thereof sequentially with the hydrophilic polymer, the nonionic surfactant, and the solvent.

47. The method of any of claims 25-46, wherein mixing comprises sonication, vortexing, or a combination of sonication and vortexing.

48. The method of claim any of claims 25-47, wherein 2-(4-chlorophenyl)-2-oxo-l-phenylethyl quinoline-2-carboxylate is chemically stabilized for about 1 hour under ambient conditions.

49. A unit dosage form configured for administration to an individual in need thereof, comprising a composition of any one of claims 1-24.

50. The unit dosage form of claim 49, wherein the unit dosage form is configured for oral administration.

51. The unit dosage form of claim 50, wherein the unit dosage form is configured for daily administration.

52. The unit dosage form of claim 49, wherein the unit dosage form is configured for intravenous administration.

53. The unit dosage form of claim 49, wherein the unit dosage form is configured for intraperitoneal administration.

54. The unit dosage form of any of claims 49-53, comprising between about 1 mg and about 200 mg of 2-(4-chlorophenyl)-2-oxo-l-phenylethyl quinoline -2-carboxylate or a stereoisomer thereof per kilogram of the individual.

55. The unit dosage form of any of claims 49-54, comprising about 2 mg, about 50 mg, about 100 mg, or about 200 mg of 2-(4-chlorophenyl)-2-oxo-l-phenylethyl quinoline-2-carboxylate or a stereoisomer thereof per kilogram of the individual.

56. A method of treating a disease associated with overactivation of connexin hemichannels in an individual in need thereof, comprising administering to the individual a composition of any of claims 1 - 24, or a unit dosage form of any of claims 49-55.

57. The method of claim 56, wherein the composition or unit dosage form comprises between about 1 mg and about 200 mg of 2-(4-chlorophenyl)-2-oxo-l-phenylethyl quinoline-2-carboxylate or a stereoisomer thereof per kilogram of the individual

58. The method of claim 56 or 57, wherein the composition or unit dosage form comprises about 2 mg, about 50 mg, about 100 mg, or about 200 mg of 2-(4-chlorophenyl)-2-oxo-l-phenylethyl quinoline - 2-carboxylate or a stereoisomer thereof per kilogram of the individual.

59. The method of any one of claims 56-58, wherein the disease is selected from the group consisting of: inflammatory diseases, neurodegenerative disorders, vascular disorder, arrhythmia, chronic injury, retinal neuroprotection, treatment of pain, skeletal muscle denervation, scarred tissue, muscular dystrophy, post-ischemia reperfusion injury, damage to the spinal cord, and any genetic disease characterized by increased activity of connexin hemichannels.

60. The method of claim 59, wherein the inflammatory disease is selected from the group consisting of: epilepsy, type II diabetes, shock, and inflammatory ocular disorders.

61. The method of claim 59, wherein the neurodegenerative disorder is selected from the group consisting of Alzheimer’s disease and Parkinson’s disease.

62. The method of any of claims 56-59, wherein the disease is selected from the group consisting of: vascular disorder, arrhythmia, chronic injury, post-ischemia reperfusion injury, spinal damage, and genetic disease characterized by increased activity of connexin hemichannels.

63. The method of any of claims 56-59, wherein the disease is muscular dystrophy.

64. The method of any of claims 56-59 or 63, wherein the disease is Duchenne muscular dystrophy.

65. A composition comprising quinaldic acid, a hydrophilic polymer, a nonionic surfactant, and a solvent.

66. The composition of claim 65, wherein the hydrophilic polymer is selected from the group consisting of polyacrylamide, polyvinyl pyrrolidone, polyvinyl alcohol, polyacrylic acid, polyethylene glycol, and polypropylene glycol.

67. The composition of claim 65 or 66, wherein the hydrophilic polymer comprises polyethylene glycol.

68. The composition of any of claims 65-67, wherein the hydrophilic polymer has an average molecular weight of between about 100 and about 2000.

69. The composition of any of claims 65-68, wherein the hydrophilic polymer has an average molecular weight of between about 200 and about 600.

70. The composition of any of claims 65-69, wherein the hydrophilic polymer has an average molecular weight of between about 380 and about 420.

71. The composition of any of claims 65-70, wherein the hydrophilic polymer comprises between about 1 % and about 50% of the composition by volume.

72. The composition of any of claims 65-71, wherein the hydrophilic polymer comprises between about 10% and about 50% of the composition by volume.

73. The composition of any of claims 65-72, wherein the hydrophilic polymer comprises between about 20% and about 40% of the composition by volume.

74. The composition of any of claims 65-73, wherein the hydrophilic polymer comprises about 30% of the composition by volume.

75. The composition of any of claims 65-74, wherein the nonionic surfactant is a poloxamer, a polysorbate, or an ethoxylated fatty acid.

76. The composition of claim 75, wherein the poloxamer is polyethoxylated castor oil.

77. The composition of any of claims 65-76, wherein the nonionic surfactant comprises between about 1 % and about 50% of the composition by volume.

78. The composition of any of claims 65-77, wherein the nonionic surfactant comprises between about 5% and about 40% of the composition by volume.

79. The composition of any of claims 65-78, wherein the nonionic surfactant comprises between about 10% and about 30% of the composition by volume.

80. The composition of any of claims 65-79, wherein the nonionic surfactant comprises about 20% of the composition by volume.

81. The composition of any of claims 65-80, wherein the solvent comprises water, saline, or a buffer solution.

82. The composition of any of claims 65-81, wherein the solvent comprises phosphate buffered saline.

83. The composition of any of claims 65-82, wherein the solvent comprises between about 50% and about 98% of the composition by volume.

84. The composition of any of claims 65-83, wherein the solvent comprises about 50% of the composition by volume.

85. The composition of any of claims 65-84, wherein quinaldic acid is present at a concentration between about 0.0001 mg/mL and about 10 mg/mL.

86. The composition of any of claims 65-85, wherein quinaldic acid is present at a concentration of about 5 mg/mL.

87. A unit dosage form configured for administration to an individual in need thereof, comprising a composition of any one of claims 65-86.

88. The unit dosage form of claim 87, wherein the unit dosage form is configured for oral administration.

89. The unit dosage form of claim 88, wherein the unit dosage form is configured for daily administration.

90. The unit dosage form of claim 87, wherein the unit dosage form is configured for intravenous administration.

91. The unit dosage form of claim 87, wherein the unit dosage form is configured for intraperitoneal administration.

92. The unit dosage form of any of claims 87-91, comprising between about 1 mg and about 100 mg of quinaldic acid per kilogram of the individual.

93. The unit dosage form of any of claims 87-92, comprising about 2 mg or about 50 mg of quinaldic acid per kilogram of the individual.

94. A method of treating a disease associated with the overactivation of connexin hemichannels in an individual, comprising administering to the individual in need thereof a therapeutically effective amount of quinaldic acid, a composition comprising quinaldic acid of any of claims 65-86, or a unit dosage form of quinaldic acid of any of claims 87-93.

95. The method of claim 94, wherein the disease is selected from the group consisting of: inflammatory diseases, neurodegenerative disorders, vascular disorder, arrhythmia, chronic injury, retinal neuroprotection, treatment of pain, skeletal muscle denervation, scarred tissue, muscular dystrophy, post- ischemia reperfusion injury, damage to the spinal cord, and any genetic disease characterized by increased activity of connexin hemichannels.

96. The method of claim 95, wherein the inflammatory disease is selected from the group consisting of: epilepsy, type II diabetes, shock, and inflammatory ocular disorders.

97. The method of claim 95, wherein the neurodegenerative disorder is selected from the group consisting of Alzheimer’s disease and Parkinson’s disease.

98. The method of claim 94 or 95, wherein the disease is selected from the group consisting of: vascular disorder, arrhythmia, chronic injury, post-ischemia reperfusion injury, spinal damage, and genetic disease characterized by increased activity of connexin hemichannels.

99. The method of any of claims 94-95, wherein the disease is muscular dystrophy.

100. The method of any of claims 94-95 or 99, wherein the disease is Duchenne muscular dystrophy.

101. The method of any of claims 94-100, wherein the individual has an elevated level of connexin hemichannel activity in at least one tissue, compared to a healthy state of that tissue.

102. The method of claim 101, wherein elevated connexin hemichannel activity is characterized by elevated serum creatine kinase activity.

103. The method of claim 102, wherein elevated serum creatine kinase activity in an individual is characterized by a 10- to 100-fold increase relative to normal levels for that individual.

104. The method of any of claims 94-103, wherein elevated connexin hemichannel activity is characterized by elevated atrogen levels in at least one tissue of the individual, relative to a healthy tissue.

105. The method of any of claims 101-104, further comprising determining whether the individual has an elevated level of connexin hemichannel activity in at least one tissue, compared to a healthy state of that tissue, prior to administering a therapeutically effective amount of quinaldic acid, or a composition or a dosage form comprising quinaldic acid, to the individual in need thereof.

106. The method of claim 105, wherein determining whether the individual has an elevated level of connexin hemichannel in at least one tissue comprises isolating serum from the individual and measuring creatine kinase activity using a creatine kinase activity assay.

107. The method of claim 105, wherein determining whether the individual has an elevated level of connexin hemichannel in at least one tissue comprises isolating muscle tissue from the individual and measuring atrogen protein levels by Western blot.

108. A method of modulating connexin hemichannel activity in an individual, comprising administering to the individual an effective amount of quinaldic acid, a composition comprising quinaldic acid of any of claims 65-86 or a unit dosage form of quinaldic acid of any of claims 87-93.

109. The method of claim 108, wherein the individual has a disease selected from the group consisting of: vascular disorder, arrhythmia, chronic injury, post-ischemia reperfusion injury, spinal damage, and genetic disease characterized by increased activity of connexin hemichannels.

110. Use of quinaldic acid for modulating connexin hemichannel activity in an individual in need thereof.

111. The use according to claim 110, wherein the individual has an elevated level of connexin hemichannel activity in at least one tissue, compared to a healthy state of that tissue.

112. The use according to claim 111, wherein the elevated level of connexin hemichannel activity is associated with a disease selected from the group consisting of: vascular disorder, arrhythmia, chronic injury, post-ischemia reperfusion injury, spinal damage, and genetic disease characterized by increased activity of connexin hemichannels.

113. The use according to claim 112, wherein the disease is muscular dystrophy.

114. The use according to claim 112 or 113, wherein the disease is Duchenne muscular dystrophy.

115. A genetically engineered yeast capable of producing connexin 43, wherein the yeast comprises a gene encoding human connexin 43.

116. The genetically engineered yeast of claim 115, wherein the yeast is derived from Saccharomyces cerevisiae BY4741.

117. The genetically engineered yeast of claim 115 or 116, wherein the gene encoding human connexin 43 is GJA1.

118. The genetically engineered yeast of any of claims 115-117, wherein the yeast has been genetically engineered to lack functional potassium channels.

119. The genetically engineered yeast of any of claims 115-118, wherein genes encoding the functional potassium channels have been deleted.

120. The genetically engineered yeast of claim 118 or 119, wherein the potassium channels are encoded by trkl and trk2.

121. A method of screening compounds to identify compounds that modulate connexin hemichannel activity, comprising conducting an assay comprising the steps of:

(a) providing to a yeast cell of any of claims 115-120 a condition which favors an open state of the connexin hemichannel;

(b) applying to the yeast cell a reporter that is yeast-impermeable or poorly permeable;

(c) applying to the yeast cell a compound of interest; and (d) quantifying the accumulation of the reporter in the yeast cell; wherein the assay is suitable for screening more than one compound simultaneously.

122. The method of claim 121, further comprising conducting a first control assay comprising the steps of:

(a) providing to a yeast cell of any of claims 115-120 a condition which favors an open state of the connexin hemichannel;

(b) applying to the yeast cell a reporter that is yeast-impermeable or poorly permeable; and

(c) quantifying the accumulation of the reporter in the yeast cell.

123. The method of claim 121 or 122, further comprising conducting a second control assay comprising the steps of:

(a) providing to a yeast cell of any of claims 115-120 a condition which disfavors an open state of the connexin hemichannel;

(b) applying to the yeast cell a reporter that is yeast-impermeable or poorly permeable; and

(c) quantifying the accumulation of the reporter in the yeast cell.

124. The method of any of claims 121-123, wherein at least one step of each assay is configured in a multiwell plate or array.

125. The method of claim 124, wherein the at least one step of each assay is configured in the same multiwell plate or array.

126. The method of any of claims 121-125, wherein the method is suitable for high throughput screening.

127. The method of any of claims 121-126, wherein the condition which favors the open state of the connexin hemichannel comprises divalent cation-free solution.

128. The method of any of claims 123-127, wherein the condition which disfavors the open state of the connexin hemichannel comprises a divalent cation solution.

129. The method of any claims 121-128, wherein the reporter comprises a fluorescent or colorimetric dye.

130. The method of any of claims 121-129, wherein the reporter comprises ethidium bromide.

131. The method of any of claims 121-130, wherein quantifying the accumulation of the reporter in the yeast cell comprises measuring absorption intensity.

132. The method of any of claims 121-130, wherein quantifying the accumulation of the reporter in the yeast cell comprises measuring fluorescence intensity.

133. The method of any of the preceding claims, wherein the assay is completed in between about 1 minutes and about 5 minutes.